ISOLATION AND CHARACTERISATION OF CRYPTOCOCCUS NEOFORMANS AND CRYPTOCOCCUS GATTII FROM ENVIRONMENTAL SOURCES IN NAIROBI, KENYA.

UNLABELLED: Objective: To establish the environmental reservoirs of Cryptococcus neoformans and Cryptococcus gattii in Nairobi, Kenya. Design: Prospective study. SETTING: Kenya Medical Research Institute, Mycology laboratory, Nairobi, Kenya. SUBJECTS: A total of 400 environmental samples from different sites were analysed including; avian droppings, tree swabs, soil contaminated with avian droppings and swabs from garbage damping sites. Samples were subjected to various phenotypic tests including microscopic morphology, physiological and biochemical tests, pigmentation on bird seed agar and reaction on Canavanine-Glycine-Bromothymol Blue agar. RESULTS: Cryptococcus neoformans was isolated from 23/200 (11.5%) dropping samples and Cryptococcus gattii in 5/200 (2.5%) of the same samples. Cryptococcus gattii was isolated from 7/60 (11.7%) tree swabs and Cryptococcus neoformans in 5/60 (8.5%) of the same samples. From other sites there was no Cryptococcus gattii recovered with (5/50: 10%), (6/60: 10%), (2/30: 6.7%) Cryptococcus neoformans recovered from chicken cage, garbage damping site and soil respectively. CONCLUSION: Findings clearly showed a high presence of Cryptococcus neoformans and Cryptococcus gattii from several environmental sites in Nairobi, Kenya. This could probably explain the high incidence of cryptococcal meningitis in HIV/AIDS patients in Kenya.

PRESENCE OF CRYPTOCOCCUS SPECIES IN DOMESTIC CHICKEN (GALLUS GALLUS) DROPPINGS AND THE POSSIBLE RISK IT POSED TO HUMANS IN KABIGERIET VILLAGE, NAKURU COUNTY, KENYA.

OBJECTIVE: To isolate and identify Cryptococcus from domestic Chicken dropping. DESIGN: cross sectional study. SETTING: Kabigeriet village, Olenguorone Division, Nakuru county, approximately 282 km from Nairobi, Kenya. SUBJECTS: Sixty four domestic chicken droppings were sampled in thirty two homesteads after obtaining the farmers consent. RESULTS: Two species of Cryptococcus were isolated. CONCLUSION: Domestic chicken (Gallus gallus) harbor Pathogenic Cryptococcus in their dropping and their close proximity to human habitation poses a risk of AIDS to immunocompromised persons.

Fungal infections among diabetic foot ulcer- patients attending diabetic clinic in Kenyatta National Hospital, Kenya.

OBJECTIVE: To isolate and identify fungal pathogens associated with dermatophytoses in diabetic patients and identify the spectrum of yeasts colonising diabetic foot ulcers at Kenyatta National Hospital. DESIGN: A cross sectional Laboratory based study. SETTING: The Kenyatta National Hospital diabetic clinic. SUBJECTS: Sixty one patients with diabetic foot ulcers from August to November 2009. RESULTS: The five most occurring pathogens were Biopolaris hawaiiensis (5.5%), Trichophyton schoenleinii (3.7%), Aspergillus niger (3.0%), Trichophyton rubrum (3.0%), Fusarium oxysporum (3.0%). Other moulds accounted forless than 3.0%. One suspected case (0.6%) of Penicilium marneffei was isolated although it couldnotbe ascertained due to its high containment requirement. Among the dermatophytes, the most occurring mould was Trichophyton schoenleinii (3.7%) while innon-dermatophyte was Biopolaris hawaiiensis (5.5%). Eight pathogenic yeasts were identified with C. parapsilosis (6.1%) being the most common followed by C. famata (3.0%). Fungal infestation was highest in callus formation (78.6%). CONCLUSION: Fungal aetiological agents are significant cause of diabetic woundinfection and may require antifungal intervention for successful management of diabetic foot ulcers.

Oxacillin resistant Staphylococcus aureus among HIV infected and non-infected Kenyan patients.

