Botswana is one of the four front line malaria elimination countries in Southern Africa, with malaria control activities that include routine vector control. Past and recent studies have shown that Anopheles arabiensis is the only known vector of Plasmodium parasites in the country. This report presents a preliminary evaluation on Anopheles species composition in seven districts of Botswana with some inferences on their vectorial role. Overall, 404 Anopheles mosquito females were collected, of which 196 were larvae collected from several breeding sites, and 208 were adults obtained from indoor pyrethrum spray catches (PSC). Anopheles arabiensis (58.9%) accounted for the highest relative frequency in 5 out of 7 districts sampled. The other species collected, among those identified, were barely represented: Anopheles longipalpis type C (16.3%), Anopheles parensis (8.9%), Anopheles quadriannulatus (5.4%), and Anopheles leesoni (0.2%). PCR test for human β-globin on mosquitoes collected by PSC showed that An. arabiensis and An. parensis had bitten human hosts. Moreover, An. arabiensis showed a non-negligible Plasmodium falciparum infection rate in two sites (3.0% and 2.5% in Chobe and Kweneng West districts, respectively). This work provides first time evidence of Anopheles diversity in several areas of Botswana. Anopheles arabiensis is confirmed to be widespread in all the sampled districts and to be vector of P. falciparum. Moreover, the presence of Anopheles funestus group in Botswana has been assessed. Further research, entomological surveillance activities and possibly vector control programmes need to be better developed and implemented as well as targeting outdoors resting vectors.

The variation of human papillomavirus (HPV) genotypes shapes the risks of cervical cancer and these variations are not well defined in Africa. Nucleotide changes within the L1 gene, nucleotide variability, and phylogeny were explored in relation to HIV in samples from Botswana and Kenya. A total of 98 HPV-positive cervical samples were sequenced to identify different HPV variants. Phylogenetic inferences were used to determine HPV genotypes and investigate the clustering of sequences between women living with HIV (WLWHIV) and -women not living with HIV (WNLWHIV). Out of 98 generated sequences, 83.7% (82/98) participants had high-risk (HR) HPV genotypes while 16.3% (16/98) had low-risk (LR) HPV genotypes. Among participants with HR-HPV genotypes, 47.6% (39/82) were coinfected with HIV. The prevalence of HR-HPV genotypes was statistically higher in the Botswana population compared to Kenya (p-value < 0.001). Multiple amino acid mutations were identified in both countries. Genetic diversity differed considerably among WLWHIV and WNLWHIV. The mean pairwise distances between HPV-16 between HIV and HIV/HPV as well as for HPV-18 were statistically significant. Six (6) new deleterious mutations were identified in the HPV genotypes based on the sequencing of the L1 region, HPV-16 (L441P, S343P), HPV-18 (S424P), HPV-45 (Q366H, Y365F), and HPV-84 (F458L). The majority of the patients with these mutations were co-infected with HIV. Genomic diversity and different genomic variants of HPV sequences were demonstrated. Candidate novel mutations within the L1 gene were identified in both countries which can be further investigated using functional assays.

Drug-resistant Plasmodium falciparum is a major threat to global malaria control and elimination efforts. In Botswana, a southern African country approaching malaria elimination, P. falciparum molecular data are not available. Parasites were assessed through pollymerase chain reaction (PCR) for confirmation of positive rapid diagnostic tests, multiplicity of infection (MOI), and drug resistance markers among isolates from clinical uncomplicated malaria cases collected at health facilities. Of 211 dried blood spot samples selected for the study, 186 (88.2%) were PCR positive for P. falciparum. The mean MOI based on MSP1 genotyping was 2.3 and was not associated with age. A high prevalence of wild-type parasites for pfcrt and pfmdr1 was found, with a haplotype frequency (K76/N86) of 88.8% and 17.7% of the isolates having two copies of the pfmdr1 gene. For pfATPase6, all the parasites carried the wild-type S769 allele. Sequencing showed no evidence of non-synonymous mutations associated with reduced artemisinin derivative sensitivity in the P. falciparum k13 gene. In conclusion, we found that P. falciparum parasites in Botswana were mostly wild type for the drug resistance markers evaluated. Yet, there was a high rate of a molecular marker associated to reduced sensitivity to lumefantrine. Our results indicate the need for systematic drug efficacy surveillance to complement malaria elimination efforts.

