Adverse Drug Reactions Related with Antibiotic Medicines in Malawi: A Retrospective Analysis of Prevalence and Associated Factors.

Objective: We aimed to assess the occurrence and characteristics of antibiotic-associated adverse drug reactions (ADRs) in Malawi. Methods: We retrospectively reviewed 304 patient records from medical wards in three hospitals in Southern Malawi. A global trigger tool was applied for the detection of suspected ADRs, and we used the Naranjo scale, the World Health Organization classification and the Schumock and Thornton scale for causality, seriousness and preventability assessment respectively. ADRs were also further characterized according to anatomical systems. Statistical analysis was done in STATA 14.1. The Chi-square test was used to determine the association between categorical variables and logistic regression analysis was used to measure the strength of the association between various independent variables and the occurrence of ADRs. Results: Suspected ADRs were detected in 24% (73/304) of patients, of which 1.4% were definite, 15.1% were probable and 83.6% were possible ADRs. Most of the sADRs were gastrointestinal events (42.5%), followed by: musculoskeletal (26.3%); cardiovascular (16.3%); central nervous system (13.8%; and urinary events (1.3%). About 27% of the sADRs were serious events such as convulsions. The geriatric age group (≥65 years) was more likely to experience sADRs as compared to the younger age group, with an adjusted odds ratio (aOR) of 4.53, 95% CI (2.21-9.28), P<0.001. Patients taking more than one antibiotic medicine had a higher risk of developing sADRs as compared to patients who were administered one type of antibiotic medicine, aOR 2.14, 95% CI (1.18-3.90), p < 0.012. A long hospital stay of >3days was associated with a higher risk of sADRs with aOR of 5.11, 95% CI (2.47-10.55), p < 0.001 than those who stayed ≤ 3 days in the hospital. Conclusion: We found a higher prevalence of serious sADRs associated with antibiotic medicines than reported elsewhere. This may, among others, contribute to high patient mortality, poor treatment adherence, antibiotic resistance and increased cost of care.

What is already known and why we did the study? Most health care workers and patients are less likely to voluntarily report suspected adverse drug reactions in low- and middle-income countries such as Malawi.Studies have revealed a high usage of antibiotic medicines in Malawi, but there is limited data on the associated adverse drug reactions. What did we do? We assessed the occurrence and characteristics of ADRs associated with antibiotics. What are the new findings? We found a higher prevalence (24%) of adverse drug reactions associated with antibiotic therapy than reported elsewhere using the global trigger tool.About 27.4% of the events were serious ADRs such as convulsions, arrhythmia and hypotension.We observed a higher rate of convulsions which could be a potential safety signal. What do the new findings imply? The high prevalence of serious ADRs leads to complicated treatment strategies and contribute to patient mortality, poor treatment adherence and antibiotic resistance.ADR risk factors need to be considered when prescribing and monitoring patients on antibiotic therapy.

SARS-CoV-2 Seroprevalence and Vaccine Uptake among Pregnant Women at First Antenatal Care Visits in Malawi.

Many SARS-CoV-2 infections are asymptomatic, thus reported cases underestimate actual cases. To improve estimates, we conducted surveillance for SARS-CoV-2 seroprevalence among pregnant women attending their first antenatal care visit (ANC1) from June 2021 through May 2022. We administered a questionnaire to collect demographic, risk factors, and COVID-19 vaccine status information and tested dried blood spots for SARS-CoV-2 antibodies. Although <1% of ANC1 participants reported having had COVID-19, monthly SARS-CoV-2 seroprevalence increased from 15.4% (95% CI: 10.5-21.5) in June 2021 to 65.5% (95% CI: 55.5-73.7) in May 2022. Although COVID-19 vaccination was available in March 2021, uptake remained low, reaching a maximum of 9.5% (95% CI: 5.7-14.8) in May 2022. Results of ANC1 serosurveillance provided prevalence estimates helpful in understanding this population case burden that was available through self-report and national case reports. To improve vaccine uptake, efforts to address fears and misconceptions regarding COVID-19 vaccines are needed.

Effect of antibiotic medicines availability on adherence to standard treatment guidelines among hospitalized adult patients in southern Malawi.