BACKGROUND: Infections due to methicillin resistant S. aureus (MRSA) present global challenges to clinicians since therapeutic options are limited and suboptimal dosing contributes to heightened mortality and increased length of hospital stay particularly among the HIV infected patients. OBJECTIVES: To assess the prevalence and relative risk of MRSA infections in HIV infected patients. DESIGN: Cross sectional analytical study. SETTING: Kenya Medical Research Institute, Opportunistic Infection Laboratories in Nairobi. SUBJECTS: Four hundred and thirty six male and female patients aged one to 65 years, of whom 220 were HIV-infected and 216 were non-infected. RESULTS: There was 436 male (57.1%) and female (42.9%) respondents. The prevalence of MRSA was 26.3% with majority infecting the HIV infected patients (P=0.046). Likewise, the overall Staphylococcal infections were more common in HIV patients (P <0.001). The common test for MRSA oxacillin disk diffusion had a sensitivity and specificity of 100% and 92%. CONCLUSION: HIV is a predisposing factor to Staphylococcal infection and there are indications that treatment with beta-lactam antibiotics may no longer be relied on as sole empiric therapy for several ill HIV patients whose infections may be of MRSA in origin. There is need for an informed choice in administration of appropriate antibiotics in order to minimise treatment failures due to the multidrug resistance and Vanvomycin intermediate S. aureus (VISA) strains. Molecular epidemiology of MRSA strains in understanding new and emerging trends is recommended.

Dermatophyte infections in primary school children in Kibera slums of Nairobi.

OBJECTIVE: To determine the prevalence and aetiology of dermatophyte infections in relation to social economic factors in primary school children in Kibera. DESIGN: A cross-sectional descriptive study. SETTING: City council sponsored schools namely Olympic, Kibera, Ayany and Mbagathi way all in Kibera, the largest of the informal settlement within Nairobi which is home to between 700,000-1,000,000 inhabitamts. The study was conducted between September 2006 and February 2007. SUBJECTS: A total of 422 primary school children from the ages of five years to 15 years were selected for the study. RESULTS: The prevalence of dermatophytoses was 11.2% with tinea capitis being the most common type while the grey patch form being the dominant clinical manifestation. There was a significant difference (p = 0.001) in dermatophytoses in different schools with Olympic primary school registering the highest prevalence (22.6%). The highest infection rate occurred among six to eight years age bracket in both sexes compared to other age brackets (p = 0.002). The genera of fungi associated with dermatophytoses were isolated indicating the number in each species as follows; T. violecium (35), T. mentagrophytes(3), T. terestre(3), T. schoenleinii(2), and T. interdigitale(1), M. canis(2), M. equinum(1) and E. flocossum(1). T. violecium was the predominant species isolated, at 35/48 (71%) followed by T. mentagrophytes and T. terrestre at 3/48 (6%) each. CONCLUSION: The study indicates high prevalence of 11.2% dermatophyte infection among the school children in Kibera. Factors contributing to the high frequency and chronic occurrences of ring worm in this area may include poor living environment, children interaction patterns and poor health seeking behaviour. There is need for health education and public awareness campaigns among the communities in urban informal settlements on healthy seeking behaviors and hygiene in order to reduce transmission and severe clinical manifestations.

Antibiotic resistance and virulence factors in Escherichia coli from broiler chicken slaughtered at Tigoni processing plant in Limuru, Kenya.

BACKGROUND: Antibiotics and disinfectant use in broiler farms is a common practice and an important risk factor for promoting the emergence, selection and spread of antimicrobial-resistant micro-organisms in environment, veterinary and human medicine. OBJECTIVES: To investigate multi-drug resistance and presence of virulence related genes in Escherichia coli isolates from healthy broiler chicken at slaughter time. DESIGN: Cross sectional and laboratory based study. SETTING: Tigoni processing plant, Limuru, Kenya. RESULTS: High resistance levels were detected for most commonly used drugs like tetracycline (75.9%) and cotrimoxazole (72.4%). Other antibiotics like ampicillin (39%), chloramphenicol (13.2%) and ciprofloxacin (19%) recorded resistance levels although they are rarely used in poultry farming. One hundred and seventeen isolates showed resistance to two and more antibiotics. Different farm treatments were a significant factor for multi-drug resistance (p < or = 0.001). The E. coli isolates showed twenty-one different multi-drug resistant patterns with tetracycline/cotrimoxazole being the most common. Sixty samples were analysed for virulence related genes using multiplex PCR. Seven virulence related genes were investigated but ten isolates were positive for verotoxin and three for intimin. Serotype 0111, 0126, 06 and 078 were positive for verotoxin, 0126 and 0111 were positive for intimin. There was no significant relationship between virulence and multi-drug resistance (p < or = 0.05). CONCLUSION: The present study highlights the presence of multi-drug resistant and virulent E. coli among healthy broiler chicken in Kenya. The possible source of antibiotic resistance in the broilers is the use of recommended antibiotics which co-select resistance for other antibiotics. Surveillance for drug resistance pathogens in food products is recommended.

