Low-Level Viremia among Adults Living with HIV on Dolutegravir-Based First-Line Antiretroviral Therapy Is a Predictor of Virological Failure in Botswana.

We evaluated subsequent virologic outcomes in individuals experiencing low-level virem ia (LLV) on dolutegravir (DTG)-based first-line antiretroviral therapy (ART) in Botswana. We used a national dataset from 50,742 adults who initiated on DTG-based first-line ART from June 2016-December 2022. Individuals with at least two viral load (VL) measurements post three months on DTG-based first-line ART were evaluated for first and subsequent episodes of LLV (VL:51-999 copies/mL). LLV was sub-categorized as low-LLV (51-200 copies/mL), medium-LLV (201-400 copies/mL) and high-LLV (401-999 copies/mL). The study outcome was virologic failure (VF) (VL ≥ 1000 copies/mL): virologic non-suppression defined as single-VF and confirmed-VF defined as two-consecutive VF measurements after an initial VL < 1000 copies/mL. Cox regression analysis identified predictive factors of subsequent VF. The prevalence of LLV was only statistically different at timepoints >6-12 (2.8%) and >12-24 (3.9%) (p-value < 0.01). LLV was strongly associated with both virologic non-suppression (adjusted hazards ratio [aHR] = 2.6; 95% CI: 2.2-3.3, p-value ≤ 0.001) and confirmed VF (aHR = 2.5; 95% CI: 2.4-2.7, p-value ≤ 0.001) compared to initially virally suppressed PLWH. High-LLV (HR = 3.3; 95% CI: 2.9-3.6) and persistent-LLV (HR = 6.6; 95% CI: 4.9-8.9) were associated with an increased hazard for virologic non-suppression than low-LLV and a single-LLV episode, respectively. In a national cohort of PLWH on DTG-based first-line ART, LLV > 400 copies/mL and persistent-LLV had a stronger association with VF. Frequent VL testing and adherence support are warranted for individuals with VL > 50 copies/mL.

No Difference in the Prevalence of HIV-1 gag Cytotoxic T-Lymphocyte-Associated Escape Mutations in Viral Sequences from Early and Late Parts of the HIV-1 Subtype C Pandemic in Botswana.

HIV is known to accumulate escape mutations in the gag gene in response to the immune response from cytotoxic T lymphocytes (CTLs). These mutations can occur within an individual as well as at a population level. The population of Botswana exhibits a high prevalence of HLA*B57 and HLA*B58, which are associated with effective immune control of HIV. In this retrospective cross-sectional investigation, HIV-1 gag gene sequences were analyzed from recently infected participants across two time periods which were 10 years apart: the early time point (ETP) and late time point (LTP). The prevalence of CTL escape mutations was relatively similar between the two time points-ETP (10.6%) and LTP (9.7%). The P17 protein had the most mutations (9.4%) out of the 36 mutations that were identified. Three mutations (A83T, K18R, Y79H) in P17 and T190A in P24 were unique to the ETP sequences at a prevalence of 2.4%, 4.9%, 7.3%, and 5%, respectively. Mutations unique to the LTP sequences were all in the P24 protein, including T190V (3%), E177D (6%), R264K (3%), G248D (1%), and M228L (11%). Mutation K331R was statistically higher in the ETP (10%) compared to the LTP (1%) sequences (p < 0.01), while H219Q was higher in the LTP (21%) compared to the ETP (5%) (p < 0.01). Phylogenetically, the gag sequences clustered dependently on the time points. We observed a slower adaptation of HIV-1C to CTL immune pressure at a population level in Botswana. These insights into the genetic diversity and sequence clustering of HIV-1C can aid in the design of future vaccine strategies.

Near-complete genome of SARS-CoV-2 Delta variant of concern identified in a symptomatic dog (Canis lupus familiaris) in Botswana.

