The possible implication of selected Fusarium Mycotoxins in the aetiology of brain cancer.

Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004.The central nervous system is a potential site of action for the Fusarium mycotoxin Fumonisin B1 (FB1), and is exemplified in horses by the disease equine leukoencephalomalacia. Structurally resembling sphingoid bases, FB1 inhibits ceramide synthase, an enzyme involved in sphingolipid metabolism, leading to accumulation of free sphinganine (Sa) and sphingosine (So). This investigation focused on FB1, Sa, So and the Fusarium mycotoxins fusaric acid (FA), moniliformin (MaN), zearalenone (ZEA), deoxynivalenol (DON), and T-2 toxin (T2). Effects of the Fusarium mycotoxins and sphingoid bases on the N2a neuroblastoma cell line were assessed using the methylthiazol tetrazolium (MIT) and ApoGlow™ assays. The MIT assay revealed significant differences between the viability of N28 control cells and the cytotoxic effects of FB1 (p=0.001), So (p=1.1 x10-6 ), Sa (p=1.9x10-6 ), MON (p=0.002), DON (p=0.04) and ZEA (p=0.003) on N28 cells between 5-250µM. The cytotoxic effects of FA did not differ significantly from controls (p=0.1). The ApoGlow™ assay revealed that in N28 cells, FB1 at 8µg.ml-1, FA at 128µg.ml-1, and (FBI+FA) combined induced growth arrest at 2 and 4µg·ml-1. Assessment of the effects of FBI and FA on the Jurkat leukaemic suspension cell line revealed that FB1 induced apoptosis at 1.56,12.5 and 50µ.ml-1, growth arrest at 100, 200 and 800µg.ml-1 and proliferation at 400µg.mg-1. Fusaric acid induced proliferation at 1. 56µg.ml-1, apoptosis at 3.15µg.mrl, growth arrest at 100 and 200µg.mrl, and necrosis at 800µg.ml-1. Combined, (FB1+FA) induced apoptosis at 1.56, 3.15,12.5 and 800µg.ml-1. Flow cytometry and fluorescence microscopy revealed that mycotoxins, Sa and So induced varying levels of apoptosis and necrosis in N28 cells. Acridine orange and ethidium bromide staining facilitated discrimination between viable, apoptotic and necrotic cells. Transition of the mitochondrial transmembrane potential was measured using Rhodamine 123 with propidium iodide, and the dual emission potential sensitive stain JC-1. Changes in mitochondrial membrane potential and plasma membrane integrity were expressed as increases or decreases in fluorescence intensity. An increase in mycotoxin concentration from 50 to 200µM was usually paralleled by a decrease in J-aggregate formation, suggesting a decrease in the ?¦¥m. Staining with Rh 123/PI indicated at specific concentrations whether N28 cells were either late apoptotic or necrotic reflected by the levels of PI uptake. No dose dependant mechanism of cell death was established using either method, as fluctuations were evident. Immunolocalisation of T2, ZEA and FB1 within cellular organelles that exhibited ultrastructural pathology provided correlation between mycotoxin exposure and effects. Multinucleate giant cells and retraction of cellular processes were observed. At the electron microscope (EM) level, FB1 was immunolocalised within microsegregated and peripherally condensed nucleoli, the nucleoplasm, distorted mitochondria and dilated endoplasmic reticulum (ER). The capacity of cells to incorporate mycotoxins and effect cytological changes represents a major factor in the potential for initiation of malignant transformation. Exposure of N2a cells to FB1 for 72 hours increased intracellular free Sa and depleted complex sphingolipids. Using High Performance Liquid chromatography (HPLC), acid hydrolysis revealed reduction in Sa from a level of O.6±0.12µM in control cells, to 02±0.lµM in cells exposed to 50µM and lOOµM FB1. Base hydrolyses revealed increase in free Sa: So ratios from 0.52±0.2 in control cells, to 1.14±0.2 and 1.4±0.3 in cells exposed to 50 and l00µM FB1 respectively. The Sa: So ratio in the complete culture media (CCM) increased from 1. 7±0. 3 for control cells to 2.0±0.2 and 2.50±0.4 for cells exposed to 50 and lOOµM FB1 respectively. Correlation coefficients between Sa: So ratios to FB1 exposure in CCM (R=0.75) and within cells (R=0.85), imply that the free Sa: So ratio within cells appears to be a better biomarker for FB1-induced disruption of sphingolipid metabolism in vitro, than the Sa: So ratio in CCM. Optimisation of HPLC analytical procedures improved recovery of FB I from spiked human sera to 95.8% (n=15) and detection limits to -5ng.ml-1 at a signal to noise ratio of 5:1. Optimisation of methods for recovery of Sa and So from spiked sera, led to recoveries of 77.9% and 85.0%, for So and Sa respectively at levels of spiking with lOng per 500µl of serum. Matched sera Sa:So ratios and FB1 levels in brain cancer and non-cancer subjects in KwaZulu-Natal were determined using these optimised methods. Fumonisin B1 was detected in sera of non-cancer (76.7±62.2nM) and brain cancer subjects (l07.38±116nM). Mean serum Sa:So ratios of 21 non-cancer subjects was 1.7±0.7. There was no correlation (R=0.26) between these variables in non-cancer subjects. The mean serum FB1 level in brain cancer subjects was 107.4±116nM (range 10.5-298nM) (n=50) and the mean Sa:So ratio (n=50) was 1.9±1.7 (range 0.40-8.16). No correlation was found between these variables in the brain cancer subjects either (R = -0.23). Fumonisin B1 was irnmunolocalised in 49 of 76 brain tumour tissue samples analysed using immunohistochemistry (IHC). Thirty-eight of the 76 specimens had matched serum FBI levels and Sa: So ratios, and 23 of these were positive for FB1 presence. Although not significantly different (p=0.ll), the FBI sera levels in the cancer group with FBI within the tumour tissue had higher levels of FB1 in sera than the IHC FB1 negative group. Fumonisin B1 was localised within irregular profiles of nuclei, elongated and swollen mitochondria and ER. Immunolocalisation of FB1 within organelles in the brain showing ultrastructural cellular pathology suggests FBI may be implicated in the aetiology of human brain carcinogenesis

