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 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-1(HXB2), 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

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

Cell-free (RNA) and cell-associated (DNA) HIV-1 and postnatal transmission through breastfeeding

&lt;p&gt;Introduction - Transmission through breastfeeding remains important for mother-to-child transmission (MTCT) in resource-limited settings. We quantify the relationship between cell-free (RNA) and cell-associated (DNA) shedding of HIV-1 virus in breastmilk and the risk of postnatal HIV-1 transmission in the first 6 months postpartum.&lt;/p&gt; &lt;p&gt;Materials and Methods - Thirty-six HIV-positive mothers who transmitted HIV-1 by breastfeeding were matched to 36 non-transmitting HIV-1 infected mothers in a case-control study nested in a cohort of HIV-infected women. RNA and DNA were quantified in the same breastmilk sample taken at 6 weeks and 6 months. Cox regression analysis assessed the association between cell-free and cell-associated virus levels and risk of postnatal HIV-1 transmission.&lt;/p&gt; &lt;p&gt;Results - There were higher median levels of cell-free than cell-associated HIV-1 virus (per ml) in breastmilk at 6 weeks and 6 months. Multivariably, adjusting for antenatal CD4 count and maternal plasma viral load, at 6 weeks, each 10-fold increase in cell-free or cell-associated levels (per ml) was significantly associated with HIV-1 transmission but stronger for cell-associated than cell-free levels [2.47 (95% CI 1.33–4.59) vs. aHR 1.52 (95% CI, 1.17–1.96), respectively]. At 6 months, cell-free and cell-associated levels (per ml) in breastmilk remained significantly associated with HIV-1 transmission but was stronger for cell-free than cell-associated levels [aHR 2.53 (95% CI 1.64–3.92) vs. 1.73 (95% CI 0.94–3.19), respectively].&lt;/p&gt; &lt;p&gt;Conclusions - The findings suggest that cell-associated virus level (per ml) is more important for early postpartum HIV-1 transmission (at 6 weeks) than cell-free virus. As cell-associated virus levels have been consistently detected in breastmilk despite antiretroviral therapy, this highlights a potential challenge for resource-limited settings to achieve the UNAIDS goal for 2015 of eliminating vertical transmission. More studies would further knowledge on mechanisms of HIV-1 transmission and help develop more effective drugs during lactation.&lt;/p&gt

Dolutegravir twice-daily dosing in children with HIV-associated tuberculosis: a pharmacokinetic and safety study within the open-label, multicentre, randomised, non-inferiority ODYSSEY trial

Background: Children with HIV-associated tuberculosis (TB) have few antiretroviral therapy (ART) options. We aimed to evaluate the safety and pharmacokinetics of dolutegravir twice-daily dosing in children receiving rifampicin for HIV-associated TB. Methods: We nested a two-period, fixed-order pharmacokinetic substudy within the open-label, multicentre, randomised, controlled, non-inferiority ODYSSEY trial at research centres in South Africa, Uganda, and Zimbabwe. Children (aged 4 weeks to <18 years) with HIV-associated TB who were receiving rifampicin and twice-daily dolutegravir were eligible for inclusion. We did a 12-h pharmacokinetic profile on rifampicin and twice-daily dolutegravir and a 24-h profile on once-daily dolutegravir. Geometric mean ratios for trough plasma concentration (Ctrough), area under the plasma concentration time curve from 0 h to 24 h after dosing (AUC0–24 h), and maximum plasma concentration (Cmax) were used to compare dolutegravir concentrations between substudy days. We assessed rifampicin Cmax on the first substudy day. All children within ODYSSEY with HIV-associated TB who received rifampicin and twice-daily dolutegravir were included in the safety analysis. We described adverse events reported from starting twice-daily dolutegravir to 30 days after returning to once-daily dolutegravir. This trial is registered with ClinicalTrials.gov (NCT02259127), EudraCT (2014–002632-14), and the ISRCTN registry (ISRCTN91737921). Findings: Between Sept 20, 2016, and June 28, 2021, 37 children with HIV-associated TB (median age 11·9 years [range 0·4–17·6], 19 [51%] were female and 18 [49%] were male, 36 [97%] in Africa and one [3%] in Thailand) received rifampicin with twice-daily dolutegravir and were included in the safety analysis. 20 (54%) of 37 children enrolled in the pharmacokinetic substudy, 14 of whom contributed at least one evaluable pharmacokinetic curve for dolutegravir, including 12 who had within-participant comparisons. Geometric mean ratios for rifampicin and twice-daily dolutegravir versus once-daily dolutegravir were 1·51 (90% CI 1·08–2·11) for Ctrough, 1·23 (0·99–1·53) for AUC0–24 h, and 0·94 (0·76–1·16) for Cmax. Individual dolutegravir Ctrough concentrations were higher than the 90% effective concentration (ie, 0·32 mg/L) in all children receiving rifampicin and twice-daily dolutegravir. Of 18 children with evaluable rifampicin concentrations, 15 (83%) had a Cmax of less than the optimal target concentration of 8 mg/L. Rifampicin geometric mean Cmax was 5·1 mg/L (coefficient of variation 71%). During a median follow-up of 31 weeks (IQR 30–40), 15 grade 3 or higher adverse events occurred among 11 (30%) of 37 children, ten serious adverse events occurred among eight (22%) children, including two deaths (one tuberculosis-related death, one death due to traumatic injury); no adverse events, including deaths, were considered related to dolutegravir. Interpretation: Twice-daily dolutegravir was shown to be safe and sufficient to overcome the rifampicin enzyme-inducing effect in children, and could provide a practical ART option for children with HIV-associated TB