We sought to investigate whether SARS-CoV-2 was present, and to perform full-length genomic sequencing, in a 5-year-old male crossbreed dog from Gaborone, Botswana that presented overt clinical signs (flu-like symptoms, dry hacking cough and mild dyspnoea). It was only sampled a posteriori, because three adult owners were diagnosed with SARS-CoV-2 infection. Next-generation sequencing based on Oxford Nanopore Technology (ONT) was performed on amplicons that were generated using a reverse transcriptase real-time polymerase chain reaction (RT-qPCR) of confirmed positive SARS-CoV-2 nasopharyngeal and buccal swabs, as well as a bronchoalveolar lavage with mean real cycle threshold (qCt) value of 36 based on the Nucleocapsid (N) gene. Descriptive comparisons to known sequences in Botswana and internationally were made using mutation profiling analysis and phylogenetic inferences. Human samples were not available. A near-full length SARS-CoV-2 genome (∼90% coverage) was successfully genotyped and classified under clade 20 O and Pango-Lineage AY.43 (Pango v.4.0.6 PLEARN-v1.3; 2022-04-21), which is a sublineage of the Delta variant of concern (VOC) (formerly called B.1.617.2, first detected in India). We did not identify novel mutations that may be used to distinguish SARS-CoV-2 isolates from the dog and humans. In addition to Spike (S) region mutation profiling, we performed phylogenetic analysis including 30 Delta sequences publicly available reference also isolated from dogs. In addition, we performed another exploratory analysis to investigate the phylogenetic relatedness of sequence isolated from dog with those from humans in Botswana (n = 1303) as of 31 March 2022 and of same sublineage. Expectedly, the sequence formed a cluster with Delta sublineages - AY.43, AY.116 and B.1.617.2 - circulating in same time frame. This is the first documented report of human-associated SARS-CoV-2 infection in a dog in Botswana. Although the direction of transmission remains unknown, this study further affirms the need for monitoring pets during different COVID-19 waves for possible clinically relevant SARS-CoV-2 transmissions between species.

Bibliometric analysis of cancer research outputs in Botswana between 2009 and 2021.

INTRODUCTION: Cancer research is critical for cancer control policies; however, the state of cancer research activities in Botswana is largely unknown. The goal of this review was to describe trends and patterns of cancer research outputs in Botswana. METHODS: PubMed, Web of Science, EBSCOhost, African Journals Online, and African Index Medicus databases were systematically searched for peer-reviewed, primary cancer-related research articles published on the Botswana population or by Botswana institutions between January 2009 and June 2021. RESULTS: Of the 86 publications included, 39 (45 %) were about cervical cancer, followed by breast cancer (10 %) and Kaposi sarcoma (7 %). The remainder (27 %) were not focused on any specific cancer type. The research activities were skewed towards three main areas of scientific interest: early detection, diagnosis, and prognosis; cancer control, survivorship, and outcomes; and treatment. Botswana was represented by authors in the first (54 %), last (53 %), and any authorship (53 %) positions. The United States of America had the strongest collaborative partnerships with Botswana, followed by the United Kingdom and South Africa. The majority of funding institutions were American (76 %) and the National Institutes of Health was the most mentioned funding organization, accounting for 33 % of all financial acknowledgments. Only 9 % of the funding acknowledgments came from Botswana. CONCLUSION AND POLICY SUMMARY: Although cancer research in Botswana is expanding because of substantial foreign assistance, it is also hampered by a lack of local funding, minimal participation by Botswana-affiliated researchers, and research that is not aligned with disease burden. Our study highlights the need to strengthen local research capacity in Botswana.

HIV-1C in-House RNA-Based Genotyping Assay for Detection of Drug Resistance Mutations in Samples with Low-Level Viral Loads.