BACKGROUND: Antibiotic resistance is a global public health problem. High and inappropriate use of antibiotic therapy exacerbate the risk of antibiotic resistance. We assessed the effect of availability of antibiotic medicines on adherence to standard treatment guidelines among hospitalized adult patients in Southern Malawi. METHODS: A cross-sectional study was done to assess the availability of 16 antibiotics among the first-line recommended treatments for common bacterial infections in Malawi. Data for up to six-month duration was extracted from stock card records in Machinga and Nsanje District Hospitals and Zomba Central Hospital. This was complemented by a retrospective review of 322 patient management files from medical wards to assess adherence to the Malawi Standard Treatment Guidelines (MSTG). Investigators abstracted data such as patient demographics, diagnoses, and prescribed therapy using a data collection form that resulted in analyzing 304 patient files. Data was entered into Microsoft excel and analyzed using STATA 14.1. Point availability, stock-out duration and adherence to treatment guidelines were presented in terms of frequencies and percentages. Chi-square test or Fisher's exact test was applied to assess the association between variables and adherence to treatment guidelines. RESULTS: Point availability of antibiotics was 81.5%, 87.7%, and 42.8% for Zomba Central, Machinga and Nsanje District Hospitals respectively. Over a period of six months, 12.5% of antibiotic medicines were stocked out for at least one day at Zomba (Median stock out days = 0, (IQR 0-0 days), while 64.3% were stocked out at Machinga (Median stock out days = 21, IQR 0-31 days) and 85.7% were stocked out at Nsanje District Hospital (Median stock out days = 66.5, IQR 18-113 days). Overall, adherence to MSTG was 79.6%, (95% CI, 73.3-84.9%). By facilities, adherence to guidelines at Zomba Central Hospital was 95.9% (95% CI, 89.7-98.9%) while at Nsanje and Machinga District Hospitals was 73.2% (95% CI, 59.7-84.2%) and 54.2% (95% CI, 39.2-68.6%) respectively. Adherence to treatment guidelines was associated with health facility, presence of laboratory test results, antibiotic spectrum, and WHO-AWaRe category of the medicine, p<0.005. Adherence was lower for antibiotics that were stocked out than antibiotics that were not stocked out during the study period (63.8%, 95% CI 48.5-77.3% vs 84.4%, 95% CI 77.7-89.8%), p< 0.002. CONCLUSION: We found unstable availability of antibiotic medicines in hospitals which might contribute to the sub-optimal adherence to standard treatment guidelines. This is a setback to efforts aimed at curbing antibiotic resistance in Malawi.

Towards Evidence-Based Implementation of Pharmacogenomics in Southern Africa: Comorbidities and Polypharmacy Profiles across Diseases.

Pharmacogenomics may improve patient care by guiding drug selection and dosing; however, this requires prior knowledge of the pharmacogenomics of drugs commonly used in a specific setting. The aim of this study was to identify a preliminary set of pharmacogenetic variants important in Southern Africa. We describe comorbidities in 3997 patients from Malawi, South Africa, and Zimbabwe. These patient cohorts were included in pharmacogenomic studies of anticoagulation, dyslipidemia, hypertension, HIV and breast cancer. The 20 topmost prescribed drugs in this population were identified. Using the literature, a list of pharmacogenes vital in the response to the top 20 drugs was constructed leading to drug-gene pairs potentially informative in translation of pharmacogenomics. The most reported morbidity was hypertension (58.4%), making antihypertensives the most prescribed drugs, particularly amlodipine. Dyslipidemia occurred in 31.5% of the participants, and statins were the most frequently prescribed as cholesterol-lowering drugs. HIV was reported in 20.3% of the study participants, with lamivudine/stavudine/efavirenz being the most prescribed antiretroviral combination. Based on these data, pharmacogenes of immediate interest in Southern African populations include ABCB1, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, SLC22A1, SLCO1B1 and UGT1A1. Variants in these genes are a good starting point for pharmacogenomic translation programs in Southern Africa.

Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 Countries.

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries.

Burden of and factors associated with poor quality antibiotic, antimalarial, antihypertensive and antidiabetic medicines in Malawi.

