BACKGROUND: We developed the HIV Triplex multiplex bead assay to identify and serotype HIV infection with high sensitivity and specificity; and distinguish recent from long-term HIV-1 infections. It can facilitate accurate incidence estimation, while reducing the number of tests and blood collected, which is highly desirable for use in future studies and surveys. Using previously collected, treatment-naive longitudinal seroconversion HIV-1 positive panels and specimens from individuals infected for >12 months, we determined the assay's mean duration of recent infection (MDRI) and false-recency rate (FRR) respectively, at various mean fluorescent intensity (MFI) cutoffs. METHODS: We tested seroconversion specimens (N = 814) from 142 individuals infected with HIV-1 subtypes B, C, or AE, and 1341 cross-sectional specimens from individuals infected >12 months. The MFI cutoffs of 1000 to 2000 were evaluated for recency classification, including an MFI of 1250 corresponding to the limiting antigen avidity enzyme immunoassay (LAg-EIA) cutoff of 1.5 normalized optical density for MDRI and FRR. We used four statistical methods: Methods 1 and 2 used the empirically balanced observation time approach. Method 2 MFI values were raised to power = 1.33, based on a repeated measures model to linearize the relationship between MFI and time points, whereas Method 1 was not linearized. Methods 3 and 4 employed quadratic and linear interpolations for each seroconversion panel. FRR was calculated by dividing the number of specimens misclassified as recent by the total number of specimens tested. RESULTS: MDRI values ranged from 135-146 days at MFI = 1000 to 229-279 days at MFI = 2000 by the 4 methods. FRR varied from 0.15%-1.27% with increasing MFI cutoff. At MFI = 1250, the average MDRI of 4 methods was 169 days and ranged from 159-183 with overlapping 95% CIs and FRR = 0.52%. CONCLUSION: The HIV Triplex assay demonstrates a longer dynamic range compared to current HIV recency assays with a low FRR for cutoffs examined. With a longer dynamic range and low FRR, the MDRI for recent infection can be extended as appropriate to detect more recent infections, increasing the value of incidence assays benefiting public health surveillance and future surveys.

The Sedia Biosciences Asanté rapid test for recent infection (RTRI) can identify HIV infections and characterize HIV-1 as recent or long-term infection via the positive verification (V) line and long-term line (LT) line, respectively. Tracking with Recency Assays to Control the Epidemic (TRACE) program uses RTRI assays. Successful implementation of TRACE requires high-quality test performance. The goal of this study is to evaluate the additional quality practices established for new kit lots prior to field use. Asanté lot quality control data from the manufacturer is reviewed by the Centers for Disease Control and Prevention International Laboratory Branch (CDC-ILB) in the Division of Global HIV and TB using. If a lot passes manufacturer quality control and CDC-ILB review, test kits are sent to CDC-ILB for further evaluation. Evaluation by CDC includes inter-rater reliability and linear regressions comparing the V and LT lines against reference data as well as V and LT line data between testers. A Bland-Altman analysis was conducted to assess bias and systematic error. Overall, CDC-ILB passed 29 (91%) out of 32 Sedia Biosciences Asanté kit lots that initially passed manufacturing quality control from July 2017 to May 2020. Regression analyses demonstrate that test kits are performing as expected with consistent R2≥0.92 for both V and LT lines. On average, inter-rater reliability kappa was 0.9, indicating a strong level of agreement. Bland-Altman analyses demonstrate high agreement with little to no systematic error and bias. Ongoing evaluation of new RTRI kit lots is important to ensure high quality test performance. Rejecting 9% of kit lots highlight the importance of continuing to work with manufacturers to ensure consistent kit production and quality assurance (QA) activities. Investing in effective QA measures, conducting both pre- and post-market performance data reviews, could help improve RTRI accuracy and outcomes in similar testing programs.