HIV testing, care and viral suppression among men who have sex with men and transgender individuals in Johannesburg, South Africa.

INTRODUCTION: Men who have sex with men and transgender individuals (MSM/TG) carry a disproportionately high burden of HIV, including in South Africa. However, there are few empirical population-representative estimates of viral suppression and the HIV care cascade including HIV testing among this population, nor of factors associated with these outcomes. METHODS: We conducted a respondent driven sampling (RDS) survey among 301 MSM/TG in Johannesburg in 2017. Participants gave blood samples for HIV testing and viral load. Participants self-completed a survey including sociodemographics, HIV testing history, and engagement in care. We calculated RDS-II weighted estimates of the percentage of HIV-negative MSM/TG reporting HIV testing in the previous 6 months, their testing experience and preferences. Among those HIV-positive, we estimated the percentage status-aware, on ART, and virally suppressed (<50 viral copies/ml plasma). We conducted RDS-weighted robust Poisson regression to obtain weighted prevalence ratios of factors associated with 1) HIV testing among those HIV-negative; and 2) viral suppression among those HIV-positive. RESULTS: There were 118/300 HIV-positive MSM/TG, (37.5%). Of the HIV-negative MSM/TG, 61.5% reported that they had tested for HIV in the previous 6 months, which was associated with selling sex to men (Prevalence Ratio = 1.67, 95% CI 1.36-2.05). There were 76/118 HIV-positive MSM/TG (56.5%) who reported having previously tested positive for HIV and 39/118 (30.0%) who reported current ART. There were 58/118 HIV-positive MSM/TG with viral loads <50 copies/ml plasma (46.9%). Viral suppression was associated with older age (adjusted PR = 1.03, 95% CI 1.00-1.06 for each year), neighbourhood, and having bought sex from men (adjusted PR = 1.53, 95% CI 1.12-2.08). CONCLUSIONS: HIV prevalence was very high. Viral suppression among those HIV-positive was similar to the general male population in South Africa, but remains far short of national and international targets. A majority of HIV-negative MSM/TG had HIV tested in the previous 6 months, though there is room for improvement

Online socializing among men who have sex with men and transgender people in Nairobi and Johannesburg and implications for public health-related research and health promotion: an analysis of qualitative and respondent-driven sampling survey data.

