Cervical and systemic innate immunity predictors of HIV risk linked to genital herpes acquisition and time from HSV-2 seroconversion.

OBJECTIVES: To examine innate immunity predictors of HIV-1 acquisition as biomarkers of HSV-2 risk and biological basis for epidemiologically established HIV-1 predisposition in HSV-2 infected women. METHODS: We analysed longitudinal samples from HIV-1 negative visits of 1019 women before and after HSV-2 acquisition. We measured cervical and serum biomarkers of inflammation and immune activation previously linked to HIV-1 risk. Protein levels were Box-Cox transformed and ORs for HSV-2 acquisition were calculated based on top quartile or below/above median levels for all HSV-2 negative visits. Bivariate analysis determined the likelihood of HSV-2 acquisition by biomarker levels preceding infection. Linear mixed-effects models evaluated if biomarkers differed by HSV-2 status defined as negative, incident or established infections with an established infection cut-off starting at 6 months. RESULTS: In the cervical compartment, two biomarkers of HIV-1 risk (low SLPI and high BD-2) also predicted HSV-2 acquisition. In addition, HSV-2 acquisition was associated with IL-1ß, IL-6, IL-8, MIP-3α, ICAM-1 and VEGF when below median levels. Systemic immunity predictors of HSV-2 acquisition were high sCD14 and IL-6, with highest odds when concomitantly increased (OR=2.23, 1.49-3.35). Concomitant systemic and mucosal predictors of HSV-2 acquisition risk included (1) serum top quartile sCD14 with cervical low SLPI, VEGF and ICAM-1, or high BD-2; (2) serum high IL-6 with cervical low VEGF and ICAM-1, SLPI, IL-1ß and IL-6; and (3) serum low C reactive protein with cervical high BD-2 (the only combination also predictive of HIV-1 acquisition). Most cervical biomarkers were decreased after HSV-2 acquisition compared with the HSV-2 negative visits, with incident infections associated with a larger number of suppressed cervical biomarkers and lower serum IL-6 levels compared with established infections. CONCLUSIONS: A combination of systemic immunoinflammatory and cervical immunosuppressed states predicts HSV-2 acquisition. A persistently suppressed innate immunity during incident HSV-2 infection may add to the increased HIV-1 susceptibility.

Evaluation of multi-assay algorithms for cross-sectional HIV incidence estimation in settings with universal antiretroviral treatment.

BACKGROUND: Multi-assay algorithms (MAAs) are used to estimate population-level HIV incidence and identify individuals with recent infection. Many MAAs use low viral load (VL) as a biomarker for long-term infection. This could impact incidence estimates in settings with high rates of early HIV treatment initiation. We evaluated the performance of two MAAs that do not include VL. METHODS: Samples were collected from 219 seroconverters (infected < 1 year) and 4376 non-seroconverters (infected > 1 year) in the HPTN 071 (PopART) trial; 28.8% of seroconverter samples and 73.2% of non-seroconverter samples had VLs ≤ 400 copies/mL. Samples were tested with the Limiting Antigen Avidity assay (LAg) and JHU BioRad-Avidity assays. Antibody reactivity to two HIV peptides was measured using the MSD U-PLEX assay. Two MAAs were evaluated that do not include VL: a MAA that includes the LAg-Avidity assay and BioRad-Avidity assay (LAg + BR) and a MAA that includes the LAg-Avidity assay and two peptide biomarkers (LAg + PepPair). Performance of these MAAs was compared to a widely used MAA that includes LAg and VL (LAg + VL). RESULTS: The incidence estimate for LAg + VL (1.29%, 95% CI: 0.97-1.62) was close to the observed longitudinal incidence (1.34% 95% CI: 1.17-1.53). The incidence estimates for the other two MAAs were higher (LAg + BR: 2.56%, 95% CI 2.01-3.11; LAg + PepPair: 2.84%, 95% CI: 1.36-4.32). LAg + BR and LAg + PepPair also misclassified more individuals infected > 2 years as recently infected than LAg + VL (1.2% [42/3483 and 1.5% [51/3483], respectively, vs. 0.2% [6/3483]). LAg + BR classified more seroconverters as recently infected than LAg + VL or LAg + PepPair (80 vs. 58 and 50, respectively) and identified ~ 25% of virally suppressed seroconverters as recently infected. CONCLUSIONS: The LAg + VL MAA produced a cross-sectional incidence estimate that was closer to the longitudinal estimate than two MAAs that did not include VL. The LAg + BR MAA classified the greatest number of individual seroconverters as recently infected but had a higher false recent rate.

HIV Antibody Profiles in HIV Controllers and Persons With Treatment-Induced Viral Suppression.

