HIV-1 subtype B and CRF01_AE are the predominant infecting subtypes among men who have sex with men (MSM) in Singapore. The genetic history, population dynamics and pattern of transmission networks of these genotypes remain largely unknown. We delineated the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and the recently characterized CRF51_01B strains circulating among the MSM population in Singapore. A total of 105 (49.5%) newly-diagnosed treatment-naïve MSM were recruited between February 2008 and August 2009. Phylogenetic reconstructions of the protease gene (HXB2: 2239 – 2629), gp120 (HXB2: 6942 – 7577) and gp41 (HXB2: 7803 – 8276) of the env gene uncovered five monophyletic transmission networks (two each within subtype B and CRF01_AE and one within CRF51_01B lineages) of different sizes (involving 3 – 23 MSM subjects, supported by posterior probability measure of 1.0). Bayesian coalescent analysis estimated that the emergence and dissemination of multiple sub-epidemic networks occurred between 1995 and 2005, driven largely by subtype B and later followed by CRF01_AE. Exponential increase in effective population size for both subtype B and CRF01_AE occurred between 2002 to 2007 and 2005 to 2007, respectively. Genealogical estimates suggested that the novel CRF51_01B lineages were probably generated through series of recombination events involving CRF01_AE and multiple subtype B ancestors. Our study provides the first insight on the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and CRF51_01B viral strains circulating among MSM in Singapore.
We analyzed antiretroviral drug susceptibility in HIV-infected adults failing first- and second-line antiretroviral treatment (ART) in Rakai, Uganda. Samples obtained from participants at baseline (pretreatment) and at the time of failure on first-line ART and second-line ART were analyzed using genotypic and phenotypic assays for antiretroviral drug resistance. Test results were obtained from 73 samples from 38 individuals (31 baseline samples, 36 first-line failure samples, and six second-line failure samples). Four (13%) of the 31 baseline samples had mutations associated with resistance to nucleoside or nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs, respectively). Among the 36 first-line failure samples, 31 (86%) had NNRTI resistance mutations and 29 (81%) had lamivudine resistance mutations; only eight (22%) had other NRTI resistance mutations. None of the six individuals failing a second-line protease inhibitor (PI)-based regimen had PI resistance mutations. Six (16%) of the participants had discordant genotypic and phenotypic test results. Genotypic resistance to drugs included in first-line ART regimens was detected prior to treatment and among participants failing first-line ART. PI resistance was not detected in individuals failing second-line ART. Surveillance for transmitted and acquired drug resistance remains a priority for scale-up of ART.
Accurate methods of HIV incidence determination are critically needed to monitor the epidemic and determine the population level impact of prevention trials. One such trial, Project Accept, a Phase III, community-randomized trial, evaluated the impact of enhanced, community-based voluntary counseling and testing on population-level HIV incidence. The primary endpoint of the trial was based on a single, cross-sectional, post-intervention HIV incidence assessment.
Methods and Findings
Test performance of HIV incidence determination was evaluated for 403 multi-assay algorithms [MAAs] that included the BED capture immunoassay [BED-CEIA] alone, an avidity assay alone, and combinations of these assays at different cutoff values with and without CD4 and viral load testing on samples from seven African cohorts (5,325 samples from 3,436 individuals with known duration of HIV infection [1 month to >10 years]). The mean window period (average time individuals appear positive for a given algorithm) and performance in estimating an incidence estimate (in terms of bias and variance) of these MAAs were evaluated in three simulated epidemic scenarios (stable, emerging and waning). The power of different test methods to detect a 35% reduction in incidence in the matched communities of Project Accept was also assessed. A MAA was identified that included BED-CEIA, the avidity assay, CD4 cell count, and viral load that had a window period of 259 days, accurately estimated HIV incidence in all three epidemic settings and provided sufficient power to detect an intervention effect in Project Accept.
In a Southern African setting, HIV incidence estimates and intervention effects can be accurately estimated from cross-sectional surveys using a MAA. The improved accuracy in cross-sectional incidence testing that a MAA provides is a powerful tool for HIV surveillance and program evaluation.