INTRODUCTION: There is little published literature about gay, bisexual and other men who have sex with men and transgender individuals (MSM and TG)'s use of social media in sub-Saharan Africa, despite repressive social and/or criminalizing contexts that limit access to physical HIV prevention. We sought to describe MSM and TG's online socializing in Nairobi and Johannesburg, identifying the characteristics of those socializing online and those not, in order to inform the development of research and health promotion in online environments. METHODS: Respondent-driven sampling surveys were conducted in 2017 in Nairobi (n = 618) and Johannesburg (n = 301) with those reporting current male gender identity or male sex assigned at birth and sex with a man in the last 12 months. Online socializing patterns, sociodemographic, sexual behaviour and HIV-testing data were collected. We examined associations between social media use and sociodemographic characteristics and sexual behaviours among all, and only those HIV-uninfected, using logistic regression. Analyses were RDS-II weighted. Thirty qualitative interviews were conducted with MSM and TG in each city, which examined the broader context of and motivations for social media use. RESULTS: Most MSM and TG had used social media to socialize with MSM in the last month (60% Johannesburg, 71% Nairobi), mostly using generic platforms (e.g. Facebook), but also gay-specific (e.g. Grindr). HIV-uninfected MSM and TG reporting riskier recent sexual behaviours had raised odds of social media use in Nairobi, including receptive anal intercourse (adjusted OR = 2.15, p = 0.006), buying (aOR = 2.24, p = 0.015) and selling sex with men (aOR = 2.17, p = 0.004). Evidence for these associations was weaker in Johannesburg, though socializing online was associated with condomless anal intercourse (aOR = 3.67, p = 0.003) and active syphilis (aOR = 13.50, p = 0.016). Qualitative findings indicated that while online socializing can limit risk of harm inherent in face-to-face interactions, novel challenges were introduced, including context collapse and a fear of blackmail. CONCLUSIONS: Most MSM and TG in these cities socialize online regularly. Users reported HIV acquisition risk behaviours, yet this space is not fully utilized for sexual health promotion and research engagement. Effective, safe and acceptable means of using online channels to engage with MSM/TG that account for MSM and TG's strategies and concerns for managing online security should now be explored, as complements or alternatives to existing outreach

Experiences in conducting multiple community-based HIV prevention trials among women in KwaZulu-Natal, South Africa

<p>Abstract</p> <p>Background</p> <p>South Africa, with its scientific capacity, good infrastructure and high HIV incidence rates, is ideally positioned to conduct large-scale HIV prevention trials. The HIV Prevention Research Unit of the South African Medical Research Council conducted four phase III and one phase IIb trials of women-initiated HIV prevention options in KwaZulu-Natal between 2003 and 2009. A total of 7046 women participated, with HIV prevalence between 25% and 45% and HIV incidence ranging from 4.5-9.1% per year. Unfortunately none of the interventions tested had any impact on reducing the risk of HIV acquisition; however, extremely valuable experience was gained, lessons learned and capacity built, while the communities gained associated benefits.</p> <p>Experience</p> <p>Our experience in conducting these trials ranged from setting up community partnerships to developing clinical research sites and dissemination of trial results. Community engagement included setting up community-based research sites with approval from both political and traditional leaders, and developing community advisory groups to assist with the research process. Community-wide education on HIV/sexually transmitted infection prevention, treatment and care was provided to over 90 000 individuals. Myths and misconceptions were addressed through methods such as anonymous suggestion boxes in clinic waiting areas and intensive education and counselling. Attempts were made to involve male partners to foster support and facilitate recruitment of women. Peer educator programmes were initiated to provide ongoing education and also to facilitate recruitment of women to the trials. Recruitment strategies such as door-to-door recruitment and community group meetings were initiated. Over 90% of women enrolled were retained.</p> <p>Community benefits from the trial included education on HIV prevention, treatment and care and provision of ancillary care (such as Pap smears, reproductive health care and referral for chronic illnesses). Social benefits included training of home-based caregivers and sustainable ongoing HIV prevention education through peer educator programmes.</p> <p>Challenges</p> <p>Several challenges were encountered, including manipulation by participants of their eligibility criteria in order to enroll in the trial. Women attempted to co-enroll in multiple trials to benefit from financial reimbursements and individualised care. The trials became ethically challenging when participants refused to take up referrals for care due to stigma, denial of their HIV status and inadequate health infrastructure. Lack of disclosure of HIV status to partners and family members was particularly challenging. Some of the ethical dilemmas put to the test our responsibility as researchers and our obligation to provide health care to research participants.</p> <p>Conclusion</p> <p>Conducting these five trials in a period of six years provided us with invaluable insights into trial implementation, community participation, recruitment and retention, provision of care and dissemination of trial results. The critical mass of scientists trained as clinical trialists will continue to address the relentless HIV epidemic in our setting and ensure our commitment to finding a biomedical HIV prevention option for women in the future.</p