Introduction: Low HIV viral load is associated with delayed disease progression and reduced HIV transmission. HIV controllers suppress viral load to low levels in the absence of antiretroviral treatment (ART). We used an antibody profiling system, VirScan, to compare antibody reactivity and specificity in HIV controllers, non-controllers with treatment-induced viral suppression, and viremic non-controllers. Methods: The VirScan library contains 3,384 phage-displayed peptides spanning the HIV proteome. Antibody reactivity to these peptides was measured in plasma from a Discovery Cohort that included 13 elite controllers, 27 viremic controllers, 12 viremic non-controllers, and 21 non-controllers who were virally suppressed on ART. Antibody reactivity to selected peptides was also assessed in an independent cohort of 29 elite controllers and 37 non-controllers who were virally suppressed on ART (Validation Cohort) and in a longitudinal cohort of non-controllers. Results: In the Discovery Cohort, 62 peptides were preferentially targeted in HIV controllers compared to non-controllers who were virally suppressed on ART. These specificities were not significantly different when comparing controllers versus viremic non-controllers. Aggregate reactivity to these peptides was also high in elite controllers from the independent Validation Cohort. The 62 peptides formed seven clusters of homologous epitopes in env, gag, integrase, and vpu. Reactivity to one of these clusters located in gag p17 was inversely correlated with viral load set point in an independent cohort of non-controllers. Conclusions: Antibody reactivity was low in non-controllers suppressed on ART, but remained high in viremic controllers despite viral suppression. Antibodies in controllers and viremic non-controllers were directed against epitopes in diverse HIV proteins; higher reactivity against p17 peptides was associated with lower viral load set point. Further studies are needed to determine if these antibodies play a role in regulation of HIV viral load.

Brief Report: Herpes Simplex Virus Type-2 Shedding and Genital Ulcers During Early HIV in Zimbabwean Women.

BACKGROUND: Herpes simplex virus type-2 (HSV-2) seropositive persons have a 3- to 5-fold higher risk of acquiring HIV, possibly because of HSV-2-induced inflammation and recruitment of susceptible immune cells to exposure sites. We hypothesized that cervical HSV-2 activation (ie, viral DNA shedding and/or ulcers) preceded HIV acquisition in the hormonal contraception and HIV cohort. METHODS: Zimbabwean women who acquired HIV were matched to HIV-negative women on visit, age, and bacterial sexually transmitted infections. Up to 5 cervical swabs bracketing first polymerase chain reaction detection of HIV DNA (the index visit) were selected (t-6months, t-3months, tindex, t+3months, t+6months). Women with HSV-2 immunoglobulin G+ before tindex were polymerase chain reaction tested for viral shedding. Self-reported and clinician-diagnosed ulcers were documented. Multivariable logistic regression, accounting for matching, estimated adjusted odds ratios (aOR) and 95% confidence intervals (CIs) at each visit. RESULTS: Of 387 HSV-2 seropositive women, most had prevalent as compared with incident HSV-2 (91% vs. 9%, respectively). HSV-2 viral shedding was more common among HIV seroconverters than HIV-negative women (26% vs. 14%, P < 0.01). Shedding occurred around HIV acquisition (t-3months aOR, 2.7; 95% CI, 0.8 to 8.8; tindex aOR, 2.6; 95% CI, 1.1 to 6.5; t+3months aOR, 2.6; 95% CI, 1.0 to 6.6). Genital ulcers were reported more often among HIV seroconverters than HIV-negative women (13% vs. 7%; P = 0.06) and detection was after HIV acquisition (t+6months aOR, 14.5; 95% CI, 1.6 to 133.9). CONCLUSIONS: HSV-2 shedding appeared synergistic with HIV acquisition followed by presentation of ulcers. Evaluating all sexually transmitted infections rather than HSV-2 alone may clarify the relationship between inflammation and HIV acquisition.

Age-Specific Risk Scores Do Not Improve HIV-1 Prediction Among Women in South Africa.

BACKGROUND: HIV-1 risk scoring tools could help target provision of prevention modalities such as pre-exposure prophylaxis. Recent research suggests that risk scores for women aged 18-45 may not predict risk well among young women aged 18-24. We evaluated the predictive performance of age-specific risk scores compared with the existing non-age-specific VOICE risk score, developed for women aged 18-45. METHODS: We conducted a secondary analysis of the Evidence for Contraceptive Options and HIV Outcomes Trial to develop and internally validate HIV-1 risk scores for women aged 18-24 and 25-35 in South Africa. Candidate predictors included baseline demographic, clinical, behavioral, and contextual characteristics readily available in clinical settings. The VOICE risk score was applied to women aged 18-35. We evaluated predictive performance of each risk score by area under the receiver operating characteristic curve (AUC). RESULTS: Predictive performance of all risk scores was moderate, with AUC (95% confidence interval) of 0.64 (0.60 to 0.67) among women aged 18-24, 0.68 (0.62 to 0.73) among those aged 25-35, and 0.61 (0.58 to 0.65) for the VOICE risk score applied to women aged 18-35; The AUC was similar in internal validation. Among women aged 18-24, HIV-1 incidence was high even at low risk scores, at 3.9 per 100 person-years (95% confidence interval: 3.2 to 4.7). CONCLUSIONS: All risk scores were moderately predictive of HIV-1 acquisition, and age-specific risk scores performed only marginally better than the VOICE non-age-specific risk score. Approaches for targeted pre-exposure prophylaxis provision to women in South Africa may require more extensive data than are currently available to improve prediction.

