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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., (© 2022. The Author(s).)

Background: Globally, key subpopulations such as healthcare workers (HCW) may have a higher risk of contracting SARS-CoV-2. In Uganda, limited access to Personal Protective Equipment and lack of clarity on the extent/pattern of community spread may exacerbate this situation. The country established infection prevention/control measures such as lockdowns and proper hand hygiene. However, due to resource limitations and fatigue, compliance is low, posing continued onward transmission risk. This study aimed to describe extent of SARS-CoV-2 seroprevalence in selected populations within the Rakai region of Uganda., Methods: From 30th November 2020 to 8th January 2021, we collected venous blood from 753 HCW at twenty-six health facilities in South-Central Uganda and from 227 population-cohort participants who reported specific COVID-19 like symptoms (fever, cough, loss of taste and appetite) in a prior phone-based survey conducted (between May and August 2020) during the first national lockdown. 636 plasma specimens collected from individuals considered high risk for SARS-CoV-2 infection, prior to the first confirmed COVID-19 case in Uganda were also retrieved. Specimens were tested for antibodies to SARS-CoV-2 using the CoronaChek™ rapid COVID-19 IgM/IgG lateral flow test assay. IgM only positive samples were confirmed using a chemiluminescent microparticle immunoassay (CMIA) (Architect AdviseDx SARS-CoV-2 IgM) which targets the spike protein. SARS-CoV-2 exposure was defined as either confirmed IgM, both IgM and IgG or sole IgG positivity. Overall seroprevalence in each participant group was estimated, adjusting for test performance., Results: The seroprevalence of antibodies to SARS-CoV-2 in HCW was 26.7% [95%CI: 23.5, 29.8] with no difference by sex, age, or cadre. We observed no association between PPE use and seropositivity among exposed healthcare workers. Of the phone-based survey participants, 15.6% [95%CI: 10.9, 20.3] had antibodies to SARS-CoV-2, with no difference by HIV status, sex, age, or occupation. Among 636 plasma specimens collected prior to the first confirmed COVID-19 case, 2.3% [95%CI: 1.2, 3.5] were reactive., Conclusions: Findings suggest high seroprevalence of antibodies to SARS-CoV-2 among HCW and substantial exposure in persons presenting with specific COVID-19 like symptoms in the general population of South-Central Uganda. Based on current limitations in serological test confirmation, it remains unclear whether seroprevalence among plasma specimens collected prior to confirmation of the first COVID-19 case implies prior SARS-CoV-2 exposure in Uganda., (© 2022. The Author(s).)

Background: The availability of effective, oral direct acting antivirals (DAAs) for hepatitis C virus (HCV) treatment has put elimination of HCV as a public health challenge within reach. However, little is known about the characteristics of transmission networks of people who inject drugs (PWID)., Methods: Sequencing of a segment of the HCV genome was performed on samples collected from a community-based cohort of PWID between August 2005 and December 2016. Phylogenetic trees were inferred, and clusters were identified (70% bootstrap threshold; 0.04 maximum genetic distance threshold). We describe sex, race, age difference, and HIV infection status of potential transmission partners. Logistic regression was used to assess factors associated with being in an HCV cluster., Results: Of 508 HCV genotype 1 viremic PWID, 8% (n = 41) were grouped into 20 clusters, consisting of 19 pairs and 1 triad. In adjusted analyses, female sex (odds ratio [OR] 2.3 [95% confidence interval (CI) 1.2-4.5]) and HIV infection (OR 5.7 [CI 2.7-11.9]) remained independently associated with being in an HCV infection cluster., Conclusions: Molecular epidemiological analysis reveals that, in this cohort of PWID in Baltimore, HIV infection and female sex were associated with HCV clustering. Combination HCV prevention interventions targeting HIV infected PWID and addressing HCV infection prevention needs of women have potential to advance HCV elimination efforts.

Background: The commercial Kalon HSV-2 IgG ELISA is currently recommended for research use in sub-Saharan Africa because of its superior accuracy compared to other serologic assays. However, there are no data on key precision parameters of Kalon such as inter-operator variation, repeatability, and reproducibility, thus contributing to a barrier for its acceptance and use in clinical trials in sub-Saharan Africa. We evaluated the analytical and field precision of the Kalon HSV-2 IgG ELISA., Methods: A total of 600 HIV-infected and uninfected serum samples from South Africa and Zambia, previously tested by the gold standard University of Washington HSV western blot (UW-WB), were tested using Kalon by two technologists in an United States reference laboratory. Aliquots of 183 samples were retested using Kalon by an on-site technologist in a South African laboratory and a Zambian laboratory., Results: Intra-assay variation was below 10 %. Intra-assay, intra-laboratory, and inter-laboratory correlation and agreement were significantly high (p < 0.01). In comparison to the UW-WB, accurate performance of Kalon was reproducible by each operator and laboratory. Receiver operating characteristic curve analysis indicated high selectivity of Kalon in the overall study population (area under the curve = 0.95, 95%CI = 0.92-0.97)., Discussion: Kalon is a robust assay with high precision and reproducibility. Accordingly, operator errorlikely does not contribute to the variability observed in Kalon's specificity throughout sera from sub-Saharan Africa., Conclusions: In populations with optimal diagnostic accuracy, Kalon is a reliable stand-alone method for on-site HSV-2 IgG antibody detection.