Neuropsychiatric manifestations and sleep disturbances with dolutegravir-based antiretroviral therapy versus standard of care in children and adolescents: a secondary analysis of the ODYSSEY trial

BACKGROUND: Cohort studies in adults with HIV showed that dolutegravir was associated with neuropsychiatric adverse events and sleep problems, yet data are scarce in children and adolescents. We aimed to evaluate neuropsychiatric manifestations in children and adolescents treated with dolutegravir-based treatment versus alternative antiretroviral therapy. METHODS: This is a secondary analysis of ODYSSEY, an open-label, multicentre, randomised, non-inferiority trial, in which adolescents and children initiating first-line or second-line antiretroviral therapy were randomly assigned 1:1 to dolutegravir-based treatment or standard-of-care treatment. We assessed neuropsychiatric adverse events (reported by clinicians) and responses to the mood and sleep questionnaires (reported by the participant or their carer) in both groups. We compared the proportions of patients with neuropsychiatric adverse events (neurological, psychiatric, and total), time to first neuropsychiatric adverse event, and participant-reported responses to questionnaires capturing issues with mood, suicidal thoughts, and sleep problems. FINDINGS: Between Sept 20, 2016, and June 22, 2018, 707 participants were enrolled, of whom 345 (49%) were female and 362 (51%) were male, and 623 (88%) were Black-African. Of 707 participants, 350 (50%) were randomly assigned to dolutegravir-based antiretroviral therapy and 357 (50%) to non-dolutegravir-based standard-of-care. 311 (44%) of 707 participants started first-line antiretroviral therapy (ODYSSEY-A; 145 [92%] of 157 participants had efavirenz-based therapy in the standard-of-care group), and 396 (56%) of 707 started second-line therapy (ODYSSEY-B; 195 [98%] of 200 had protease inhibitor-based therapy in the standard-of-care group). During follow-up (median 142 weeks, IQR 124–159), 23 participants had 31 neuropsychiatric adverse events (15 in the dolutegravir group and eight in the standard-of-care group; difference in proportion of participants with ≥1 event p=0·13). 11 participants had one or more neurological events (six and five; p=0·74) and 14 participants had one or more psychiatric events (ten and four; p=0·097). Among 14 participants with psychiatric events, eight participants in the dolutegravir group and four in standard-of-care group had suicidal ideation or behaviour. More participants in the dolutegravir group than the standard-of-care group reported symptoms of self-harm (eight vs one; p=0·025), life not worth living (17 vs five; p=0·0091), or suicidal thoughts (13 vs none; p=0·0006) at one or more follow-up visits. Most reports were transient. There were no differences by treatment group in low mood or feeling sad, problems concentrating, feeling worried or feeling angry or aggressive, sleep problems, or sleep quality. INTERPRETATION: The numbers of neuropsychiatric adverse events and reported neuropsychiatric symptoms were low. However, numerically more participants had psychiatric events and reported suicidality ideation in the dolutegravir group than the standard-of-care group. These differences should be interpreted with caution in an open-label trial. Clinicians and policy makers should consider including suicidality screening of children or adolescents receiving dolutegravir

