Assessing the Variability of Cell-Associated HIV DNA Quantification through a Multicenter Collaborative Study.

Reliable and accurate quantification of cell-associated HIV DNA (CA HIV DNA) is critical for early infant diagnosis, clinical management of patients under therapy, and to inform new therapeutics efficacy. The present study assessed the variability of CA HIV DNA quantification obtained from various assays and the value of using reference materials to help harmonize the measurements. Using a common set of reagents, our multicenter collaborative study highlights significant variability of CA HIV DNA quantification and lower limit of quantification across assays. The quantification of CA HIV DNA from a panel of infected PBMCs can be harmonized through cross-subtype normalization but assay calibration with the commonly used 8E5 cell line failed to reduce quantification variability between assays, demonstrating the requirement to thoroughly evaluate reference material candidates to help improve the comparability of CA HIV DNA diagnostic assay performance. IMPORTANCE Despite a global effort, HIV remains a major public health burden with an estimated 1.5 million new infections occurring in 2020. HIV DNA is an important viral marker, and its monitoring plays a critical role in the fight against HIV: supporting diagnosis in infants and underpinning clinical management of patients under therapy. Our study demonstrates that HIV DNA measurement of the same samples can vary significantly from one laboratory to another, due to heterogeneity in the assay, protocol, and reagents used. We show that when carefully selected, reference materials can reduce measurement variability and harmonize HIV DNA quantification across laboratories, which will help contribute to improved diagnosis and clinical management of patients living with HIV.

HIV serostatus, inflammatory biomarkers and the frailty phenotype among older people in rural KwaZulu-Natal, South Africa.

Objective: We compared the prevalence of frailty by HIV serostatus and related biomarkers to the modified frailty phenotype among older individuals in a rural population in South Africa. Methods: Questionnaire data were from a cohort of people living with HIV (PWH) on antiretroviral therapy (ART) and HIV-uninfected people aged 50 years and older sampled from the Africa Health Research Institute Demographic Health and Surveillance area in northern KwaZulu-Natal. The prevalence of frailty was compared using five categories: (1) physical activity; (2) mobility; (3) fatigue; (4) gait speed; and (5) grip strength, and assessed for demographic, clinical, and inflammatory correlates of frailty. Results: Among 614 individuals in the study, 384 (62.5%) were women. The median age at study enrolment was 64 years [Interquartile range (IQR) (58.6-72.0)]. 292 (47.6%) were PWH. 499 (81%) were classified as either pre-frail or frail. 43 (7%) were frail and HIV positive, 185 (30%) were pre-frail and HIV positive, 57 were frail and HIV negative and 214 (35%) were pre-frail and HIV negative. Frailty was similar for HIV negative and PWH (17.7% vs 14.7%, p = 0.72). Women were more likely to be frail (18.3% vs 13.04%, p = 0.16). The prevalence of frailty increased with age for both HIV groups. In the multivariable analysis, the odds of being frail were higher in those aged 70 years and above than those aged between 50 and 59 years (p < 0.001). Females were less likely to be pre-frail than males (p < 0.001). There was no association between any of the inflammatory biomarkers and frailty and pre-frailty. Conclusion: In this population, the prevalence of frailty is similar for PWH and people without HIV, but higher for women than men. These data suggest that the odds of developing frailty is similar for PWH over the age of 50 years, who survive into older age, as for people without HIV.

Analysis of Dominant HIV Quasispecies Suggests Independent Viral Evolution Within Spinal Granulomas Coinfected with Mycobacterium tuberculosis and HIV-1 Subtype C.

