Charles Muthoga

Comparative analyses of clinical and environmental populations of Cryptococcus neoformans in Botswana

Cryptococcus neoformans var. grubii (Cng) is the most common cause of fungal meningitis, and its prevalence is highest in sub‐Saharan Africa. Patients become infected by inhaling airborne spores or desiccated yeast cells from the environment, where the fungus thrives in avian droppings, trees and soil. To investigate the prevalence and population structure of Cng in southern Africa, we analysed isolates from 77 environmental samples and 64 patients. We detected significant genetic diversity among isolates and strong evidence of geographic structure at the local level. High proportions of isolates with the rare MATa allele were observed in both clinical and environmental isolates; however, the mating‐type alleles were unevenly distributed among different subpopulations. Nearly equal proportions of the MATa and MATα mating types were observed among all clinical isolates and in one environmental subpopulation from the eastern part of Botswana. As previously reported, there was evidence of both clonality and recombination in different geographic areas. These results provide a foundation for subsequent genomewide association studies to identify genes and genotypes linked to pathogenicity in humans.

The Nasopharyngeal Microbiota of Children with Respiratory Infections in Botswana.

Background: Nearly half of child pneumonia deaths occur in sub-Saharan Africa. Microbial communities in the nasopharynx are a reservoir for pneumonia pathogens and remain poorly described in African children. Methods: Nasopharyngeal swabs were collected from children with pneumonia (N = 204), children with upper respiratory infection symptoms (N = 55) and healthy children (N = 60) in Botswana between April 2012 and April 2014. We sequenced the V3 region of the bacterial 16S ribosomal RNA gene and used partitioning around medoids to cluster samples into microbiota biotypes. We then used multivariable logistic regression to examine whether microbiota biotypes were associated with pneumonia and upper respiratory infection symptoms. Results: Mean ages of children with pneumonia, children with upper respiratory infection symptoms and healthy children were 8.2, 11.4 and 8.0 months, respectively. Clustering of nasopharyngeal microbiota identified 5 distinct biotypes: Corynebacterium/Dolosigranulum-dominant (23%), Haemophilus-dominant (11%), Moraxella-dominant (24%), Staphylococcus-dominant (13%) and Streptococcus-dominant (28%). The Haemophilus-dominant [odds ratio (OR): 13.55; 95% confidence interval (CI): 2.10–87.26], the Staphylococcus-dominant (OR: 8.27; 95% CI: 2.13–32.14) and the Streptococcus-dominant (OR: 39.97; 95% CI: 6.63–241.00) biotypes were associated with pneumonia. The Moraxella-dominant (OR: 3.71; 95% CI: 1.09–12.64) and Streptococcus-dominant (OR: 12.26; 95% CI: 1.81–83.06) biotypes were associated with upper respiratory infection symptoms. In children with pneumonia, HIV infection was associated with a lower relative abundance of Dolosigranulum (P = 0.03). Conclusions: Pneumonia and upper respiratory infection symptoms are associated with distinct nasopharyngeal microbiota biotypes in African children. A lower abundance of the commensal genus Dolosigranulum may contribute to the higher pneumonia risk of HIV-infected children.

Cytochrome P450 2C8*2 allele in Botswana: Human genetic diversity and public health implications

Human cytochrome P450 2C8 is a highly polymorphic gene and shows variation according to ethnicity. The CYP2C8*2 is a slow drug metabolism allele and shows 10-24% frequency in Black populations. The objective of this study was to assess the prevalence of CYP2C8*2 allele in Botswana among the San (or Bushmen) and the Bantu ethnic groups. For that purpose we recruited 544 children of the two ethnicities in three districts of Botswana from primary schools, collected blood samples, extracted DNA and genotyped them through PCR-based restriction fragment length polymorphism analysis. The results demonstrated that in the San the prevalence of the CYP2C8*2 allele is significantly higher than among the Bantu-related ethnic groups (17.5% and 8.5% for San and Bantu, respectively; P=0.00002). These findings support the evidence of a different genetic background of the San with respect to Bantu-related populations, and highlight a possible higher risk of longer drug clearance or poor level of activation of pro-drugs among the San group.

