We investigated for the first time the subtype distribution, prevalence of multiple HIV-1 infections, sexual networks, and partnership histories in a cohort of women engaged in high-risk sexual behavior such as female sex workers (FSWs) and women employed in entertainment facilities. Viral RNA was extracted from blood samples collected from 324 HIV-1-positive women; the gp-41 and pol-IN genes were directly sequenced. Women found to have closely related viruses and those with recombinant viruses were further analyzed in the pol-IN gene by clonal sequencing to determine HIV-1 multiple infections. Individual partnership histories were used to provide information on when sex work was undertaken and where. Subtyping in both gp-41 and pol-IN was successfully done in 210/324 (64.8%) women. Subtype distribution in these two genes was 54.3% (n=114) A/A, 2.9% (n=6) C/C, 24.3% (n=51) D/D, 11.9% (n=25) A/D, 4.8% (n=10) D/A, 0.5% (n=1) C/A, 1.0% (n=2) B/A, and 0.5% (n=1) B/D. Sexual networks were identified in six pairs and one triplet of women with closely related subtype A viruses. Partnership histories showed that women having phylogenetically similar viruses had worked in the same localities. Five cases of multiple infections were confirmed: four dual infections and one triple infection. In this first molecular epidemiology study among FSWs in Kampala, subtype A was the predominant subtype. About 9% of a subgroup had multiple infections. Partnership histories and multiple infections observed in this population suggest sexual mixing of the FSWs and their clients confirming their high-risk characteristics.
Reagents for evaluating non-clade B HIV-specific T cell responses are uncommon. Peptides based on highly conserved HIV-1 consensus group M sequences that are phylogenetically closer to most circulating strains may provide potential alternative reagents in populations with diverse infections, and may be relevant for vaccine design. Recognition of such reagents in clade A1-and D-infected populations has not been previously evaluated. Interferon (IFN)-γ ELISpot assay was used to evaluate T cell recognition of Gag and Nef peptides based on consensus group M sequences in 50 treatment-naive adults predominantly infected with HIV-1 clades A1 and D. Gag-induced T cell responses were correlated with gag sequence diversity. Infecting clades were determined from gag sequences for 45 of the 50 subjects as 40% clade A1 (18/45), 45% clade D (20/45), 2% clade C (1/45), 2% A1/C recombinant (1/45), 2% A1/D (1/45), 7% CRF10_CD (3/45), and 2% U (unclassifiable) (1/45). The mean genetic divergence and diversity of clade A and D gag region compared to group M consensus sequences at synonymous and nonsynonymous nucleotide and amino acid levels were not always significant. Gag peptides were targeted at significantly higher frequency [88% (44/50)] than Nef [64% (32/50)]; p=0.014, although their mean IFN-γ magnitudes were comparable ([3703 (95% CI 2567-4839)] vs. [2120 (95% CI 478-3762)]), respectively. Measurable virus-induced IFN-γ responses were detected in 96% (48/50) individuals, primarily targeting the more conserved Gag p24 and Nef central core regions. Use of these reagents to screen for HIV-specific IFN-γ responses may mitigate the challenge of viral diversity; although this targeting is apparently biased toward a few highly conserved epitopes.
We investigated the rate of transmitted drug resistance (TDR) among HIV-1 seroconverters identified from the Rakai Community Cohort Study (RCCS) survey, a population-based cohort in Rakai District, Uganda. Participants aged 15–49 are interviewed at study visits approximately every 12–18 months and provided a serological sample. Antiretroviral therapy (ART) has been provided free of charge since 2004. RCCS participants with documented negative HIV-1 serology between January 2011 and August 2012 and confirmed seroconversion between November 2012 and October 2013 were included in this analysis. Serum was genotyped for HIV drug resistance mutations in reverse transcriptase and protease genes. Mutations were classified according to the 2009 World Health Organization surveillance of transmitted HIV-1 drug resistance update. Seventy-five (75) seroconverters were identified and genotyped. The mean age was 28 years (range 18–49) and the majority were male, n = 44 (58%). The HIV-1 subtype frequencies were A = 19 (25%), D = 44 (59%), C = 4 (5%), A/D recombinant = 5 (7%), and C/D recombinant = 3 (4%). The majority (72/75, 96%) of individuals were infected with wild-type virus with no evidence of TDR. Two individuals had a single non-nucleoside reverse transcriptase inhibitor mutation each, K101E and K103N, and one had a single protease inhibitor mutation, M46I. No mutations were identified involving nucleoside reverse transcriptase inhibitors. In conclusion, almost 10 years after the introduction of ART in rural Uganda, rates of TDR remain low. Ongoing surveillance for TDR remains an important public health priority and should be conducted among known seroconverters to estimate TDR.
