Christine Bii

Antifungal drug susceptibility of Cryptococcus neoformans from clinical sources in Nairobi, Kenya.

The serotypes and mating types of 80 clinical isolates of Cryptococcus neoformans from Kenya were studied and subjected to broth microdilution susceptibility testing to amphotericin B (AMP), flucytosin, fluconazole (FLC), itraconazole (ITC) and miconazole (MCZ). The isolates included C. neoformans var. grubii– 75 of 80 (serotype A; 93.7%), C. neoformans var. neoformans– three of 80 (3.8%) and C. neoformans var. gattii– two (serotype B; 2.5%). Mating experiment confirmed all the isolates to be α‐mating type. Seventy‐eight (97.5%) of the isolates had minimum inhibitory concentration (MIC) of ≤0.5 μg ml−1 to AMP and at 1 μg ml−1, 100% of the isolates were inhibited. Flucytosin resistance was observed in 21% with MIC in which 90% of the isolates were inhibited (MIC90) of 64 μg ml−1. Only 23.8% of the strains were susceptible to FLC with 65% susceptible dose‐dependent (SDD) and 11.2% resistant. Itraconazole susceptibility was 61.3% while the rest were either SDD or resistant. The MIC90 for ITC and MCZ were 0.5 and 2 μg ml−1 respectively.

Antimicrobial activity of Acacia mellifera extracts and lupane triterpenes.

ETHNOPHARMACOLOGICAL RELEVANCE Acacia mellifera (Vahl) Benth (Leguminosae) is a subtropical medicinal plant that is widely used in traditional African medicines against various diseases such as pneumonia and malaria. AIM OF THE STUDY The present study was performed to evaluate the antimicrobial effects of extracts from the stem bark of Acacia mellifera. MATERIALS AND METHODS The extracts were examined for antibacterial and antifungal activity using the disc-diffusion method against bacterial strains: Streptococcus pneumoniae (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35218), Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) and fungal strains Candida albicans (ATCC 90028), Candida krusei (ATCC 6255), Cryptococcus neoformans (ATCC 6603) and clinical isolates of Microsporum gypseum and Trichophyton mentagrophytes. RESULTS Some of these extracts were found to be active against some bacterial and fungal strains and were further fractionated to give 12 pure compounds. The methanolic and methanol:dichloromethane (1:1) extracts exhibited antibacterial and antifungal activity. The two were active against Staphylococcus aureus (ATCC 25923), Microsporum gypseum, and Trichophyton mentagrophytes. Activity guided fractionation led to isolation of two active compounds: 3-(Z)-cis coumaroylbetulin and 30-hydroxylup-20 (29)-en-3beta-ol which were against Staphylococcus aureus (ATCC 25923), Microsporum gypseum, Trichophyton mentagrophytes and Pseudomonas aeruginosa (ATCC 27853). CONCLUSIONS These results may partly explain and support the use of Acacia mellifera stem barks for the treatment of infectious diseases in traditional Kenya medicine.

Antimicrobial activity and probable mechanisms of action of medicinal plants of Kenya: Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus.

Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus are used traditionally in Kenya for treatment of microbial infections and cancer. Information on their use is available, but scientific data on their bioactivity, safety and mechanisms of action is still scanty. A study was conducted on the effect of organic extracts of these plants on both bacterial and fungal strains, and their mechanisms of action. Extracts were evaluated through the disc diffusion assay. Bacteria and yeast test strains were cultured on Mueller-Hinton agar and on Sabouraud dextrose agar for the filamentous fungi. A 0.5 McFarland standard suspension was prepared. Sterile paper discs 6 mm in diameter impregnated with 10 µl of the test extract (100 mg/ml) were aseptically placed onto the surface of the inoculated media. Chloramphenicol (30 µg) and fluconazole (25 µg) were used as standards. Discs impregnated with dissolution medium were used as controls. Activity of the extracts was expressed according to zone of inhibition diameter. MIC was determined at 0.78–100 mg/ml. Safety studies were carried using Cell Counting Kit 8 cell proliferation assay protocol. To evaluate extracts mechanisms of action, IEC-6 cells and RT-PCR technique was employed in vitro to evaluate Interleukin 7 cytokine. Investigated plants extracts have both bactericidal and fungicidal activity. W. ugandensis is cytotoxic at IC50<50 µg/ml with MIC values of less than 0.78 mg/ml. Prunus africana shuts down expression of IL 7 mRNA at 50 µg/ml. W. somnifera has the best antimicrobial (1.5625 mg/ml), immunopotentiation (2 times IL 7 mRNA expression) and safety level (IC50>200 µg/ml). Fractions from W. ugandensis and W. somnifera too demonstrated antimicrobial activity. Mechanisms of action can largely be attributed to cytotoxicity, Gene silencing and immunopotentiation. Use of medicinal plants in traditional medicine has been justified and possible mechanisms of action demonstrated. Studies to isolate and characterize the bioactive constituents continue.