Effects of Depot Medroxyprogesterone Acetate, Copper Intrauterine Devices, and Levonorgestrel Implants on Early HIV Disease Progression.

Limited data exist on the effects of contraceptives on HIV disease progression. We studied the association between intramuscular injectable depot medroxyprogesterone acetate (DMPA-IM), the copper intrauterine device (IUD), and the levonorgestrel (LNG) implant on markers of HIV disease progression at the time of HIV detection and 3 months postdetection and time from detection to CD4 count <350 cells/mm3. Among women initiating antiretroviral therapy (ART), we studied the effect of contraceptive group on time from ART initiation to viral load (VL) <40 copies/mL. We included women 16-35 years randomized to DMPA-IM, copper IUD, or LNG implant with incident HIV infection during the Evidence for Contraceptive Options and HIV Outcomes (ECHO) trial (n = 382). We analyzed HIV VL and CD4 cell count according to participants' randomized method and also conducted a "continuous use" analysis that excluded follow-up time after method discontinuation. We used adjusted linear models to compare mean VL and CD4 cell levels by contraceptive group up to the time of ART initiation. We compared time from HIV detection to CD4 count <350 cells/mm3 and, following ART initiation, time to viral suppression (VL <40 copies/mL) using Cox proportional hazards models. At HIV detection, women allocated to DMPA-IM had lower VL relative to copper IUD (-0.28 log10 copies/mL; 95% confidence interval [CI]: -0.55 to -0.01) and LNG implant (-0.27, CI: -0.55 to 0.02) and higher mean CD4 than copper IUD users by 66 cells/mm3 (CI: 11-121). In continuous use analyses women allocated to DMPA-IM progressed to CD4 < 350 cells/mm3 slower than copper IUD users (hazard ratio [HR] = 0.6, CI: 0.3-1.1), whereas copper IUD users progressed faster than LNG implant users (HR = 1.8, CI: 1.0-3.3). Time to viral suppression was faster for DMPA-IM than copper IUD (HR = 1.5, CI: 1.0-2.3) and LNG implant 1.4 (CI: 0.9-2.2) users. We found no evidence of more rapid early HIV disease progression among women using DMPA-IM than among women using copper IUD or LNG implant. Our finding of more rapid progression among copper IUD compared with DMPA-IM users should be interpreted cautiously.

Concomitant Imbalances of Systemic and Mucosal Immunity Increase HIV Acquisition Risk.

BACKGROUND: We previously reported association of increased cervical RANTES and decreased secretory leukocyte protease inhibitor (SLPI) with higher risk of HIV acquisition in reproductive-age women. We now examine the interaction of concomitantly altered systemic and cervical immunity on such risk. METHODS: We measured immune biomarkers in 4390 cervical and 2390 paired serum specimens at quarterly visits in 218 HIV seroconverters and 784 seronegative women. We assessed proinflammatory (IL-1ß, IL-6, IL-8, MIP-3α, and RANTES), anti-inflammatory (IL-1RA and SLPI), vascular activation (vascular endothelial growth factor and Intercellular Adhesion Molecule-1) and defensin (BD2) cervical biomarkers and systemic (peripheral blood) C reactive protein (CRP), IL-6, IL-7, and sCD14 as indicators of immune dysregulation. Biomarker levels were Box-Cox transformed and odds ratios for HIV acquisition calculated based on top quartile or higher/lower than median levels for all HIV-negative visits. RESULTS: Subsequent HIV acquisition was associated with 5 of 14 individual biomarkers: low systemic CRP [odds ratio (OR) = 1.49, 1.21-1.83] and IL-6 (OR = 1.23, 1.00-1.51), high cervical BD-2 (OR = 1.33, 1.11-1.58) and RANTES (OR = 1.20, 1.01-1.43), and low cervical IL-1RA (OR = 0.65, 0.48-0.86). Low systemic CRP concomitant with altered cervical immunity, especially high BD2, conveyed highest HIV risk (1.63, 1.29-2.05). Additional markers of increased risk emerged when low systemic CRP coincided with: low systemic IL-6 and IL-7 (OR = 1.53, 1.18-1.97); high cervical IL-8 and MIP-3α (OR = 1.40, 1.07-1.83); high cervical IL-1ß and IL-6 (OR = 1.43, 1.09-1.86); or low cervical SLPI (OR = 1.36, 1.08-1.71). CONCLUSIONS: Changes in both peripheral and mucosal immunity may precede and predispose women to HIV infection. Suppressed systemic immunity (ie, low CRP) alone or in combination with imbalanced cervical innate immunity (high proinflammatory and low anti-inflammatory mediators) indicated increased vulnerability to infection. Understanding these combined effects on HIV susceptibility is essential to preventing new infections.