Background: Mucosally acquired human immunodeficiency virus type 1 (HIV-1) infection results from a limited number of variants, and these infecting strains potentially have unique properties, such as increased susceptibility to entry blockers, relative interferon-alpha (IFN-α) resistance, and replication differences in some primary cells. There is no data about the phenotypic properties of HIV-1 envelope variants found early after acquisition among subjects infected through injection drug use (IDU). For the first time, we compared the characteristics of virus envelopes among injection drug users sampled prior to seroconversion (HIV RNA+/Ab-), within 1 year (early), and more than 2 years (chronic) after estimated acquisition., Results: Virus envelopes from 7 HIV RNA+/Ab- subjects possessed lower genetic diversity and divergence compared to 7 unrelated individuals sampled during the chronic phase of disease. Replication competent recombinant viruses incorporating the HIV RNA+/Ab- as compared to the chronic phase envelopes were significantly more sensitive to a CCR5 receptor inhibitor and IFN-α and showed a statistical trend toward greater sensitivity to a fusion blocker. The early as compared to chronic infection envelopes also demonstrated a statistical trend or significantly greater sensitivity to CCR5 and fusion inhibitor and IFN- α. The HIV RNA+/Ab- as compared to chronic envelope viruses replicated to a lower extent in mature monocyte derived dendritic cells - CD4+ T cell co-cultures, but there were no significant replication differences in other primary cells among the viruses with envelopes from the 3 different stages of infection., Conclusions: Similar to mucosal acquisition, HIV-1 envelope quasispecies present in injection drug users prior to seroconversion have unique phenotypic properties compared to those circulating during the chronic phase of disease.

Background: Previous studies suggest that active selection limits the number of HIV-1 variants acquired by a newly infected individual from the diverse variants circulating in the transmitting partner. We compared HIV-1 envelopes from 9 newly infected subjects and their linked transmitting partner to explore potential mechanisms for selection., Results: Recipient virus envelopes had significant genotypic differences compared to those present in the transmitting partner. Recombinant viruses incorporating pools of recipient and transmitter envelopes showed no significant difference in their sensitivity to receptor and fusion inhibitors, suggesting they had relatively similar entry capacity in the presence of low CD4 and CCR5 levels. Aggregate results in primary cells from up to 4 different blood or skin donors showed that viruses with envelopes from the transmitting partner as compared to recipient envelopes replicated more efficiently in CD4+ T cells, monocyte derived dendritic cell (MDDC) - CD4+ T cell co-cultures, Langerhans cells (LCs) - CD4+ T cell co-cultures and CD4+ T cells expressing high levels of the gut homing receptor, α4β7, and demonstrated greater binding to α4β7 high / CD8+ T cells. These transmitter versus recipient envelope virus phenotypic differences, however, were not always consistent among the primary cells from all the different blood or skin donation volunteers., Conclusion: Although genotypically unique variants are present in newly infected individuals compared to the diverse swarm circulating in the chronically infected transmitting partner, replication in potential early target cells and receptor utilization either do not completely dictate this genetic selection, or these potential transmission phenotypes are lost very soon after HIV-1 acquisition.

Background: 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., Methods: 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., Results: 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&#95;AE, 10 CRF51&#95;01B, and 9 other subtypes. At FPR=10%, 10 (100%) CRF51&#95;01B-infected subjects and 26 (40.6%) CRF01&#95;AE-infected subjects had CXCR4-using virus, compared to 7 (25.9%) subtype B subjects and 1 (11.1%) CRF33&#95;01B-infected subject (P < 0.001). At FPR=5.75%, 10 (100%) CRF51&#95;01B-infected subjects and 20 (31.3%) CRF01&#95;AE-infected subjects had CXCR4-using virus, compared to 4 (14.8%) subtype B and 1 (11.1%) CRF33&#95;01B-infected subjects (P < 0.001). Among those with evidence of seroconversion within 2 years prior to study enrolment, 100% of CRF51&#95;01B-infected subjects had CXCR4-using virus, independent of Geno2pheno FPR., Conclusion: CRF51&#95;01B and CRF01&#95;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.