Purpose: Monitoring HIV-1 drug resistance mutations (DRM) in treated patients on combination antiretroviral therapy (cART) with a detectable HIV-1 viral load (VL) is important for the selection of appropriate cART. Currently, there is limited data on HIV DRM at low-level viremia (LLV) (VL 401-999 copies/mL) due to the use of a threshold of VL ≥1000 copies/mL for HIV DRM testing. We here assess the performance of an in-house HIV drug resistance genotyping assay using plasma for the detection of DRM at LLV. Methods: We used a total of 96 HIV plasma samples from the population-based Botswana Combination Prevention Project (BCPP). The samples were stratified by VL groups: 50 samples had LLV, defined as 401-999 copies/mL, and 46 had ≥1000 copies/mL. HIV pol (PR and RT) region was amplified and sequenced using an in-house genotyping assay with BigDye sequencing chemistry. Known HIV DRMs were identified using the Stanford HIV Drug Resistance Database. Genotyping success rate between the two groups was estimated and compared using the comparison of proportions test. Results: The overall genotyping success rate was 79% (76/96). For VL groups, the genotyping success was 72% (36/50) at LLV and 87% (40/46) at VL ≥1000 copies/mL. Among generated sequences, the overall prevalence of individuals with at least 1 major or intermediate-associated DRM was 24% (18/76). The proportions of NNRTI-, NRTI- and PI-associated resistance mutations were 28%, 24%, and 0%, respectively. The most predominant mutations detected were K103N (18%) and M184V (12%) in NNRTI- and NRTI-associated mutations, respectively. The prevalence of DRM was 17% (6/36) at LLV and 30% (12/40) at VL ≥1000 copies/mL. Conclusion: The in-house HIV genotyping assay successfully genotyped 72% of LLV samples and was able to detect 17% of DRM amongst them. Our results highlight the possibility and clinical significance of genotyping HIV among individuals with LLV.

HIV-1 drug resistance mutations among individuals with low-level viraemia while taking combination ART in Botswana.

OBJECTIVES: To assess whether a single instance of low-level viraemia (LLV) is associated with the presence of drug resistance mutations (DRMs) and predicts subsequent virological failure (VF) in adults receiving ART in 30 communities participating in the Botswana Combination Prevention Project. METHODS: A total of 6078 HIV-1 C pol sequences were generated and analysed using the Stanford HIV drug resistance database. LLV was defined as plasma VL = 51-999 copies/mL and VF was defined as plasma VL ≥ 1000 copies/mL. RESULTS: Among 6078 people with HIV (PWH), 4443 (73%) were on ART for at least 6 months. Of the 332 persons on ART with VL > 50 copies/mL, 175 (4%) had VL ≥ 1000 copies/mL and 157 (4%) had LLV at baseline. The prevalence of any DRM was 57 (36%) and 78 (45%) in persons with LLV and VL ≥ 1000 copies/mL, respectively. Major DRMs were found in 31 (20%) with LLV and 53 (30%) with VL ≥ 1000 copies/mL (P = 0.04). Among the 135 PWH with at least one DRM, 17% had NRTI-, 35% NNRTI-, 6% PI- and 3% INSTI-associated mutations. Among the 3596 participants who were followed up, 1709 (48%) were on ART for ≥6 months at entry and had at least one subsequent VL measurement (median 29 months), 43 (3%) of whom had LLV. The OR of experiencing VF in persons with LLV at entry was 36-fold higher than in the virally suppressed group. CONCLUSIONS: A single LLV measurement while on ART strongly predicted the risk of future VF, suggesting the use of VL > 50 copies/mL as an indication for more intensive adherence support with more frequent VL monitoring.

Lack of RH2 gene expression may have influenced the HIV pandemic in sub-Saharan Africa.

OBJECTIVE: To evaluate the association between the Rhesus system RH2-blood group expression and susceptibility to HIV infection, viral load, CD4+ cell count and rate of CD4+ decline. We also aimed to determine if a country's HIV prevalence may be predicted from its RH2 relative frequency. DESIGN: Our previous studies did not find any HIV-infected RH2 homozygotes. Therefore, the current cross-sectional study analysed a larger sample to determine whether HIV-infection also occurs in homozygotes. We also conducted a cross-sectional analysis of RH2 expression in an HIV natural history cohort in Botswana. Lastly, we analysed published data from 60 countries around the world to interrogate the link between RH2 frequency and HIV prevalence. METHODS: One thousand and six hundred anticoagulated blood samples (800 HIV-positive and 800 HIV-negative) were phenotyped for RH2 using serological methods. The proportion of RH2-positive samples was compared across categories of HIV status and odds ratios calculated. Mean viral load and CD4+ cell counts from a natural history cohort study were also compared across categories of RH2. Kaplan--Meier plots were generated for 4-year CD4+-decline to 350 cells/µl. RESULTS: No RH2 homozygotes were found among HIV-positives. Moreover, RH2-negatives were 1.37 times more likely to be HIV-positive than heterozygotes (P = 0.02) and 33 times more likely than RH2 homozygotes (P = 0.01). RH2-positive patients showed significantly higher mean CD4+ cell counts (P < 0.0001), lower viral load (P = 0.024) and slower CD4+ decline (P = 0.038). CONCLUSION: RH2 is potentially a critical host genetic factor determining susceptibility of any population to HIV infection, and probably transcends most other factors in importance for HIV risk of infection.