Aberrant cervical innate immunity predicts onset of dysbiosis and sexually transmitted infections in women of reproductive age.

Sexually transmitted infections (STIs) and vaginal dysbiosis (disturbed resident microbiota presenting with abnormal Nugent score or candidiasis) have been associated with mucosal inflammation and risk of HIV-1 infection, cancer and poor reproductive outcomes. To date, the temporal relationships between aberrant cervical innate immunity and the clinical onset of microbial disturbance have not been studied in a large population of reproductive age women. We examined data from a longitudinal cohort of 934 Ugandan and Zimbabwean women contributing 3,274 HIV-negative visits who had complete laboratory, clinical and demographic data. Among those, 207 women later acquired HIV, and 584 women were intermittently diagnosed with C. trachomatis (CT), N. gonorrhoeae (NG), genital herpes (HSV-2), T. vaginalis (TV), candidiasis, and abnormal intermediate (4-6) or high (7-10) Nugent score, i.e. bacterial vaginosis (BV). Immune biomarker concentrations in cervical swabs were analyzed by generalized linear and mixed effect models adjusting for site, age, hormonal contraceptive use (HC), pregnancy, breastfeeding, genital practices, unprotected sex and overlapping infections. High likelihood ratios (1.5-4.9) denoted the values of cervical immune biomarkers to predict onset of abnormal Nugent score and candidiasis at the next visits. When controlling for covariates, higher levels of ß-defensin-2 were antecedent to BV, CT and HSV-2, lower anti-inflammatory ratio IL-1RA:IL-1ß-to intermediate Nugent scores and candida, lower levels of the serine protease inhibitor SLPI-to candida, lower levels of the adhesion molecule ICAM-1 -to TV, and lower levels of the oxidative stress mitigator and endothelial activation marker VEGF-to NG. Changes in innate immunity following onset of dysbiosis and infections were dependent on HC use when controlling for all other covariates. In conclusion, imminent female genital tract dysbiosis or infection can be predicted by distinct patterns of innate immunity. Future research should characterize biotic and abiotic determinants of this pre-existing innate immunity state.

A Longitudinal Assessment of Cervical Inflammation and Immunity Associated with HIV-1 Infection, Hormonal Contraception, and Pregnancy.

Hormonal contraception (HC), particularly injectable depot-medroxyprogesterone acetate (DMPA), has been associated with increased HIV acquisition and higher levels of cervical regulated upon activation, normal T-cell expressed, and secreted (RANTES), also associated with HIV seroconversion. Longitudinal changes in cervical immunity associated with DMPA and combined oral contraceptives (COCs) have not been studied. Cervical samples from 216 HIV seroconverters in Uganda and Zimbabwe with matched samples from 727 HIV-uninfected controls were collected at two quarterly visits before (t - 2, t - 1), at (t0), and two visits following (t + 1, t + 2) HIV seroconversion and corresponding visits for HIV-negative controls. We measured 10 biomarkers of inflammation and immunity and used generalized linear models to estimate and compare biomarker levels across HIV status, contraceptive, and pregnancy groups. Biomarkers remained relatively stable across visits for controls, while in HIV-infected women cervical immunity started to change before seroconversion with RANTES and BD-2 increased and secretory leukocyte protease inhibitor (SLPI) decreased at t - 1 and continued to change at t0 with ICAM-1 up and IL-8 down and with more biomarkers after seroconversion (IL-1ß, IL-6, MIP-3α, VEGF, and IL-1RA down and IL-1RA:IL-1ß ratio up). In multivariable analyses, seroconverters had higher BD-2 at t - 1, higher RANTES and lower SLPI from t - 1 through t + 2, and lower IL-8 and IL-1RA at and/or after seroconversion compared to nonseroconverters. Compared to non-HC users, DMPA users had higher RANTES at all visits and lower BD-2 at t - 2 through t0, while COC users and pregnant women had higher IL-8 and SLPI at all visits; COC users also had lower BD-2 preseroconversion; pregnant women had lower RANTES at t0 - t + 2. Longitudinal patterns of cervical immunity differ between HIV seroconverters and HIV-negative women; seroconverters demonstrate increased RANTES and decreased SLPI starting before and continuing postseroconversion. Furthermore, these patterns are differentially regulated by DMPA, COC, and pregnancy.

