Genetic polymorphisms in xenobiotic (or drug) metabolizing enzyme genes among 18 sub-Saharan African populations: a window into genetic diversity

A dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Masters of Science in Medicine, 2014Many loci coding for xenobiotic metabolising enzymes, especially those involved in carcinogen metabolism, confer susceptibility to various types of cancers. These genes have been poorly investigated in sub-Saharan African populations, where the genetic variation that exists is relatively unknown. The primary objectives of the study are to determine the frequency variation among 15 loci in sub-Saharan Africans, the level of genetic diversity, and the genetic affinities among sub-Saharan Africans. Secondary, the study aims to evaluate the implication of these variants in disease susceptibility, especially cancer. The study population comprised of 1880 unrelated individuals from 18 sub-Saharan African populations. DNA samples were used to examine genetic variation for phase I metabolism genes CYP1A1, CYP1A2, CYP2A6, CYP2D6, CYP2E1, and phase II metabolism genes GSTM1, GSTT1, GSTP1 and NAT2. A single base extension (SBE) method was designed and used to genotype single nucleotide polymorphisms (SNP): CYP1A1*2A and *2C; CYP1A2*1C and *1F; CYP2A6*7 and *8; CYP2D6*3A (2549delA) and CYP2D6*4(1846G>A); CYP2E1*5B(PstI) and CYP2E1*5B(RsaI); GSTP1*Ile105Val and *Ala114Val; and NAT2*14A. To investigate the presence of null mutations GSTM1*0 and GSTT1*0 a previously reported multiplex PCR method was used. The distribution of mutations in the sample was interpreted and compared with data from literature respectively. Mutations CYP1A1*2A, CYP1A2*1C, CYP1A2*1F, CYP2A6*7, CYP2D6*4 and GSTP1*Ile105Val mutations was found in most sub-Saharan Africans, while CYP1A1*2C, CYP2A6*8, CYP2D6*3A and GSTP1*Ala114Val mutations were almost non-existent. Both GSTM1*0 and GSTT1*0 mutations were present in all populations, with GSTT1*0 most frequent. The distribution of NAT2*14A confirms previous reports of its exclusive existence in Africans. Hardy-Weinberg Equilibrium (HWE) and Tajima’s D statistic tests showed none of the mutations were under selection. The genetic affinities of sub-Saharans were analysed. Bantu-speakers were closely related with little correlation to their geographic locations. Khoisan-speakers were closely related, genetically most distinct and oldest among populations. Pygmies were similarly distinct from most populations and one of the oldest surviving populations. The data further supports previous reports that the Khwe are descendants of an east African pastoralist group. AMOVA analyses revealed language as a major confounder among sub-Saharans. Haplotypes were inferred to determine their distribution and to understand their significance in populations with respect to their functional relevance. The study has confirmed previous reports of genetic histories of these sub-Saharan African populations. In unravelling the distribution of these mutations, the study has added to the global picture of these mutations. In doing so, the data may add value to the design of future cancer studies and pharmacological studies. The study also highlights the importance of elucidating ancestral relations of populations, more specifically linguistic and anthropological relationships, and to include in the design of future clinical trials in Africa

Pregnancy incidence and correlates in a clinical trial preparedness study, North West Province South Africa.