Background. A genetic bottleneck is known to exist for human immunodeficiency virus (HIV) at the point of sexual transmission. However, the nature of this bottleneck and its effect on viral diversity over time is unclear.
Methods. Interhost and intrahost HIV diversity was analyzed in a stable population in Rakai, Uganda, from 1994 to 2002. HIV-1 envelope sequences from both individuals in initially HIV-discordant relationships in which transmission occurred later were examined using Sanger sequencing of bulk polymerase chain reaction (PCR) products (for 22 couples), clonal analysis (for 3), and next-generation deep sequencing (for 9).
Results. Intrahost viral diversity was significantly higher than changes in interhost diversity (P < .01). The majority of HIV-1–discordant couples examined via bulk PCR (16 of 22 couples), clonal analysis (3 of 3), and next-generation deep sequencing (6 of 9) demonstrated that the viral populations present in the newly infected recipient were more closely related to the donor partner's HIV-1 variants found earlier during infection as compared to those circulating near the estimated time of transmission (P = .03).
Conclusions. These findings suggest that sexual transmission constrains viral diversity at the population level, partially because of the preferential transmission of ancestral as opposed to contemporary strains circulating in the transmitting partner. Future successful vaccine strategies may need to target these transmitted ancestral strains.
Accurate and reliable laboratory-based assays are needed for estimating HIV-1 incidence from cross-sectional samples. We recently described the development of a customized, HIV-1-specific Bio-Plex assay that allows for the measurement of HIV-specific antibody levels and avidity to multiple analytes for improved HIV-1 incidence estimates.
To assess intra- and inter-laboratory assay performance, prototype multiplex kits were developed and evaluated by three distinct laboratories. Longitudinal seroconversion specimens were tested in parallel by each laboratory and kit performance was compared to that of an in-house assay. Additionally, the ability of the kit to distinguish recent from long-term HIV-1 infection, as compared to the in-house assay, was determined by comparing the reactivity of known recent (infected <6 months) and long-term (infected >12 months) drug naïve specimens.
Although the range of reactivity for each analyte varied between the prototype kit and in-house assay, a measurable distinction in reactivity between recent and long-term specimens was observed with both assays in all three laboratories. Additionally, kit performance was consistent between all three laboratories. The intra-assay coefficient of variation (CV), between sample replicates for all laboratories, ranged from 0.5% to 6.1%. The inter-laboratory CVs ranged from 8.5% to 21.3% for gp160-avidity index (a) and gp120-normalized mean fluorescent intensity (MFI) value (n), respectively.
We demonstrate the feasibility of producing a multiplex kit for measuring HIV antibody levels and avidity, with the potential for improved incidence estimates based on multi-analyte algorithms. The availability of a commercial kit will facilitate the transfer of technology among diverse laboratories for widespread assay use.
Assays to determine cross-sectional HIV incidence misclassify some individuals with nonrecent HIV infection as recently infected, overestimating HIV incidence. We analyzed factors associated with false-recent misclassification in five African countries. Samples from 2197 adults from Botswana, Kenya, South Africa, Tanzania, and Uganda who were HIV infected >12 months were tested using the (1) BED capture enzyme immunoassay (BED), (2) avidity assay, (3) BED and avidity assays with higher assay cutoffs (BED+avidity screen), and (4) multiassay algorithm (MAA) that includes the BED+avidity screen, CD4 cell count, and HIV viral load. Logistic regression identified factors associated with misclassification. False-recent misclassification rates and 95% confidence intervals were BED alone: 7.6% (6.6, 8.8); avidity assay alone: 3.5% (2.7, 4.3); BED+avidity screen: 2.2% (1.7, 2.9); and MAA: 1.2% (0.8, 1.8). The misclassification rate for the MAA was significantly lower than the rates for the other three methods (each p<0.05). Misclassification rates were lower when the analysis was limited to subtype C-endemic countries, with the lowest rate obtained for the MAA [0.8% (0.2, 1.9)]. Factors associated with misclassification were for BED alone: country of origin, antiretroviral treatment (ART), viral load, and CD4 cell count; for avidity assay alone: country of origin; for BED+avidity screen: country of origin and ART. No factors were associated with misclassification using the MAA. In a multivariate model, these associations remained significant with one exception: the association of ART with misclassification was completely attenuated. A MAA that included CD4 cell count and viral load had lower false-recent misclassification than the BED or avidity assays (alone or in combination). Studies are underway to compare the sensitivity of these methods for detection of recent HIV infection.