Detection of virulence-related genes by multiplex PCR in multidrug-resistant diarrhoeagenic Escherichia coli isolates from Kenya and Japan

We compared serotypes, drug susceptibility and presence of virulence-related genes in diarrhoeagenic Escherichia coli isolates from children < 5 years from Kenya (n = 82) and Japan (n = 47). Multiplex PCR was used to detect genes coding for enteroaggregative adherence (aggR), heat-stable toxin (st), heat-labile toxin (It), verotoxin (vt), attaching and effacing mechanism (eaeA), enteroaggregative E. coli heat-stable enterotoxin 1 (astA) and enteroinvasive mechanism (invE). Kenyan E. coli O-serotypes were more diverse than those from Japan (29 vs. 12 serotypes) and exhibited high level multidrug resistance to World Health Organization (WHO) recommended antibiotics. Resistance rates to tetracycline, ampicillin and sulphamethoxazole-trimethoprim were 70.7, 65.9 and 68.3% respectively, but resistance to sulphamethoxazole-trimethoprim among the E. coli isolates from Japan was low (21%). Kenyan isolates harboured virulence-related genes in high frequency (82.9%) compared to those from Japan (25.5%) with aggR and astA being the most frequently detected genes. The presence of multiple virulence genes was associated with multidrug resistance and this merits further investigation.

Molecular types of Cryptococcus gattii/Cryptococcus neoformans species complex from clinical and environmental sources in Nairobi, Kenya

Cryptococcal meningitis infections cause high mortality rates among HIV‐infected patients in Sub‐Saharan Africa. The high incidences of cryptococcal infections may be attributed to common environmental sources which, if identified, could lead to institution of appropriate control strategies. To determine the genotypes of Cryptococcus gattii/C. neoformans‐ species complex from Nairobi, Kenya, 123 clinical and environmental isolates were characterised. Typing was done using orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphism (URA5‐RFLP). The majority of the isolates [105/123; 85.4%] were C. neoformans genotype (AFLPI/VNI) and 1.6% AFLP1A/VNB/VNII, whereas (13%) were C. gattii (AFLP4/VGI). This is the first report on the genotypes of C. gattii/C. neoformans species complex from clinical and environmental sources in Nairobi, Kenya and the isolation of C. gattii genotype AFLP4/VGI from the environment in Kenya.

High Oral Carriage of Non-albicans Candida spp. among HIV-Infected Individuals.

BACKGROUND Non-albicans Candida (NAC) spp. in immunocompromised patients are linked to invasive infections with narrow treatment choice. This study aimed at comparing the oral colonization of NAC spp. between HIV and non-HIV infected individuals in Mwanza, Tanzania. METHOD Oral rinse of 351 HIV-infected and 639 non-HIV infected individuals were collected between March and July 2015. Phenotypic identifications of Candida spp. was done using Candida Chromogenic agar and confirmed by MALDI-TOF MS. RESULTS NAC spp. were detected in 36/351 (10.3%) HIV-infected individuals compared to 28/639 (4.4%) of non-HIV infected individuals; P=0.0003. In HIV infected individuals, commonly isolated NAC spp. were Candida tropicalis, 10(2.8%), C. krusei (Issatschenki orientalis) 9(2.6%) and C. glabrata 8(2.3%). While for non-HIV infected individuals C. dubliniensis 8(1.3%) and C. tropicalis 5(0.9%) were commonly detected. As CD4 count/μl decreases by one unit the risk of being colonized by NAC spp. among HIV infected individuals increases by 1% (OR 1.01, 95% CI; 1.001-1.004, P=0.001). CONCLUSION The prevalence of NAC spp. is high among HIV-infected individuals with low CD4 count placing them at higher risk of invasive infections. Further studies to investigate the role of NAC spp. in causing invasive infections among immunocompromised patients are recommended.

Total aflatoxin, fumonisin and deoxynivalenol contamination of busaa in Bomet county, Kenya

Mycotoxin contamination is a common problem in developing countries, particularly in cereals, and this poses a serious health risk to its consumers. Busaa is a Kenyan traditional brew whose cereal ingredients are prone to mycotoxin contamination. This study aimed at detecting the presence and subsequently quantifying aflatoxin, fumonisin and deoxynivalenol (DON), in busaa in Bomet county, Kenya. Busaa samples were collected from homesteads involved in brewing in the north eastern part of Bomet East constituency. Mycotoxins were detected in the samples using the Envirologix QuickTox kits and quantified using the QuickScan machine according to the manufacturers instructions. Among the 61 samples tested, 93, 9.8 and 23% were contaminated with aflatoxin, fumonisin and DON, respectively, (mean: 5.2±0.2 µg/kg, range: 2.8 to 11 µg/kg; mean 1460±188 µg/kg, range 280 to 4000 µg/kg, mean 259±5.2 µg/kg, range 200 to 360 µg/kg, respectively). Although traditional brews are not directly included in the European law on mycotoxins, it is important to consider their mycotoxin levels. In this study, busaa is a mainly a maize product and also the European Union (EU) guidelines on mycotoxins in maize were used as reference. It was found out that 65.6% of busaa had aflatoxin levels above the limit set in the EU guideline (4 µg/kg). Although, the average levels of fumonisin and DON were within the set limits (fumonisins: 4000 µg/kg; DON: 1750 µg/kg), studies have shown that chronic exposure to multiple mycotoxins has detrimental health effects. Therefore, there is need for mycotoxicological quality control of traditionally produced brews for public mycotoxicological safety. Keywords: Mycotoxin, traditional brew African Journal of Biotechnology Vol 13(26) 2675-2678

