CD14(+) macrophages that accumulate in the colon of African AIDS patients express pro-inflammatory cytokines and are responsive to lipopolysaccharide.

BACKGROUND: Intestinal macrophages are key regulators of inflammatory responses to the gut microbiome and play a central role in maintaining tissue homeostasis and epithelial integrity. However, little is known about the role of these cells in HIV infection, a disease fuelled by intestinal inflammation, a loss of epithelial barrier function and increased microbial translocation (MT). METHODS: Phenotypic and functional characterization of intestinal macrophages was performed for 23 African AIDS patients with chronic diarrhea and/or weight loss and 11 HIV-negative Africans with and without inflammatory bowel disease (IBD). AIDS patients were treated with cotrimoxazole for the prevention of opportunistic infections (OIs). Macrophage phenotype was assessed by flow cytometry and immuno-histochemistry (IHC); production of proinflammatory mediators by IHC and Qiagen PCR Arrays; in vitro secretion of cytokines by the Bio-Plex Suspension Array System. Statistical analyses were performed using Spearman's correlation and Wilcoxon matched-pair tests. Results between groups were analyzed using the Kruskal-Wallis with Dunn's post-test and the Mann-Whitney U tests. RESULTS: None of the study participants had evidence of enteric co-infections as assessed by stool analysis and histology. Compared to healthy HIV-negative controls, the colon of AIDS patients was highly inflamed with increased infiltration of inflammatory cells and increased mRNA expression of proinflammatory cytokine (tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IFN-γ, and IL-18), chemokines (chemokine (C-C motif) ligand (CCL)2 and chemokine (C-X-C) motif ligand (CXCL)10) and transcription factors (TNF receptor-associated factor (TRAF)6 and T-box (TXB)21). IHC revealed significant co-localization of TNF-α and IL-1ß with CD68(+) cells. As in IBD, HIV was associated with a marked increase in macrophages expressing innate response receptors including CD14, the co-receptor for lipopolysaccharide (LPS). The frequency of CD14(+) macrophages correlated positively with plasma LPS, a marker of MT. Total unfractionated mucosal mononuclear cells (MMC) isolated from the colon of AIDS patients, but not MMC depleted of CD14(+) cells, secreted increased levels of proinflammatory cytokines ex vivo in response to LPS. CONCLUSIONS: Intestinal macrophages, in the absence of overt OIs, play an important role in driving persistent inflammation in HIV patients with late-stage disease and diarrhea. These results suggest intensified treatment strategies that target inflammatory processes in intestinal macrophages may be highly beneficial in restoring the epithelial barrier and limiting MT in HIV-infected patients.

Impaired CD4+ T-cell restoration in the small versus large intestine of HIV-1-positive South Africans receiving combination antiretroviral therapy.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infection is associated with a massive depletion of intestinal CD4(+) T cells that is only partially reversed by combination antiretroviral therapy (cART). Here, we assessed the ability of nucleoside reverse-transcriptase inhibitor/nonnucleoside reverse-transcriptase inhibitor treatment to restore the CD4(+) T-cell populations in the intestine of South African patients with AIDS. METHODS: Thirty-eight patients with advanced HIV-1 infection who had chronic diarrhea (duration, >4 weeks) and/or unintentional weight loss (>10% decrease from baseline) of uncertain etiology were enrolled. Blood specimens were collected monthly, and gastrointestinal tract biopsy specimens were collected before cART initiation (from the duodenum, jejunum, ileum, and colon), 3 months after cART initiation (from the duodenum), and 6 months after cART initiation (from the duodenum and colon). CD4(+), CD8(+), and CD38(+)CD8(+) T cells were quantified by flow cytometry and immunohistochemistry analyses, and the HIV-1 RNA load was determined by the Nuclisens assay. RESULTS: CD4(+) T-cell and HIV-1 RNA levels were significantly lower, whereas CD8(+) T-cell levels, including activated CD38(+)CD8(+) T cell levels, were higher in the duodenum and jejunum, compared with the colon. After 6 months of cART, a significant but incomplete recovery of CD4(+) T cells was detected in the colon and peripheral blood but not in the duodenum. Failed restoration of the CD4(+) T-cell count in the duodenum was associated with nonspecific enteritis and CD8(+) T-cell activation. CONCLUSIONS: Strategies that target inflammation and immune activation in the small intestine may be required to expedite CD4(+) T-cell recovery and improve therapeutic outcomes.