Correction: Prevalence of sexually transmitted infections and bacterial vaginosis among women in sub-Saharan Africa: An individual participant data meta-analysis of 18 HIV prevention studies.

[This corrects the article DOI: 10.1371/journal.pmed.1002511.].

Identification and validation of a multi-assay algorithm for cross-sectional HIV incidence estimation in populations with subtype C infection.

INTRODUCTION: Cross-sectional methods can be used to estimate HIV incidence for surveillance and prevention studies. We evaluated assays and multi-assay algorithms (MAAs) for incidence estimation in subtype C settings. METHODS: We analysed samples from individuals with subtype C infection with known duration of infection (2442 samples from 278 adults; 0.1 to 9.9 years after seroconversion). MAAs included 1-4 of the following assays: Limiting Antigen Avidity assay (LAg-Avidity), BioRad-Avidity assay, CD4 cell count and viral load (VL). We evaluated 23,400 MAAs with different assays and assay cutoffs. We identified the MAA with the largest mean window period, where the upper 95% confidence interval (CI) of the shadow was <1 year. This MAA was compared to the LAg-Avidity and BioRad-Avidity assays alone, a widely used LAg algorithm (LAg-Avidity <1.5 OD-n + VL >1000 copies/mL), and two MAAs previously optimized for subtype B settings. We compared these cross-sectional incidence estimates to observed incidence in an independent longitudinal cohort. RESULTS: The optimal MAA was LAg-Avidity <2.8 OD-n  +  BioRad-Avidity <95% + VL >400 copies/mL. This MAA had a mean window period of 248 days (95% CI: 218, 284), a shadow of 306 days (95% CI: 255, 359), and provided the most accurate and precise incidence estimate for the independent cohort. The widely used LAg algorithm had a shorter mean window period (142 days, 95% CI: 118, 167), a longer shadow (410 days, 95% CI; 318, 491), and a less accurate and precise incidence estimate for the independent cohort. CONCLUSIONS: An optimal MAA was identified for cross-sectional HIV incidence in subtype C settings. The performance of this MAA is superior to a testing algorithm currently used for global HIV surveillance.

Prevalence of sexually transmitted infections and bacterial vaginosis among women in sub-Saharan Africa: An individual participant data meta-analysis of 18 HIV prevention studies.

BACKGROUND: Estimates of sexually transmitted infection (STI) prevalence are essential for efforts to prevent and control STIs. Few large STI prevalence studies exist, especially for low- and middle-income countries (LMICs). Our primary objective was to estimate the prevalence of chlamydia, gonorrhea, trichomoniasis, syphilis, herpes simplex virus type 2 (HSV-2), and bacterial vaginosis (BV) among women in sub-Saharan Africa by age, region, and population type. METHODS AND FINDINGS: We analyzed individual-level data from 18 HIV prevention studies (cohort studies and randomized controlled trials; conducted during 1993-2011), representing >37,000 women, that tested participants for ≥1 selected STIs or BV at baseline. We used a 2-stage meta-analysis to combine data. After calculating the proportion of participants with each infection and standard error by study, we used a random-effects model to obtain a summary mean prevalence of each infection and 95% confidence interval (CI) across ages, regions, and population types. Despite substantial study heterogeneity for some STIs/populations, several patterns emerged. Across the three primary region/population groups (South Africa community-based, Southern/Eastern Africa community-based, and Eastern Africa higher-risk), prevalence was higher among 15-24-year-old than 25-49-year-old women for all STIs except HSV-2. In general, higher-risk populations had greater prevalence of gonorrhea and syphilis than clinic/community-based populations. For chlamydia, prevalence among 15-24-year-olds was 10.3% (95% CI: 7.4%, 14.1%; I2 = 75.7%) among women specifically recruited from higher-risk settings for HIV in Eastern Africa and was 15.1% (95% CI: 12.7%, 17.8%; I2 = 82.3%) in South African clinic/community-based populations. Among clinic/community-based populations, prevalence was generally greater in South Africa than in Southern/Eastern Africa for most STIs; for gonorrhea, prevalence among 15-24-year-olds was 4.6% (95% CI: 3.3%, 6.4%; I2 = 82.8%) in South Africa and was 1.7% (95% CI: 1.2%, 2.6%; I2 = 55.2%) in Southern/Eastern Africa. Across the three primary region/population groups, HSV-2 and BV prevalence was high among 25-49-year-olds (ranging from 70% to 83% and 33% to 44%, respectively). The main study limitation is that the data are not from random samples of the target populations. CONCLUSIONS: Combining data from 18 HIV prevention studies, our findings highlight important features of STI/BV epidemiology among sub-Saharan African women. This methodology can be used where routine STI surveillance is limited and offers a new approach to obtaining critical information on STI and BV prevalence in LMICs.

