Monalisa N. Kalimashe

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.

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.

Background: Identification of all possible HIV reservoirs is an important aspect in HIV eradication efforts. The urinary tract has however not been well studied as a potential HIV reservoir. In this pilot study we molecularly characterized HIV-1 viruses in urine and plasma samples to investigate HIV-1 replication, compartmentalization and persistence in the urinary tract. Methods: Prospectively collected urine and blood samples collected over 12–36 months from 20 HIV-1 infected individuals were analysed including sampling points from prior to and after ART initiation. HIV-1 pol gene RNA and DNA from urine supernatant and urine pellets respectively were analysed and compared to plasma RNA viruses from the same individual. Results: HIV-1 nucleic acid was detected in urine samples from at least one time point in 8/20 (40%) treatment-naïve subjects compared to 1/13 (7.7%) individuals on antiretroviral treatment (ART) during periods of plasma viral suppression and 1/7 (14.3%) individuals with virological failure. HIV-1 RNA was undetectable in urine samples after ART initiation but HIV-1 DNA was detectable in one patient more than 6 months after treatment initiation. There was co-clustering of urine-derived pol sequences but some urine-derived sequences were interspersed among the plasma-derived sequences. Conclusions: Suppressive ART reduces HIV-1 replication in the urinary tract but HIV-1 DNA may persist in these cells despite treatment. A larger number of sequences would be required to confirm HIV compartmentalization in the urinary tract.

Background: South Africa (SA) has expanded efforts to reduce mother-to-child transmission of HIV (MTCT) to less than 2% at six weeks after birth and to less than 5% at 18 months postpartum by 2016. Despite improved antiretroviral regimens and coverage between 2001 and 2016, there is little data on infant HIV drug resistance. This paper tracks the prevalence of HIV drug resistance patterns amongst HIV infected infants from three nationally representative studies that assessed the effectiveness of national programs to prevent MTCT (PMTCT). The first study was conducted in 2010 (under the dual therapy PMTCT policy), the second from 2011 to 12 (PMTCT Option A policy) and the third from 2012 to 13 (PMTCT Option A policy). From 2010 to 2013, infant non-nucleoside reverse transcriptase inhibitor (NNRTI) exposure increased from single dose to daily throughout breastfeeding; maternal nucleoside reverse transcriptase inhibitor (NRTI) and NNRTI exposure increased with initiation of NNRTI-and NRTI- containing triple antiretroviral therapy (ART) earlier in gestation and at higher CD4 cell counts. Methods: Three nationally representative surveys were conducted in 2010, 2011–12 and 2012–13. During the surveys, mothers with known, unknown, or no exposure to antiretrovirals for PMTCT and their infants were included, and MTCT was measured. For this paper, infant dried blood spots (iDBS) from HIV PCR positive infants aged 4–8 weeks, with consent for additional iDBS testing, were analysed for HIV drug resistance at the National Institute of Communicable Diseases (NICD), SA, using an in-house assay validated by the Centers for Disease Control and Prevention (CDC). Total viral nucleic acid was extracted from 2 spots and amplified by nested PCR to generate a ~ 1 kb amplicon that was sequenced using Sanger sequencing technologies. Sequence assembly and editing was performed using RECall v3. Results: Overall, HIV-1 drug resistance was detected in 51% (95% Confidence interval (CI) [45–58%]) of HIV PCR positive infants, 37% (95% CI [28–47%]) in 2010, 64% (95% CI [53–74%]) in 2011 and 63% (95% CI [47–77%]) in 2012 (p < 0.0001), particularly to the NNRTI drug class. Pooled analyses across all three surveys demonstrated that infants whose mothers received ART showed the highest prevalence of resistance (74%); 26% (21/82) of HIV PCR positive infants with no or undocumented antiretroviral drug (ARV) exposure harboured NNRTI resistance. Conclusions: These data demonstrate increasing NNRTI resistance amongst newly-diagnosed infants in a high HIV prevalence setting where maternal ART coverage increased across the years, starting earlier in gestation and at higher CD4 cell counts. This is worrying as lifelong maternal ART coverage for HIV positive pregnant and lactating women is increasing. Also of concern is that resistant virus was detected in HIV positive infants whose mothers were not exposed to ARVs, raising questions about circulating resistant virus. Numbers in this group were too small to assess trends over the three years.