The two non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz (EFV) and nevirapine (NVP), are currently the core antiretroviral drugs for treatment of HIV in sub-Saharan Africa including Botswana. The drugs are metabolized by Cytochrome P450 2B6 (CYP2B6) liver enzyme. The CYP2B6 gene that encodes for metabolism of these drugs is known to be highly polymorphic. One of the polymorphism in the CYP2B6 gene, 516G>T, particularly the 516T allele, is known to confer poor metabolism of EFV and NVP. This may lead to high levels of plasma drug concentrations and development of treatment toxicities, like central nervous system toxicities, and cutaneous and hepatic toxicities, for EFV and NVP, respectively. The CYP2B6 516G allele on the other hand is associated with an extensive metabolism of the two NNRTIs drugs. We sought to establish association between possible developments of NNRTIs toxicities with CYP2B6 516G>T variation in Botswana. A total of 316 peripheral blood mononuclear cells samples were used in a retrospective view. All the samples were from participants on EFV/NVP-containing regimen with known toxicity output. TaqMan Real-Time PCR approach was applied for assessing CYP2B6 516 allele variation in cases with treatment toxicity and those without. Analysis was performed by chi-square statistics and logistic regression analysis. The rate of poor metabolizers among participants with toxicity and those without toxicity was 18.4% and 15.1%, respectively. The CYP2B6 516 genotype distribution comparisons between the participants with toxicity and those without were not statistically different (chi-square = .326; P = .568). CYP2B6 516 variation was not associated with NNRTI toxicity. No other factors were associated with toxicity when considering age, baseline body mass index, baseline CD4, baseline HIV viral load and adherence. The results were discussed in the context of all the studies done in Botswana to date.

Background: Cervical cancer is an important global health problem, including in Botswana. Human papillomavirus (HPV) and human immunodeficiency virus (HIV) are responsible for a considerable burden of the disease globally. The significance of HPV viral load in the detection of pre-invasive cervical lesions in patients with and without HIV is still controversial. The present study aimed to explore the correlation between HPV-16 and HPV-18 genotype viral loads and the severity of cervical lesions in pre-cervical cancer patients with and without HIV in Botswana. Methods: A cross-sectional convenience sampling was adopted using 109 archived residual tissue blocks collected from pre-cervical cancer patients in Gaborone, Botswana, during 2017-2019. Viral load for HPV-16 and HPV-18 has been quantified in pre-invasive cervical lesions using quantitative PCR to evaluate its potential as a predictor of disease severity, and examined correlations with HIV status and pre-cancer stage. Results: Of the 109 patients, HPV-16 was detected in 33.94% and HPV-18 in 14.68%. Only 3 (2.75%) patients were co-infected with both genotypes, while the remaining patients 56 (48.6%) tested negative for both genotypes. In this study 70.6% of the 109 samples were infected with HIV while 29.4% were HIV-uninfected. For HPV-16, higher cervical intraepithelial neoplasia (CIN) grade was associated with increased HPV viral load, while HPV-18 exhibited a more variable pattern, with the highest HPV viral load observed in CIN II. The presence of HPV-16 and elevated HPV-16 viral load were significant risk factors for CIN III. In contrast, the presence of HPV-18 and higher HPV-18 viral load were associated with increased risk for CIN II, but did not have a significant impact on the risk for CIN III. HIV status did not have a significant impact on viral load of these HPV genotypes. Conclusions: Higher HPV-16 viral loads are linked to more severe cervical lesions while HPV-18 is more associated with CIN II than CIN III. However, HIV status was not associated with higher viral loads. Larger studies are needed to confirm these findings and improve screening.

Children living with HIV (CLWH) are at high risk of colonization and infection by bacterial respiratory pathogens, though this risk can be reduced by other microbes in the upper respiratory microbiome. The impact of HIV infection on development of the upper respiratory microbiome during childhood is poorly understood, and we sought to address this knowledge gap by identifying associations between HIV infection and the nasopharyngeal microbiomes of children in Botswana. We enrolled Batswana CLWH (<5 years) and age- and sex-matched HIV-exposed, uninfected (HEU) and HIV-unexposed, uninfected (HUU) children in a cross-sectional study. We used shotgun metagenomic sequencing to compare the nasopharyngeal microbiomes of children by HIV status. Among the 143 children in this study, HIV infection and HIV-associated immunosuppression were associated with alterations in nasopharyngeal microbiome composition, including lower abundances of Corynebacterium species associated with respiratory health. These findings suggest that the upper respiratory microbiome may contribute to the high risk of bacterial respiratory infections among CLWH.