Higher sequence diversity in the vaginal tract than in blood at early HIV-1 infection.

In the majority of cases, human immunodeficiency virus type 1 (HIV-1) infection is transmitted through sexual intercourse. A single founder virus in the blood of the newly infected donor emerges from a genetic bottleneck, while in rarer instances multiple viruses are responsible for systemic infection. We sought to characterize the sequence diversity at early infection, between two distinct anatomical sites; the female reproductive tract vs. systemic compartment. We recruited 72 women from Uganda and Zimbabwe within seven months of HIV-1 infection. Using next generation deep sequencing, we analyzed the total genetic diversity within the C2-V3-C3 envelope region of HIV-1 isolated from the female genital tract at early infection and compared this to the diversity of HIV-1 in plasma. We then compared intra-patient viral diversity in matched cervical and blood samples with three or seven months post infection. Genetic analysis of the C2-V3-C3 region of HIV-1 env revealed that early HIV-1 isolates within blood displayed a more homogeneous genotype (mean 1.67 clones, range 1-5 clones) than clones in the female genital tract (mean 5.7 clones, range 3-10 clones) (p<0.0001). The higher env diversity observed within the genital tract compared to plasma was independent of HIV-1 subtype (A, C and D). Our analysis of early mucosal infections in women revealed high HIV-1 diversity in the vaginal tract but few transmitted clones in the blood. These novel in vivo finding suggest a possible mucosal sieve effect, leading to the establishment of a homogenous systemic infection.

Assays for estimating HIV incidence: updated global market assessment and estimated economic value.

INTRODUCTION: Accurate incidence estimates are needed to characterize the HIV epidemic and guide prevention efforts. HIV Incidence assays are cost-effective laboratory assays that provide incidence estimates from cross-sectional surveys. We conducted a global market assessment of HIV incidence assays under three market scenarios and estimated the economic value of improved incidence assays. METHODS: We interviewed 27 stakeholders, and reviewed journal articles, working group proceedings, and manufacturers' sales figures. We determined HIV incidence assay use in 2014, and estimated use in 2015 to 2017 and in 5 to 10-years under three market scenarios, as well as the cost of conducting national and key population surveys using an HIV incidence assay with improved performance. RESULTS: Global 2014 HIV incidence assay use was 308,900 tests, highest in Asia and mostly for case- and population-based surveillance. Estimated 2015 to 2017 use was 94,475 annually, with declines due to China and the United States discontinuing incidence assay use for domestic surveillance. Annual projected 5 to 10 year use under scenario 1 - no change in technology - was 94,475. For scenario 2 - a moderately improved incidence assay - projected annual use was 286,031. Projected annual use for scenario 3 - game-changing technologies with an HIV incidence assay part of (a) standard confirmatory testing, and (b) standard rapid testing, were 500,000 and 180 million, respectively. As HIV incidence assay precision increases, decreased sample sizes required for incidence estimation resulted in $5 to 23 million annual reductions in survey costs and easily offset the approximately $3 million required to develop a new assay. CONCLUSIONS: Improved HIV incidence assays could substantially reduce HIV incidence estimation costs. Continued development of HIV incidence assays with improved performance is required to realize these cost benefits.

Rationale and design of a multi-center, open-label, randomised clinical trial comparing HIV incidence and contraceptive benefits in women using three commonly-used contraceptive methods (the ECHO study).

Background:In vitro, animal, biological and observational clinical studies suggest that some hormonal methods, particularly depot medroxyprogesterone acetate - DMPA, may increase women's risk of HIV acquisition. DMPA is the most common contraceptive used in many countries worst affected by the HIV epidemic. To provide robust evidence for contraceptive decision-making among women, clinicians and planners, we are conducting the Evidence for Contraceptive Options and HIV Outcomes (ECHO) study in four countries with high HIV incidence and DMPA use: Kenya, South Africa, Swaziland, and Zambia (Clinical Trials.gov identifier NCT02550067). Study design: We randomized HIV negative, sexually active women 16-35 years old requesting effective contraception and agreeing to participate to either DMPA, the copper T 380A intrauterine device or levonorgestrel implant. Participants attend a contraception support visit after 1 month and quarterly visits thereafter for up to 18 months. Participants receive a standard HIV prevention package and contraceptive side-effect management at each visit. The primary outcome is HIV seroconversion. Secondary outcomes include pregnancy, serious adverse events and method discontinuation. The sample size of 7800 women provides 80% power to detect a 50% relative increase in HIV risk between any of the three method pairs, assuming 250 incident infections per comparison. Ethical considerations: Several WHO consultations have concluded that current evidence on HIV risk associated with DMPA is inconclusive and that a randomized trial is needed to guide policy, counselling and choice. Previous studies suggest that women without a specific contraceptive preference are willing to accept randomization to different contraceptive methods. Stringent performance standards are monitored by an independent data and safety monitoring board approximately every 6 months. The study has been conducted with extensive stakeholder engagement. Conclusions: The ECHO study is designed to provide robust evidence on the relative risks (HIV acquisition) and benefits (pregnancy prevention) between three effective contraceptive methods.

