Irith De Baetselier

Drug resistance and plasma viral RNA level after ineffective use of oral pre-exposure prophylaxis in women

Background:Pre-exposure prophylaxis (PrEP) with daily oral emtricitabine (FTC)/tenofovir disoproxil fumarate may select for drug resistance if there is low adherence. Methods:Plasma viral HIV-1 RNA level, CD4+ T-cell counts, and drug resistance were evaluated among seroconverting women in the FEM-PrEP trial (clinicaltrials.gov NCT00625404) using standard clinical tests, allele-specific PCR (ASPCR), and by deep sequencing. Tenofovir, FTC, and their intracellular metabolites were measured in plasma and cells. Results:There was no difference in plasma HIV-1 RNA level or CD4+ cell count among seroconverters in the active arm versus those receiving placebo. Tenofovir resistance was not observed. FTC resistance was detected using clinical assays in five seroconverters (four in the active arm and one in the placebo arm); two in the active arm occurred among women having moderate concentrations of PrEP drugs in the blood. The first evidence of infection occurred at the first postenrollment visit in three of the four with FTC resistance, although none had detectable viral nucleic acids at enrollment. FTC-resistant minor variants were detected in an additional four seroconverters (one in the active arm and three in the placebo arm). Conclusions:Drug resistance detected during ineffective PrEP use had characteristics suggesting transmitted infection or incubating infection prior to starting PrEP.

Tenofovir Diphosphate and Emtricitabine Triphosphate Concentrations in Blood Cells Compared with Isolated Peripheral Blood Mononuclear Cells: A New Measure of Antiretroviral Adherence?

Background:The active metabolites of tenofovir (TFV) and emtricitabine (FTC) in peripheral blood mononuclear cells (PBMCs) have been used as markers of long-term antiretroviral (ARV) adherence. However, the process of isolating PBMCs is expensive, complex, and not feasible in many settings. We compared concentrations of TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in the upper layer packed cells (ULPCs) obtained after whole blood centrifugation to isolated PBMCs as a possible alternative marker of adherence. Methods:Ten HIV+ adults with HIV RNA <50 copies/mL on a TDF/FTC-containing regimen provided 5 paired PBMC and ULPC samples over 6 hours. TFV-DP and FTC-TP concentrations were analyzed by liquid chromatography/mass spectrometry. Partial areas under the curve were calculated using noncompartmental methods and Spearman Rank Correlations (rho) between PBMC and ULPC were determined. Results:The median (25th–75th percentile) concentration of TFV-DP in PBMCs was 143 (103–248) fmol/106 cells and in ULPC was 227 (160–394) fmol/106 cells (rho = 0.65; P < 0.0001). The concentration of FTC-TP in PBMCs was 6660 (5650–10,000) fmol/106 cells and in ULPC was 19.0 (12.0–27.8) fmol/106 cells (rho = 0.55; P < 0.0001). Compared to PBMCs, ULPC TFV-DP was 64% higher and FTC-TP was 99.7% lower. ULPC concentrations of TFV-DP and FTC-TP in one additional subject receiving a single dose of TDF/FTC were only 0.05% and 25%, of the other 10 subjects, respectively. Conclusions:ULPC concentrations significantly correlated with PBMC concentrations. Preliminary single-dose data suggest some discrimination between intermittent versus consistent dosing. ULPC concentrations of TFV-DP and FTC-TP should be further investigated as a simply collected surrogate measure of ARV adherence.

A fruitful alliance: the synergy between Atopobium vaginae and Gardnerella vaginalis in bacterial vaginosis-associated biofilm

Objectives Bacterial vaginosis (BV) is characterised by a change in the microbial composition of the vagina. The BV-associated organisms outnumber the health-associated Lactobacillus species and form a polymicrobial biofilm on the vaginal epithelium, possibly explaining the difficulties with antibiotic treatment. A better understanding of vaginal biofilm with emphasis on Atopobium vaginae and Gardnerella vaginalis may contribute to a better diagnosis and treatment of BV. Methods To this purpose, we evaluated the association between the presence of both bacteria by fluorescence in situ hybridisation (FISH) and BV by Nugent scoring in 463 vaginal slides of 120 participants participating in a clinical trial in Rwanda. Results A bacterial biofilm was detected in half of the samples using a universal bacterial probe. The biofilm contained A. vaginae in 54.1% and G. vaginalis in 82.0% of the samples. A. vaginae was accompanied by G. vaginalis in 99.5% of samples. The odds of having a Nugent score above 4 were increased for samples with dispersed G. vaginalis and/or A. vaginae present (OR 4.5; CI 2 to 10.3). The probability of having a high Nugent score was even higher when a combination of adherent G. vaginalis and dispersed A. vaginae was visualised (OR 75.6; CI 13.3 to 429.5) and highest when both bacteria were part of the biofilm (OR 119; CI 39.9 to 360.8). Conclusions Our study, although not comprehensive at studying the polymicrobial biofilm in BV, provided a strong indication towards the importance of A. vaginae and the symbiosis of A. vaginae and G. vaginalis in this biofilm. Trial registration number NCT01796613.