INTRODUCTION: Women in HIV prevention trials often must typically agree to avoid pregnancy. Regardless, some become pregnant. Screening tools predicting pregnancy risk could maximize trial safety and efficiency. OBJECTIVES: We assessed incidence and correlates of pregnancy among women at high HIV risk. METHODS: We enrolled sexually-active, HIV-negative women into an observational cohort (2008-2011). At enrollment demographic, contraceptive, reproductive, pregnancy intention and behavioural data were collected. Women reported if one or both partners wanted or intended for the couple to become pregnant. We measured gender role beliefs using a locally validated eight-point index. We tested HIV and pregnancy, and inquired about sexually transmitted infection symptoms (STIs) at enrollment and monthly. HIV testing included behavioural counselling and condom provision, but did not specifically counsel women to avoid pregnancy. Cox proportional hazard modelling evaluated the associations with pregnancy. The multivariate model included the following variables "Recent pregnancy attempts", "Gender Roles Beliefs", "Self-reported STIs" and "Age". RESULTS: We screened 1068 women and excluded (24.6%, 263/1068) who did not report risk behaviour. Non-pregnant, non-sterilized women aged 18-35 (median = 21 years) enrolled (n = 438). Most women reported one partner (74.7%) and a prior live birth (84.6%). Median follow-up time was 6 months (range 0.7-15.5). Pregnancy incidence was 25.1 per 100 women-years (n = 57 pregnancies). Conservative beliefs on gender roles (Adjusted Hazard Ratio (aHR) 1.8; 95% confidence interval [CI] 1.1-2.9), recent pregnancy attempts (aHR 1.9; 95% CI 1.1-3.4) and baseline self-reported STI (aHR 2.5; 95% CI 1.4-4.4) were associated with increased incident pregnancy. Report of no pregnancy intention was associated with lowered pregnancy risk (aHR 0.3; 95% CI 0.1-0.7). CONCLUSIONS: We identified new and confirmed existing factors that can facilitate screening for pregnancy risk

Development of a single base extension method to resolve Y chromosome haplogroups in sub-Saharan African populations

<p>Abstract</p> <p>Background</p> <p>The ability of the Y chromosome to retain a record of its evolution has seen it become an essential tool of molecular anthropology. In the last few years, however, it has also found use in forensic genetics, providing information on the geographic origin of individuals. This has been aided by the development of efficient screening methods and an increased knowledge of geographic distribution. In this study, we describe the development of single base extension assays used to resolve 61 Y chromosome haplogroups, mainly within haplogroups A, B and E, found in Africa.</p> <p>Results</p> <p>Seven multiplex assays, which incorporated 60 Y chromosome markers, were developed. These resolved Y chromosomes to 61 terminal branches of the major African haplogroups A, B and E, while also including a few Eurasian haplogroups found occasionally in African males. Following its validation, the assays were used to screen 683 individuals from Southern Africa, including south eastern Bantu speakers (BAN), Khoe-San (KS) and South African Whites (SAW). Of the 61 haplogroups that the assays collectively resolved, 26 were found in the 683 samples. While haplogroup sharing was common between the BAN and KS, the frequencies of these haplogroups varied appreciably. Both groups showed low levels of assimilation of Eurasian haplogroups and only two individuals in the SAW clearly had Y chromosomes of African ancestry.</p> <p>Conclusions</p> <p>The use of these single base extension assays in screening increased haplogroup resolution and sampling throughput, while saving time and DNA. Their use, together with the screening of short tandem repeat markers would considerably improve resolution, thus refining the geographic ancestry of individuals.</p

Failure of A Novel, Rapid Antigen and Antibody Combination Test to Detect Antigen-Positive HIV Infection in African Adults with Early HIV Infection

BACKGROUND: Acute HIV infection (prior to antibody seroconversion) represents a high-risk window for HIV transmission. Development of a test to detect acute infection at the point-of-care is urgent. METHODS: Volunteers enrolled in a prospective study of HIV incidence in four African cities, Kigali in Rwanda and Ndola, Kitwe and Lusaka in Zambia, were tested regularly for HIV by rapid antibody test and p24 antigen ELISA. Five subgroups of samples were also tested by the Determine Ag/Ab Combo test 1) Antigen positive, antibody negative (acute infection); 2) Antigen positive, antibody positive; 3) Antigen negative, antibody positive; 4) Antigen negative, antibody negative; and 5) Antigen false positive, antibody negative (HIV uninfected). A sixth group included serial dilutions from a p24 antigen-positive control sample. Combo test results were reported as antigen positive, antibody positive, or both. RESULTS: Of 34 group 1 samples with VL between 5x105 and >1.5x107 copies/mL (median 3.5x106), 1 (2.9%) was detected by the Combo antigen component, 7 (20.6%) others were positive by the Combo antibody component. No group 2 samples were antigen positive by the Combo test (0/18). Sensitivity of the Combo antigen test was therefore 1.9% (1/52, 95% CI 0.0, 9.9). One false positive Combo antibody result (1/30, 3.3%) was observed in group 4. No false-positive Combo antigen results were observed. The Combo antigen test was positive in group 6 at concentrations of 80 pg/mL, faintly positive at 40 and 20 pg/mL, and negative thereafter. The p24 ELISA antigen test remained positive at 5 pg/mL. CONCLUSIONS: Although the antibody component of the Combo test detected antibodies to HIV earlier than the comparison antibody tests used, less than 2% of the cases of antigen-positive HIV infection were detected by the Combo antigen component. The development of a rapid point-of-care test to diagnose acute HIV infection remains an urgent goal

