Ewaldé Cutler

Quantifying viral load, a key indicator required to achieve control and elimination of the HIV epidemic, requires cell-free plasma or serum to ensure measurements are not biased by proviral DNA contained in infected CD4 T lymphocytes. Plasma separation cards (PSC) collect and preserve a dried specimen, which makes them practical solutions for decentralized sample collection and transport in limited-resource settings. However, physiological variations in hematocrit levels can introduce significant variability in the quality of plasma generated by commercial PSCs and can lead to inaccurate test results and clinical decisions. In addition to hematocrit-dependent sampling, the Roche PSC, a standard for dried plasma collection, is known to induce considerable hemolysis, which further impacts specimen quality, concordance with liquid plasma, and the overall benefit of microsampling. We address these gaps with a patterned dried plasma spot (pDPS) card, which generates plasma with improved hematocrit independence and minimal hemolysis. This study directly compares pDPS cards to the Roche PSC to measure HIV viral load. Analysis of viral load from 75 donors revealed strong agreement in sensitivity, specificity, overall accuracy, and viral load band placement between devices, with quantitative metrics suggesting improved performance for pDPS cards. In reflexive genotyping, remnant dried blood from pDPS cards exhibited greater success than Roche PSC in amplification and sequencing (71% vs. 62%) and detecting drug resistance mutations (63% vs. 42%). Based on this performance, pDPS cards can be versatile across multiple analytical platforms, integrate seamlessly into existing clinical laboratory workflows, and aid clinicians in making accurate treatment decisions.

Quantifying viral load, a key measure required to achieve control and elimination of the HIV epidemic, requires cell-free plasma or serum to ensure measurements are not biased by proviral DNA contained in infected CD4 T lymphocytes. Plasma separation cards (PSC) collect and preserve a dried specimen, which makes them practical solutions for decentralized sample collection and transport in limited-resource settings. However, physiological variations in hematocrit levels can introduce significant variability in sample quality and lead to inaccurate test results. In addition to hematocrit-dependent sampling, the Roche PSC, the commercial standard for dried plasma collection, is also known to induce hemolysis, which further reduces specimen quality and impacts concordance with liquid plasma. We address these gaps with the development of a new patterned dried plasma spot (pDPS) card, which generates cell-free plasma with improved hematocrit independence and minimal hemolysis. This study directly compares pDPS cards to the Roche PSC to measure HIV viral load. Analysis of HIV RNA from 75 different donors revealed strong agreement in sensitivity, specificity, overall accuracy, and viral load band placement between collection devices, with quantitative metrics suggesting improved performance for pDPS cards. In reflexive genotyping, remnant dried blood from pDPS cards exhibited greater success in amplification and sequencing (71% v. 62%) and in detecting drug resistance mutations (63% v. 42%) than Roche PSC. These results suggest that pDPS cards could be versatile across multiple analytical platforms, can integrate seamlessly into existing clinical laboratory workflows, and aid clinicians in making informed treatment decisions.

Background: Dual HIV/syphilis testing may be an acceptable intervention to identify men with sexually transmitted infections (STIs) and at risk of HIV acquisition. Objectives: We sought to determine the acceptability, and performance of dual HIV/syphilis testing among men attending voluntary medical male circumcision (VMMC) services at six public sector facilities in Gauteng. Method: This was a cross-sectional study at VMMC facilities. Men ≥ 18 years were enrolled. The men had (1) a questionnaire administered, (2) on-site dual HIV/syphilis testing with First Response HIV1+2/Syphilis Combo Card Test by routine lay counsellors, and (3) a blood specimen collected for centralised laboratory testing for HIV and syphilis serology. We evaluated pre-test and post-test acceptability and performance compared to serological testing. Results: Of the 679 men analysed (median age 32.1 years), 96.7% of HIV-negative men preferred testing for HIV and syphilis simultaneously. Of the 675 men tested for syphilis, 28 (4.7%) tested positive (past or recent). In the laboratory, 43/609 (7.1%) had syphilis infection detected, with 9/609 (1.5%) having recent syphilis. There was sub-optimal sensitivity for HIV detection (90.9%; 95% confidence interval [CI]: 88.5% – 93.3%), and for past/recent syphilis (55.8%; 95% CI: 51.9% – 59.8%), improving to 88.9% (95% CI: 86.4% – 91.4%) for recent syphilis. Specificities were > 99% for HIV and syphilis (past or recent). Post-test acceptability was 96.6% and willingness to pay for future testing was 86.1%. Conclusion: Dual HIV/syphilis testing was acceptable but had sub-optimal sensitivity for HIV and syphilis. Syphilis detection was adequate for recent infection.

Background. Point-of-care (POC) rapid recency testing can be used as a cost-effective tool to identify recently infected individuals (i.e. infected within the last 12 months) in near-real time, support epidemic control and identify hotspots for transmission as part of recent infection surveillance. Objective. To evaluate the performance of the Asanté (HIV-1) rapid recency assay as a POC rapid test among blood donors in South Africa (SA). Methods. The study was a cross-sectional and validity study of the Asanté HIV-1 Rapid Recency Assay performed on 715 consecutively archived plasma donor specimens from the SA National Blood Services to determine their recency and established HIV infection status. ELISA and rapid assays for HIV antibody detection were used as the reference-testing standard for confirming an infection, while the Maxim HIV-1 limiting antigen (LAg) avidity assay was used as a reference for comparing HIV recency status. Validity tests (sensitivity, specificity, negative and positive predictive values) and Cohen-Kappa tests of the agreement were conducted to compare the Asanté HIV-1 rapid recency assay results with the reference tests. Results. Of the 715 studied blood samples, 63.1% (n=451/715) were confirmed to be HIV-positive based on the reference standard. The sensitivity and specificity of the Asanté HIV-1 rapid recency assay in diagnosing established HIV infection compared to the ELISA were 98.4% (95% CI 96.7 - 99.3) and 99.6% (95% CI 97.6 - 100), respectively. Compared with HIV rapid assay, the sensitivity and specificity of the Asanté HIV-1 rapid recency assay was 98.7% (95% CI 97.0 - 99.4) and 99.2% (95% CI 97.1 - 100), respectively. Of the 451 HIV-positive blood samples, 43% were confirmed as recent HIV infections by the Maxim HIV-1 LAg avidity assay. There was high agreement between the Asanté HIV-1 rapid recency assay and the Maxim HIV-1 LAg avidity assay (94.1%, k=0.879, p90%) compared with the HIV ELISA and rapid assays for determining established infection and the Maxim HIV-1 LAg avidity assay for classifying recent HIV-1 infections. The assay’s sensitivity for established infections was below the World Health Organization criteria (<99%) for POC devices. The Asanté HIV-1 rapid recency assay can be used to distinguish between recent and long-term infections, but may not be considered a POC test for determining HIV infection.

Monitoring of HIV drug resistance (HIVDR) remains critical for ensuring countries attain and sustain the global goals for ending HIV as a public health threat by 2030. On an individual patient level, drug resistance results assist in ensuring unnecessary treatment switches are avoided and subsequent regimens are tailored on a case-by-case basis, should resistance be detected. Although there is a disparity in access to HIVDR testing in high-income countries compared to low- and middle-income countries (LMICS), more LMICs have now included HIVDR testing for individual patient management in some groups of patients. In this review, we describe different strategies for surveillance as well as where HIVDR testing can be implemented for individual patient management. In addition, we briefly review available technologies for HIVDR testing in LMICs, including Sanger sequencing, next-generation sequencing, and some point-of-care options. Finally, we describe how South Africa has implemented HIVDR testing in the public sector.