Cost-effectiveness of single-visit cervical cancer screening in KwaZulu-Natal, South Africa: a model-based analysis accounting for the HIV epidemic

Introduction Women living with human immunodeficiency virus (WLHIV) face elevated risks of human papillomavirus (HPV) acquisition and cervical cancer (CC). Coverage of CC screening and treatment remains low in low-and-middle-income settings, reflecting resource challenges and loss to follow-up with current strategies. We estimated the health and economic impact of alternative scalable CC screening strategies in KwaZulu-Natal, South Africa, a region with high burden of CC and HIV. Methods We parameterized a dynamic compartmental model of HPV and HIV transmission and CC natural history to KwaZulu-Natal. Over 100 years, we simulated the status quo of a multi-visit screening and treatment strategy with cytology and colposcopy triage (South African standard of care) and six single-visit comparator scenarios with varying: 1) screening strategy (HPV DNA testing alone, with genotyping, or with automated visual evaluation triage, a new high-performance technology), 2) screening frequency (once-per-lifetime for all women, or repeated every 5 years for WLHIV and twice for women without HIV), and 3) loss to follow-up for treatment. Using the Ministry of Health perspective, we estimated costs associated with HPV vaccination, screening, and pre-cancer, CC, and HIV treatment. We quantified CC cases, deaths, and disability-adjusted life-years (DALYs) averted for each scenario. We discounted costs (2022 US dollars) and outcomes at 3% annually and calculated incremental cost-effectiveness ratios (ICERs). Results We projected 69,294 new CC cases and 43,950 CC-related deaths in the status quo scenario. HPV DNA testing achieved the greatest improvement in health outcomes, averting 9.4% of cases and 9.0% of deaths with one-time screening and 37.1% and 35.1%, respectively, with repeat screening. Compared to the cost of the status quo ($12.79 billion), repeat screening using HPV DNA genotyping had the greatest increase in costs. Repeat screening with HPV DNA testing was the most effective strategy below the willingness to pay threshold (ICER:$3,194/DALY averted). One-time screening with HPV DNA testing was also an efficient strategy (ICER: $1,398/DALY averted). Conclusions Repeat single-visit screening with HPV DNA testing was the optimal strategy simulated. Single-visit strategies with increased frequency for WLHIV may be cost-effective in KwaZulu-Natal and similar settings with high HIV and HPV prevalence

Weight change among women using intramuscular depot medroxyprogesterone acetate, a copper intrauterine device, or a levonorgestrel implant for contraception : findings from a randomised, multicentre, open-label trial

BACKGROUND : There is limited evidence on the impact of the use of progestin-only hormonal contraception (POC) on weight change. We conducted a secondary analysis of prospective weight change among women enrolled in the Evidence for Contraceptive options and HIV Outcomes (ECHO) trial. METHODS : The ECHO trial was conducted at 12 sites in eSwatini, Kenya, South Africa and Zambia between December 2015 and October 2018. HIV negative, women aged 16 35 years, desiring contraception, were randomised (1:1:1) to either 3-monthly intramuscular depot medroxyprogesterone acetate (DMPA-IM), levonorgestrel (LNG) implant or copper intrauterine device (IUD). Follow-up was up to 18 months. Weight (kg) was measured at baseline and study exit. Analysis was performed as intention to treat (ITT) and time on continuous contraceptive use. The primary outcome of this secondary analysis is weight change from study enrolment to the final visit at study month 12 18. The ECHO trial is registered with ClinicalTrials.gov, NCT02550067. FINDINGS : 7829 women were randomly assigned to DMPA-IM (n = 2609), copper IUD (n = 2607) or LNG implant (n = 2613). The ITT population included 7014 women 2293 DMPA-IM group, 2372 copper IUD group and 2349 LNG group) who were not lost to follow-up, pregnant on study, or missing weight data. The mean weight increased in all groups but was significantly different in magnitude: 3.5 kg (SD = 6.3), 2.4 kg (SD = 5.9) and 1.5 kg (SD = 5.7) in the DMPA-IM, LNG implant and copper IUD groups, respectively. Comparative differences between groups were (2.02 kg (95% CI, 1.68, 2.36, p < 0.001) for DMPA-IM versus copper IUD, 0.87 kg (0.53,1.20 p < 0.001) for LNG implant compared to copper IUD and 1.16 kg (0.82, 1.50, p < 0.001) for DMPAIM compared with LNG implant. Results for continuous contraceptive use were similar. INTERPRETATION : We found differences in weight gain between POC users compared to the non-hormonal copper IUD group over 12 18 months of use. Women using POCs should be counselled about this potential side effect when choosing a contraceptive method.Bill & Melinda Gates Foundation, US Agency for International Development (USAID) and the President’s Emergency Plan for AIDS Relief, Swedish International Development Cooperation Agency, South African Medical Research Council, and UNFPA.http://www.journals.elsevier.com/eclinicalmedicineam2022Medical Microbiolog