Infecting HIV-1 Subtype Predicts Disease Progression in Women of Sub-Saharan Africa.

INTRODUCTION: Long-term natural history cohorts of HIV-1 in the absence of treatment provide the best measure of virulence by different viral subtypes. METHODS: Newly HIV infected Ugandan and Zimbabwean women (N=303) were recruited and monitored for clinical, social, behavioral, immunological and viral parameters for 3 to 9.5years. RESULTS: Ugandan and Zimbabwean women infected with HIV-1 subtype C had 2.5-fold slower rates of CD4 T-cell declines and higher frequencies of long-term non-progression than those infected with subtype A or D (GEE model, P<0.001), a difference not associated with any other clinical parameters. Relative replicative fitness and entry efficiency of HIV-1 variants directly correlated with virulence in the patients, subtype D>A>C (P<0.001, ANOVA). DISCUSSION: HIV-1 subtype C was less virulent than either A or D in humans; the latter being the most virulent. Longer periods of asymptomatic HIV-1 subtype C could explain the continued expansion and dominance of subtype C in the global epidemic.

Serum vitamin D status and bacterial vaginosis prevalence and incidence in Zimbabwean women.

BACKGROUND: Bacterial vaginosis, a highly prevalent vaginal condition, is correlated with many adverse reproductive outcomes. In some studies, low vitamin D status (measured as serum 25-hydroxyvitamin D, 25[OH]D) has been associated with increased prevalence of bacterial vaginosis. OBJECTIVES: We examined the cross-sectional association between vitamin D status and prevalence of bacterial vaginosis, separately for pregnant and nonpregnant women. Using prospectively collected data, we also characterized the effect of time-varying vitamin D status on incident bacterial vaginosis. STUDY DESIGN: We quantified 25(OH)D in stored sera collected quarterly from 571 Zimbabwean women participating in the Hormonal Contraception and Risk of HIV Acquisition Study. The analysis was restricted to women not using hormonal contraception. We characterized associations between vitamin D insufficiency (defined as 25[OH]D ≤ 30 ng/mL vs > 30 ng/mL) and prevalence of bacterial vaginosis among nonpregnant women at the enrollment visit and among pregnant women at the first follow-up visit that pregnancy was detected. Among women who were negative for bacterial vaginosis at enrollment (n = 380), we also assessed the effect of time-varying vitamin D status on incident bacterial vaginosis. We used the Liaison 25(OH)D total assay to measure 25(OH)D. Bacterial vaginosis was diagnosed via Nugent score. RESULTS: At enrollment, the prevalence of bacterial vaginosis was 31% and overall median 25(OH)D was 29.80 ng/mL (interquartile range, 24.70-34.30 ng/mL): 29.75 ng/mL (interquartile range, 25.15-33.95 ng/mL) among women with bacterial vaginosis, and 29.90 ng/mL (interquartile range, 24.70-34.50 ng/mL) among women without bacterial vaginosis. Among pregnant women, the prevalence of bacterial vaginosis was 27% and overall median 25(OH)D was 29.90 ng/mL (interquartile range, 24.10-34.00 ng/mL): 30.80 ng/mL (interquartile range, 26.10-36.90 ng/mL) among women with bacterial vaginosis, and 29.10 ng/mL (interquartile range, 23.80-33.45 ng/mL) among women without bacterial vaginosis. Vitamin D levels ≤ 30 ng/mL were not associated with a prevalence of bacterial vaginosis in nonpregnant women (adjusted prevalence ratio, 1.04; 95% confidence interval, 0.81-1.34) or pregnant women (adjusted prevalence ratio, 0.88, 95% confidence interval, 0.51-1.54). Vitamin D levels ≤ 30 ng/mL were similarly not associated with incident bacterial vaginosis (adjusted hazard ratio, 0.98, 95% confidence interval, 0.73-1.31). Our findings were robust to alternative specifications of vitamin D status including using a cut point for vitamin D deficiency of < 20 ng/mL vs ≥ 20 ng/mL and modeling 25(OH)D as a continuous variable. CONCLUSION: Among reproductive-age Zimbabwean women, insufficient vitamin D was not associated with increased bacterial vaginosis prevalence or incidence. Given established associations between bacterial vaginosis and poor reproductive outcomes, identification of factors leading to high bacterial vaginosis prevalence is urgently needed.

Biomarkers of Cervical Inflammation and Immunity Associated with Cervical Shedding of HIV-1.