OBJECTIVE: To assess the prevalence and factors associated with substandard and falsified (SF) medicines among antibiotic, antimalarial, antihypertensive and antidiabetic medicines in Malawi. METHODS: We conducted a cross-sectional study in 23 public, faith-based and private health facilities in Zomba, Machinga and Nsanje districts. We analyzed oral medicine samples of commonly used medicines among antibiotics, antimalarial, antihypertensive and antidiabetics in accordance with Malawi Essential Medicines List and local treatment guidelines. These medicines were subjected to visual inspection for any defects and screening for the content of active pharmaceutical ingredient and disintegration of dosage units. Samples that failed during screening and at least 10% of those that passed were subjected to pharmacopeia assay and dissolution test for confirmation. We used thin layer chromatography and disintegration test methods provided in the Global Pharma Health Fund minilab® for the screening purposes. We conducted confirmatory test using High-Performance Liquid Chromatography (HPLC) or ultra-violet/visible spectrophotometer and dissolution. RESULTS: Of the 293 medicine samples collected, 14.3% were SF medicines. Among the SF medicines were 12.5% of Amlodipine (1/8), 19.2% of Amoxicillin (5/26), 72.2% of Atenolol (8/11), 21.2% of Ciprofloxacin (7/33), 14.3% of Enalapril (1/7), 44.4% of Flucloxacillin (4/9), and 35.7% of sulfadoxine/ pyrimethamine (10/28). Medicine quality was associated with therapeutic medicine class, stated origin of manufacturer, primary packaging material and geographical location. Antimalarial and antidiabetic medicines were of better quality as compared to antibiotics, odds ratio OR 4.2 (95% CI 1.7-9.49), p < 0.002 and OR 5.6 (95% CI 1.21-26.09), p < 0.028 respectively. In terms of stated country of origin, the prevalence of SF medicines was 30% (15/50), 33% (9/27), 26.7% (4/15) and 6.6% (8/122) for medicines stated to be manufactured in Malawi, China, Kenya and India respectively. CONCLUSION: This study presents the first findings on the assessment of quality of medicines since the establishment of the national pharmacovigilance center in 2019 in Malawi. It is revealed that the problem of SF medicines is not improving and hence the need for further strengthening of quality assurance systems in Malawi.

Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 Countries

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries.

Approaches to Deployment of Molecular Testing for SARS-CoV-2 in Resource-Limited Settings.

Deployment of molecular testing for SARS-CoV-2 in resource-limited settings is challenging. Scale-up of molecular had to be conducted with a laboratory system strengthening approach that emphasize laboratory integration. National reference laboratories play a central role. In Malawi the molecular testing was underpinned by existing pathogen control programs for human immunodeficiency virus and tuberculosis that use Abbott and GeneXpert machines and reagents. Despite this, the impact on these programs was well managed. Antigen testing increased access to testing. Pooled testing and direct-to-polymerase chain reaction methods have the potential to save costs and further increase access to molecular tests.

Ethnobotanical Study of Medicinal Plants Used for Management of Cancer in Karonga District, Northern Malawi.

BACKGROUND: In Malawi, cancer is one of the leading causes of morbidity and mortality. This has led to increased use of herbal medicines for cancer management. OBJECTIVE: This study aimed at identifying medicinal plants that are used for the management of cancer in the southern area of Karonga district, Northern Malawi. METHODS: Semi-structured individual questionnaire interviews were used to collect ethnobotanical data from traditional herbal practitioners in the study area. RESULTS: A total of twenty six (26) plant species from seventeen (17) botanical families were reported by Traditional Herbal Practitioners to be effective in the management of cancer. The botanical families with representation of more than one plant species were Fabaceae with five species, followed by Combretaceae and the Anacardiaceae with three species each, and Meliaceaewith with two species. The relative frequency of citation (RFC) showed that Senna singueana (RFC = 0.833), Lannea discolour (RFC = 0.833), Melia azedarach (RFC = 0.667), and Moringa oleifera (RFC = 0.667) were the medicinal plant species which were frequently mentioned and used in the study. The recipes could be a mixture of plant species or plant parts such as the leaves, barks, roots, rhizomes, seeds, flowers, and fruits. CONCLUSION: The study showed that a potential cancer management drug could be developed from the medicinal plant species found in the area. The results of this study could provide baseline information on medicinal plant species for further phytochemical studies and other studies to validate their use.

HIV-1 Recent Infection Testing Algorithm With Antiretroviral Drug Detection to Improve Accuracy of Incidence Estimates.