Evaluation of the efficacy, acceptability and palatability of calcium montmorillonite clay used to reduce aflatoxin B1 dietary exposure in a crossover study in Kenya

ABSTRACT Acute aflatoxin exposure can cause death and disease (aflatoxicosis) in humans. Aflatoxicosis fatality rates have been documented to be as high as 40% in Kenya. The inclusion in the diet of calcium silicate 100 (ACCS100), a calcium montmorillonite clay, may reduce aflatoxin bioavailability, thus potentially decreasing the risk of aflatoxicosis. We investigated the efficacy, acceptability and palatability of ACCS100 in a population in Kenya with recurring aflatoxicosis outbreaks. Healthy adult participants were enrolled in this double-blinded, crossover clinical trial in 2014. Following informed consent, participants (n = 50) were randomised to receive either ACCS100 (3 g day–1) or placebo (3 g day–1) for 7 days. Treatments were switched following a 5-day washout period. Urine samples were collected daily and assessed for urinary aflatoxin M1 (AFM1). Blood samples were collected at the beginning and end of the trial and assessed for aflatoxin B1-lysine adducts from serum albumin (AFB1-lys). AFM1 concentrations in urine were significantly reduced while taking ACCS100 compared with calcium carbonate placebo (β = 0.49, 95% confidence limit = 0.32–0.75). The 20-day interval included both the placebo and ACCS100 treatments as well as a washout period. There were no statistically significant differences in reported taste, aftertaste, appearance, colour or texture by treatment. There were no statistically significant differences in self-reported adverse events by treatment. Most participants would be willing to take ACCS100 (98%) and give it to their children (98%). ACCS100 was effective, acceptable and palatable. More work is needed to test ACCS100 among vulnerable populations and to determine if it remains effective at the levels of aflatoxin exposure that induce aflatoxicosis.

Oral candidiasis among African human immunodeficiency virus-infected individuals: 10 years of systematic review and meta-analysis from sub-Saharan Africa

ABSTRACT Oral candidiasis (OC) is the most common opportunistic fungal infection among immunocompromised individuals. This systematic review and meta-analysis reports on the contribution of non-albicans Candida species in causing OC among human immunodeficiency virus (HIV)-infected individuals in sub-Saharan Africa between 2005 and 2015. Thirteen original research articles on oral Candida infection/colonization among HIV-infected African populations were reviewed. The prevalence of OC ranged from 7.6% to 75.3%. Pseudomembranous candidiasis was found to range from 12.1% to 66.7%. The prevalence of non-albicans Candida species causing OC was 33.5% [95% confidence interval (CI) 30.9–36.39%]. Of 458 non-albicans Candida species detected, C. glabrata (23.8%; 109/458) was the most common, followed by C. tropicalis (22%; 101/458) and C. krusei (10.7%; 49/458). The overall fluconazole resistance was 39.3% (95% CI 34.4–44.1%). Candida albicans was significantly more resistant than non-albicans Candida species to fluconazole (44.7% vs 21.9%; p < 0.001). One-quarter of the cases of OC among HIV-infected individuals in sub-Saharan Africa were due to non-albicans Candida species. Candida albicans isolates were more resistant than the non-albicans Candida species to fluconazole and voriconazole. Strengthening the capacity for fungal diagnosis and antifungal susceptibility testing in sub-Saharan Africa is mandatory in order to track the azole resistance trend.

Risk of Fungi Associated with Aflatoxin and Fumonisin in Medicinal Herbal Products in the Kenyan Market

Utilization of herbal products is a major concern due to the possibility of contamination by toxigenic fungi that are mycotoxin producers such as Aspergillus species during processing and packaging. Research was carried out to determine the presence of aflatoxins and fumonisins in herbal medicinal products sold in Eldoret and Mombasa towns in Kenya. The study employed both exploratory and laboratory experimental design. The herbal products were purchased from the market and transported to Kenya Medical Research Institute for processing and analysis. Fungal contaminants were determined according to Pharmacopoeia specifications. The toxins were quantified using ELISA based technique. The genus Aspergillus was the most dominant followed by Penicillium. Fungal counts ranged between 1 CFU/g and >1000 cfu/g. Analysis of variance showed that the rate of fungal contaminants for Eldoret and Mombasa samples had significant association (p ≤ 0.001). Aflatoxin levels ranged from 1 to 24 ppb, while fumonisin levels ranged from 1 to >20 ppb. Only 31% of samples met the standards for microbial limits as specified in Pharmacopoeia. There is need for product microbial quality improvement through proper harvesting, processing, storage, and marketing. It is recommended that a policy be enacted to enable regulation of herbal products in Kenya.