Short-course High-dose Liposomal Amphotericin B for Human Immunodeficiency Virus–associated Cryptococcal Meningitis: A Phase 2 Randomized Controlled Trial

Background We performed a phase 2 noninferiority trial examining the early fungicidal activity (EFA) of 3 short-course, high-dose liposomal amphotericin B (L-AmB) regimens for cryptococcal meningitis (CM) in Tanzania and Botswana. Methods Human immunodeficiency virus (HIV)-infected adults with CM were randomized to (i) L-AmB 10 mg/kg on day 1 (single dose); (ii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on day 3 (2 doses); (iii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on days 3 and 7 (3 doses); or (iv) L-AmB 3 mg/kg/day for 14 days (control). All patients also received oral fluconazole 1200 mg/day for 14 days. Primary endpoint was mean rate of clearance of cerebrospinal fluid cryptococcal infection (EFA). Noninferiority was defined as an upper limit of the 2-sided 95% confidence interval (CI) of difference in EFA between intervention and control <0.2 log10 colony-forming units (CFU)/mL/day. Results Eighty participants were enrolled. EFA for daily L-AmB was -0.41 log10 CFU/mL/day (standard deviation, 0.11; n = 17). Difference in mean EFA from control was -0.11 (95% CI, -.29 to .07) log10 CFU/mL/day faster with single dose (n = 16); -0.05 (95% CI, -.20 to .10) log10 CFU/mL/day faster with 2 doses (n = 18); and -0.13 (95% CI, -.35 to .09) log10 CFU/mL/day faster with 3 doses (n = 18). EFA in all short-course arms was noninferior to control. Ten-week mortality was 29% (n = 23) with no statistical difference between arms. All arms were well tolerated. Conclusions Single-dose 10 mg/kg L-AmB was well tolerated and led to noninferior EFA compared to 14 days of 3 mg/kg/day L-AmB in HIV-associated CM. Induction based on a single 10 mg/kg L-AmB dose is being taken forward to a phase 3 clinical endpoint trial. Clinical Trials Registration ISRCTN 10248064.Cryptococcal meningitis (CM) causes 10-20% of HIV-related deaths in Africa. We performed a phase-II non-inferiority trial examining the Early Fungicidal Activity (EFA) of three short-course, high-dose liposomal amphotericin B (L-AmB) regimens for CM in Tanzania and Botswana. HIV-infected adults with CM were randomized to: (i) L-AmB 10mg/kg day 1 (single dose); (ii) L-AmB 10mg/kg day 1, 5mg/kg day 3 (two doses); (iii) L-AmB 10mg/kg day 1, 5 mg/kg days 3 and 7 (three doses); (iv) standard 14-day L-AmB 3mg/kg/day (control); all given with fluconazole 1200mg/day for 14 days. Primary endpoint was mean rate of clearance of cerebrospinal fluid (CSF) cryptococal infection (EFA). Non-inferiority was defined as an upper limit of the two-sided 95% confidence interval (CI) of difference in EFA between intervention and control less than 0.2 log10CFU/ml/day. 80 participants were enrolled. EFA for daily L-AmB was -0.41 (standard deviation 0.11, n=17) log10CFU/mL/day. Difference in mean EFA from control was -0.11 (95%CI -0.29,0.07) log10CFU/mL/day faster with single dose (n=16); -0.05 (95%CI -0.20,0.10) log10CFU/mL/day faster with two doses (n=18); and -0.13 (95%CI -0.35,0.09) log10CFU/mL/day faster with three doses (n=18). EFA in all short-course arms was non-inferior to control at the predefined non-inferiority margin. Overall 10-week mortality was 29% (n=23) with no statistical difference between arms. All arms were well tolerated. Single dose 10mg/kg L-AmB was well tolerated and led to non-inferior EFA compared to 14 days of 3mg/kg/d L-AmB in HIV-associated CM. Induction based on single 10mg/kg L-AmB dose is being taken forward to a phase-III clinical endpoint trial.

Staphylococcus aureus nasal colonization among HIV-infected adults in Botswana: prevalence and risk factors

ABSTRACT We sought to determine the clinical and epidemiologic determinants of Staphylococcus aureus nasal colonization in HIV-infected individuals at two outpatient centers in southern Botswana. Standard microbiologic techniques were used to identify S. aureus and methicillin-resistant S. aureus (MRSA). In a sample of 404 HIV-infected adults, prevalence of S. aureus nasal carriage was 36.9% (n = 152) and was associated with domestic overcrowding and lower CD4 cell count. MRSA prevalence was low (n = 13, 3.2%), but more common among individuals with asthma and eczema. The implications of these findings for HIV management are discussed.