BACKGROUND: HIV-1 incidence calculation currently includes recency classification by HIV-1 incidence assay and unsuppressed viral load (VL ≥ 1000 copies/mL) in a recent infection testing algorithm (RITA). However, persons with recent classification not virally suppressed and taking antiretroviral (ARV) medication may be misclassified. SETTING: We used data from 13 African household surveys to describe the impact of an ARV-adjusted RITA on HIV-1 incidence estimates. METHODS: HIV-seropositive samples were tested for recency using the HIV-1 Limiting Antigen (LAg)-Avidity enzyme immunoassay, HIV-1 viral load, ARVs used in each country, and ARV drug resistance. LAg-recent result was defined as normalized optical density values ≤1.5. We compared HIV-1 incidence estimates using 2 RITA: RITA1: LAg-recent + VL ≥ 1000 copies/mL and RITA2: RITA1 + undetectable ARV. We explored RITA2 with self-reported ARV use and with clinical history. RESULTS: Overall, 357 adult HIV-positive participants were classified as having recent infection with RITA1. RITA2 reclassified 55 (15.4%) persons with detectable ARV as having long-term infection. Those with detectable ARV were significantly more likely to be aware of their HIV-positive status (84% vs. 10%) and had higher levels of drug resistance (74% vs. 26%) than those without detectable ARV. RITA2 incidence was lower than RITA1 incidence (range, 0%-30% decrease), resulting in decreased estimated new infections from 390,000 to 341,000 across the 13 countries. Incidence estimates were similar using detectable or self-reported ARV (R2 > 0.995). CONCLUSIONS: Including ARV in RITA2 improved the accuracy of HIV-1 incidence estimates by removing participants with likely long-term HIV infection.

Tuberculosis bacillary load, an early marker of disease severity: the utility of tuberculosis Molecular Bacterial Load Assay.

In this comparative biomarker study, we analysed 1768 serial sputum samples from 178 patients at 4 sites in Southeast Africa. We show that tuberculosis Molecular Bacterial Load Assay (TB-MBLA) reduces time-to-TB-bacillary-load-result from days/weeks by culture to hours and detects early patient treatment response. By day 14 of treatment, 5% of patients had cleared bacillary load to zero, rising to 58% by 12th week of treatment. Fall in bacillary load correlated with mycobacterial growth indicator tube culture time-to-positivity (Spearmans r=-0.51, 95% CI (-0.56 to -0.46), p<0.0001). Patients with high pretreatment bacillary burdens (above the cohort bacillary load average of 5.5log10eCFU/ml) were less likely to convert-to-negative by 8th week of treatment than those with a low burden (below cohort bacillary load average), p=0.0005, HR 3.1, 95% CI (1.6 to 5.6) irrespective of treatment regimen. TB-MBLA distinguished the bactericidal effect of regimens revealing the moxifloxacin-20 mg rifampicin regimen produced a shorter time to bacillary clearance compared with standard-of-care regimen, p=0.008, HR 2.9, 95% CI (1.3 to 6.7). Our data show that the TB-MBLA could inform clinical decision making in real-time and expedite drug TB clinical trials.

Adverse drug reactions experienced by out-patients taking chlorpromazine or haloperidol at Zomba Mental Hospital, Malawi.

OBJECTIVE: Drugs for managing mental disorders can cause adverse drug reactions (ADRs) that have negative impacts on patients yet, in Malawi, epidemiological data on the drug-related problems are limited. This study assessed the prevalence and severity of ADRs in out-patients at Zomba Mental Hospital. RESULTS: Twenty-six of forty patients (65.0%) were taking haloperidol and 14 (35.0%) chlorpromazine. The commonest diagnosis was schizophrenia (n = 23, 57.5%) followed by epileptic psychosis (n = 4, 10.0%) and general psychosis (n = 4, 10.0%) with one of psychotic depression and one psychosis secondary to general medical condition. Comorbidities were also found with epilepsy being the commonest (n = 4, 10.0%). All patients reported at least one ADR of varying severity (mild, moderate and severe). Polydipsia was the most prevalent (24, 60.0%) followed by weight gain (20, 50.0%), spasm (15, 37.5%) and xerostomia (15, 37.5%). Some ADRs were gender specific and these included impotence (6/27, 29.6%) for males and menstrual changes (3/14, 21.4%) for females. Severe ADRs were more common in the older aged group (> 35 years 8.3% vs 7.1%), in males (11.1% vs 0.0%) and on chlorpromazine (14.3% vs 3.8%). Patients taking chlorpromazine and haloperidol are at risk of experiencing a wide range of ADRs with varying degrees of severity.