Objective To investigate the role of HIV-1 envelope subtypes on disease progression in a rural cohort of Ugandan adults where two major HIV-1 subtypes (A and D) exist. Methods Participants of a clinical cohort seen between December 1995 and December 1998 had blood collected for HIV-1 subtyping. These included prevalent cases (people already infected with HIV at the start of the study in 1990) and incident cases (those who seroconverted between 1990 and December 1998). HIV-1 subtyping was carried out by heteroduplex mobility assay and DNA sequencing in the V3 env region. Disease progression was measured by the rate of CD4 lymphocyte count decline, clinical progression for the incident cases as time from seroconversion to AIDS or death, to first CD4 lymphocyte count < 200 × 106/l and to the World Health Organization clinical stage 3. All analyses were adjusted for age and sex. Results One hundred and sixty-four individuals, including 47 prevalent and 117 incident cases, had V3 env subtype data of which 65 (40%) were subtyped as A and 99 as D. In the incident cases, 44 (38%) were subtyped as A and 73 as D. There was a suggestion that for most end-points A had a slower progression than D. The cumulative probability of remaining free from AIDS or death at 6 years post-seroconversion was 0.72 [95% confidence interval (CI), 0.50 to 0.85] for A and 0.58 (95% CI, 0.42 to 0.71) for D, and the adjusted hazard ratio of subtype D compared to A was estimated to be 1.39 (95% CI, 0.66 to 2.94;P = 0.39). The estimated difference in rates of decline in square root CD4 lymphocyte counts was −0.41 per year (95% CI, −0.98 to 0.15;P = 0.15). Conclusion This study suggests that although subtype A may have a slower progression than D, HIV-1 envelope subtype is not a major factor in determining the progression of HIV-1 disease in a rural population in Uganda.
HIV care programs in resource-limited settings have hitherto concentrated on antiretroviral therapy (ART) access, but HIV drug resistance is emerging. In a cross-sectional study of HIV-positive adults on ART for ≥6 months enrolled into a prospective cohort in Uganda, plasma HIV RNA was measured and genotyped if ≥1000 copies/ml. Identified Drug resistance mutations (DRMs) were interpreted using the Stanford database, 2009 WHO list of DRMs and the IAS 2014 update on DRMs, and examined and tabulated by ART drug classes. Between July 2013 and August 2014, 953 individuals were enrolled, 119 (12.5%) had HIV-RNA ≥1000 copies/ml and 110 were successfully genotyped; 74 (67.3%) were on first-line and 36 (32.7%) on second-line ART regimens. The predominant HIV-1 subtypes were D (34.5%), A (33.6%) and Recombinant forms (21.8%). The commonest clinically significant major resistance mutations associated with the highest levels of reduced susceptibility or virological response to the relevant Nucleoside Reverse Transcriptase Inhibitor (NRTI) were; the Non-thymidine analogue mutations (Non-TAMS) M184V—20.7% and K65R—8.0%; and the TAMs M41L and K70R (both 8.0%). The major Non-NRTI (NNRTI) mutations were K103N—19.0%, G190A—7.0% and Y181C—6.0%. A relatively nonpolymorphic accessory mutation A98G—12.0% was also common. Seven of the 36 patients on second line ART had major Protease Inhibitor (PI) associated DRMS including; V82A—7.0%, I54V, M46I and L33I (all 5.0%). Also common were the accessory PI mutations L10I—27%, L10V—12.0% and L10F—5.0% that either reduce PI susceptibility or increase the replication of viruses containing PI-resistance mutations. Of the 7 patients with major PI DRMs, five had high level resistance to ritonavir boosted Lopinavir and Atazanavir, with Darunavir as the only susceptible PI tested. In resource-limited settings, HIV care programs that have previously concentrated on ART access, should now consider availing access to routine HIV viral load monitoring, targeted HIV drug resistance testing and availability of third-line ART regimens.