High levels of pretreatment HIV-1 drug resistance mutations among South African women who acquired HIV during a prospective study

Access to data from the ECHO Study may be requested through submission of a research concept to [email protected]. The concept must include the research question, data requested, analytic methods, and steps taken to ensure ethical use of the data. Access will be granted if the concept is evaluated to have scientific merit and if sufficient data protections are in place. As of the time of publication, data access applications are in process with the governing institutional review boards of the ECHO Study to make de-identified data publicly available.BACKGROUND : Pretreatment HIV drug resistance (PDR) undermines individual treatment success and threatens the achievement of UNAIDS 95-95-95 targets. In many African countries, limited data are available on PDR as detection of recent HIV infection is uncommon and access to resistance testing is limited. We describe the prevalence of PDR among South African women with recent HIV infection from the Evidence for Contraceptive Options and HIV Outcomes (ECHO) Trial. METHODS : HIV-uninfected, sexually active women, aged 18–35 years, and seeking contraception were enrolled in the ECHO Trial at sites in South Africa, from 2015 to 2018. HIV testing was done at trial entry and repeated quarterly. We tested stored plasma samples collected at HIV diagnosis from women who seroconverted during follow-up and had a viral load >1000 copies/mL for antiretroviral resistant mutations using a validated laboratory-developed population genotyping assay, which sequences the full protease and reverse transcriptase regions. Mutation profiles were determined using the Stanford Drug Resistance Database. RESULTS : We sequenced 275 samples. The median age was 23 years, and majority (98.9%, n = 272) were infected with HIV-1 subtype C. The prevalence of surveillance drug resistance mutations (SDRMs) was 13.5% (n = 37). Nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations were found in 12.4% of women (n = 34). Few women had NRTI (1.8%, n = 5) and protease inhibitor (1.1%, n = 3) mutations. Five women had multiple NRTI and NNRTI SDRMs. CONCLUSIONS : The high levels of PDR, particularly to NNRTIs, strongly support the recent change to the South African national HIV treatment guidelines to transition to a first-line drug regimen that excludes NNRTIs.The Bill & Melinda Gates Foundation, the American people through the United States Agency for International Development, the Swedish International Development Cooperation Agency, the South Africa Medical Research Council, and the United Nations Population Fund.http://journals.lww.com/jaidshj2023Family MedicineMedical Microbiolog

Control of the HIV-1 Load Varies by Viral Subtype in a Large Cohort of African Adults With Incident HIV-1 Infection.