BACKGROUND: We examined the epidemiology and transmission potential of HIV population viral load (VL) in 12 sub-Saharan African countries. METHODS: We analyzed data from Population-based HIV Impact Assessments (PHIAs), large national household-based surveys conducted between 2015 and 2019 in Cameroon, Cote d'Ivoire, Eswatini, Kenya, Lesotho, Malawi, Namibia, Rwanda, Tanzania, Uganda, Zambia, and Zimbabwe. Blood-based biomarkers included HIV serology, recency of HIV infection, and VL. We estimated the number of people living with HIV (PLHIV) with suppressed viral load (<1,000 HIV-1 RNA copies/mL) and with unsuppressed viral load (viremic), the prevalence of unsuppressed HIV (population viremia), sex-specific HIV transmission ratios (number female incident HIV-1 infections/number unsuppressed male PLHIV per 100 persons-years [PY] and vice versa) and examined correlations between a variety of VL metrics and incident HIV. Country sample sizes ranged from 10,016 (Eswatini) to 30,637 (Rwanda); estimates were weighted and restricted to participants 15 years and older. RESULTS: The proportion of female PLHIV with viral suppression was higher than that among males in all countries, however, the number of unsuppressed females outnumbered that of unsuppressed males in all countries due to higher overall female HIV prevalence, with ratios ranging from 1.08 to 2.10 (median: 1.43). The spatial distribution of HIV seroprevalence, viremia prevalence, and number of unsuppressed adults often differed substantially within the same countries. The 1% and 5% of PLHIV with the highest VL on average accounted for 34% and 66%, respectively, of countries' total VL. HIV transmission ratios varied widely across countries and were higher for male-to-female (range: 2.3-28.3/100 PY) than for female-to-male transmission (range: 1.5-10.6/100 PY). In all countries mean log10 VL among unsuppressed males was higher than that among females. Correlations between VL measures and incident HIV varied, were weaker for VL metrics among females compared to males and were strongest for the number of unsuppressed PLHIV per 100 HIV-negative adults (R2 = 0.92). CONCLUSIONS: Despite higher proportions of viral suppression, female unsuppressed PLHIV outnumbered males in all countries examined. Unsuppressed male PLHIV have consistently higher VL and a higher risk of transmitting HIV than females. Just 5% of PLHIV account for almost two-thirds of countries' total VL. Population-level VL metrics help monitor the epidemic and highlight key programmatic gaps in these African countries.

OBJECTIVE: Lesotho does not have reliable data on HIV prevalence in children, relying on estimates generated from program data. The 2016 Lesotho Population-based HIV Impact Assessment (LePHIA) aimed to determine HIV prevalence among children 0-14 years to assess the effectiveness of the prevention of mother to child transmission (PMTCT) program and guide future policy. METHODS: A nationally representative sample of children under 15 years underwent household-based, two-stage HIV testing from November 2016-May 2017. Children <18 months with a reactive screening test were tested for HIV infection using total nucleic acid (TNA) PCR. Parents (61.1%) or legal guardians (38.9%) provided information on children's clinical history. Children aged 10-14 years also answered a questionnaire on knowledge and behaviors. RESULTS: HIV prevalence was 2.1% (95% CI: 1.5-2.6%). Prevalence in 10-14 year olds (3.2%; 95% CI: 2.1%, 4.2%) was significantly greater compared to 0-4 year olds (1.0%; 95% CI: 0.5%, 1.6%). HIV prevalence in girls and boys was 2.6% (95% CI: 1.8% - 3.3%) and 1.5% (95% CI: 1.0% - 2.1%), respectively. Based on reported status and/or the presence of detectable antiretrovirals, 81.1% (95% CI: 71.7-90.4%) of HIV-positive children were aware of their status, 98.2% (95% CI: 90.7 - 100.0%) of those aware were on ART and 73.9% (95% CI: 62.1-85.8%) of those on ART were virally suppressed. CONCLUSIONS: Despite the roll-out of Option B+ in Lesotho in 2013, pediatric HIV prevalence remains high. Further research is required to understand the greater prevalence among girls, barriers to PMTCT, and how to better achieve viral suppression in children living with HIV.