Histopathology and genotyping in infectious spondylitis of HIV- and HIV+ patients

Approximately 2 million South Africans are HIV/TB coinfected, and many develop skeletal disease. The resurgence of spinal tuberculosis, including atypical forms, is due largely to HIV-associated immune suppression. We investigated the impact of HIV coinfection on the histological features of the disease and the occurrence of atypical opportunistic organisms in infectious spondylitis in an HIV/TB endemic region. We analyzed blood and tissue biopsies from 60 patients with tuberculous spondylitis. Investigations included full blood counts, CD4/CD8 counts, HIV-1 serology and RNA quantification (tissue and plasma), acid-fast bacilli localization and routine TB culture, histopathologic evaluation of biopsies, and bacterial genotyping using the 16S rDNA gene. Twenty-two patients (37%) were HIV positive with a mean age of 29 years (range, 2-65 years). Forty-one (68%) tissue specimens were culture negative for Mycobacterium tuberculosis (Mtb), although nontuberculous mycobacteria (NTM) were identified in three HIV-negative patients. Histopathologic features were characteristic of TB infection in 91.4% of all specimens tested and 100% of the HIV-infected group. Genotyping of 10 culture-positive isolates identified Mtb (3/10), NTMs (2/10), and environmental bacilli (3/10). Our observations suggest HIV-induced immune suppression impacts the histological and clinical features of infectious spondylitis but has no impact on the incidence of NTMs in this setting.The authors thank Prof. T. Naicker (HOD, Optics and Imaging Unit, UKZN), Mrs. R. Reddy (Laboratory Technologist, Dept. of Pathology, NALCH), and Dr. A. Bramdev (Pathologist, Lancet Laboratories)

Prevalence, incidence, and mother-to-child transmission of HIV-1 in rural South Africa.

Simple, robust approaches are needed to monitor prevalence, incidence, and mother-to-child transmission of HIV-1 in rural Africa. We have designed a method that uses antibody and viral RNA testing of dried blood spots obtained from mother-infant pairs attending routine immunisation clinics. In our study, prevalence and incidence of HIV-1 was highest in young women in their late teens and early twenties. In children born to infected mothers, prevalence increased from 14% in infants younger than 6 weeks of age to 24% at 3-6 months. The blood-spot approach is an effective method for surveillance of HIV-1 in women and children, and for early identification of incidence of this infection in women of child-bearing age

HIV drug resistance levels in adults failing first-line antiretroviral therapy in an urban and a rural setting in South Africa.

OBJECTIVES: Urban and rural HIV treatment programmes face different challenges in the long-term management of patients. There are few studies comparing drug resistance profiles in patients accessing treatment through these programmes. The aim of this study was to perform such a comparison. METHODS: HIV drug resistance data and associated treatment and monitoring information for adult patients failing first-line therapy in an urban and a rural programme were collected. Data were curated and managed in SATuRN RegaDB before statistical analysis using Microsoft Excel 2013 and stata Ver14, in which clinical parameters, resistance profiles and predicted treatment responses were compared. RESULTS: Data for 595 patients were analysed: 492 patients from a rural setting and 103 patients from an urban setting. The urban group had lower CD4 counts at treatment initiation than the rural group (98 vs. 126 cells/μL, respectively; P = 0.05), had more viral load measurements performed per year (median 3 vs. 1.4, respectively; P < 0.01) and were more likely to have no drug resistance mutations detected (35.9% vs. 11.2%, respectively; P < 0.01). Patients in the rural group were more likely to have been on first-line treatment for a longer period, to have failed for longer, and to have thymidine analogue mutations. Notwithstanding these differences, the two groups had comparable predicted responses to the standard second-line regimen, based on the genotypic susceptibility score. Mutations accumulated in a sigmoidal fashion over failure duration. CONCLUSIONS: The frequency and patterns of drug resistance, as well the intensity of virological monitoring, in adults with first-line therapy failure differed between the urban and rural sites. Despite these differences, based on the genotypic susceptibility scores, the majority of patients across the two sites would be expected to respond well to the standard second-line regimen