The BED capture enzyme immunoassay (BED-CEIA) was developed for estimating HIV incidence from cross-sectional data. This assay misclassifies some individuals with nonrecent HIV infection as recently infected, leading to overestimation of HIV incidence. We analyzed factors associated with misclassification by the BED-CEIA. We analyzed samples from 383 men who were diagnosed with HIV infection less than 1 year after a negative HIV test (Multicenter AIDS Cohort Study). Samples were collected 2–8 years after HIV seroconversion, which was defined as the midpoint between the last negative and first positive HIV test. Samples were analyzed using the BED-CEIA with a cutoff of OD-n ≤0.8 for recent infection. Logistic regression was used to identify factors associated with misclassification. Ninety-one (15.1%) of 603 samples were misclassified. In multivariate models, misclassification was independently associated with highly active antiretroviral treatment (HAART) for >2 years, HIV RNA <400 copies/ml, and CD4 cell count <50 or <200 cells/mm3; adjusted odds ratios (OR) and 95% confidence intervals (CI) were 4.72 (1.35–16.5), 3.96 (1.53–10.3), 6.85 (2.71–17.4), and 11.5 (3.64–36.0), respectively. Among 220 men with paired samples, misclassification 2–4 years after seroconversion was significantly associated with misclassification 6–8 years after seroconversion [adjusted OR: 25.8 (95% CI: 8.17–81.5), p<0.001] after adjusting for race, CD4 cell count, HIV viral load, and HAART use. Low HIV viral load, low CD4 cell count, and >2 years of HAART were significantly associated with misclassification using the BED-CEIA. Some men were persistently misclassified as recently infected up to 8 years after HIV seroconversion.
Background. Human immunodeficiency virus (HIV) superinfection has been documented in high-risk individuals; however, the rate of superinfection among HIV-infected individuals within a general population remains unknown.
Methods. A novel next-generation ultra-deep sequencing technique was utilized to determine the rate of HIV superinfection in a heterosexual population by examining two regions of the viral genome in longitudinal samples from recent HIV seroconverters (n = 149) in Rakai District, Uganda.
Results. The rate of superinfection was 1.44 per 100 person years (PYs) (95% confidence interval [CI], .4–2.5) and consisted of both inter- and intrasubtype superinfections. This was compared to primary HIV incidence in 20 220 initially HIV-negative individuals in the general population in Rakai (1.15 per 100 PYs; 95% CI, 1.1–1.2; P = .26). Propensity score matching (PS) was used to control for differences in sociodemographic and behavioral characteristics between the HIV-positive individuals at risk for superinfection and the HIV-negative population at baseline and follow-up. After PS matching, the estimated rate of primary incidence was 3.28 per 100 PYs (95% CI, 2.0–5.3; P = .07) controlling for baseline differences and 2.51 per 100 PYs (95% CI, 1.5–4.3; P = .24) controlling for follow-up differences.
Conclusions. This suggests that the rate of HIV superinfection in a general population is substantial, which could have a significant impact on future public health and HIV vaccine strategies.
National Institute of Mental Health Project Accept (HIV Prevention Trials Network [HPTN] 043) is a large, Phase III, community-randomized, HIV prevention trial conducted in 48 matched communities in Africa and Thailand. The study intervention included enhanced community-based voluntary counseling and testing. The primary endpoint was HIV incidence, assessed in a single, cross-sectional, post-intervention survey of >50,000 participants.
HIV rapid tests were performed in-country. HIV status was confirmed at a central laboratory in the United States. HIV incidence was estimated using a multi-assay algorithm (MAA) that included the BED capture immunoassay, an avidity assay, CD4 cell count, and HIV viral load.
Data from Thailand was not used in the endpoint analysis because HIV prevalence was low. Overall, 7,361 HIV infections were identified (4 acute, 3 early, and 7,354 established infections). Samples from established infections were analyzed using the MAA; 467 MAA positive samples were identified; 29 of those samples were excluded because they contained antiretroviral drugs. HIV prevalence was 16.5% (range at study sites: 5.93% to 30.8%). HIV incidence was 1.60% (range at study sites: 0.78% to 3.90%).