Hyperuricaemia is associated with dyslipidemia but not HbA1c among type 2 diabetes mellitus patients in Botswana.

Medical records and residual samples from 334 type 2 diabetes mellitus patients attending a clinic in Gaborone, Botswana, during the period September-December 2016 were analysed for the effects of hyperuricaemia on biochemical markers of adverse outcomes. The patients were stratified as having hyperuricaemia (> 400 µmol/L) or normal serum uric acid (≤ 400 µmol/L). We compared glycated haemoglobin, triglycerides, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, total cholesterol and serum creatinine between the two serum uric acid categories. Hyperuricaemia was detected in 28% of patients (95% confidence interval 23.1-32.9) and was associated with increased serum triglycerides, triglyceride to high-density lipoprotein-cholesterol ratio and creatinine concentration, but not with glycated haemoglobin.

Potential role of Lu/BCAM in HIV-related atherosclerosis.

Atheromatous lesions are formed by macrophages and low-density lipoprotein cholesterol invading the vascular intima. Here we show that increasing cholesterol levels are associated with peripheral monocyte depletion and this imbalance is aggravated by carriage of Lu/BCAM leukocyte adhesion molecules. This is true only in HIV infection and probably explains the risk of atherosclerosis observed in HIV-positive patients.

Nontuberculous Mycobacteria, Botswana, 2011-2014.

We documented a 6-fold increase in the frequency of nontuberculous mycobacteria isolated from clinical samples in Botswana during 2011-2014. Because antituberculosis treatment is often initiated only on the basis of acid-fast bacilli smear-positive microscopy results, some patients with nontuberculous mycobacterial infections might have received inappropriate treatment.

Blood Group Antigens C, Lub and P1 May Have a Role in HIV Infection in Africans.

BACKGROUND: Botswana is among the world's countries with the highest rates of HIV infection. It is not known whether or not this susceptibility to infection is due to genetic factors in the population. Accumulating evidence, however, points to the role of erythrocytes as potential mediators of infection. We therefore sought to establish the role, if any, of some erythrocyte antigens in HIV infection in a cross-section of the population. METHODS: 348 (346 HIV-negative and 2 HIV-positive) samples were obtained from the National Blood Transfusion Service as residual samples, while 194 HIV-positive samples were obtained from the Botswana-Harvard HIV Reference Laboratory. Samples were grouped for twenty three antigens. Chi-square or Fischer Exact analyses were used to compare the frequencies of the antigens in the two groups. A stepwise, binary logistic regression was used to study the interaction of the various antigens in the light of HIV-status. RESULTS: The Rh antigens C and E were associated with HIV-negative status, while blood group Jka, P1 and Lub were associated with HIV-positive status. A stepwise binary logistic regression analysis yielded group C as the most significant protective blood group while Lub and P1 were associated with significantly higher odds ratio in favor of HIV-infection. The lower-risk-associated group C was significantly lower in Africans compared to published data for Caucasians and might partially explain the difference in susceptibility to HIV-1. CONCLUSION: The most influential antigen C, which also appears to be protective, is significantly lower in Africans than published data for Caucasians or Asians. On the other hand, there appear to be multiple antigens associated with increased risk that may override the protective role of C. A study of the distribution of these antigens in other populations may shed light on their roles in the HIV pandemic.

Serum ferritin concentration is affected by ferroportin Q248H mutation in Africans.