OBJECTIVE: Viral load suppression (VLS) is critical in reducing morbidity and mortality associated with HIV as well as minimizing the likelihood of HIV transmission to uninfected persons. The objective of this study was to identify factors associated with VLS among people living with HIV (PLWH) on antiretroviral (ARV) therapy to inform HIV programme strategies in Nigeria. METHODS: Adult participants, aged 15-64 years, from the 2018 Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS), who self-reported to be a PLWH or had detectable ARVs, were analysed to examine factors associated with VLS defined as HIV RNA <1000 copies/mL. NAIIS measured HIV prevalence, viral load, ARV and hepatitis B in PLWH. Logistic regression models were used and reported weighted prevalence. RESULTS: Of 1322 participants, 949 (68.25%) were women and 1287 (96.82%) had detectable ARVs. The median age was 39.31 [interquartile range (IQR): 31.47-47.63] years. Prevalence of VLS was 80.88%. Compared with participants with detectable ARVs, those with undetectable ARVs in their blood specimens had lower odds of VLS [adjusted odds ratio (aOR) = 0.24, 95% confidence interval (CI): 0.08-0.64). Coinfection with hepatitis B and nonnucleoside reverse transcriptase inhibitor metabolites were also associated with lower odds of VLS. Older people (45-54 vs 15-24 years) had increased odds of VLS (aOR = 2.81, 95% CI: 1.14-6.90). CONCLUSION: Young people and those with undetectable ARVs had lower odds of virological suppression. Targeted interventions focusing on young people and adherence to medication are needed to achieve the UNAIDS 95-95-95 goals for HIV epidemic control.

BACKGROUND: In the population-based HIV impact assessment surveys, early infant diagnosis (EID) was provided to infants <18 months without a prior diagnosis. For the Namibia population-based HIV impact assessment (NAMPHIA), the GeneXpert platform was assessed for the feasibility of near POC EID testing compared with the standard Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) platform. Quality assurance measures and turnaround time were compared to improve EID results reporting. METHODS: NAMPHIA participants were screened for HIV exposure using Determine HIV-1/2 rapid test; samples reactive on Determine received EID testing on the GeneXpert instrument and Xpert HIV-1 Qual assay using whole blood. Results were confirmed at the Namibia Institute of Pathology using dried blood spots on the Roche CAP/CTM platform per national guidelines. RESULTS: Of the 762 screened infants, 61 (8.0%) were Determine-reactive and considered HIV-exposed. Of the 61 exposed infants, 2 were found to be HIV-infected whereas 59 were negative on both GeneXpert and Roche platforms, achieving 100% concordance. Average turnaround time was 3.4 days for the Xpert HIV-1 Qual assay, and average time from collection to testing was 1.0 days for GeneXpert compared with 10.7 days for Roche. No samples failed using GeneXpert whereas 1 sample failed using Roche and was repeated. CONCLUSION: Quality POC EID testing is feasible in a national survey through extensive training and external quality assurance measures. The use of decentralized POC EID for national testing would provide rapid diagnosis and improve TATs which may prevent loss to follow-up, ensure linkage to care, and improve clinical outcomes for infants.

BACKGROUND: Conducting HIV surveys in resource-limited settings is challenging because of logistics, limited availability of trained personnel, and complexity of testing. We described the procedures and systems deemed critical to ensure high-quality laboratory data in the population-based HIV impact assessments and large-scale household surveys. METHODS: Laboratory professionals were engaged in every stage of the surveys, including protocol development, site assessments, procurement, training, quality assurance, monitoring, analysis, and reporting writing. A tiered network of household, satellite laboratories, and central laboratories, accompanied with trainings, optimized process for blood specimen collection, storage, transport, and real-time monitoring of specimen quality, and test results at each level proved critical in maintaining specimen integrity and high-quality testing. A plausibility review of aggregate merged data was conducted to confirm associations between key variables as a final quality check for quality of laboratory results. RESULTS: Overall, we conducted a hands-on training for 3355 survey staff across 13 surveys, with 160-387 personnel trained per survey on biomarker processes. Extensive training and monitoring demonstrated that overall, 99% of specimens had adequate volume and 99.8% had no hemolysis, indicating high quality. We implemented quality control and proficiency testing for testing, resolved discrepancies, verified >300 Pima CD4 instruments, and monitored user errors. Aggregate data review for plausibility further confirmed the high quality of testing. CONCLUSIONS: Ongoing engagement of laboratory personnel to oversee processes at all levels of the surveys is critical for successful national surveys. High-quality population-based HIV impact assessments laboratory data ensured reliable results and demonstrated the impact of HIV programs in 13 countries.