In this community-randomized trial, a MAA was used to estimate HIV incidence in a single, cross-sectional post-intervention survey. Results from this analysis were subsequently used to compare HIV incidence in the control and intervention communities.
The foreskin contains a subset of dendritic cells, macrophages, and CD4+ and CD8+ T cells that may be targets for initial HIV infection in female-to-male sexual transmission of HIV-1. We present analyses comparing HIV-1 sequences isolated from foreskin DNA and serum RNA in 12 heterosexual men enrolled in an adult male circumcision trial performed in Rakai, Uganda. Phylogenetic analysis demonstrated three topologies: (1) little divergence between foreskin and serum, (2) multiple genetic bottlenecks occurring in both foreskin and serum, and (3) complete separation of foreskin and serum populations. The latter tree topology provided evidence that foreskin may serve as a reservoir for distinct HIV-1 strains. Distance and recombination analysis also demonstrated that viral genotypes in the foreskin might segregate independently from the circulating pool of viruses.
Recent-infection testing assays/algorithms (RITAs) have been developed to exploit the titer and avidity of HIV antibody evolution following seroconversion for incidence estimation. The Vitros Anti-HIV 1+2 assay (Ortho-Clinical Diagnostics) was approved by the FDA to detect HIV-1 and HIV-2 infections. We developed a less-sensitive (LS) and an avidity-modified version of this assay to detect recent HIV infection. Seroconversion panels (80 subjects, 416 samples) were tested to calculate the mean duration of recent infection (MDR) for these assays. A panel from known long-term (2+ years) HIV-infected subjects on highly active antiretroviral therapy (HAART) (n = 134) and subjects with low CD4 counts (AIDS patients [n = 140]) was used to measure the false-recent rate (FRR) of the assays. Using a signal-to-cutoff ratio of 20 and the LS-Vitros assay gave a RITA MDR of 215 days (95% confidence interval [95% CI], ±65 days) and using an avidity index (AI) of 0.6 gave an MDR of 170 days (±44 days), while a combination of the two assays yielded a MDR of 146 days (±38.6) and an FRR of 8%. Misclassifying subjects with known long-term infection as recently infected occurred in 14% of AIDS patients and 29% (95% CI, 22, 38) of HAART subjects and 3% (95% CI, 0.8, 7.2) and 42% (95% CI, 33, 51), respectively, for the LS- and avidity-modified Vitros assays, with a misclassification rate of 15% (95% CI, 11, 20) overall using a dual-assay algorithm. Both modified Vitros assays can be used to estimate the length of time since seroconversion and in calculations for HIV incidence. Like other RITAs, they are subject to high FRR in subjects on HAART or with AIDS.
A recent HIV-1 molecular epidemiology survey in Singapore identified a novel CRF01_AE/B recombinant form, which accounted for 13 (11.9%) of 109 patient samples. Peripheral blood mononuclear cell DNA from three of these 13 patients was used to generate near full-length sequences to characterize the novel CRF01_AE/B recombinant form. The three isolates had a recombinant structure composed of CRF01_AE and subtype B, and shared identical breakpoints. As the three patients were not epidemiologically linked, this recombinant form has been designated CRF51_01B. Identification of the novel recombinant forms indicates ongoing active HIV-1 transmission in Singapore.
CCR5 is the primary coreceptor for HIV entry. Early after infection, the HIV viral population diversifies rapidly into a quasispecies. It is not known whether the initial efficiency of the viral quasispecies to utilize CCR5 is associated with HIV disease progression or if it changes in an infected individual over time. The CCR5 and CXCR4 utilization efficiencies (R5-UE and X4-UE) of the HIV quasispecies were examined using a pseudovirus, single-round infection assay for samples obtained from known seroconverters from Rakai district, Uganda (n=88). Initial and longitudinal R5-UE values were examined to assess the association of R5-UE with HIV disease progression using multivariate Cox proportional hazard models. Longitudinal samples were analyzed for 35 seroconverters who had samples available from multiple time points. There was no association between initial or longitudinal changes in R5-UE and the hazard of HIV disease progression (p=0.225 and p=0.942, respectively). In addition, R5-UE increased significantly over time after HIV seroconversion (p<0.001), regardless of HIV subtype or the emergence of CXCR4-tropic virus. These data demonstrate that the R5-UE of the viral quasispecies early in HIV infection is not associated with disease progression, and that R5-UE levels increase in HIV-infected individuals over time.