Human cytochrome P450 2B6 genetic variability in Botswana: a case of haplotype diversity and convergent phenotypes

Identification of inter-individual variability for drug metabolism through cytochrome P450 2B6 (CYP2B6) enzyme is important for understanding the differences in clinical responses to malaria and HIV. This study evaluates the distribution of CYP2B6 alleles, haplotypes and inferred metabolic phenotypes among subjects with different ethnicity in Botswana. A total of 570 subjects were analyzed for CYP2B6 polymorphisms at position 516 G > T (rs3745274), 785 A > G (rs2279343) and 983 T > C (rs28399499). Samples were collected in three districts of Botswana where the population belongs to Bantu (Serowe/Palapye and Chobe) and San-related (Ghanzi) ethnicity. The three districts showed different haplotype composition according to the ethnic background but similar metabolic inferred phenotypes, with 59.12%, 34.56%, 2.10% and 4.21% of the subjects having, respectively, an extensive, intermediate, slow and rapid metabolic profile. The results hint at the possibility of a convergent adaptation of detoxifying metabolic phenotypes despite a different haplotype structure due to the different genetic background. The main implication is that, while there is substantial homogeneity of metabolic inferred phenotypes among the country, the response to drugs metabolized via CYP2B6 could be individually associated to an increased risk of treatment failure and toxicity. These are important facts since Botswana is facing malaria elimination and a very high HIV prevalence.

Prevalence of G6PD deficiency and associated haematological parameters in children from Botswana

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is commonly seen in malaria endemic areas as it is known to confer a selective advantage against malaria. Recently, we reported a high proportion of asymptomatic reservoir of Plasmodium vivax in Botswana, that calls for intervention with primaquine to achieve radical cure of vivax malaria. Considering that individuals with this enzyme deficiency are at risk of haemolysis following primaquine treatment, assessment of the population for the relative frequency of G6PD deficiency is imperative. Samples from 3019 children from all the districts of Botswana were successfully genotyped for polymorphisms at positions 202 and 376 of the G6PD gene. Haematological parameters were also measured. The overall population allele frequency (based on the hemizygous male frequency) was 2.30% (95% CI, 1.77-2.83), while the overall frequency of G6PD-deficient genotypes A- (hemizygote and homozygote genotypes only) was 1.26% (95% CI, 0.86-1.66). G6PD deficiency is spread in Botswana according to the historical prevalence of malaria with a North-West to South-East decreasing gradient trend. There was no association between G6PD status and P. vivax infection. G6PD A- form was found to be associated with decreased RBC count and haemoglobin levels without a known cause or illness. In conclusion, we report for the first time the prevalence of G6PD deficiency in Botswana which is relevant for strategies in the malaria elimination campaign. Further work to examine the activities of the enzyme in the Botswana population at risk for malaria is warranted.

Molecular Surveillance of Plasmodium falciparum Drug Resistance Markers in Clinical Samples from Botswana

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.

Prevalence of Staphylococcus aureus Nasal Carriage in Human Immunodeficiency Virus–Infected and Uninfected Children in Botswana: Prevalence and Risk Factors

AbstractStaphylococcus aureus is an important cause of morbidity and mortality in children in sub-Saharan Africa (SSA). A major risk factor for staphylococcal infection is S. aureus colonization of the anterior nares. We sought to define risk factors for S. aureus carriage and characterize antimicrobial resistance patterns in children in Botswana. A cross-sectional study was conducted at two clinical sites in southern Botswana. Patients under 18 years of age underwent two nasal swabs and brief interviews, 4 weeks apart. Standard microbiological techniques were used. For persistent carriers, S. aureus was isolated from swabs at both time points, and for intermittent carriers, S. aureus was isolated from only one swab. Poisson regression with robust variance estimator was used to compare prevalence of carriage and the resistance phenotypes. Among 56 enrollees, prevalence of S. aureus colonization was 55% (N = 31), of whom 42% (N = 13) were persistent carriers. Of human immunodeficiency virus-infected children, 64% (N = 9) were carriers. Risk factors for nasal carriage included a history of tuberculosis (prevalence ratio [PR] = 1.60; 95% confidence interval [CI] = 1.02, 2.51; P = 0.040) and closer proximity to health care (PR = 0.89; 95% CI = 0.80, 0.99; P = 0.048). Prior pneumonia was more common among persistent rather than intermittent carriers (PR = 2.64; 95% CI = 1.64, 4.23; P < 0.001). Methicillin-resistant S. aureus (MRSA) prevalence was 13%. Of isolates tested, 16% were resistant to three or more drugs (N = 7/44). In summary, children in southern Botswana are frequently colonized with S. aureus. Antibiotic resistance, especially MRSA, is also widespread. Antibiotic recommendations for treatment of staphylococcal infections in SSA should take cognizance of these resistance patterns.