Prevalence and incidence estimation of HSV-2 by two IgG ELISA methods among South African women at high risk of HIV.

Introduction Previous comparison studies of the Kalon and HerpeSelect 2 ELISA IgG assays on sub-Saharan samples have found differences in the sensitivity and specificity of these assays. Using longitudinal samples from an HIV prevention study, we compared both assays and determined the HSV-2 prevalence and incidence in a South African young female population at elevated risk of acquiring HIV. Methods Samples at baseline were tested in both assays using the manufacturers’ guidelines (cut-off > 1.10). When non-reactive in one assay, the final visit samples were tested to determine the incidence rate. Using correlation and regression analyses, the intra- and inter-assay variabilities were assessed. Results The prevalence rate was 41.1% and 44.9% for Kalon and HerpeSelect using the manufacturer guidelines, respectively. Agreement between the two tests were high (kappa = 0.92). The original optical density values of both assays were highly correlated (R = 0.94), but the calibrator and correspondingly cut-off index values differed between the assays. Lowering the index value cut-off for the Kalon assay by 40% (to 0.66) resulted in a HSV-2 prevalence of 43.2%, and increased agreement between the assays (to kappa = 0.96). The incidence rate was 16.3/100 Person Years using the lower cut-off for the Kalon assay. Discussion In this longitudinal study, we showed that the performance of the two assays was very similar. After lowering the cut-off for the Kalon assay to 0.66 early infections were detected without impairing its specificity. The prevalence and incidence rates are in line with previously described rates for sub-Saharan African cohorts.

Liver and renal safety of tenofovir disoproxil fumarate in combination with emtricitabine among African women in a pre-exposure prophylaxis trial.

BackgroundSafety of tenofovir disoproxil fumarate/emtricitabine (TDF-FTC) has been studied more extensively among HIV-infected patients than among HIV-uninfected people. Using data from a pre-exposure trial – FEM-PrEP –, we determined the cumulative probabilities of grade 1+ ALT, AST and creatinine and grade 2+ phosphorus toxicities; ALT/AST toxicities by baseline hepatitis B status; and change in mean creatinine, phosphorus, ALT and AST levels controlling for TDF-FTC adherence.Methods and findingsFEM-PrEP was a randomized, blinded, placebo-controlled trial of daily TDF-FTC among women in Africa. Enrolled women were in general good health, HIV antibody negative, 18 to 35 years old, hepatitis B surface antigen negative, and had normal hepatic and renal function at baseline. AST, ALT, phosphorus and serum creatinine were measured regularly throughout the trial. TDF-FTC concentrations were measured to assess adherence to TDF-FTC. The cumulative probabilities of grade 1+ creatininemia and grade 2+ phosphatemia toxicities were not statistically different between TDF-FTC and placebo arms. The cumulative probabilities of grade 1+ ALT and AST toxicities were higher among participants in the TDF-FTC arm than in the placebo arm (p = 0.03 for both). The proportions of grade 1+ and grade 2+ ALT or AST toxicities were significantly higher in participants who were hepatitis B virus surface antibody (HBsAb) positive than in those who were HBsAb-negative. Women with good adherence had higher mean change from baseline to week 4 in their AST levels (2.90 (0.37, 5.42); p = 0.025) than women with less than good adherence.ConclusionsWe did not observe a significant relationship between randomization to TDF-FTC and creatinine or phosphorus toxicities. Women randomized to TDF-FTC had higher rates of mild to moderate ALT/AST toxicities, especially women with prior hepatitis B virus exposure. We also observed a significant increase in AST from baseline to week 4 among women who had higher adherence to TDF-FTC during that interval.Trial register#NCT00625404, February 19, 2008.

