Long-term non-progression and risk factors for disease progression among children living with HIV in Botswana and Uganda: A retrospective cohort study.

OBJECTIVES: We utilize a large retrospective study cohort derived from electronic medical records to estimate the prevalence of long-term non-progression (LTNP) and determine the factors associated with progression among children infected with HIV in Botswana and Uganda. METHODS: Electronic medical records from large tertiary HIV clinical centers in Botswana and Uganda were queried to identify LTNP children 0-18 years enrolled between June 2003 and May 2014 and extract demographic and nutritional parameters. Multivariate subdistribution hazard analyses were used to examine demographic factors and nutritional status in progression in the pre-antiretroviral therapy era. RESULTS: Between the two countries, 14,246 antiretroviral therapy-naïve children infected with HIV were enrolled into clinical care. The overall proportion of LTNP was 6.3% (9.5% in Botswana vs 5.9% in Uganda). The median progression-free survival for the cohort was 6.3 years, although this was lower in Botswana than in Uganda (6.6 vs 8.8 years; P <0.001). At baseline, the adjusted subdistribution hazard ratio (aHRsd) of progression was increased among underweight children (aHRsd 1.42; 95% confidence interval [CI]: 1.32-1.53), enrolled after 2010 (aHRsd 1.32; 95% CI 1.22-1.42), and those from Botswana (aHRsd 2; 95% CI 1.91-2.10). CONCLUSIONS: In our study, the prevalence of pediatric LTNP was lower than that observed among adult populations, but progression-free survival was higher than expected. Underweight, year of enrollment into care, and country of origin are independent predictors of progression among children.

Exome Sequencing Reveals a Putative Role for HLA-C*03:02 in Control of HIV-1 in African Pediatric Populations.

Human leucocyte antigen (HLA) class I molecules present endogenously processed antigens to T-cells and have been linked to differences in HIV-1 disease progression. HLA allelotypes show considerable geographical and inter-individual variation, as does the rate of progression of HIV-1 disease, with long-term non-progression (LTNP) of disease having most evidence of an underlying genetic contribution. However, most genetic analyses of LTNP have occurred in adults of European ancestry, limiting the potential transferability of observed associations to diverse populations who carry the burden of disease. This is particularly true of HIV-1 infected children. Here, using exome sequencing (ES) to infer HLA allelotypes, we determine associations with HIV-1 LTNP in two diverse African pediatric populations. We performed a case-control association study of 394 LTNPs and 420 rapid progressors retrospectively identified from electronic medical records of pediatric HIV-1 populations in Uganda and Botswana. We utilized high-depth ES to perform high-resolution HLA allelotyping and assessed evidence of association between HLA class I alleles and LTNP. Sixteen HLA alleles and haplotypes had significantly different frequencies between Uganda and Botswana, with allelic differences being more prominent in HLA-A compared to HLA-B and C allelotypes. Three HLA allelotypes showed association with LTNP, including a novel association in HLA-C (HLA-B∗57:03, aOR 3.21, Pc = 0.0259; B∗58:01, aOR 1.89, Pc = 0.033; C∗03:02, aOR 4.74, Pc = 0.033). Together, these alleles convey an estimated population attributable risk (PAR) of non-progression of 16.5%. We also observed novel haplotype associations with HLA-B∗57:03-C∗07:01 (aOR 5.40, Pc = 0.025) and HLA-B∗58:01-C∗03:02 (aOR 4.88, Pc = 0.011) with a PAR of 9.8%, as well as a previously unreported independent additive effect and heterozygote advantage of HLA-C∗03:02 with B∗58:01 (aOR 4.15, Pc = 0.005) that appears to limit disease progression, despite weak LD (r 2 = 0.18) between these alleles. These associations remained irrespective of gender or country. In one of the largest studies of HIV in Africa, we find evidence of a protective effect of canonical HLA-B alleles and a novel HLA-C association that appears to augment existing HIV-1 control alleles in pediatric populations. Our findings outline the value of using multi-ethnic populations in genetic studies and offer a novel HIV-1 association of relevance to ongoing vaccine studies.

Unmapped exome reads implicate a role for Anelloviridae in childhood HIV-1 long-term non-progression.

Human immunodeficiency virus (HIV) infection remains a significant public health burden globally. The role of viral co-infection in the rate of progression of HIV infection has been suggested but not empirically tested, particularly among children. We extracted and classified 42 viral species from whole-exome sequencing (WES) data of 813 HIV-infected children in Botswana and Uganda categorised as either long-term non-progressors (LTNPs) or rapid progressors (RPs). The Ugandan participants had a higher viral community diversity index compared to Batswana (p = 4.6 × 10-13), and viral sequences were more frequently detected among LTNPs than RPs (24% vs 16%; p = 0.008; OR, 1.9; 95% CI, 1.6-2.3), with Anelloviridae showing strong association with LTNP status (p = 3 × 10-4; q = 0.004, OR, 3.99; 95% CI, 1.74-10.25). This trend was still evident when stratified by country, sex, and sequencing platform, and after a logistic regression analysis adjusting for age, sex, country, and the sequencing platform (p = 0.02; q = 0.03; OR, 7.3; 95% CI, 1.6-40.5). Torque teno virus (TTV), which made up 95% of the Anelloviridae reads, has been associated with reduced immune activation. We identify an association between viral co-infection and prolonged AIDs-free survival status that may have utility as a biomarker of LTNP and could provide mechanistic insights to HIV progression in children, demonstrating the added value of interrogating off-target WES reads in cohort studies.