Modeling HIV-1 drug resistance as episodic directional selection.

The evolution of substitutions conferring drug resistance to HIV-1 is both episodic, occurring when patients are on antiretroviral therapy, and strongly directional, with site-specific resistant residues increasing in frequency over time. While methods exist to detect episodic diversifying selection and continuous directional selection, no evolutionary model combining these two properties has been proposed. We present two models of episodic directional selection (MEDS and EDEPS) which allow the a priori specification of lineages expected to have undergone directional selection. The models infer the sites and target residues that were likely subject to directional selection, using either codon or protein sequences. Compared to its null model of episodic diversifying selection, MEDS provides a superior fit to most sites known to be involved in drug resistance, and neither one test for episodic diversifying selection nor another for constant directional selection are able to detect as many true positives as MEDS and EDEPS while maintaining acceptable levels of false positives. This suggests that episodic directional selection is a better description of the process driving the evolution of drug resistance.

Persistent microbial translocation and immune activation in HIV-1-infected South Africans receiving combination antiretroviral therapy.

BACKGROUND: Microbial translocation contributes to immune activation and disease progression during chronic human immunodeficiency virus type 1 (HIV-1) infection. However, its role in the African AIDS epidemic remains controversial. Here, we investigated the relationship between markers of monocyte activation, plasma lipopolysaccharide (LPS), and HIV-1 RNA in South Africans prioritized to receive combination antiretroviral therapy (cART). METHODS: Ten HIV-1-negative African controls and 80 HIV-1-infected patients with CD4 T cell counts <200 cells/microL were sampled prior to (n=60) or during (n=20) receipt of effective cART. Viral load was measured by Nuclisens; LPS by the Limulus amoebocyte lysate assay; monocyte and T cell subsets by flow cytometry; and soluble CD14, cytokines, and chemokines by enzyme-linked immunosorbent assay and customized Bio-Plex plates. RESULTS: Three distinct sets of markers were identified. CCL2, CXCL10, and CD14(+)CD16(+) monocyte levels were positively correlated with HIV-1 viremia. This finding, together with cART-induced normalization of these markers, suggests that their upregulation was driven by HIV-1. Plasma interleukin-6 was associated with the presence of opportunistic coinfections. Soluble CD14 and tumor necrosis factor were linked to plasma LPS levels and, as observed for LPS, remained elevated in patients receiving effective cART. CONCLUSIONS: Microbial translocation is a major force driving chronic inflammation in HIV-infected Africans receiving cART. Prevention of monocyte activation may be especially effective at enhancing therapeutic outcomes.

Prevalence of HIV type-1 drug-associated mutations in pre-therapy patients in the Free State, South Africa.

BACKGROUND: We aimed to characterize the molecular epidemiology of HIV type-1 (HIV-1) and the prevalence of drug-associated mutations prior to initiating highly active antiretroviral therapy (HAART) in the Free State province, South Africa. The Free State has a population of 3 million, an antenatal HIV prevalence of approximately 34% and a well established infrastucture for antiretroviral (ARV) provision. METHODS: HIV-1 polymerase genes were sequenced from 425 HAART-naive HIV-1-positive patients at voluntary primary healthcare HIV testing centres, who were subsequently attending district centres for assessment for commencing ARVs. Patients (>18 years) were sampled randomly with no exclusion for gender or clinical criteria. Sequences were analysed according to phylogeny and drug resistance. RESULTS: Phylogenetic clustering within the cohort was suggestive of multiple introductions of subtype C virus into the region. Drug resistance mutations (according to the International AIDS Society-USA classification) were distributed randomly across the cohort phylogeny with an overall prevalence of 2.3% in the sampled patients. When stratified according to CD4(+) T-cell count, the prevalence of resistance was 3.6%, 0.9% and 1.2% for CD4(+) T-cell counts <100, 200-350 and >500 cells/microl, respectively, and was most common for non-nucleoside reverse transcriptase inhibitor resistance (3.1% in patients with CD4(+) T-cell count <100 cells/microl). We surveyed all drug-selected mutations and found further significant clustering among patients with low CD4(+) T-cell counts (P=0.003), suggesting unrecognized exposure to ARVs. CONCLUSIONS: In the Free State population, there was a statistical association between low CD4(+) T-cell counts and drug-associated viral polymorphisms. Our data advocate the benefit of detailed history taking from patients starting HAART at low CD4(+) T-cell counts with close follow-up of the virological response.