Association of vaginal dysbiosis and biofilm with contraceptive vaginal ring biomass in African women

We investigated the presence, density and bacterial composition of contraceptive vaginal ring biomass and its association with the vaginal microbiome. Of 415 rings worn by 120 Rwandese women for three weeks, the biomass density was assessed with crystal violet and the bacterial composition of biomass eluates was assessed with quantitative polymerase chain reaction (qPCR). The biomass was visualised after fluorescence in situ hybridisation (FISH) and with scanning electron microscopy (SEM). The vaginal microbiome was assessed with Nugent scoring and vaginal biofilm was visualised after FISH. All vaginal rings were covered with biomass (mean optical density (OD) of 3.36; standard deviation (SD) 0.64). Lactobacilli were present on 93% of the rings, Gardnerella vaginalis on 57%, and Atopobium vaginae on 37%. The ring biomass density was associated with the concentration of A. vaginae (OD +0.03; 95% confidence interval (CI) 0.01–0.05 for one log increase; p = 0.002) and of G. vaginalis (OD +0.03; (95% CI 0.01–0.05; p = 0.013). The density also correlated with Nugent score: rings worn by women with a BV Nugent score (mean OD +0.26), and intermediate score (mean OD +0.09) had a denser biomass compared to rings worn by participants with a normal score (p = 0.002). Furthermore, presence of vaginal biofilm containing G. vaginalis (p = 0.001) and A. vaginae (p = 0.005) correlated with a denser ring biomass (mean OD +0.24 and +0.22 respectively). With SEM we observed either a loose network of elongated bacteria or a dense biofilm. We found a correlation between vaginal dysbiosis and the density and composition of the ring biomass, and further research is needed to determine if these relationships are causal. As multipurpose vaginal rings to prevent pregnancy, HIV, and other sexually transmitted diseases are being developed, the potential impact of ring biomass on the vaginal microbiota and the release of active pharmaceutical ingredients should be researched in depth.

Pre-Exposure Prophylaxis (PrEP) as an Additional Tool for HIV Prevention Among Men Who Have Sex With Men in Belgium: The Be-PrEP-ared Study Protocol

Background Pre-exposure prophylaxis (PrEP) is a promising and effective tool to prevent HIV. With the approval of Truvada as daily PrEP by the European Commission in August 2016, individual European Member states prepare themselves for PrEP implementation following the examples of France and Norway. However, context-specific data to guide optimal implementation is currently lacking. Objective With this demonstration project we evaluate whether daily and event-driven PrEP, provided within a comprehensive prevention package, is a feasible and acceptable additional prevention tool for men who have sex with men (MSM) at high risk of acquiring HIV in Belgium. The study’s primary objective is to document the uptake, acceptability, and adherence to both daily and event-driven PrEP, while several secondary objectives have been formulated including impact of PrEP use on sexual behavior. Methods The Be-PrEP-ared study is a phase 3, single-site, open-label prospective cohort study with a large social science component embedded in the trial. A total of 200 participants choose between daily or event-driven PrEP use and may switch, discontinue, or restart their regimen at the 3-monthly visits for a duration of 18 months. Data are collected on several platforms: an electronic case report form, a Web-based tool where participants register their sexual behavior and pill use, a more detailed electronic self-administered questionnaire completed during study visits on a tablet computer, and in-depth interviews among a selected sample of participants. To answer the primary objective, the recruitment rate, (un)safe sex behavior during the last 6 months, percentage of reported intention to use PrEP in the future, retention rates in different regimens, and attitudes towards PrEP use will be analyzed. Adherence will be monitored using self-reported adherence, pill count, tenofovir drug levels in blood samples, and the perceived skills to adhere. Results All participants are currently enrolled, and the last study visit is planned to take place around Q3 2018. Conclusions As PrEP is not yet available in Belgium for use, this study will provide insights into how to optimally implement PrEP within the current health care provision and will shape national and European guidelines with regard to the place of PrEP in HIV prevention strategies. ClinicalTrial EU Clinical Trial 2015-000054-37; https://www.clinicaltrialsregister.eu/ctr-search/trial/2015-000054-37/BE (Archived by WebCite at http://www.webcitation.org/6nacjSdmM).

A cross-sectional study of hepatitis C among people living with HIV in Cambodia: Prevalence, risk factors, and potential for targeted screening

