Mosimanegape Jongman

(1) Background: Antimicrobial resistance poses a critical global public health threat, with surface water acting as a potential reservoir for pathogenic, zoonotic, and AMR determinants and transmission pathways between humans and animals. We aimed to detect bacterial DNA of clinically relevant taxa and putative AMR determinants in surface water and human beings to support the expansion of One Health environmental surveillance in the Ngamiland and Okavango Delta regions. (2) Methods: We performed a cross-sectional environmental surveillance study using metagenomic sequencing. We collected 28 surface-water samples from Thamalakane River and Moremi Game Reserve sampling sites: 12 samples from Mall of Maun, Island Safari Lodge, and Police Bridge sites along the Thamalakane River, and 16 samples from Xakanaxa, Maxwexwana, Paradise, and Jessie’s Pools sites in Moremi Game Reserve. Additionally, 72 human plasma samples from Letsholathebe II Memorial District Hospital in Maun were also sequenced. Taxonomic classification was performed using Kraken2, and predictive AMR gene identification was performed using the CZ ID AMR module. Beta-diversity was assessed using Principal Coordinates Analysis and PERMANOVA for significant differences in bacterial and AMR gene composition. A p-value of less than 0.05 was considered to be statistically significant. (3) Results: Thamalakane River was dominated by Acinetobacter schindleri (~27%), Acinetobacter lwoffii (~67%), and the toxic cyanobacterium Microcystis aeruginosa (~99%). Moremi Game Reserve sites showed more varied dominant bacterial species, including Stenotrophomonas maltophilia at two sites (~26% and ~29%), Microcystis aeruginosa (~60%), P. aeruginosa (~20%), P. putida (~13%), and A. baumannii (~10%). Beta diversity analysis suggested differences in bacterial community composition between locations (PERMANOVA: R² = 0.261, p = 0.001). Only 5 out of 28 bacterial species identified were shared between surface water and human plasma samples. No putative AMR genes were detected in human samples. However, only intrinsic efflux pump genes (mex-, sme-, ade-families, etc.) dominated surface water. Beta diversity analysis suggested differences in bacterial community composition between locations. (4) Conclusions: Detection of bacterial DNA of clinically relevant bacterial taxa in both surface water and human samples and putative AMR determinants in surface water samples only highlights the need for expanded One Health environmental surveillance. Even though no putative AMR genes were detected in human plasma, monitoring the emergence and transmission of pathogens and resistance-associated genes between One Health interfaces remains a significant research area that requires further investigation.

(1) Background: The Hepatitis B virus (HBV) is characterized by extensive genetic diversity, including low-frequency variants that contribute to disease progression. We aimed to characterize low-frequency variants and evaluate their potential clinical impact. (2) Methods: We utilized 104 HBV near-full-length sequences generated using next-generation sequencing (NGS) from people living with HIV (PLHIV). We used an in-house bioinformatics suite (HBVgenomeR v5.9.7) to filter for low-frequency variants (5–50%), which were compared to escape and drug resistance mutations (DRMs) and hepatocellular carcinoma (HCC)-associated mutations reported at the consensus level. Unclassified variants were characterized by HBV open reading frames (ORFs) to determine mutation frequency per genomic region. (3) Results: A total of six escape mutations were detected in 8/104 (7.7%) sequences, with _surfaceN131T being the most prevalent (5/8). We also observed six DRMs in 30/104 (28.8%), with _rtV173L being the most prevalent (21/30). Truncation mutations were also observed with _rtA181T/_sW172* and _rtM204I/_sW196L being the most prevalent. A total of 8/104 (7.7%) sequences had four variants associated with HCC. The _xP46S was the highest observed HCC-associated mutation at 5/8. We report 1152 unique uncharacterized variants across all ORFs, and these were found in 94/104 (90.4%) sequences. The RNaseH domain had the highest burden (330/1152, 28.6%). (4) Conclusions: Deep sequencing results identified clinically significant mutations, including those below the 20% detection limit of traditional sequencing, that would go unreported. This highlights the possible underreporting of mutational burden in people living with HBV/HIV, indicating the importance of deep sequencing to aid in HBV/HIV understanding and management.

