Serological evidence of chronic pulmonary Aspergillosis in tuberculosis patients in Kenya.

BACKGROUND: Pulmonary tuberculosis (PTB) is a significant risk factor for fungal infection. The cavitary lesions post PTB serves as a good reservoir for fungal colonization and subsequent infection. Furthermore, the severe immunosuppression associated with HIV and TB co-infection is another predisposition. The inadequate capacity to investigate and manage fungal infection in PTB patients increases their morbidity and mortality. The study aimed to provide serological evidence of chronic pulmonary aspergillosis (CPA) among PTB patients in Kenya. Towards this, we analysed 234 serum samples from patients presenting with persistent clinical features of PTB infections despite TB treatment in four referral hospitals. METHODS: This was a cross sectional laboratory based study and patients were recruited following an informed consent. Serological detection of Aspergillus fumigatus IgG was done using enzyme-linked immunosorbent assay (Bordier Affinity Products SA). Sputum samples were subjected to microscopy and standard fungal culture. The isolated fungi were subjected to macro and micro morphological identifications and confirmed by sequence analysis of calmadulin, betatubilin and ITS genes. RESULTS: Serological evidence of CPA or fungal sensitization was 46(19.7%) and equivocal or borderline was 14(6.0%). Mycological investigations of sputum resulted in 88(38%) positive for fungal culture. Aspergillus spp. accounted for 25(28%) of which A. fumigatus was 13(14.8%), A. niger 8(9.1%), A. terreus, A. flavus, A. candidus and A. clavatus 1 (1.1%) each. This was followed by Penicillium spp. 10 (11.4%), Scedosporium spp. 5 (5.7%) and Rhizopus spp. 3 (3.4%). Among the yeasts; Candida albicans accounted for 18(20.5%) followed by C. glabrata 5(5.7%). Cryptococcus spp. was isolated from 3(3.4%) of the samples while 13(14.8%) were other yeasts. CONCLUSION: Chronic pulmonary aspergillosis is a significant co-morbidity in PTB patients in Kenya that could be misdiagnosed as relapse or treatment failures in the absence of reliable diagnostic and clinical management algorithm. It could be the cause of persistent clinical symptoms despite TB treatment often misdiagnosed as TB smear/GeneXpert MTB/RIF® negative or relapse. We recommend that all patients with persistent clinical symptoms despite TB treatment should be subjected to fungal investigations before retreatment.

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.

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 >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.

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.

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 microg/ml and minimum bactericidal concentrations ranging from 31.25 to 500 microg/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 microg/ml and minimum bactericidal concentrations (MBCs) ranging between 7.8 and 500 microg/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 microg/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.

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 alpha-mating type. Seventy-eight (97.5%) of the isolates had minimum inhibitory concentration (MIC) of < or =0.5 microg ml(-1) to AMP and at 1 microg 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 microg 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 microg 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.

Antimicrobial flavonoids from the stem bark of Erythrina burttii.

The chloroform extract of the stem bark of Erythrina burttii showed antifungal and antibacterial activities using the disk diffusion method. Flavonoids were identified as the active principles. Activities were observed against fungi and Gram(+) bacteria, but the Gram(-) bacteria Escherichia coli was resistant.