The epidemiology of hepatitis C in Cambodia is not well-known. We evaluated the prevalence of hepatitis C virus (HCV) and risk factors in the HIV cohort of Sihanouk Hospital Center of Hope in Phnom Penh to strengthen the evidence for suitable HCV testing strategies among people living with HIV (PLWH) in Cambodia. All consenting adult PLWH without a history of HCV treatment were tested for HCV between November 2014 and May 2016 according to the CDC algorithm (HCV antibody II electro-chemiluminescence immunoassay, followed by COBAS® AmpliPrep/COBAS® TaqMan® HCV PCR and INNO-LIA® HCV Score immunoblot end-testing). Genotyping was performed using the line probe assay Versant HCV genotype 2.0®. The study enrolled a total of 3045 patients (43% males, median age: 42.5 years, <1% high-risk). HCV antibodies were detected in 230 (7.6%; 95% confidence interval [CI] 6.6–8.5). Upon further testing, HCV antibodies were confirmed in 157 (5.2%; 95% CI 4.4–6.0) and active HCV in 106 (3.5%; 95% CI 2.8–4.2). Viremic prevalence peaked among men aged 50–55 years (7.3%) and women aged >55 years (11.2%). Genotype 1b (45%) and 6 (41%) were predominant. Coinfected patients had a higher aspartate-to-platelet ratio index, lower platelets, a lower HBsAg positivity rate and more frequent diabetes. Based on logistic regression, blood transfusion antecedents (adjusted odds ratio 2.9; 95% CI 1.7–4.9), unsafe medical injections (2.0; 1.3–3.2), and partner (3.4; 1.5–7.6) or household member (2.4; 1.3–3.2) with liver disease were independently associated with HCV in women. However, having a tattoo/scarification (1.9; 1.1–3.4) and household member (3.1; 1.3–7.3) with liver disease were associated with HCV in men. Thus, our study found intermediate endemicity of active hepatitis C in a large Cambodian HIV cohort and provides initial arguments for targeted HCV screening (>50 years, partner/household member with liver disease, diabetes, increased aspartate-to-platelet ratio index) as efficient way forward.

Detection of new HIV infections in a multicentre HIV antiretroviral pre-exposure prophylaxis trial

BACKGROUND Monthly specimens collected from FEM-PrEP-a Phase III trial [1] were investigated for the detection of acute HIV (AHI) infection. OBJECTIVES To evaluate the efficiency of the study-specific HIV algorithm in detecting AHI, and the performance of each of the serological and molecular tests used in diagnosing new infections, and their contribution to narrowing the window period. STUDY DESIGN A total of 83 pre-seroconversion specimens from 61 seroconverters from the FEM-PrEP trial were further analyzed in a sub-study. During the trial, HIV seroconversion was diagnosed on site using a testing algorithm with simple/rapid tests (SRTs) and confirmed with a gold standard testing algorithm (see short communication: Fig. 1). The infection date was determined more accurately by the use of standard ELISAs and Nucleic Acid Amplification Tests (NAAT) in a look-back procedure. For this sub-study, the international central laboratory repeated the study algorithm using SRTs. RESULTS A total of 83 pre-seroconversions specimens from 61 seroconverters were analyzed in a look-back procedure. RNA was detected in 35/61 seroconverters at the visit before the seroconversion visit as determined at the study sites. Four seroconversion dates were inaccurate at one study site as the international central laboratory detected the HIV infection one visit earlier using the same test algorithm. Using the gold standard, an additional seroconversion was detected at an earlier visit. The combined antigen/antibody and the single antigen test had a higher sensitivity compared to the SRTs in detecting acute infections. CONCLUSIONS In the FEM-PrEP trial, the international central laboratory detected a small number of seroconversions one month earlier than the study sites using the same study algorithm. Standard tests are still the most sensitive tests in detecting pre-seroconversion or acute HIV infection, but they are costly, time consuming and not recommended for use on-site in a clinical trial.

Performance of serological and molecular tests within acute HIV infection

BACKGROUND Early diagnosis of acute HIV infection can be challenging and is critical in regards to early therapeutic decision making. OBJECTIVES To evaluate the performance of different HIV tests in detecting early infections. STUDY DESIGN A total of 83 leftover specimens of 61 study participants who seroconverted were used in this sub-study. 35 HIV RNA positive but still seronegative specimens (acute infections) were used for analysis of the sensitivity of the different assays in detecting early infections and 42 HIV RNA and antibody negative specimens were used for specificity analysis. RESULTS Four (11%) specimens out of 35 acute infections were reactive with the Enzygnost® Anti-HIV 1/2 Plus and 12/35 (34%) with the Vironostika® HIV Ag/Ab. 16 (46%) specimens were confirmed as acute by the INNOTEST® HIV antigen mAb Antigen test. Only three (9%), 10 (29%) and 9 (27%) specimens were reactive with the Determine HIV-1/2 Ag/Ab Combo, SD Bioline HIV Ag/Ab Combo test and the HIV Combo test, respectively. The specificity of the different tests were 100%, 95%, 100%, 93%, 100% and 93% for Enzygnost® Anti-HIV 1/2 Plus, Vironostika® HIV Ag/Ab, INNO-Test HIV antigen mAb, Determine HIV-1/2 Ag/Ab Combo, SD Bioline HIV Ag/Ab Combo test and HIV Combo test respectively. CONCLUSION RNA test, 4th generation ELISA and Single Ag test are the most sensitive tests for detection of an acute infection. As an alternative, the HIV Combo test is generally slightly more sensitive compared to its previous version, but the SD Bioline HIV Ag/Ab Combo tests has the best performance compared to the other simple rapid tests (SRTs) but none of them are precise in detecting Ag in the determination of acute infections.