Genome-wide association study of nevirapine hypersensitivity in a sub-Saharan African HIV-infected population.

Background: The antiretroviral nevirapine is associated with hypersensitivity reactions in 6%-10% of patients, including hepatotoxicity, maculopapular exanthema, Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Objectives: To undertake a genome-wide association study (GWAS) to identify genetic predisposing factors for the different clinical phenotypes associated with nevirapine hypersensitivity. Methods: A GWAS was undertaken in a discovery cohort of 151 nevirapine-hypersensitive and 182 tolerant, HIV-infected Malawian adults. Replication of signals was determined in a cohort of 116 cases and 68 controls obtained from Malawi, Uganda and Mozambique. Interaction with ERAP genes was determined in patients positive for HLA-C*04:01 . In silico docking studies were also performed for HLA-C*04:01 . Results: Fifteen SNPs demonstrated nominal significance ( P < 1 × 10 -5 ) with one or more of the hypersensitivity phenotypes. The most promising signal was seen in SJS/TEN, where rs5010528 ( HLA-C locus) approached genome-wide significance ( P < 8.5 × 10 -8 ) and was below HLA -wide significance ( P < 2.5 × 10 -4 ) in the meta-analysis of discovery and replication cohorts [OR 4.84 (95% CI 2.71-8.61)]. rs5010528 is a strong proxy for HLA-C*04:01 carriage: in silico docking showed that two residues (33 and 123) in the B pocket were the most likely nevirapine interactors. There was no interaction between HLA-C*04:01 and ERAP1 , but there is a potential protective effect with ERAP2 [ P = 0.019, OR 0.43 (95% CI 0.21-0.87)]. Conclusions: HLA-C*04:01 predisposes to nevirapine-induced SJS/TEN in sub-Saharan Africans, but not to other hypersensitivity phenotypes. This is likely to be mediated via binding to the B pocket of the HLA-C peptide. Whether this risk is modulated by ERAP2 variants requires further study.

Differences in genetic variants in lopinavir disposition among HIV-infected Bantu Africans.

INTRODUCTION: Variability in lopinavir (LPV) plasma concentration among patients could be due to genetic polymorphisms. This study set to evaluate significance of variants in CYP3A4/5, SLCO1B1 and ABCC2 on LPV plasma concentration among African HIV-positive patients. MATERIALS & METHODS: Eighty-six HIV-positive participants on ritonavir (LPV/r) were genetically characterized and LPV plasma concentration determined. RESULTS & DISCUSSION: LPV plasma concentrations differed >188-fold (range 0.0206-38.6 µg/ml). Both CYP3A4*22 and SLCO1B1 rs4149056G (c.521C) were not observed in this cohort. CYP3A4*1B, CYP3A5*3, CYP3A5*6 and ABCC2 c.1249G>A which have been associated with LPV plasma concentration, showed no significant association. CONCLUSION: These findings highlight the need to include African groups in genomics research to identify variants of pharmacogenomics significance.

CYP2B6 c.983T>C polymorphism is associated with nevirapine hypersensitivity in Malawian and Ugandan HIV populations.