Extrapulmonary tuberculosis (TB) is a significant public health challenge in South Africa and worldwide, largely fuelled by the HIV epidemic. In spinal TB, Mycobacteria infect the spinal column without dissemination to the spinal cord. The immune microenvironment, target cell characteristics, and other evolutionary forces within granulomas during HIV/TB coinfection are poorly characterized. We investigated whether spinal TB granulomas represent a sequestered anatomical site where independent HIV evolution occurs, and assessed the role of macrophages as a target cell for both HIV and mycobacteria. RNA was extracted from plasma and granulomatous tissue from six antiretroviral-naive HIV-1/spinal TB-coinfected patients, RT-PCR amplified, and the C2-V5 env segment was cloned and sequenced. Analysis of genetic diversity, phylogeny and coalescence patterns was performed on clonal sequences. To investigate their role in HIV sequestration, macrophages and the HIV-1 p24 protein were immune localized and ultrastructural features were studied. Intercompartment diversity measurements and phylogenetic reconstruction revealed anatomically distinct monophyletic HIV-1 clusters in four of six patients. Genotypic CCR5-tropic variants were predominant (98.9%) with conservation of putative N-linked glycosylation sites in both compartments. CD68(+) reactivity was associated with higher tissue viral load (r = 1.0; p < 0.01) but not greater intrapatient diversity (r = 0.60; p > 0.05). Ultrastructural imaging revealed the presence of bacterial and virus-like particles within membrane-bound intracellular compartments of macrophages. Spinal tuberculosis granulomas may form anatomically discreet sites of divergent viral evolution. Macrophages in these granulomas harbored both pathogens, suggesting that they may facilitate the process of viral sequestration within this compartment.

Evidence of long-lived founder virus in mother-to-child HIV transmission.

Exposure of the infant's gut to cell-associated and cell-free HIV-1 trafficking in breast milk (BM) remains a primary cause of mother-to-child transmission (MTCT). The mammary gland represents a unique environment for HIV-1 replication and host-virus interplay. We aimed to explore the origin of the virus transmitted during breastfeeding, and the link with quasi-species found in acellular and cellular fractions of breast-milk (BM) and in maternal plasma. The C2-V5 region of the env gene was amplified, cloned and sequenced from the RNA and DNA of BM, the RNA from the mother's plasma (PLA) and the DNA from infant's dried blood spot (DBS) in 11 post-natal mother-infant pairs. Sequences were assembled in Geneious, aligned in ClustalX, manually edited in SeAL and phylogenetic reconstruction was undertaken in PhyML and MrBayes. We estimated the timing of transmission (ETT) and reconstructed the time for the most recent common ancestor (TMRCA) of the infant in BEAST. Transmission of single quasi-species was observed in 9 of 11 cases. Phylogenetic analysis illustrated a BM transmission event by cell-free virus in 4 cases, and by cell-associated virus in 2 cases but could not be identified in the remaining 5 cases. Molecular clock estimates, of the infant ETT and TMRCA, corresponded well with the timing of transmission estimated by sequential infant DNA PCR in 10 of 11 children. The TMRCA of BM variants were estimated to emerge during gestation in 8 cases. We hypothesize that in the remaining cases, the breast was seeded with a long-lived lineage latently infecting resting T-cells. Our analysis illustrated the role of DNA and RNA virus in MTCT. We postulate that DNA archived viruses stem from latently infected quiescent T-cells within breast tissue and MTCT can be expected to continue, albeit at low levels, should interventions not effectively target these cells.

Drug resistance in children at virological failure in a rural KwaZulu-Natal, South Africa, cohort.