Whole-Exome Sequencing Reveals Uncaptured Variation and Distinct Ancestry in the Southern African Population of Botswana.

Large-scale, population-based genomic studies have provided a context for modern medical genetics. Among such studies, however, African populations have remained relatively underrepresented. The breadth of genetic diversity across the African continent argues for an exploration of local genomic context to facilitate burgeoning disease mapping studies in Africa. We sought to characterize genetic variation and to assess population substructure within a cohort of HIV-positive children from Botswana-a Southern African country that is regionally underrepresented in genomic databases. Using whole-exome sequencing data from 164 Batswana and comparisons with 150 similarly sequenced HIV-positive Ugandan children, we found that 13%-25% of variation observed among Batswana was not captured by public databases. Uncaptured variants were significantly enriched (p = 2.2 × 10-16) for coding variants with minor allele frequencies between 1% and 5% and included predicted-damaging non-synonymous variants. Among variants found in public databases, corresponding allele frequencies varied widely, with Botswana having significantly higher allele frequencies among rare (<1%) pathogenic and damaging variants. Batswana clustered with other Southern African populations, but distinctly from 1000 Genomes African populations, and had limited evidence for admixture with extra-continental ancestries. We also observed a surprising lack of genetic substructure in Botswana, despite multiple tribal ethnicities and language groups, alongside a higher degree of relatedness than purported founder populations from the 1000 Genomes project. Our observations reveal a complex, but distinct, ancestral history and genomic architecture among Batswana and suggest that disease mapping within similar Southern African populations will require a deeper repository of genetic variation and allelic dependencies than presently exists.

The Collaborative African Genomics Network (CAfGEN): Applying Genomic technologies to probe host factors important to the progression of HIV and HIV-tuberculosis infection in sub-Saharan Africa.

Background: Here, we describe how the Collaborative African Genomics Network ( CAfGEN) of the Human Heredity and Health in Africa (H3Africa) consortium is using genomics to probe host genetic factors important to the progression of HIV and HIV-tuberculosis (TB) coinfection in sub-Saharan Africa.   The H3Africa was conceived to facilitate the application of genomics technologies to improve health across Africa..          Methods: CAfGEN is an H3Africa collaborative centre comprising expertise from the University of Botswana; Makerere University; Baylor College of Medicine Children's Clinical Centers of Excellence (COEs) in Botswana, Uganda, and Swaziland; as well as Baylor College of Medicine, Texas. The COEs provide clinical expertise for community engagement, participant recruitment and sample collection while the three University settings facilitate processing and management of genomic samples and provide infrastructure and training opportunities to sustain genomics research. Results: The project has focused on utilizing whole-exome sequencing to identify genetic variants contributing to extreme HIV disease progression phenotypes in children, as well as RNA sequencing and integrated genomics to identify host genetic factors associated with TB disease progression among HIV-positive children. These cohorts, developed using the COEs' electronic medical records, are exceptionally well-phenotyped and present an unprecedented opportunity to assess genetic factors in individuals whose HIV was acquired by a different route than their adult counterparts in the context of a unique clinical course and disease pathophysiology. Conclusions: Our approach offers the prospect of developing a critical mass of well-trained, highly-skilled, continent-based African genomic scientists. To ensure long term genomics research sustainability in Africa, CAfGEN contributes to a wide range of genomics capacity and infrastructure development on the continent, has laid a foundation for genomics graduate programs at its institutions, and continues to actively promote genomics research through innovative forms of community engagement brokered by partnerships with governments and academia to support genomics policy formulation.

The collaborative African genomics network training program: a trainee perspective on training the next generation of African scientists.

PURPOSE: The Collaborative African Genomics Network (CAfGEN) aims to establish sustainable genomics research programs in Botswana and Uganda through long-term training of PhD students from these countries at Baylor College of Medicine. Here, we present an overview of the CAfGEN PhD training program alongside trainees' perspectives on their involvement. BACKGROUND: Historically, collaborations between high-income countries (HICs) and low- and middle-income countries (LMICs), or North-South collaborations, have been criticized for the lack of a mutually beneficial distribution of resources and research findings, often undermining LMICs. CAfGEN plans to address this imbalance in the genomics field through a program of technology and expertise transfer to the participating LMICs. METHODS: An overview of the training program is presented. Trainees from the CAfGEN project summarized their experiences, looking specifically at the training model, benefits of the program, challenges encountered relating to the cultural transition, and program outcomes after the first 2 years. CONCLUSION: Collaborative training programs like CAfGEN will not only help establish sustainable long-term research initiatives in LMICs but also foster stronger North-South and South-South networks. The CAfGEN model offers a framework for the development of training programs aimed at genomics education for those for whom genomics is not their "first language." Genet Med advance online publication 06 April 2017.