BACKGROUND: Ferroportin Q248H mutation is common in populations with African ancestry. Studies have reported that the mutation does not alter the ferroportin-hepcidin axis, but there is evidence suggesting that the mutation may lead to hyperferritinemia. We report on the relationship of ferroportin Q248H mutation on serum ferritin (SF) in health adults. SUBJECTS AND METHODS: A total of 174 apparently healthy adults from Botswana were studied. SF was measured using an enzyme immunoassay and ferroportin Q248H mutation was identified by polymerase chain reaction and restriction enzyme digestion. Independent sample Mann-Whitney U test was used to correlate the presence of the mutation with SF. RESULTS: Ferroportin Q248H mutation was identified in 30 individuals (17.2%) (one homozygote, 29 heterozygotes) and was absent in 144 individuals (82.8%), with Q248H allele frequency of 8.9%. In males, SF was significantly higher in ferroportin Q248H heterozygotes compared to wild types, p=0.029, but the relationship between ferroportin Q248H mutation and iron stores was blunted in females. CONCLUSION: Our study of healthy adults provides further evidence that ferroportin Q248H mutation affects SF concentration in Africans.

Continuing professional development training needs of medical laboratory personnel in Botswana.

BACKGROUND: Laboratory professionals are expected to maintain their knowledge on the most recent advances in laboratory testing and continuing professional development (CPD) programs can address this expectation. In developing countries, accessing CPD programs is a major challenge for laboratory personnel, partly due to their limited availability. An assessment was conducted among clinical laboratory workforce in Botswana to identify and prioritize CPD training needs as well as preferred modes of CPD delivery. METHODS: A self-administered questionnaire was disseminated to medical laboratory scientists and technicians registered with the Botswana Health Professions Council. Questions were organized into domains of competency related to (i) quality management systems, (ii) technical competence, (iii) laboratory management, leadership, and coaching, and (iv) pathophysiology, data interpretation, and research. Participants were asked to rank their self-perceived training needs using a 3-point scale in order of importance (most, moderate, and least). Furthermore, participants were asked to select any three preferences for delivery formats for the CPD. RESULTS: Out of 350 questionnaires that were distributed, 275 were completed and returned giving an overall response rate of 79%. The most frequently selected topics for training in rank order according to key themes were (mean, range) (i) quality management systems, most important (79%, 74-84%); (ii) pathophysiology, data interpretation, and research (68%, 52-78%); (iii) technical competence (65%, 44-73%); and (iv) laboratory management, leadership, and coaching (60%, 37-77%). The top three topics selected by the participants were (i) quality systems essentials for medical laboratory, (ii) implementing a quality management system, and (iii) techniques to identify and control sources of error in laboratory procedures. The top three preferred CPD delivery modes, in rank order, were training workshops, hands-on workshops, and internet-based learning. Journal clubs at the workplace was the least preferred method of delivery of CPD credits. CONCLUSIONS: CPD programs to be developed should focus on topics that address quality management systems, case studies, competence assessment, and customer care. The findings from this survey can also inform medical laboratory pre-service education curriculum.

Tuberculosis alters pancreatic enzymes in the absence of pancreatitis.

BACKGROUND: Lipases and phospholipases are thought to contribute to the pathogenesis of Mycobacterium tuberculosis (MTB) infection. Genes coding for lipases, phospholipases and amylase are present in MTB, enabling the bacteria to produce these enzymes. OBJECTIVE: To compare serum lipase and amylase activity levels in patients with tuberculosis (TB) against those of healthy controls. METHODS: Serum lipase and amylase activity levels were measured in 99 patients and 143 healthy controls using the Vitros 250 Chemistry analyser. Reference ranges for serum lipase and amylase were 23-300 U/L and 30-110 U/L, respectively. RESULTS: Lipase was higher in patients with MTB than in controls (81.5 IU/L versus 66.5 IU/L, p = 0.006). Similarly, amylase was higher in the MTB patient group (76 IU/L versus 60 IU/L, p < 0.001). The Pearson correlation coefficient for lipase versus amylase (R) was higher in the controls (R = 0.351, p < 0.0001) compared with MTB patients (R = 0.217, p = 0.035). Amongst MTB patients, lipase activity correlated positively with erythrocyte sedimentation rate (ESR) (R = 0.263, p = 0.013), but not with haemoglobin concentration or treatment duration. A weak inverse correlation was noted between ESR and treatment duration (R = -0.222, p = 0.028). CONCLUSION: Pancreatic enzyme levels differ between MTB patients and normal controls; however, this difference still lies within the normal range. The concomitant increase of lipase with ESR, an inflammatory marker, could conceivably suggest a causal relationship. Further research is necessary to characterise MTB-derived enzymes for diagnostic and therapeutic utility.

