Diversity of Azoles Resistant Aspergillus Species Isolated from Experience and Naïve Soils in Nairobi County and Naivasha Sub-County Kenya

New triazole antifungals voriconazole, itraconazole and posaconazole are recommended for prophylaxis and treatment of both invasive and chronic fungal infections such as aspergillosis and aspergilloma. Emergence of azole-resistant among A. fumigatus isolates have been reported in other countries including Tanzania ascribed to either previous antifungal treatment, prophylaxis or triazoles use in agriculture. The use of azole based fungicides in the robust horticulture in Kenya is a significant risk factor for antifungal resistance. The study proposes to analyze environmental isolates of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger for the presence of resistance against the triazoles antifungals. Fungicide naïve soils were obtained from uncultivated virgin fields while fungicide experience soils were collected from flower, agricultural and horticultural fields and greenhouses within Naivasha sub-county and Nairobi County. The fungal isolates were subjected to antifungal susceptibility to triazoles using broth micro dilution method. A total of 492 samples were analyzed in Nairobi, 52 isolates were identified and they resistance were as follow: A. fumigatus (32%), A. niger (26.09%), A. flavus (33.33%) and A .terreus (0%) and in Naivasha 44 isolates were isolated out of which 25 were A. fumigatus and its resistance was at 36%. Data were analyses using student T test and showed they no different between resistant and susceptible isolates from the two location. Data generated will serve to inform on the current status of triazoles resistance pattern and to raise concern emerging antifungal resistance in clinical practice

Triazole-Resistant Aspergillus fumigatus from Fungicide-Experienced Soils in Naivasha Subcounty and Nairobi County, Kenya

The mainstay in prevention and treatment of aspergillosis is the use triazole drugs. In Kenya, the use of agricultural azole is one of the predisposing factors in development of resistance. One hundred fifty-six (156) experienced soils were collected from agricultural farms and cultured on Sabouraud DextroseAagar. The study isolated 48 yielded Aspergillus fumigatus and 2 A. flavus. All the isolates were subjected to antifungal susceptibility testing against three triazoles: posaconazole, voriconazole, and itraconazole. Out of the isolates, 3 had MIC of 32 and 1 had MIC of 16 against itraconazole, and 1 isolate had MIC of 32 against posaconazole. CYP51A gene was sequenced, and TR34/L98H mutation was identified. Triazole resistance existing in Kenya calls for rational use of azole-based fungicides in agriculture over concerns of emerging antifungal resistance in clinical practice

Estimated burden of fungal infections in Kenya

Introduction: Kenya is a developing country with a high rate of tuberculosis (TB) and a moderate HIV infection burden. No estimate of the burden of fungal diseases in Kenya is published. Methodology: We used specific populations at risk and fungal infection frequencies from the literature to estimate national incidence or prevalence of serious fungal infections. Used sources were: 2010 WHO TB statistics, Kenya Acquired Immunodeficiency Syndrome (AIDS) Epidemic Update 2012, Kenya Facts and figures 2012, Kenya Demographic and Health Survey 2008-2009. Results: Of Kenya’s population of ~40 million, 43% are under 15 years old and approximately 594,660 Kenyan women get &gt;4 episodes Candida vulvovaginitis annually (2,988/100,000). The HIV/AIDS population at risk of opportunistic infections (OI) is 480,000 and the OI estimates include 306,000 patients with oral thrush (768/100,000), 114,000 with oesophageal candidiasis (286/100,000), 11,900 with cryptococcal meningitis (29/100,000) and 17,000 patients with Pneumocystis pneumonia (42/100,000). Chronic pulmonary aspergillosis following TB has a prevalence of 10,848 cases (32/100,000). The adult asthma prevalence is 3.1% and assuming 2.5% have allergic bronchopulmonary aspergillosis then 17,696 (44/100,000) are affected.  Invasive aspergillosis, candidaemia and Candida peritonitis are probably uncommon. Tinea capitis infects 9.6% of children in Kenya, while fungal keratitis and otomycoses are difficult to estimate. Conclusion: At any one time, about 7% of the Kenyan population suffers from a significant fungal infection, with recurrent vaginitis and tinea capitis accounting for 82% of the infections. These estimates require further epidemiological studies for validation.</jats:p

Antimicrobial properties and toxicity of Hagenia abyssinica (Bruce) J.F.Gmel, Fuerstia africana T.C.E. Fries, Asparagus racemosus (Willd.) and Ekebergia capensis Sparrm.

