Samuel Kyobe

Prevalence and Antimicrobial Susceptibility Patterns of Bacteria from Milkmen and Cows with Clinical Mastitis in and around Kampala, Uganda

Background Identification of pathogens associated with bovine mastitis is helpful in treatment and management decisions. However, such data from sub-Saharan Africa is scarce. Here we describe the distribution and antimicrobial susceptibility patterns of bacteria from cows with clinical mastitis in Kampala, Uganda. Due to high concern of zoonotic infections, isolates from milkmen are also described. Methodology/Principal Findings Ninety seven milk samples from cows with clinical mastitis and 31 nasal swabs from milkmen were collected (one sample per cow/human). Fifty eight (60%) Gram-positive isolates namely Staphylococci (21), Enterococci (16), Streptococci (13), Lactococci (5), Micrococci (2) and Arcanobacteria (1) were detected in cows; only one grew Staphylococcus aureus. Furthermore, 24 (25%) coliforms namely Escherichia coli (12), Klebsiella oxytoca (5), Proteus vulgaris (2), Serratia (2), Citrobacter (1), Cedecea (1) and Leclercia (1) were identified. From humans, 24 Gram-positive bacteria grew, of which 11 were Staphylococci (35%) including four Staphylococcus aureus. Upon susceptibility testing, methicillin-resistant coagulase-negative staphylococci (CoNS) were prevalent; 57%, 12/21 in cows and 64%, 7/11 in humans. However, methicillin-resistant Staphylococcus aureus was not detected. Furthermore, methicillin and vancomycin resistant CoNS were detected in cows (Staphylococcus hominis, Staphylococcus lugdunensis) and humans (Staphylococcus scuiri). Also, vancomycin and daptomycin resistant Enterococci (Enterococcus faecalis and Enterococcus faecium, respectively) were detected in cows. Coliforms were less resistant with three pan-susceptible isolates. However, multidrug resistant Klebsiella, Proteus, Serratia, Cedecea, and Citrobacter were detected. Lastly, similar species grew from human and bovine samples but on genotyping, the isolates were found to be different. Interestingly, human and bovine Staphylococcus aureus were genetically similar (spa-CC435, spa-type t645 corresponding to ST121) but with different susceptibility patterns. Conclusions/Significance CoNS, Enterococci, Streptococci, and Escherichia coli are the predominant pathogens associated with clinical bovine-mastitis in Kampala, Uganda. Multidrug resistant bacteria are also prevalent. While similar species occurred in humans and cows, transmission was not detected.

Pertussis prevalence and its determinants among children with persistent cough in urban Uganda.

Background We determined prevalence of pertussis infection and its associated host and environmental factors to generate information that would guide strategies for disease control. Methods In a cross-sectional study, 449 children aged 3 months to 12 years with persistent cough lasting ≥14 days were enrolled and evaluated for pertussis using DNA polymerase chain reaction (PCR) and ELISA serology tests. Results Pertussis prevalence was 67 (15% (95% Confidence Interval (CI): 12–18)) and 81 (20% (95% CI: 16–24)) by PCR and ELISA, respectively among 449 participating children. The prevalence was highest in children with >59 months of age despite high vaccination coverage of 94% in this age group. Study demographic and clinical characteristics were similar between pertussis and non-pertussis cases. Of the 449 children, 133 (30%) had a coughing household member and 316 (70%) did not. Among 133 children that had a coughing household member, sex of child, sharing bed with a coughing household member and having a coughing individual in the neighborhood were factors associated with pertussis. Children that had shared a bed with a coughing household individual had seven-fold likelihood of having pertussis compared to children that did not (odds ratio (OR) 7.16 (95% CI: 1.24–41.44)). Among the 316 children that did not have a coughing household member, age <23 months, having or contact with a coughing individual in neighborhood, a residence with one room, and having a caretaker with >40 years of age were the factors associated with pertussis. Age <23months was three times more likely to be associated with pertussis compared to age 24–59 months (OR 2.97 (95% CI: 1.07–8.28)). Conclusion Findings suggest high prevalence of pertussis among children with persistent cough at a health facility and it was marked in children >59 months of age, suggesting the possibility of waning immunity. The factors associated with pertussis varied by presence or absence of a coughing household member.