The role of red blood cells in enhancing or preventing HIV infection and other diseases.

AIM: To highlight the apparently neglected role of erythrocyte antigens in the epidemiology of infectious diseases, especially HIV, with the prime objective of stimulating research in this area. METHOD: A literature search was performed on the PubMed for relevant papers from 1984 to 2013, the era covering active HIV research. This was achieved by using the phrases "erythrocyte blood groups HIV" (81 papers) or "red cell antigen, blood groups, and HIV" (60 papers). A manual Google Scholar search was done and supplemented by original papers referenced by various authors. However, the review was limited by the relative scarcity of papers on the subject, and only papers written in English were reviewed during the period October 2012 to September 2013. RESULTS: Many communicable and noncommunicable diseases are associated with specific blood groups. Examples of these diseases are discussed in detail. HIV has been shown to bind to erythrocytes, and candidate erythrocyte-binding molecules and mechanisms are also discussed. Moreover, erythrocyte-HIV binding is associated with increased viral infectivity, thus, underscoring the need to study this phenomenon and its implications for HIV epidemiology. CONCLUSION: Erythrocyte antigens may be important in the pathogenesis and epidemiology of many diseases, including HIV.

Immunohaematological reference values for HIV-negative healthy adults in Botswana.

BACKGROUND: Clinical laboratories in Botswana have relied entirely on the reference intervals for normal immunohaematological values provided by manufacturers' kits and textbooks. OBJECTIVES: The aim of this study was to determine the means, medians, 2.5th and 97.5th percentile reference intervals, for normal immunohaematological values in healthy adults in Botswana. METHOD: A total of 261 healthy participants comprising 126 men (48%) and 135 (52%) women were enrolled in the southern part of Botswana, and immunological and haematological laboratory parameters were measured. RESULTS: The mean age was 28.8 (95% Confidence Interval [CI] 27.7-29.8) years, with a median of 27 years and a range 18-66 years. The mean haemoglobin level was significantly lower for women (12.4 g/dL; 95% CI 12.1% - 12.7%) than men (15.1 g/dL; 95% CI 14.9% - 15.3%). The women's haemoglobin reference values (9.0 g/dL - 15.0 g/dL) levels were lower than observed in predominantly White populations (12.0 g/dL - 16.0 g/dL), but comparable with regional consensus reference intervals (9.5 g/dL - 15.8 g/dL) recently defined for East and Southern Africa. CONCLUSION: The established values provide an important tool for patient management and could influence decisions on inclusion of participants and adverse events in clinical trials conducted locally.

Immunohaematological reference values for HIV-negative healthy adults in Botswana.

BACKGROUND: Clinical laboratories in Botswana have relied entirely on the reference intervals for normal immunohaematological values provided by manufacturers' kits and textbooks. OBJECTIVES: The aim of this study was to determine the means, medians, 2.5th and 97.5th percentile reference intervals, for normal immunohaematological values in healthy adults in Botswana. METHOD: A total of 261 healthy participants comprising 126 men (48%) and 135 (52%) women were enrolled in the southern part of Botswana, and immunological and haematological laboratory parameters were measured. RESULTS: The mean age was 28.8 (95% Confidence Interval [CI] 27.7-29.8) years, with a median of 27 years and a range 18-66 years. The mean haemoglobin level was significantly lower for women (12.4 g/dL; 95% CI 12.1% - 12.7%) than men (15.1 g/dL; 95% CI 14.9% - 15.3%). The women's haemoglobin reference values (9.0 g/dL - 15.0 g/dL) levels were lower than observed in predominantly White populations (12.0 g/dL - 16.0 g/dL), but comparable with regional consensus reference intervals (9.5 g/dL - 15.8 g/dL) recently defined for East and Southern Africa. CONCLUSION: The established values provide an important tool for patient management and could influence decisions on inclusion of participants and adverse events in clinical trials conducted locally.