Background With the scale-up of antiretroviral therapy (ART), monitoring programme performance is needed to maximize ART efficacy and limit HIV drug resistance (HIVDR). Methods We implemented a WHO HIVDR prospective survey protocol at three treatment centers between 2012 and 2013. Data were abstracted from patient records at ART start (T1) and after 12 months (T2). Genotyping was performed in the HIV pol region at the two time points. Results Of the 425 patients enrolled, at T2, 20 (4.7%) had died, 66 (15.5%) were lost to follow-up, 313 (73.6%) were still on first-line, 8 (1.9%) had switched to second-line, 17 (4.0%) had transferred out and 1 (0.2%) had stopped treatment. At T2, 272 out of 321 on first and second line (84.7%) suppressed below 1000 copies/ml and the HIV DR prevention rate was 70.1%, just within the WHO threshold of ≥70%. The proportion of participants with potential HIVDR was 20.9%, which is higher than the 18.8% based on pooled analyses from African studies. Of the 35 patients with mutations at T2, 80% had M184V/I, 65.7% Y181C, and 48.6% (54.8% excluding those not on Tenofovir) had K65R mutations. 22.9% had Thymidine Analogue Mutations (TAMs). Factors significantly associated with HIVDR prevention at T2 were: baseline viral load (VL) <100,000 copies/ml [Adjusted odds ratio (AOR) 3.13, 95% confidence interval (CI): 1.36–7.19] and facility. Independent baseline predictors for HIVDR mutations at T2 were: CD4 count <250 cells/μl (AOR 2.80, 95% CI: 1.08–7.29) and viral load ≥100,000 copies/ml (AOR 2.48, 95% CI: 1.00–6.14). Conclusion Strengthening defaulter tracing, intensified follow-up for patients with low CD4 counts and/or high VL at ART initiation together with early treatment initiation above 250 CD4 cells/ul and adequate patient counselling would improve ART efficacy and HIVDR prevention. The high rate of K65R and TAMs could compromise second line regimens including NRTIs.
A pilot study was undertaken with the objective of developing a simple, economical, and efficient algorithm through which to subtype HIV-1 in a large epidemiological cohort study in Uganda. A peptide enzyme immunoassay (PEIA) employing both V3 and gp41 regions and a heteroduplex mobility assay (HM A) were evaluated in comparison with DNA sequencing. Of 146 samples selected, 115 (79%) were successfully sequenced. Taking sequence data as the "gold standard" other assays were compared with these data. The HMA correctly identified 95 (83%) of the samples, and only 1 sample was wrongly identified. The V3 PEIA alone and in combination with gp41 peptides correctly identified 76 and 78% of the samples, respectively; however, the number of wrongly identified samples was four times less with the combination compared with V3 peptides alone (4 versus 16%). The sensitivity, specificity, and positive and negative predictive values for serotype A and D samples were greater for the combination than V3 peptides alone. We have described a new algorithm to segregate subtypes A and D. This algorithm uses the two peptide assays followed by HMA and then DNA sequencing for untypable samples, giving an accuracy of 95% at a cost of 37 and 21% for consumables compared with subtyping all the samples by HMA or DNA sequencing, respectively. This proposed approach is suitable for epidemiological studies in Uganda and other regions with a predominance of A and D subtypes.
Background Fishing communities around Lake Victoria in sub-Saharan Africa have been characterised as a population at high risk of HIV-infection. Methods Using data from a cohort of HIV-positive individuals aged 13–49 years, enrolled from 5 fishing communities on Lake Victoria between 2009–2011, we sought to identify factors contributing to the epidemic and to understand the underlying structure of HIV transmission networks. Clinical and socio-demographic data were combined with HIV-1 phylogenetic analyses. HIV-1 gag-p24 and env-gp-41 sub-genomic fragments were amplified and sequenced from 283 HIV-1-infected participants. Phylogenetic clusters with ≥2 highly related sequences were defined as transmission clusters. Logistic regression models were used to determine factors associated with clustering. Results Altogether, 24% (n = 67/283) of HIV positive individuals with sequences fell within 34 phylogenetically distinct clusters in at least one gene region (either gag or env). Of these, 83% occurred either within households or within community; 8/34 (24%) occurred within household partnerships, and 20/34 (59%) within community. 7/12 couples (58%) within households clustered together. Individuals in clusters with potential recent transmission (11/34) were more likely to be younger 71% (15/21) versus 46% (21/46) in un-clustered individuals and had recently become resident in the community 67% (14/21) vs 48% (22/46). Four of 11 (36%) potential transmission clusters included incident-incident transmissions. Independently, clustering was less likely in HIV subtype D (adjusted Odds Ratio, aOR = 0.51 [95% CI 0.26–1.00]) than A and more likely in those living with an HIV-infected individual in the household (aOR = 6.30 [95% CI 3.40–11.68]). Conclusions A large proportion of HIV sexual transmissions occur within house-holds and within communities even in this key mobile population. The findings suggest localized HIV transmissions and hence a potential benefit for the test and treat approach even at a community level, coupled with intensified HIV counselling to identify early infections.
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