BACKGROUND: Better understanding of drug resistance patterns in HIV-infected children on antiretroviral therapy (ART) is required to inform public health policies in high prevalence settings. The aim of this study was to characterise the acquired drug resistance in HIV-infected children failing first-line ART in a decentralised rural HIV programme. METHODS: Plasma samples were collected from 101 paediatric patients (≤15 yrs of age) identified as failing ART. RNA was extracted from the plasma, reverse transcribed and a 1.3 kb region of the pol gene was amplified and sequenced using Sanger sequencing protocols. Sequences were edited in Geneious and drug resistance mutations were identified using the RegaDB and the Stanford resistance algorithms. The prevalence and frequency of mutations were analysed together with selected clinical and demographic data in STATA v11. RESULTS: A total of 101 children were enrolled and 89 (88%) were successfully genotyped; 73 on a non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based regimen and 16 on a protease inhibitor (PI)-based regimen at the time of genotyping. The majority of patients on an NNRTI regimen (80%) had both nucleoside reverse-transcriptase inhibitor (NRTI) and NNRTI resistance mutations. M184V and K103N were the most common mutations amongst children on NNRTI-based and M184V among children on PI-based regimens. 30.1% had one or more thymidine analogue mutation (TAM) and 6% had ≥3 TAMs. Only one child on a PI-based regimen harboured a major PI resistance mutation. CONCLUSIONS: Whilst the patterns of resistance were largely predictable, the few complex resistance patterns seen with NNRTI-based regimens and the absence of major PI mutations in children failing PI-based regimens suggest the need for wider access to genotypic resistance testing in this setting.

Dried blood spot HIV-1 RNA quantification using open real-time systems in South Africa and Burkina Faso.

There is an urgent need to assess the accuracy/feasibility of using dried blood spots (DBS) for monitoring of HIV-1 viral load in resource-limited settings. A total of 892 DBS from HIV-1-positive pregnant women and their neonates enrolled in the Kesho Bora prevention of mother-to-child transmission trial conducted in Durban (South Africa) and Bobo-Dioulasso (Burkina Faso) between May 2005 and July 2008 were tested for HIV-1 RNA. The combination Nuclisens extraction method (BioMérieux)/Generic HIV Viral Load assay (Biocentric) was performed using one DBS (in Durban) versus 2 DBS (in Bobo-Dioulasso) on 2 distinct open real-time polymerase chain reaction instruments. DBS HIV-1 RNA results were compared with plasma HIV-1 RNA and HIV serology results used as the gold standards. The limits of detection of assays on DBS were 3100 and 1550 copies per milliliter in Durban and Bobo-Dioulasso, respectively. DBS HIV-1 RNA values correlated significantly with plasma levels (n = 327; R = 0.7351) and were uniformly distributed according to duration of DBS storage at -20°C (median duration, 280 days). For early infant diagnosis, the sensitivity and specificity were 100% (95% confidence interval: 97.2 to 100.0 and 96.5 to 100.0, respectively). HIV-1 viral load kinetics in DNase-pretreated DBS were similar to those obtained in plasma specimens among 13 patients receiving antiretroviral treatment. HIV-1 RNA findings from serial infant DBS collected prospectively (n = 164) showed 100% concordance with HIV serology at 18 months of life. Our findings strongly advocate the implementation of DBS HIV-1 RNA testing in remote areas from low-income and middle-income countries.

Comparison of the Generic HIV Viral Load assay with the Amplicor HIV-1 monitor v1.5 and Nuclisens HIV-1 EasyQ v1.2 techniques for plasma HIV-1 RNA quantitation of non-B subtypes: the Kesho Bora preparatory study.

The implementation of cost effective HIV-1 RNA quantitation assays in resource-poor settings is of paramount importance for monitoring HV-1 infection. A study comparing the analytical performance of three HIV-1 RNA assays (Generic HIV Viral Load, Amplicor v1.5 and Nuclisens EasyQ v1.2) was performed on 160 plasma samples from 160 consecutive antiretroviral treatment naive HIV-1-infected pregnant women assessed for eligibility in the Kesho Bora trial aimed at prevention of mother-to-child transmission of HIV-1 in three African countries (Burkina Faso, Kenya and South Africa). Correlation and agreement of results of the three assays were assessed for plasma HIV-1 RNA quantitation in specimens harbouring mainly sub-subtype A1, subtype C, and circulating recombinant form (CRF) 02_AG and CRF06_cpx. Good degrees of correlation and agreement were observed between these HIV-1 RNA assays. However, nine (9/160, 5.6%) strains detectable with the Generic HIV Viral Load assay were not detected by either the Amplicor (n=7) or EasyQ (n=2) test. One strain (0.6%) was missed with the Generic HIV Viral Load assay. Further, concordantly positive plasma samples harbouring CRF02_AG and CRF06_cpx yielded significantly higher HIV-1 RNA concentrations when tested by Generic HIV Viral Load, as compared to Amplicor v1.5 (mean differences, +0.33 and +0.67 log(10) copies/ml; P=0.0004 and P=0.002, respectively). The Generic HIV Viral Load assay accurately quantified the majority of the non-B HIV-1 subtypes assessed in this study. Due to its low cost (approximately 10 US $/test), this assay performed with open real-time PCR instruments is now used routinely in the Kesho Bora trial and may be recommended in other African settings.