Effectiveness of antiretroviral treatment in a South African program: a cohort study.

BACKGROUND: The effectiveness of the South African government's expanding antiretroviral treatment program is unknown. Observational studies of treatment effectiveness are prone to selection bias, rarely compare patients receiving antiretroviral treatment with similar patients not receiving antiretroviral treatment, and underestimate mortality rates unless patients are actively followed up. METHODS: We followed up 14 267 patients in the Public Sector Anti-Retroviral Treatment project in Free State, South Africa, for up to 20 months after enrollment. A total of 3619 patients received highly active triple antiretroviral treatment (HAART) for up to 19 months (median, 6 months; interquartile range, 3-9 months) after enrollment. Patients' clinical data were linked with the national mortality register. Marginal structural regression models adjusted for baseline and time-varying covariates. RESULTS: Of 4570 patients followed up for at least 1 year, 53.2% died. Eighty-seven percent of patients who died had not received HAART. HAART was associated with lower mortality (hazard ratio, 0.14; 95% confidence interval [CI], 0.11-0.18) and with the presence of tuberculosis (hazard ratio, 0.61; 95% CI, 0.46-0.81) after adjusting for age, sex, weight, clinic, district, CD4 cell count, cotrimoxazole therapy, tuberculosis at baseline, and previous antiretroviral therapy. Cotrimoxazole therapy was associated with lower mortality (hazard ratio, 0.37; 95% CI, 0.32-0.42). Each month of HAART was associated with an increase in CD4 cell count of 15.1 cells/microL (95% CI, 14.7-15.5 cells/microL) and with an increase in body weight of 602 g (95% CI, 548-658 g). CONCLUSIONS: HAART provided through these South African government health services seems as effective as that provided in high-income countries. Delays starting HAART contributed to high mortality rates. Faster expansion and timely commencement of HAART are needed.

Self-enrolment antenatal health promotion data as an adjunct to maternal clinical information systems in the Western Cape Province of South Africa.

Information systems designed to support health promotion in pregnancy, such as the MomConnect programme, are potential sources of clinical information which can be used to identify pregnancies prospectively and early on. In this paper we demonstrate the feasibility and value of linking records collected through the MomConnect programme, to an emergent province-wide health information exchange in the Western Cape Province of South Africa, which already enumerates pregnancies from a range of other clinical data sources. MomConnect registrations were linked to pregnant women known to the public health services using the limited identifiers collected by MomConnect. Three-quarters of MomConnect registrations could be linked to existing pregnant women, decreasing over time as recording of the national identifier decreased. The MomConnect records were usually the first evidence of pregnancy in pregnancies which were subsequently confirmed by other sources. Those at lower risk of adverse pregnancy outcomes were more likely to register. In some cases, MomConnect was the only evidence of pregnancy for a patient. In addition, the MomConnect records provided gestational age information and new and more recently updated contact numbers to the existing contact registry. The pilot integration of the data in the Western Cape Province of South Africa demonstrates how a client-facing system can augment clinical information systems, especially in contexts where electronic medical records are not widely available.

Experience in implementing the OpenMRS medical record system to support HIV treatment in Rwanda.

The challenge of scaling up HIV treatment in Africa has led to a new emphasis on improving health systems in impoverished areas. One aspect of this is the development and deployment of electronic medical record systems to support HIV and TB treatment. In this paper we describe the design and implementation of a new medical record architecture to support an HIV treatment program in rural Rwanda. The architecture is called OpenMRS and it has been developed to address the problem of configuring EMR systems to suit new sites, languages and diseases. OpenMRS uses a data dictionary called the concept dictionary to represent all the possible data items that can be collected. This allows new items to be added to the system by non-programmers. In addition, there are form creation tools that use drag and drop web technologies to simplify form construction. The OpenMRS system was first implemented in Kenya in February 2006 and then in Rwanda in August 2006. The system is now functioning well and we are developing extensions to improve the support for the clinic. These include improved, easy to use reporting tools, support for additional clinical problems including nutrition and child health, better database synchronization tools, and modules to collect laboratory data and support the pharmacy. The system is also in use in South Africa and Lesotho and is being deployed in Tanzania and Uganda.