BACKGROUND: Nevirapine, an NNRTI used in HIV treatment, can cause hypersensitivity reactions in 6%-10% of patients. In the most serious cases (1.3%) this can manifest as Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN). METHODS: DNA samples were obtained and analysed from a total of 209 adult patients with nevirapine hypersensitivity (57 from a prospective cohort and 152 routine clinic patients) and compared with 463 control patients on nevirapine without any hypersensitivity. The case group included 70 patients with SJS/TEN. All individuals were genotyped for two SNPs in the CYP2B6 gene [c.516G>T (CYP2B6*9) and c.983T>C (CYP2B6*18)] using the TaqMan real-time genotyping platform. The replication cohort comprised 29 controls and 55 nevirapine hypersensitive patients, including 8 SJS/TEN cases. RESULTS: An association between the CYP2B6 c.983T>C polymorphism and nevirapine-induced SJS/TEN was observed. In the SJS/TEN group, 30% of individuals possessed at least one c.983T>C versus 16% in the tolerant group [P = 0.006; OR (95% CI) 2.24 (1.27-3.94)]. This association was not significant in the replication cohort [P = 0.075; OR (95% CI) 4.33 (0.80-23.57)]. Combined analysis resulted in an OR of 2.52 (95% CI 1.48-4.20; P = 0.0005) for the association of c.983T>C with SJS/TEN. No association was observed for c.983T>C with other hypersensitivity phenotypes and for CYP2B6 c.516G>T with any hypersensitivity phenotypes. CONCLUSIONS: Our data show an association between the c.983T>C polymorphism and nevirapine-induced SJS/TEN. CYP2B6 c.983T>C has a frequency of 5%-10% in a variety of African populations, but is not observed in Caucasians, thus representing an ethnic-specific predisposing factor.

Peripheral blood mitochondrial DNA/nuclear DNA (mtDNA/nDNA) ratio as a marker of mitochondrial toxicities of stavudine containing antiretroviral therapy in HIV-infected Malawian patients.

Mitochondrial toxicity is a major concern related to nucleoside reverse transcriptase inhibitors. Common manifestations are peripheral neuropathy and lipodystrophy. Depletion of mitochondria has been associated with mitochondrial dysfunction. We investigated whether mitochondria DNA (mtDNA) levels in peripheral blood can be used as biomarker of stavudine-associated mitochondrial toxicities. We enrolled 203 HIV-infected Malawian adult patients on stavudine-containing ART and 64 healthy controls of Bantu origin in a cross-sectional study. Total DNA was extracted from whole blood.The glyceraldehyde-3-phosphate dehydrogenase gene was used to estimate nuclear DNA (nDNA) levels and the ATP synthase-8 mitochondrial DNA gene to estimate mtDNA levels, from which mtDNA/nDNA ratios were determined. MtDNA subhaplogroups were established by sequencing. Among patients, peripheral neuropathy was present in 21% (43/203), lipodystrophy in 18% (20/112), elevated lactate level (>2.5 mmol/L) in 17% (19/113). Healthy controls had a higher median mtDNA/nDNA ratio when compared to HIV/AIDS patients (6.64 vs. 5.08; p=0.05), patients presenting with peripheral neuropathy (6.64 vs. 3.40, p=0.039), and patients with high lactate levels (6.64 vs. 0.68, p=0.024), respectively. Significant differences in median mtDNA/nDNA ratios were observed between patients with high and normal lactate levels (5.88 vs. 0.68, p=0.018). The median mtDNA/nDNA ratio of patients in subhaplogroup L0a2 was much lower (0.62 vs. 8.50, p=0.01) than that of those in subhaplogroup L2a. Our data indicate that peripheral blood mtDNA/nDNA ratio is a marker of mitochondrial toxicities of stavudine and is associated with elevated lactate levels and mtDNA subhaplogroups. This could open the prospect to select a substantial group of patients who will not have problematic side effects from stavudine, an affordable and effective antiretroviral drug that is being phased out in Africa due to its toxicity.

Frequency variation among sub-Saharan populations in virus restriction gene, BST-2 proximal promoter polymorphisms: implications for HIV-1 prevalence differences among African countries.

The present study reports promoter variants in four sub-Saharan African populations that may affect BST-2 gene regulation. Recently, an in/del within the BST-2 promoter has been associated with HIV-1 disease progression in a Spanish cohort. Hence, we sequenced the proximal promoter region of the BST-2 gene in 581 individuals from South Africa, Zimbabwe, Malawi, and Cameroon. Seven SNPs were identified: rs28413176 (+26i6/Δ6); rs28413175 (-160i1/Δ1), -187A>G (nucleotide position -17516614); rs28413174 (-193G>A); rs73921425 (-199G>A); rs12609479 (-201C>T); and rs112492472 (-225C>T). The -199A and -225T alleles showed interesting trends across the sub-Saharan continent. Using predictive bioinformatics tools, we show that allelic variation at -199 and -201 potentially affect key transcription factor binding sites including bHLH, c-Myb, and E47. Importantly, data available from the ENCODE study gave further credence to our hypothesis of transcriptional regulation of BST-2 by a bHLH TF such as Mxi1. The possible repressive transcriptional effect of Mxi1 combined with the allelic frequency trend seen at -199 between African populations overlays well with current HIV-1 prevalence data, and may be a contributing factor to this phenomenon. The differences in HIV-1 prevalence in African countries could be, in part, due to distribution of genetic variants that affect susceptibility to HIV-1. Our findings therefore have substantive value for the design of future diagnostics for global health oriented diagnostics for HIV-1 susceptibility, and rational therapeutics on the critical path to personalized medicine in the African continent. As HIV-1 epidemiology vastly impacts human populations around the world, the population genomics strategy we have utilized herein can have value for other global regions as well.