BACKGROUND: Cervicovaginal HIV shedding is associated with increased female-to-male and mother-to-child transmission. Genital inflammation may increase shedding through cytokines/chemokines which recruit and activate HIV target cells. We evaluated whether cervical immune mediators present before seroconversion affected HIV shedding and whether mediators differed between shedders and nonshedders. METHODS: We used cervical samples from 187 African women with documented HIV seroconversion in the Hormonal Contraception and HIV study. Samples were from the two visits before seroconversion (T-2 and/or T-1), and/or at seroconversion (T0), and/or the two visits (T + 1 and/or T + 2) after seroconversion. We measured interleukin (IL)-1ß, IL-1 Receptor Antagonist (IL-1RA), IL-6, IL-8, RANTES (Regulated on Activation, Normal T-Cell Expressed and Secreted), MIP-3α, vascular endothelial growth factor (VEGF), Intercellular Adhesion Molecule-1 (ICAM-1), secretory leukocyte protease inhibitor (SLPI), and BD-2 and used the Wilcoxon test and generalized linear models to evaluate the association between mediators and shedding. RESULTS: The only immune mediator that differed at T-1 was RANTES, which was higher among shedders (p ≤ .05). HIV seroconversion was followed by significant decreases in many mediators, but a significant increase in RANTES. The magnitude of the change was significantly different for shedders versus nonshedders with regard to RANTES (increased in both groups, significantly more so in shedders), SLPI (decreased in both groups, significantly more so in shedders), and MIP-3α (decreased in shedders and increased in nonshedders). At T0, shedders had lower levels of SLPI and MIP-3α than nonshedders. CONCLUSIONS: In this study, a specific immune mediator profile was associated with risk of cervical HIV shedding. Higher and increasing levels of RANTES and lower and decreasing levels of SLPI and MIP-3α were associated with increased risk of HIV shedding.

The Contribution of Cervicovaginal Infections to the Immunomodulatory Effects of Hormonal Contraception.

UNLABELLED: Particular types of hormonal contraceptives (HCs) and genital tract infections have been independently associated with risk of HIV-1 acquisition. We examined whether immunity in women using injectable depot medroxyprogesterone acetate (DMPA), combined oral contraceptives (COC), or no HCs differs by the presence of cervicovaginal infections. Immune mediators were quantified in cervical swabs from 832 HIV-uninfected reproductive-age Ugandans and Zimbabweans. Bacterial infections and HIV were diagnosed by PCR, genital herpes serostatus by enzyme-linked immunosorbent assay (ELISA), altered microflora by Nugent score, and Trichomonas vaginalis and Candida albicans infection by wet mount. Generalized linear models utilizing Box-Cox-Power transformation examined associations between levels of mediators, infection status, and HCs. In no-HC users, T. vaginalis was associated with broadest spectrum of aberrant immunity (higher interleukin 1ß [IL-1ß], IL-8, macrophage inflammatory protein 3α [MIP-3α], ß-defensin 2 [BD2], and IL-1 receptor antigen [IL-1RA]). In women with a normal Nugent score and no genital infection, compared to the no-HC group, COC users showed higher levels of IL-1ß, IL-6, IL-8, and IL-1RA, while DMPA users showed higher levels of RANTES and lower levels of BD2, both associated with HIV seroconversion. These effects of COC were blunted in the presence of gonorrhea, chlamydia, trichomoniasis, candidiasis, and an abnormal Nugent score; however, RANTES was increased among COC users with herpes, chlamydia, and abnormal Nugent scores. The effect of DMPA was exacerbated by lower levels of IL-1RA in gonorrhea, chlamydia, or herpes, SLPI in gonorrhea, and IL-1ß, MIP-3α, and IL-1RA/IL1ß ratio in trichomoniasis. Thus, the effects of HC on cervical immunity depend on the genital tract microenvironment, and a weakened mucosal barrier against HIV may be a combined resultant of genital tract infections and HC use. IMPORTANCE: In this article, we show that in young reproductive-age women most vulnerable to HIV, hormonal contraceptives are associated with altered cervical immunity in a manner dependent on the presence of genital tract infections. Through altered immunity, hormones may predispose women to bacterial and viral pathogens; conversely, a preexisting specific infection or disturbed vaginal microbiota may suppress the immune activation by levonorgestrel or exacerbate the suppressed immunity by DMPA, thus increasing HIV risk by their cumulative action. Clinical studies assessing the effects of contraception on HIV susceptibility and mucosal immunity may generate disparate results in populations that differ by microbiota background or prevalence of undiagnosed genital tract infections. A high prevalence of asymptomatic infections among HC users that remain undiagnosed and untreated raises even more concerns in light of their combined effects on biomarkers of HIV risk. The molecular mechanisms of the vaginal microbiome's simultaneous interactions with hormones and HIV remain to be elucidated.