This study reassessed the medicinal potential of Harpagophytum procumbens and related taxa in Botswana using ethnobotanical, phytochemical, microbiological, and AI-assisted approaches. Field surveys revealed a broader than expected distribution of Harpagophytum across Botswana, and evidence of hybridisation between H. procumbens and H. zeyheri that contributes to chemotypic variability. In selected accessions, Microbiological assays demonstrated strong antimicrobial activity, particularly against resistant Klebsiella pneumoniae, while additional activity was observed against Escherichia coli and Pseudomonas aeruginosa. However, limited efficacy was recorded against methicillin-resistant Staphylococcus aureus and Mycobacterium tuberculosis. Phytochemical integration indicated pathway-dependent medicinal functions, with phenylethanoid glycosides associated with antioxidant activity and iridoid and terpenoid metabolites linked to anti-inflammatory and antimicrobial effects. Artificial intelligence-assisted synthesis established chemogeographic stratification across Botswana, and identified ecological zones with taxa that have distinct therapeutic potential. The findings highlighted Harpagophytum as a chemically heterogeneous but commercially valuable medicinal resource with potential applications in precision phytomedicine and targeted bioprospecting.

Environmental pollution by hard-to-degrade polymers is on a steep rise and impacting the entire ecosystem. Microbial keratinases are pivotal in the breakdown of protein polymers that are otherwise resistant to most proteases. In this study, we isolated and identified bacteria from Sua pan soil through morphological, biochemical, and 16SrRNA sequencing approaches and further assessed these isolates for their keratinase production potential. Among the screened isolates, four bacteria, Bacillus cereus Bac 2, Bacillus sp. Bac 1, Pseudomonas aeruginosa Bac 3, and Achromobacter insuavis Bac 4, exhibited the highest degradation activity. B. cereus Bac 2 produced the widest clearance zone, whereas A. insuavis Bac 4 produced the narrowest clearance zone on feather meal agar. Protein bands observed in SDS-PAGE gels for the selected isolates further supported the presence of keratinolytic enzymes. We also investigated the effect of temperature and pH on keratinase activity and determined that all the keratinases were alkaline proteases, with B. cereus Bac 2, P. aeruginosa Bac 3, and Bacillus sp. Bac 1 showing maximum activity at a pH of 8.5, while A. insuavis Bac 4 had maximal activity at a pH of 8.0. Overall, our results indicated that B. cereus Bac 2 keratinase had significantly higher activity across all temperature and pH ranges investigated, compared to all the other isolates (p ≤ 0.0001). These findings highlight the potential application of bacterial isolates from alkalophilic environments, in the eco-friendly degradation of feather waste, as valuable by-products such as organic fertilizers, peptides, and amino acids.

Botswana's Seronga region saw a mass elephant die-off potentially linked to water sources. This study analyzes Okavango Delta metagenomes, uncovering a diversity of viruses and harmful pathogens. Findings highlight the importance of understanding viral ecology in these waters and support One Health's objective in protecting human, animal, and ecosystem health.

Hepatitis delta virus (HDV) co-infections more often result in severe hepatitis compared to hepatitis B virus (HBV) infections alone. Despite a high HDV prevalence (7.1%), information regarding circulating HDV clades is very limited in Botswana. We extracted total nucleic acid from confirmed HDV-positive samples and quantified their viral load. We then sequenced the large hepatitis delta antigen (L-HDAg) using Oxford Nanopore Technology (ONT). Genotyping was performed using the HDV Database, and HDV mutation profiling was performed on AliView. All participants with HBV genotypic information belonged to sub-genotype A1, and 80% (4/5) of them had a higher HDV viral load and a lower HBV viral load. We sequenced 75% (9/12) of the HDV-positive samples, which belonged to HDV clade 8. A total of 54 mutations were discovered, with the most prevalent being Q148R (16%), D149P (16%) and G151D (16%). Known mutations such as S117A, K131R, R139K and G151D were detected, while the other mutations were novel. Our results reveal that HDV clade 8 is the predominant clade in Botswana. The significance of all mutations remains unclear. Future studies with a larger sample size to detect other HDV clades that might be circulating in Botswana and functionally characterize the detected mutations are warranted.