Mitochondrial subhaplogroups and differential risk of stavudine-induced lipodystrophy in Malawian HIV/AIDS patients.

BACKGROUND & AIM: Lipodystrophy remains a significant problem in HIV/AIDS patients, especially those on regimens containing either protease inhibitors or thymidine analogs (stavudine or zidovudine). Many of the manifestations of lipodystrophy have been linked to mitochondrial dysfunction. We set out to investigate whether mtDNA variation is associated with the development of stavudine-induced lipodystrophy among adult Malawian HIV/AIDS patients on antiretroviral therapy that included stavudine. MATERIALS & METHODS: A total of 117 adult HIV/AIDS patients on stavudine-containing antiretroviral therapy (ART) were recruited from the ART clinic at the Queen Elizabeth Central Hospital, Malawi. The patients were categorized according to whether or not they had developed lipodystrophy after being on a stavudine-containing ART regimen for at least 6 months. Whole mtDNA-coding regions of each patient were sequenced and correlated with clinical characteristics. RESULTS: Lipodystrophy was apparent in 16% (n = 19) of the participants. In multivariate analysis, age >40 years (odds ratio: 4.43; 95% CI: 1.36-14.47; p = 0.014) was significantly associated with the presence of lipodystrophy. The mtDNA subhaplogroup L3e appeared to be protective against lipodystrophy, as none of 11 subjects with this subhaplogroup presented with lipodystrophy. CONCLUSION: mtDNA subhaplogroups seem to differentially affect susceptibility to lipodystrophy. More research is required in order to identify patients who are more or less likely to benefit from stavudine-containing ART.

Severe toxicity and polymerase-γ gene abnormalities in Malawian adults on stavudine-based antiretroviral therapy.

In a cohort study of Malawian adults who were followed up through their second year of stavudine-containing antiretroviral therapy, we sequenced the polymerase-γ gene (POLG) of 10 of the 14 patients with the most severe stavudine side effects. No mutations were observed, suggesting that monogenic POLG mutations are not a common pathogenic determinant of severe stavudine-associated mitochondrial toxicity in Malawians.

Longitudinal lactate levels from routine point-of-care monitoring in adult Malawian antiretroviral therapy patients: associations with stavudine toxicities.

INTRODUCTION: Stavudine is still widely used in under-resourced settings such as Malawi due to its low price. It frequently causes peripheral neuropathy and lipodystrophy and increases the risk of lactic acidosis and other high lactate syndromes. METHODS: We studied the association of longitudinal lactate levels, obtained by routine, 3-monthly point-of-care monitoring, with peripheral neuropathy, lipodystrophy and high lactate syndromes in adult Malawians who were in the second year of stavudine containing antiretroviral therapy (ART). RESULTS: Point-of-care lactate measurements were feasible in a busy urban ART clinic. Of 1170 lactate levels collected from 253 patients over the course of one year, 487 (41.8%) were elevated (>2.2mg/dl), 58 (5.0%) were highly elevated (>3.5mg/dl). At least one elevated lactate level occurred in 210 (83.0%) of patients and sustained hyperlactatemia in 65 (26.4%). In random effects analyses lipodystrophy and peripheral neuropathy were associated with higher lactate levels. Only five patients developed high lactate syndromes (one lactic acidosis) of whom no preceding lactate measurements were available because events had started before enrolment. Lactate levels significantly decreased over time and no high lactate syndromes were observed after the 15th month on ART. CONCLUSION: Lipodystrophy and peripheral neuropathy were associated with higher lactate levels. Lactate levels decreased over time, coinciding with absence of new high lactate syndromes after the 15th month on ART.