Background: Project Accept is a community randomized, controlled trial to evaluate the efficacy of community mobilization, mobile testing, same-day results, and post-test support for the prevention of HIV infection in Thailand, Tanzania, Zimbabwe, and South Africa. We evaluated the accuracy of in-country HIV rapid testing and determined HIV prevalence in the Project Accept pilot study., Methods: Two HIV rapid tests were performed in parallel in local laboratories. If the first two rapid tests were discordant (one reactive, one non-reactive), a third HIV rapid test or enzyme immunoassay was performed. Samples were designated HIV NEG if the first two tests were non-reactive, HIV DISC if the first two tests were discordant, and HIV POS if the first two tests were reactive. Samples were re-analyzed in the United States using a panel of laboratory tests., Results: HIV infection status was correctly determined based on-in country testing for 2,236 (99.5%) of 2,247 participants [7 (0.37%) of 1,907 HIV NEG samples were HIV-positive; 2 (0.63%) of 317 HIV POS samples were HIV-negative; 2 (8.3%) of 24 HIV DISC samples were incorrectly identified as HIV-positive based on the in-country tie-breaker test]. HIV prevalence was: Thailand: 0.6%, Tanzania: 5.0%, Zimbabwe 14.7%, Soweto South Africa: 19.4%, Vulindlela, South Africa: 24.4%, (overall prevalence: 14.4%)., Conclusions: In-country testing based on two HIV rapid tests correctly identified the HIV infection status for 99.5% of study participants; most participants with discordant HIV rapid tests were not infected. HIV prevalence varied considerably across the study sites (range: 0.6% to 24.4%)., Trial Registration: ClinicalTrials.gov registry number NCT00203749.

Objective: To evaluate the utility of a single quantitative PCR (qPCR) measurement of HSV (HSV-1&2) DNA in cervicovaginal lavage (CVL) specimens collected from women with predominantly chronic HSV-2 infection in assessing genital HSV shedding and the clinical course of genital herpes (GH) within a cohort with semiannual schedule of follow up and collection of specimens., Methods: Two previously described methods used for detection of HSV DNA in mucocutaneous swab samples were adapted for quantification of HSV DNA in CVLs. Single CVL specimens from 509 women were tested. Presence and quantity of CVL HSV DNA were explored in relation to observed cross-sectional and longitudinal clinical data., Results: The PCR assay was sensitive and reproducible with a limit of quantification of ~50 copies per milliliter of CVL. Overall, 7% of the samples were positive for HSV-2 DNA with median log10 HSV-2 DNA copy number of 3.9 (IQR: 2.6-5.7). No HSV-1 was detected. Presence and quantity of HSV-2 DNA in CVL directly correlated with the clinical signs and symptoms of presence of active symptomatic disease with frequent recurrences., Conclusion: Single qPCR measurement of HSV DNA in CVL fluids of women with chronic HSV-2 infection provided useful information for assessing GH in the setting of infrequent sampling of specimens. Observed positive correlation of the presence and quantity of HSV-2 DNA with the presence of active and more severe course of HSV-2 infection may have clinical significance in the evaluation and management of HSV-2 infected patients.

Background: The objective of this study was to estimate the effect of male circumcision (MC) on HIV acquisition estimated using HIV incidence assays and to compare it to the effect measured by survival analysis., Methods: We used samples collected during the MC randomized controlled trial (ANRS-1265) conducted in Orange Farm (South Africa) among men aged 18 to 24. Among the 2946 samples collected at the last follow-up visit, 194 HIV-positive samples were tested using two incidence assays: Calypte HIV-EIA (BED) and an avidity assay based on the BioRad HIV1/2+O EIA (AI). The results of the assays were also combined (BED-AI). The samples included the 124 participants (4.2% of total) who were HIV-positive at randomization. The protective effect was calculated as one minus the intention-to-treat incidence rate ratio in an uncorrected manner and with correction for misclassifications, with simple theoretical formulae. Theoretical calculations showed that the uncorrected intention-to-treat effect was approximately independent of the value of the incidence assay window period and was the ratio of the number tested recent seroconverters divided by the number tested HIV-negative between the randomization groups. We used cut-off values ranging from 0.325 to 2.27 for BED, 31.6 to 96 for AI and 0.325-31.6 to 1.89-96 for BED-AI. Effects were corrected for long-term specificity using a previously published formula. 95% Confidence intervals (CI) were estimated by bootstrap resampling., Results: With the highest cut-off values, the uncorrected protective effects evaluated by BED, AI and BED-AI were 50% (95%CI: 27% to 66%), 50% (21% to 69%) and 63% (36% to 81%). The corrections for misclassifications were lower than 50% of the number of tested recent. The corrected effects were 53% (30% to 70%), 55% (25% to 77%) and 67% (38% to 86%), slightly higher than the corresponding uncorrected values. These values were consistent with the previously reported protective effect of 60% (34% to 76%) obtained with survival analysis., Conclusions: HIV incidence assays may be employed to assess the effect of interventions using cross-sectional data.