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.

Progression to AIDS in South Africa is associated with both reverting and compensatory viral mutations.

We lack the understanding of why HIV-infected individuals in South Africa progress to AIDS. We hypothesised that in end-stage disease there is a shifting dynamic between T cell imposed immunity and viral immune escape, which, through both compensatory and reverting viral mutations, results in increased viral fitness, elevated plasma viral loads and disease progression. We explored how T cell responses, viral adaptation and viral fitness inter-relate in South African cohorts recruited from Bloemfontein, the Free State (n = 278) and Durban, KwaZulu-Natal (n = 775). Immune responses were measured by γ-interferon ELISPOT assays. HLA-associated viral polymorphisms were determined using phylogenetically corrected techniques, and viral replication capacity (VRC) was measured by comparing the growth rate of gag-protease recombinant viruses against recombinant NL4-3 viruses. We report that in advanced disease (CD4 counts <100 cells/µl), T cell responses narrow, with a relative decline in Gag-directed responses (p<0.0001). This is associated with preserved selection pressure at specific viral amino acids (e.g., the T242N polymorphism within the HLA-B*57/5801 restricted TW10 epitope), but with reversion at other sites (e.g., the T186S polymorphism within the HLA-B*8101 restricted TL9 epitope), most notably in Gag and suggestive of "immune relaxation". The median VRC from patients with CD4 counts <100 cells/µl was higher than from patients with CD4 counts ≥ 500 cells/µl (91.15% versus 85.19%, p = 0.0004), potentially explaining the rise in viral load associated with disease progression. Mutations at HIV Gag T186S and T242N reduced VRC, however, in advanced disease only the T242N mutants demonstrated increasing VRC, and were associated with compensatory mutations (p = 0.013). These data provide novel insights into the mechanisms of HIV disease progression in South Africa. Restoration of fitness correlates with loss of viral control in late disease, with evidence for both preserved and relaxed selection pressure across the HIV genome. Interventions that maintain viral fitness costs could potentially slow progression.

Brief report: methods for collecting sexual behaviour information from South African adolescents--a comparison of paper versus personal digital assistant questionnaires.

Reporting bias in adolescent behavioural research may be overcome with the use of personal digital assistants (PDA) or other computer based technologies. However, there is little insight into the use of these tools among adolescents in low resource settings. We compared self-administered paper questionnaires with PDA questionnaires to collect sexual behaviour data from a sample of 11-19 year olds living in a periurban, Xhosa-speaking community in South Africa. There was a high level of agreement between sexual risk behaviour data collected via each method (kappas> or = 0.50). Data collected from the PDA questionnaires were more complete. Subjectively, adolescents found the use of PDA to be simple and confidential. PDA may be a useful method to collect sensitive, self-reported information from adolescents in resource-limited settings.

An automated genotyping system for analysis of HIV-1 and other microbial sequences.

MOTIVATION: Genetic analysis of HIV-1 is important not only for vaccine development, but also to guide treatment strategies, track the emergence of new viral variants and ensure that diagnostic assays are contemporary and fully optimized. However, most genotyping methods are laborious and complex, and involve the use of multiple software applications. Here, we describe the development of an automated genotyping system that can be easily applied to HIV-1 and other rapidly evolving viral pathogens. RESULTS: The new REGA subtyping tool, developed using Java programming and PERL scripts, combines phylogenetic analyses with boot-scanning methods for the genetic subtyping of full-length and subgenomic fragments of HIV-1. When used to investigate the subtype of previously published reference datasets that were analysed using manual phylogenetic methods, the automated method correctly identified 97.5-100% of non-recombinant and circulating recombinant forms of HIV-1, including 108 full-length, 108 gag and 221 env sequences downloaded from the Los Alamos database.