ART initiation among women newly diagnosed with HIV in a contraceptive trial in sub-Saharan Africa

Current guidelines recommend starting antiretroviral therapy (ART) as soon as possible after HIV diagnosis to reduce morbidity, mortality and onward HIV transmission. We examined factors influencing ART initiation by women who seroconverted during the Evidence for Contraceptive Options and HIV Outcomes (ECHO) Trial. ECHO, conducted between 2015 and 2018, enrolled HIV-negative, sexually active women, aged 16–35 years, from four African countries. Follow-up was 12–18 months, with quarterly HIV testing. Women with incident HIV infection received extensive counselling by trial staff and referral to local facilities for HIV care. Of 304 women with ≥90 days follow-up time since HIV diagnosis, 186(61.2%) initiated ART within 90 days, 69(22.7%) initiated after 90 days, and 49(16.1%) had not initiated by the end of the study. There were no statistically significant differences in characteristics among women who initiated ART ≤90 days versus those who did not. Frequent reasons for delayed or non-initiation of ART included not feeling ready to start ART and being newly diagnosed. In a large clinical trial, ART initiation was modest within 90 days of HIV diagnosis and grew to 84% with longer observation. Despite extensive counselling on the importance of early ART initiation, personal barriers delayed some women from starting ART.https://www.tandfonline.com/loi/caic20pm2021Medical Microbiolog

Integrating oral PrEP delivery among African women in a large HIV endpoint-driven clinical trial

INTRODUCTION : Global guidelines emphasize the ethical obligation of investigators to help participants in HIV-endpoint trials reduce HIV risk by offering an optimal HIV prevention package. Oral pre-exposure prophylaxis (PrEP) has increasingly become part of state-of-the-art HIV prevention. Here we describe the process of integrating oral PrEP delivery into the HIV prevention package of the Evidence for Contraceptive Options and HIV Outcomes (ECHO) Trial. METHODS : ECHO was an open-label randomized clinical trial that compared HIV incidence among women randomized to one of three effective contraceptives. In total, 7830 women aged 16 to 35 years from 12 sites in four African countries (Eswatini, Kenya, South Africa and Zambia) were enrolled and followed for 12 to 18 months, from 2015 to 2018. Part-way through the course of the trial, oral PrEP was provided to study participants either off-site via referral or on site via trained trial staff. PrEP uptake was compared between different contraceptive users using Chi-squared tests or t-tests. HIV seroincidence rates were compared between participants who never versus ever initiated PrEP using exact Poisson regression. RESULTS : PrEP access in ECHO began through public availability in Kenya in May 2017 and was available at all sites by June 2018. When PrEP became available, 3626 (46.3%) eligible women were still in follow-up in the study, and of these, 622 (17.2%) initiated PrEP. Women initiating PrEP were slightly older; more likely to be unmarried, not living with their partner, having multiple partners; and less likely to be earning their own income and receiving financial support from partners (all p < 0.05). PrEP initiation did not differ across study randomized groups (p = 0.7). Two-thirds of PrEP users were continuing PrEP at study exit. CONCLUSIONS : There is a need for improved HIV prevention services in clinical trials with HIV endpoints, especially trials among African women. PrEP as a component of a comprehensive HIV prevention package provided to women in a large clinical trial is practical and feasible. Provision of PrEP within clinical trials with HIV outcomes should be standard of prevention.The ECHO Trial was funded by Bill & Melinda Gates Foundation, US Agency for International Development and the President’s Emergency Plan for AIDS Relief, Swedish International Development Cooperation Agency, South African Medical Research Council and UN Population Fund. Contraceptive supplies were donated by the Government of South Africa and US Agency for International Development. IB received funding from the South African Medical Research Council under the SAMRC Clinician Researcher MD PhD Development Programme.https://onlinelibrary.wiley.com/journal/17582652am2020Family Medicin

HIV disease progression among women following seroconversion during a tenofovir-based HIV prevention trial.

CAPRISA, 2017.Abstract available in pdf