In-house HIV-1 RNA real-time RT-PCR assays: principle, available tests and usefulness in developing countries.

The principle of currently available licensed HIV-1 RNA assays is based on real-time technologies that continuously monitor the fluorescence emitted by the amplification products. Besides these assays, in-house quantitative (q) real-time reverse transcription (RT)-PCR (RT-qPCR) tests have been developed and evaluated particularly in developing countries, for two main reasons. First, affordable and generalized access to HIV-1 RNA viral load is urgently needed in the context of expected universal access to prevention and antiretroviral treatment programs in these settings. Second, since many non-B subtypes, circulating recombinant forms and unique recombinant forms circulate in these areas, in-house HIV-1 RNA RT-qPCR assays are ideal academic tools to thoroughly evaluate the impact of HIV-1 genetic diversity on the accuracy of HIV-1 RNA quantification, as compared with licensed techniques. To date, at least 15 distinct in-house assays have been designed. They differ by their chemistry and the HIV-1 target sequence (located in gag, Pol-IN or LTR gene). Analytical performances of the tests that have been extensively evaluated appear at least as good as (or even better than) those of approved assays, with regard to HIV-1 strain diversity. Their clinical usefulness has been clearly demonstrated for early diagnosis of pediatric HIV-1 infection and monitoring of highly active antiretroviral therapy efficacy. The LTR-based HIV-1 RNA RT-qPCR assay has been evaluated by several groups under the auspices of the Agence Nationale de Recherches sur le SIDA et les hépatites virales B et C. It exists now as a complete standardized commercial test.

BioAfrica's HIV-1 proteomics resource: combining protein data with bioinformatics tools.

Most Internet online resources for investigating HIV biology contain either bioinformatics tools, protein information or sequence data. The objective of this study was to develop a comprehensive online proteomics resource that integrates bioinformatics with the latest information on HIV-1 protein structure, gene expression, post-transcriptional/post-translational modification, functional activity, and protein-macromolecule interactions. The BioAfrica HIV-1 Proteomics Resource http://bioafrica.mrc.ac.za/proteomics/index.html is a website that contains detailed information about the HIV-1 proteome and protease cleavage sites, as well as data-mining tools that can be used to manipulate and query protein sequence data, a BLAST tool for initiating structural analyses of HIV-1 proteins, and a proteomics tools directory. The Proteome section contains extensive data on each of 19 HIV-1 proteins, including their functional properties, a sample analysis of HIV-1HXB2, structural models and links to other online resources. The HIV-1 Protease Cleavage Sites section provides information on the position, subtype variation and genetic evolution of Gag, Gag-Pol and Nef cleavage sites. The HIV-1 Protein Data-mining Tool includes a set of 27 group M (subtypes A through K) reference sequences that can be used to assess the influence of genetic variation on immunological and functional domains of the protein. The BLAST Structure Tool identifies proteins with similar, experimentally determined topologies, and the Tools Directory provides a categorized list of websites and relevant software programs. This combined database and software repository is designed to facilitate the capture, retrieval and analysis of HIV-1 protein data, and to convert it into clinically useful information relating to the pathogenesis, transmission and therapeutic response of different HIV-1 variants. The HIV-1 Proteomics Resource is readily accessible through the BioAfrica website at: http://bioafrica.mrc.ac.za/proteomics/index.html.