Few human immunodeficiency virus (HIV)-infected persons can maintain low viral levels without therapeutic intervention. We evaluate predictors of spontaneous control of the viral load (hereafter, "viral control") in a prospective cohort of African adults shortly after HIV infection. Viral control was defined as ≥2 consecutively measured viral loads (VLs) of ≤10 000 copies/mL after the estimated date of infection, followed by at least 4 subsequent measurements for which the VL in at least 75% was ≤10 000 copies/mL in the absence of ART. Multivariable logistic regression characterized predictors of viral control. Of 590 eligible volunteers, 107 (18.1%) experienced viral control, of whom 25 (4.2%) maintained a VL of 51-2000 copies/mL, and 5 (0.8%) sustained a VL of ≤50 copies/mL. The median ART-free follow-up time was 3.3 years (range, 0.3-9.7 years). Factors independently associated with control were HIV-1 subtype A (reference, subtype C; adjusted odds ratio [aOR], 2.1 [95% confidence interval {CI}, 1.3-3.5]), female sex (reference, male sex; aOR, 1.8 [95% CI, 1.1-2.8]), and having HLA class I variant allele B*57 (reference, not having this allele; aOR, 1.9 [95% CI, 1.0-3.6]) in a multivariable model that also controlled for age at the time of infection and baseline CD4+ T-cell count. We observed strong associations between infecting HIV-1 subtype, HLA type, and sex on viral control in this cohort. HIV-1 subtype is important to consider when testing and designing new therapeutic and prevention technologies, including vaccines

HIV incidence and predictors of inconsistent condom use among adult men enrolled into an HIV vaccine preparedness study, Rustenburg, South Africa.

IntroductionUnderstanding HIV incidence and risk behaviour among populations being considered for HIV vaccine studies is necessary for the appropriate design of trials.MethodsBetween May 2012 and June 2015, we recruited men aged 18-49 years from urban and peri-urban areas of Rustenburg, a mining town in the North West Province, South Africa. Men who reported HIV-risk behaviour were followed for nine to 12 months to determine HIV incidence and factors associated with condom use.ResultsA total of 400 HIV uninfected men were enrolled; 366 (91.5%) had at least one follow-up visit and were included in the analysis; 47.6% were under 25 years of age. HIV incidence was 1.9 per 100 person-years (95% CI: 0.79-4.56). Among heterosexual men (N = 339), 80.8% reported having vaginal intercourse with multiple partners in the past three months, among whom 74.1% reported inconsistent condom use. Sixty-eight percent reported vaginal intercourse with new female partners, of whom 40.6% reported inconsistent condom use. Over half (55.6%) of men who had sex with men (N = 27) reported anal intercourse with multiple male partners in the past three months, of whom 68.2% reported using condoms inconsistently. Men who had more than two female partners in the last three months (n = 121) were more likely to use condoms inconsistently (aOR 4.31, 95% CI: 1.34-13.8); in contrast, those with more than one new female sex partner (aOR 0.13, 94% CI 0.04-0.44), and whose sexual debut was after 19 years of age (aOR 0.39, 95% CI: 0.15-1.01) were less likely to use condoms inconsistently.ConclusionHIV incidence was low and similar to other studies of heterosexual men in South Africa. To identify men at high risk for HIV for enrolment in prevention trials, future researchers may need to focus on those who report early sexual debut and who report having multiple sexual partners. Men in newer relationships appear to use condoms more frequently

Multivariable model of the association of past pregnancy attempts, gender roles beliefs, STI symptoms and age with pregnancy incidence (n = 409; 54 pregnancies)¹.

<p>Footnotes: ¹Number of observations reduced to 409 and number of pregnancies 54 due to missing data on the trying to become pregnant variable. ²This was the female participant's perception. n = 29 unknown, not applicable and missing data. ³Test for linear trend; relationship belief coded as progressive view = 1, neutral = 2, conservative view = 3. * From likelihood ratio test. ** All variables have been adjusted for each other.</p

Cohort description, pregnancy rate and associations with pregnancy incidence in HIV negative women (n = 438; 57 pregnancies).

<p>Footnotes: *Assuming a linear dose response where gender roles beliefs coded as progressive view = 1 neutral = 2, conservative view = 3 ¹n = 1 participant who spoke isiXhosa was removed from the analysis; ²This was the female participant's perception. n = 29 unknown, not applicable and missing data; ³n = 87 unknown, not applicable and missing data; ⁴n = 80 unknown, not applicable and missing data; ⁵n = 104 unknown, not applicable and missing data; ⁶n = 17 unknown, not applicable and missing data; ⁷n = 307 unknown, not applicable and missing data.</p