The Post-exposure Prophylaxis in Infants (PEPI)-Malawi trial evaluated infant antiretroviral regimens for prevention of post-natal HIV transmission. A multi-assay algorithm (MAA) that includes the BED capture immunoassay, an avidity assay, CD4 cell count, and viral load was used to identify women who were vs. were not recently infected at the time of enrollment (MAA recent, N = 73; MAA non-recent, N = 2,488); a subset of the women in the MAA non-recent group known to have been HIV infected for at least 2 years before enrollment (known non-recent, N = 54). Antibody maturation and viral diversification were examined in these women.
Samples collected at enrollment (N = 2,561) and 12–24 months later (N = 1,306) were available for serologic analysis using the BED and avidity assays. A subset of those samples was used for analysis of viral diversity, which was performed using a high resolution melting (HRM) diversity assay. Viral diversity analysis was performed using all available samples from women in the MAA recent group (61 enrollment samples, 38 follow-up samples) and the known non-recent group (43 enrollment samples, 22 follow-up samples). Diversity data from PEPI-Malawi were also compared to similar data from 169 adults in the United States (US) with known recent infection (N = 102) and known non-recent infection (N = 67).
In PEPI-Malawi, results from the BED and avidity assays increased over time in the MAA recent group, but did not change significantly in the MAA non-recent group. At enrollment, HIV diversity was lower in the MAA recent group than in the known non-recent group. HRM diversity assay results from women in PEPI-Malawi were similar to those from adults in the US with known duration of HIV infection.
Antibody maturation and HIV diversification patterns in African women provide additional support for use of the MAA to identify populations with recent HIV infection.
Viral suppression and viral breakthrough impact the humoral immune response to HIV infection. We evaluated the impact of viral suppression and viral breakthrough on results obtained with two cross-sectional HIV incidence assays.
All samples were collected from adults in the US who were HIV infected for >2 years. Samples were tested with the BED capture enzyme immunoassay (BED-CEIA) which measures the proportion of IgG that is HIV-specific, and with an antibody avidity assay based on the Genetic Systems 1/2+ O ELISA. We tested 281 samples: (1) 30 samples from 18 patients with natural control of HIV-1 infection known as elite controllers or suppressors (2) 72 samples from 18 adults on antiretroviral therapy (ART), with 1 sample before and 2–6 samples after ART initiation, and (3) 179 samples from 20 virally-suppressed adults who had evidence of viral breakthrough receiving ART (>400 copies/ml HIV RNA) and with subsequent viral suppression.
For elite suppressors, 10/18 had BED-CEIA values <0.8 normalized optical density units (OD-n) and these values did not change significantly over time. For patients receiving ART, 14/18 had BED-CEIA values that decreased over time, with a median decrease of 0.42 OD-n (range 0.10 to 0.63)/time point receiving ART. Three patterns of BED-CEIA values were observed during viral breakthrough: (1) values that increased then returned to pre-breakthrough values when viral suppression was re-established, (2) values that increased after viral breakthrough, and (3) values that did not change with viral breakthrough.
Viral suppression and viral breakthrough were associated with changes in BED-CEIA values, reflecting changes in the proportion of HIV-specific IgG. These changes can result in misclassification of patients with long-term HIV infection as recently infected using the BED-CEIA, thereby influencing a falsely high value for cross-sectional incidence estimates.
Recent studies suggest HIV-1 inter-subtype differences in co-receptor usage. We examined the correlation between HIV-1 subtype and co-receptor usage among treatment-naïve HIV-1 subjects in Singapore. Additionally, we investigated whether the subtype co-receptor association was influenced by stage of infection.