Background: The world health organization (WHO) estimates that 80% of population in Africa relies on traditional remedies for their healthcare. However, very few studies have been carried out to establish the therapeutic effects of these remedies. Objective: Four medicinal plants were investigated for antimicrobial activity and toxicity. Materials and Methods: Plants were collected from their natural habitat, dried, and extracted with organic and aqueous solvents. Antimicrobial activity was determined by the disc diffusion assay technique. In vitro cytotoxicity studies were carried out on extracts using MTT assay on Vero cell lines while acute toxicity in Swiss mice. Results: Extracts from H. abyssinica, F. africana and A. racemosus exhibited antibacterial activity with minimum inhibitory concentration of ≤ 6.25mg/ml against S. aureus, MRSA and P. aeruginosa. However, the plants studied had weak antifungal activity. H. abyssinica and F. africana extracts were found to be cytotoxic with CC50 of ˂ 90 µg/ml. These extracts were tested for acute toxicity and found to be safe at 5000 mg/kg body weight per day. Conclusion: The results of the study support the medicinal use of these plants and indicate that useful compounds from Hagenia abyssinica and Fuerstia africana can be isolated for further exploitation. Keywords: Medicinal plants, Antimicrobial activity, Cytotoxicity, Acute toxicit

Postharvest Storage Practices of Maize in Rift Valley and Lower Eastern Regions of Kenya: A Cross-Sectional Study

An assessment of local farmers’ knowledge, attitude, and practices on postharvest maize storage and management was carried out with a view of understanding its role in maize contamination with mycotoxins and postharvest losses in Rift Valley and Lower Eastern Regions of Kenya among 165 and 149 farmers, respectively. Differences between the two regions were analyzed using the Chi-square test, Fisher exact test, and two-sample t-test. The median quantity of maize harvested by farmers in the two regions after shelling was 585 kg. A median of 20 kg of maize was put aside as a result of rotting before shelling, and there was a significant mean difference in maize set aside as a result of rotting between the two regions (107.88 kg vs. 31.96 kg; t (306.25) = 5.707, P value <0.001). The quantity of discoloured and mouldy maize consumed ranged from 0 to 90 kg; 7 (2.2%) respondents consumed mouldy maize, 36 (11.5%) fed it to cows, and 19 (6.1%) fed it to poultry. A small percentage (3.5%) believed mouldy maize is safe for human consumption, 23.6% for animal consumption, while 15.0% considered it safe for brewing, with the differences between the two regions being statistically significant (P value <0.05). Nearly half of the respondents (48.4%) kept maize on cobs indoors, 47.1% left it in the field without covering, and 33.1% consumed and sold maize while still green, with more farmers from Lower Eastern practicing this. The results of the study suggest that there were poor postharvest practices and low awareness levels among maize farmers and that this can lead to postharvest losses due to Fusarium spp. infection and mycotoxin contamination that poses a threat to human and animal food safety. This calls for interventions on better postharvest practices

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

Antibacterial Activity of Tabernaemontana Stapfiana Britten (Apocynaceae) Extracts

Antibacterial and phytochemical screening of methanolic, sequential extracts (hexane, dichloromethane, ethyl acetate and methanol) and alkaloid rich fractions of Tabernaemontana stapfiana Britten was carried out. The phytochemical screening showed the presence of alkaloids, flavonoids, coumarins, tannins and saponins that have been associated with antimicrobial activity. The stem and root bark methanolic extracts showed good activity against the bacterial strains used including the multiple drug resistant Staphylococcus aureus strain with minimum inhibitory concentrations ranging from 15.6 to 500 µg/ml and minimum bactericidal concentrations ranging from 31.25 to 500 µg/ml. The sequential extracts of the root and stem bark had high antimicrobial activity with minimum inhibitory concentrations (MICs) ranging between 3.9 and 250 µg/ml and minimum bactericidal concentrations (MBCs) ranging between 7.8 and 500 µg/ml against the tested microorganisms. The dichloromethane extract of the alkaloid rich fractions however exhibited reduced antibacterial activities as compared to methanol and sequential extracts but the dichloromethane:methanol (4:1) mixture showed high activity with MICs ranging between 15.6 and 250 µg/ml. These antibacterial efficacy studies suggest that Tabernaemontana stapfiana Britten could be a source of antibacterial agents

Antifungal Drug Susceptibility of Cryptococcus Neoformans from Clinical Sources in Nairobi, Kenya

Journal ArticleThe 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 a-mating type. Seventy-eight (97.5%) of the isolates had minimum inhibitory concentration (MIC) of £0.5 lg ml)1 to AMP and at 1 lg 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 lg 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 lg ml)1 respectively. The study reports the serotypes, mating types and highlights the existence of azoles resistance in C. neoformans in Nairobi which calls for antifungal drug resistance surveillance as prophylactic use of FLC increases because of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic in sub-Saharan Africa

72 hr expression of GAPDH and IL 7 on treatment with different combinations of the 4 plant extracts.

<p>A & B represent GAPDH & IL 7 respectively. Combination ratios, WU/PA 1∶2, WU/PB 1∶4, WU/WS 1∶1, PA/PB 1∶2, PA/WS 2∶1, PB/WS 2∶1, PB/WS 4∶1, WU/WS/PB, 1∶1:4. P. africana was able to shut down the expression of IL 7 irrespective of the combination used.</p

Graphical representation of the results of IEC-6 cell proliferation/cytotoxicity assay of 4 plant extracts.

<p>W.ugandesis (Wu), P. africana (Pa) & P. barbatus (Pb) at concentrations ranging from 0.0005–5000 µg/ml and, W. somnifera (Ws) at concentrations ranging from 0.001–10,000 µg/ml. W. somnifera (Ws), P. africana (Pa) & P. barbatus (Pb) are relatively safe for use even in dose levels exceeding 200 µg/ml. W. ugandensis has a much lower safety level below 50 µg/ml.</p