Rhomboids of Mycobacteria: Characterization Using an aarA Mutant of Providencia stuartii and Gene Deletion in Mycobacterium smegmatis

Background Rhomboids are ubiquitous proteins with unknown roles in mycobacteria. However, bioinformatics suggested putative roles in DNA replication pathways and metabolite transport. Here, mycobacterial rhomboid-encoding genes were characterized; first, using the Providencia stuartii null-rhomboid mutant and then deleted from Mycobacterium smegmatis for additional insight in mycobacteria. Methodology/Principal Findings Using in silico analysis we identified in M. tuberculosis genome the genes encoding two putative rhomboid proteins; Rv0110 (referred to as “rhomboid protease 1”) and Rv1337 (“rhomboid protease 2”). Genes encoding orthologs of these proteins are widely represented in all mycobacterial species. When transformed into P. stuartii null-rhomboid mutant (ΔaarA), genes encoding mycobacterial orthologs of “rhomboid protease 2” fully restored AarA activity (AarA is the rhomboid protein of P. stuartii). However, most genes encoding mycobacterial “rhomboid protease 1” orthologs did not. Furthermore, upon gene deletion in M. smegmatis, the ΔMSMEG_4904 single mutant (which lost the gene encoding MSMEG_4904, orthologous to Rv1337, “rhomboid protease 2”) formed the least biofilms and was also more susceptible to ciprofloxacin and novobiocin, antimicrobials that inhibit DNA gyrase. However, the ΔMSMEG_5036 single mutant (which lost the gene encoding MSMEG_5036, orthologous to Rv0110, “rhomboid protease 1”) was not as susceptible. Surprisingly, the double rhomboid mutant ΔMSMEG_4904–ΔMSMEG_5036 (which lost genes encoding both homologs) was also not as susceptible suggesting compensatory effects following deletion of both rhomboid-encoding genes. Indeed, transforming the double mutant with a plasmid encoding MSMEG_5036 produced phenotypes of the ΔMSMEG_4904 single mutant (i.e. susceptibility to ciprofloxacin and novobiocin). Conclusions/Significance Mycobacterial rhomboid-encoding genes exhibit differences in complementing aarA whereby its only genes encoding “rhomboid protease 2” orthologs that fully restore AarA activity. Additionally, gene deletion data suggests inhibition of DNA gyrase by MSMEG_4904; however, the ameliorated effect in the double mutant suggests occurrence of compensatory mechanisms following deletion of genes encoding both rhomboids.

The collaborative African genomics network training program: a trainee perspective on training the next generation of African scientists

Purpose:The Collaborative African Genomics Network (CAfGEN) aims to establish sustainable genomics research programs in Botswana and Uganda through long-term training of PhD students from these countries at Baylor College of Medicine. Here, we present an overview of the CAfGEN PhD training program alongside trainees’ perspectives on their involvement.Background:Historically, collaborations between high-income countries (HICs) and low- and middle-income countries (LMICs), or North–South collaborations, have been criticized for the lack of a mutually beneficial distribution of resources and research findings, often undermining LMICs. CAfGEN plans to address this imbalance in the genomics field through a program of technology and expertise transfer to the participating LMICs.Methods:An overview of the training program is presented. Trainees from the CAfGEN project summarized their experiences, looking specifically at the training model, benefits of the program, challenges encountered relating to the cultural transition, and program outcomes after the first 2 years.Conclusion:Collaborative training programs like CAfGEN will not only help establish sustainable long-term research initiatives in LMICs but also foster stronger North–South and South–South networks. The CAfGEN model offers a framework for the development of training programs aimed at genomics education for those for whom genomics is not their “first language.” Genet Med advance online publication 06 April 2017

Prevalence and patterns of rifampicin and isoniazid resistance conferring mutations in Mycobacterium tuberculosis isolates from Uganda