The interaction of multiple viruses in one host is thought to enhance the development of mutations. However, the impact of hepatitis D virus (HDV) positivity on the development of unique hepatitis B virus (HBV) mutations among people living with human immunodeficiency virus (HIV) (PLWH) remains poorly understood in African countries, including Botswana. We used HBV sequences generated from the Botswana Combination Prevention Project (BCPP), which is the largest pair-matched cluster-randomized HIV trial in Botswana. Only participants with available HBV sequences (n = 55) were included in our study ([HIV/HBV-positive (n = 50) and HIV/HBV/HDV-positive (n = 5)]. Geno2pheno was used to determine HBV genotypes, and HBV surface region sequences (all subgenotype A1) were aligned in AliView for mutational analysis, while the impact of mutations was assessed using Phyre2. Our results identified 182 common mutations between the two groups. In the HIV/HBV/HDV cohort, only three mutations (L95W, W156Q, C221Y) were classified as deleterious, with only L95W being the most frequent. In the HIV/HBV cohort, four mutations (W199R, C221A, C221S, W223G) were also classified as deleterious. Our results demonstrate the presence of unique HBV mutations among the HIV/HBV/HDV-positive cohort. Functional characterization of these mutations is recommended to determine their effect on HDV.

(1) Background: Hepatitis B virus (HBV) viral load (VL) is useful in determining treatment eligibility, however, due to high costs associated with the test, hepatitis B e antigen (HBeAg) is recommended as a proxy for HBV VL in resource-limited settings. (2) Methods: We used 299 archived plasma samples with hepatitis B surface antigen (HBsAg), HBeAg, and HBV VL (cut-off of 20,000 IU/mL and 200,000 IU/mL) results from people living with human immunodeficiency virus (PLWH). HBeAg and HBV VL results were analyzed to evaluate the use of HBeAg to determine CHB treatment eligibility and qualification of HBV prevention of mother-to-child transmission (PMTCT) prophylaxis for the first time in Botswana. (3) Results: Approximately 70.8% and 87.5% of participants with HBeAg-positive serology had HBV viral loads ≥20,000 IU/mL and 200,000 IU/mL respectively. Using logistic regression, the odds of having a qualifying HBV VL for CHB treatment and PMTCT prophylaxis were respectively 55.5 and 110.4 times higher in participants with positive HBeAg serology indicating a statistically significant association between HBeAg status and HBV VL. (4) Conclusions: HBeAg detection may not always correlate with high HBV VL, warranting further research focusing on this association, and other biomarkers that correlate better with HBV VL.

Opportunistic infections due to Cryptococcus neoformans and C. gattii species complexes continue to rise unabated among HIV/AIDS patients, despite improved antifungal therapies. Here, we collected a total of 20 environmental and 25 presumptive clinical cryptococcal isolates from cerebrospinal fluid (CSF) samples of 175 patients enrolled in an ongoing clinical trial Ambition 1 Project (Botswana-Harvard Partnership). Identity confirmation of the isolates was done using MALDI-TOF MS and PCR. We describe the diversity of the isolates by PCR fingerprinting and sequencing (Oxford Nanopore Technology) of the intergenic spacer region. Mating types of the isolates were determined by amplification of the MAT locus. We report an unusual prevalence of 42.1% of C. neoformans x C. deneoformans hybrids Serotype AD (n = 16), followed by 39.5% of C. neoformans Serotype A (n = 15), 5.3% of C. deneoformans, Serotype D (n = 2), 7.9% of C. gattii (n = 3), and 5.3% of C. tetragattii (n = 2) in 38 representative isolates that have been characterized. Mating type-specific PCR performed on 38 representative environmental and clinical isolates revealed that 16 (42.1%) were MAT a/ MATα hybrids, 17 (44.7%) were MATα, and five (13.2%) possessed MAT a mating type. We used conventional and NGS platforms to demonstrate a potential link between environmental and clinical isolates and lay a foundation to further describe mating patterns/history in Botswana.