Evaluation of the NucliSens EasyQ v2.0 assay in comparison with the Roche Amplicor v1.5 and the Roche CAP/CTM HIV-1 Test v2.0 in quantification of C-clade HIV-1 in plasma.

Human immunodeficiency virus type 1 (HIV-1) genetic diversity poses a challenge to reliable viral load monitoring. Discrepancies between different testing platforms have been observed, especially for non-clade-B virus. Therefore we compare, in antiretroviral therapy (ART)-naïve South African subjects predominantly infected with HIV-1 clade-C, three commercially available assays: the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0), the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0) and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5). Strong linear correlation was observed and Bland-Altman analyses showed overall good agreement between the assays with mean viral load differences of 0.078 log cp/ml (NucliSens v2.0 - Amplicor v1.5), 0.260 log cp/ml (CAP/CTM v2.0 - Amplicor v1.5) and 0.164 log cp/ml (CAP/CTM v2.0 - NucliSens v2.0), indicating lower mean viral load results for the Amplicor v1.5 and higher mean readings for the CAP/CTM v2.0. Consistent with observations following previous comparisons of CAP/CTM v2.0 versus Amplicor v1.5, the CAP/CTM v2.0 assay detected low-level viremia (median 65 cp/ml) in more than one-third of those in whom viremia had been undetectable (<20 cp/ml) in assays using the NucliSens platform. These levels of viremia are of uncertain clinical significance but may be of importance in early detection of ART resistance in those on treatment. Overall the three assays showed good comparability of results but with consistent, albeit relatively small, discrepancies for HIV-1 clade-C samples, especially in the low-viremic range that should be taken into account when interpreting viral load data.

Prevalence of transmitted HIV drug resistance in Botswana: lessons learned from the HIVDR-Threshold Survey conducted among women presenting for routine antenatal care as part of the 2007 national sentinel survey.

The emergence and spread of transmitted drug resistance (TDR) poses a major threat to the success of the rapidly expanding antiretroviral treatment (ART) programs in resource-limited countries. The World Health Organization recommends the use of the HIV Drug Resistance Threshold Survey (HIVDR-TS) as an affordable means to monitor the presence of TDR in these settings. We report our experiences and results of the 2007 HIVDR-TS in Botswana, a country with one of the longest-existing national public ART programs in Africa. The HIVDR-TS and HIV-1 incidence testing were performed in the two largest national sites as part of the 2007 antenatal Botswana Sentinel Survey. The HIVDR-TS showed no significant drug resistance mutations (TDR less than 5%) in one site. TDR prevalence, however, could not be ascertained at the second site due to low sample size. The agreement between HIVDR-TS eligibility criteria and laboratory-based methodologies (i.e., BED-CEIA and LS-EIA) in identifying recently HIV-1 infected adults was poor. Five years following the establishment of Botswana's public ART program, the prevalence of TDR remains low. The HIVDR-TS methodology has limitations for low-density populations as in Botswana, where the majority of antenatal sites are too small to recruit sufficient numbers of patients. In addition, the eligibility criteria (age <25 years and parity (first pregnancy)) of the HIVDR-TS performed poorly in identifying recent HIV-1 infections in Botswana. An alternative sampling strategy should be considered for the surveillance of HIVDR in Botswana and similar geographic settings.

Viral load and CD4+ T-cell dynamics in primary HIV-1 subtype C infection.

BACKGROUND: Most knowledge of primary HIV-1 infection is based on subtype B studies, whereas the evolution of viral parameters in the early phase of HIV-1 subtype C infection is not well characterized. METHODS: The kinetics of viral RNA, proviral DNA, CD4+ T-cell count, and subsets of CD4+ T cells expressing CCR5 or CXCR4 were characterized in 8 acute and 62 recent subtype C infections over the first year postseroconversion. RESULTS: The viral RNA peak was 6.25 +/- 0.92 log10 copies per milliliter. After seroconversion, heterogeneity among acute cases was evident by patterns of change in viral load and CD4+ T-cell count over time. The patterns were supported by the rate of viral RNA decline from peak (P = 0.022), viral RNA means (P = 0.005), CD4 levels (P < 0.001), and CD4 decline to 350 (P = 0.011) or 200 (P = 0.046). Proviral DNA had no apparent peak and its mean was 2.59 +/- 0.69 log10 per 106 peripheral blood mononuclear cell. In recent infections, viral RNA set point was 4.00 +/- 0.97 log10 and viral RNA correlated inversely with CD4+ T cells (P < 0.001) and directly with proviral DNA (P < 0.001). CONCLUSIONS: Distinct patterns of viral RNA evolution may exist shortly after seroconversion in HIV-1 subtype C infection. The study provides better understanding of the early phase of subtype C infection.