Background Accurate diagnosis of tuberculosis, especially by using rapid molecular assays, can reduce transmission of drug resistant tuberculosis in communities. However, the frequency of resistance conferring mutations varies with geographic location of Mycobacterium tuberculosis, and this affects the efficiency of rapid molecular assays in detecting resistance. This has created need for characterizing drug resistant isolates from different settings to investigate frequencies of resistance conferring mutations. Here, we describe the prevalence and patterns of rifampicin- and isoniazid- resistance conferring mutations in isolates from Uganda, which could be useful in the management of MDR-TB patients in Uganda and other countries in sub-Saharan Africa. Results Ninety seven M. tuberculosis isolates were characterized, of which 38 were MDR, seven rifampicin-resistant, 12 isoniazid-mono-resistant, and 40 susceptible to rifampicin and isoniazid. Sequence analysis of the rpoB rifampicin-resistance determining region (rpoB/RRDR) revealed mutations in six codons: 588, 531, 526, 516, 513, and 511, of which Ser531Leu was the most frequent (40%, 18/45). Overall, the three mutations (Ser531Leu, His526Tyr, Asp516Tyr) frequently associated with rifampicin-resistance occurred in 76% of the rifampicin resistant isolates while 18% (8/45) of the rifampicin-resistant isolates lacked mutations in rpoB/RRDR. Furthermore, sequence analysis of katG and inhA gene promoter revealed mainly the Ser315Thr (76%, 38/50) and C(-15)T (8%, 4/50) mutations, respectively. These two mutations combined, which are frequently associated with isoniazid-resistance, occurred in 88% of the isoniazid resistant isolates. However, 20% (10/50) of the isoniazid-resistant isolates lacked mutations both in katG and inhA gene promoter. The sensitivity of sequence analysis of rpoB/RRDR for rifampicin-resistance via detection of high confidence mutations (Ser531Leu, His526Tyr, Asp516Tyr) was 81%, while it was 77% for analysis of katG and inhA gene promoter to detect isoniazid-resistance via detection of high confidence mutations (Ser315Thr, C(-15)T, T(-8)C). Furthermore, considering the circulating TB genotypes in Uganda, the isoniazid-resistance conferring mutations were more frequent in M. tuberculosis lineage 4/sub-lineage Uganda, perhaps explaining why this genotype is weakly associated with MDR-TB. Conclusion Sequence analysis of rpoB/RRDR, katG and inhA gene promoter is useful in detecting rifampicin/isoniazid resistant M. tuberculosis isolates in Uganda however, about ≤20% of the resistant isolates lack known resistance-conferring mutations hence rapid molecular assays may not detect them as resistant.

The Collaborative African Genomics Network (CAfGEN): Applying Genomic technologies to probe host factors important to the progression of HIV and HIV-tuberculosis infection in sub-Saharan Africa

Background: Here, we describe how the Collaborative African Genomics Network ( CAfGEN) of the Human Heredity and Health in Africa (H3Africa) consortium is using genomics to probe host genetic factors important to the progression of HIV and HIV-tuberculosis (TB) coinfection in sub-Saharan Africa. The H3Africa was conceived to facilitate the application of genomics technologies to improve health across Africa.. Methods: CAfGEN is an H3Africa collaborative centre comprising expertise from the University of Botswana; Makerere University; Baylor College of Medicine Children’s Clinical Centers of Excellence (COEs) in Botswana, Uganda, and Swaziland; as well as Baylor College of Medicine, Texas. The COEs provide clinical expertise for community engagement, participant recruitment and sample collection while the three University settings facilitate processing and management of genomic samples and provide infrastructure and training opportunities to sustain genomics research. Results: The project has focused on utilizing whole-exome sequencing to identify genetic variants contributing to extreme HIV disease progression phenotypes in children, as well as RNA sequencing and integrated genomics to identify host genetic factors associated with TB disease progression among HIV-positive children. These cohorts, developed using the COEs’ electronic medical records, are exceptionally well-phenotyped and present an unprecedented opportunity to assess genetic factors in individuals whose HIV was acquired by a different route than their adult counterparts in the context of a unique clinical course and disease pathophysiology. Conclusions: Our approach offers the prospect of developing a critical mass of well-trained, highly-skilled, continent-based African genomic scientists. To ensure long term genomics research sustainability in Africa, CAfGEN contributes to a wide range of genomics capacity and infrastructure development on the continent, has laid a foundation for genomics graduate programs at its institutions, and continues to actively promote genomics research through innovative forms of community engagement brokered by partnerships with governments and academia to support genomics policy formulation.

Incomplete reversal of genotypic resistance of Plasmodium falciparum to chloroquine after a decade of change in malaria treatment policy in Uganda

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