V3 sequences of HIV-1 envelope protein gp120 were obtained from 110 HIV treatment-naïve patients and genotypic co-receptor tropism determination was performed using Geno2pheno. Two false-positive rate (FPR) cut-offs, 10% and 5.75% were selected for tropism testing.
Subtype assignment of viral strains from 110 HIV-infected individuals based on partial sequencing of HIV-1 pol, gp120 and gp41 were as follows: 27 subtype B, 64 CRF01_AE, 10 CRF51_01B, and 9 other subtypes. At FPR=10%, 10 (100%) CRF51_01B-infected subjects and 26 (40.6%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 7 (25.9%) subtype B subjects and 1 (11.1%) CRF33_01B-infected subject (P < 0.001). At FPR=5.75%, 10 (100%) CRF51_01B-infected subjects and 20 (31.3%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 4 (14.8%) subtype B and 1 (11.1%) CRF33_01B-infected subjects (P < 0.001). Among those with evidence of seroconversion within 2 years prior to study enrolment, 100% of CRF51_01B-infected subjects had CXCR4-using virus, independent of Geno2pheno FPR.
CRF51_01B and CRF01_AE-infected individuals have higher prevalence of CXCR4-usage compared to subtype B infected individuals. Further studies examining these differences could help optimise the use of CCR5-antagonist in populations with these subtypes, and increase our understanding of HIV-1 biology.
CXCR4 usage; HIV-1; treatment-naïve
To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).
We previously developed a multi-assay algorithm (MAA) to identify recent HIV infection that includes the BED-Capture Enzyme Immunoassay, an avidity assay based on the Genetic Systems HIV-1/HIV-2+O Enzyme Immunoassay, CD4 cell count, and HIV viral load. We used this MAA to evaluate the association between recent maternal HIV infection and in utero transmission of HIV.
Plasma samples were collected at delivery from 2,561 HIV-infected women in the PEPI-Malawi trial. The MAA described above was used to identify women with recent HIV infection. Logistic regression models assessed association between recent HIV infection and in utero HIV transmission (defined as a positive infant HIV DNA test at birth).
Seventy-three women were identified as recently infected using the MAA. Those women were younger and had lower parity than women who were identified as not recently infected using the MAA (P<0.0001 for age and parity). The frequency of in utero HIV transmission was 17.8% among women identified as recently infected, compared to 6.7% among women identified as not recently infected (13/73 vs. 166/2488, P=0.001). In a multivariate model, three factors were independently associated with in utero HIV transmission: recent infection (adjusted odds ratio [AOR]: 2.49, 95% CI: 1.30–4.78, P=0.006), log10 HIV viral load at delivery (AOR: 2.01, 95% CI: 1.60–2.51, P<0.0001), and younger age (per 10 year increase, AOR: 0.66, 95% CI: 0.43–0.93, P=0.02).
Results obtained using a MAA suggest that recent maternal HIV acquisition is strongly associated with in utero HIV transmission, independent of HIV viral load at delivery.
HIV; incidence; multiassay algorithm; mother-to-child transmission; Malawi
Herpes simplex virus type 2 (HSV-2) infection is one of the most commonly sexually transmitted infections worldwide. While glycoprotein G-2 ELISA based assays are commonly used for the serologic detection of HSV-2 infections, they have low specificity in developing countries. Euroline Western blot (WB) is a commercially available assay that is easy to perform; however, little is known about its performance characteristics. This study evaluated Euroline WB for the detection of HSV-2 antibodies compared to University of Washington Western blot in three geographically different regions, Baltimore, Maryland, Rakai, Uganda, and Kunming, China. Among the 135 American men attending an STD clinic in Baltimore, Maryland, 72% (n=97) were HSV-2 positive by Euroline WB. The Euroline WB had a sensitivity of 97.8% and a specificity of 81.8%. Among the 273 commercial sex workers in Kunming, 62.3% were HSV-2 positive by Euroline WB. The Euroline WB had a sensitivity of 96.9% and a specificity of 89.1%. Among the 437 Ugandans in Rakai, 67.3% were HSV-2 positive by Euroline WB. The Euroline WB had a sensitivity of 98.7% and a specificity of 65.4%. The Euroline WB has a consistently high sensitivity, but specificity varied significantly among the different locations.
HIV superinfection, which occurs when a previously infected individual acquires a new distinct HIV strain, has been described in a number of populations. Previous methods to detect superinfection have involved a combination of labor-intensive assays with various rates of success. We designed and tested a next-generation sequencing (NGS) protocol to identify HIV superinfection by targeting two regions of the HIV viral genome, p24 and gp41. The method was validated by mixing control samples infected with HIV subtype A or D at different ratios to determine the inter- and intrasubtype sensitivity by NGS. This amplicon-based NGS protocol was able to consistently identify distinct intersubtype strains at ratios of 1% and intrasubtype variants at ratios of 5%. By using stored samples from the Rakai Community Cohort Study (RCCS) in Uganda, 11 individuals who were HIV seroconcordant but virally unlinked from their spouses were then tested by this method to detect superinfection between 2002 and 2005. Two female cases of HIV intersubtype superinfection (18.2%) were identified. These results are consistent with other African studies and support the hypothesis that HIV superinfection occurs at a relatively high rate. Our results indicate that NGS can be used for detection of HIV superinfection within large cohorts, which could assist in determining the incidence and the epidemiologic, virologic, and immunological correlates of this phenomenon.
HIV and hepatitis B virus (HBV) co-infection poses important public health considerations in resource-limited settings. Demographic data and sera from adult participants of the Rakai Health Sciences Program Cohort in Southwestern Uganda were examined to determine HBV seroprevalence patterns in this area of high HIV endemicity prior to the introduction of antiretroviral therapy. Commercially available EIAs were used to detect prevalent HBV infection (positive for HBV core antibody [anti-HBc] and/or positive HBV surface antigen [HBsAg]), and chronic infection (positive for HBsAg). Of 438 participants, 181 (41%) had prevalent HBV infection while 21 (5%) were infected chronically. Fourteen percent of participants were infected with HIV. Fifty three percent showed evidence of prevalent HBV infection compared to 40% among participants infected with HIV (p=0.067). Seven percent of participants infected with HIV were HBsAg positive compared to 4% among participants not infected with HIV (p=0.403). The prevalence of prevalent HBV infection was 55% in adults aged >50 years old, and 11% in persons under 20 years. In multivariable analysis, older age, HIV status and serologic syphilis were significantly associated with prevalent HBV infection. Transfusion status and receipt of injections were not significantly associated with HBV infection. Contrary to expectations that HBV exposure in Uganda occurred chiefly during childhood, prevalent HBV infection was found to increase with age and was associated sexually transmitted diseases (HIV and syphilis.) Therefore vaccination against HBV, particularly susceptible adults with HIV or at risk of HIV/STDs should be a priority.
Hepatitis B virus HBV; HIV; Sexual transmission; Uganda; Africa
Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual.
HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14–540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env.
Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection.
The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined.
To determine if mishandling prior to testing would make a sample from a chronically infected subject appear recently infected when tested by cross-sectional HIV incidence assays.
Serum samples from 31 subjects with chronic HIV infection were tested. Samples were subjected to different handling conditions, including incubation at 4°C, 25°C and 37°C, for 1, 3, 7 or 15 days prior to testing. Samples were also subjected to 1,3, 7 and 15 freeze-thaw cycles prior to testing. Samples were tested using the BED capture enzyme immuno assay (BED-CEIA), Vironostika-less sensitive (V-LS), and an avidity assay using the Genetic Systems HIV-1/HIV-2 plus O EIA (avidity assay).
Compared to the sample that was not subjected to any mishandling conditions, for the BED-CEIA, V-LS and avidity assay, there was no significant change in test results for samples incubated at 4°C or 25°C prior to testing. No impact on test results occurred after 15 freeze-thaw cycles. A decrease in assay results was observed when samples were held for 3 days or longer at 37°C prior to testing.
Samples can be subjected up to 15 freeze-thaw cycles without affecting the results the BED-CEIA, Vironostika-LS, or avidity assays. Storing samples at 4°C or 25°C for up to fifteen days prior to testing had no impact on test results. However, storing samples at 37°C for three or more days did affect results obtained with these assays.