Antimicrobial activity of pentacyclic triterpenes isolated from Acacia mellifera.

Acacia mellifera has been used widely in traditional African medicines against various diseases. Among the Kipsigis community of Kenya, water extracts from the plant is used for the treatment of skin diseases, coughs and gastrointestinal ailments. The aim of the study was to provide scientific rationale for the use of the plant in traditional medicine through bioassay-guided fractionation of A. mellifera stem bark. Bioactivity testing was done against selected microbes using disc diffusion technique as outlined in Clinical Laboratory Standard Institute (CLSI). Structure elucidation of the isolated compounds was based primarily on 1D and 2D NMR analyses, including HMQC, HMBC, and NOESY correlations. Fractionation yielded three triterpenoids; (20S)-oxolupane-30-al, (20R)-oxolupane-30-al, and betulinic acid. The three compounds were active against Staphylococcus aureus ATCC 25923 and only (20S)-oxolupane-30-al against clinical isolate of Microsporum gypseum. The three compounds had no activity against Escherichia coli ATCC 25922, Enterococcus feacalis, Candida albicans ATCC 90028, Cryptococcus neoformans, Trichophyton mentagrophyte, Candida krusei, Microsporum gypseum, and Sacharomyces cerevisiae. These results explain and support the use of A. mellifera stem barks for the treatment of infectious diseases in traditional Kenya medicine. It also shows that the antimicrobial activity is concentrated in the triterpenoid fractions.

Pneumocystis jirovecii and microbiological findings in children with severe pneumonia in Nairobi, Kenya.

OBJECTIVE: To determine the significance of Pneumocystis jirovecii infection in the Kenyan paediatric population. DESIGN: Sixty samples of induced sputum from children aged < or =23 months, half of whom were human immunodeficiency virus (HIV) positive, admitted with severe pneumonia in Nairobi were subjected to immunofluorescent staining for detection of P. jirovecii and microbiological culture. RESULTS: P. jirovecii was detected in 8/60 (13%) as a copathogen with other respiratory pathogens. Five of eight samples with >5 oocysts were from HIV-positive children aged < or =6 months, while equivocally scored samples (< or =5 oocysts) were from HIV-negative children aged >6 months. Klebsiella pneumoniae was significantly recovered in 26/ 60 (43%), followed by Escherichia coli 11/60 (18%) and Staphylococcus aureus 8/60 (13%). Streptococcus pneumoniae, Haemophilus influenzae and Pseudomonas aeruginosa were isolated infrequently. Candida albicans was recovered from 27/60 (45%), while the frequency of C. tropicalis, C. glabrata and C. parapsilosis was 7%, 5% and 3% respectively. Multidrug resistance among E. coli and K. pneumoniae were: sulphamethoxazoletrimethoprim 100% vs. 69%, chloramphenicol 55% vs. 73% and ampicillin 100% vs. 89%. CONCLUSION: Paediatricians in Kenya should be aware of Pneumocystis pneumonia, irrespective of the patient's HIV status.

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.

Tuberculosis and oral Candida species surveillance in HIV infected individuals in Northern Kenya, and the implications on tuberculin skin test screening for DOPT-P.

OBJECTIVE: To determine the pattern of opportunistic infections such as TB and Candida species in HIV infected patients in Northern Kenya. DESIGN: Cross-sectional study. SETTING: Five health facilities in Moyale (n=224), Mandera (n=121) and Turkana Kakuma; (n=83), Lopiding; (n=94) districts during different periods in 2003. SUBJECTS: Five hundred and fifty two patients. RESULTS: In total 94 (18%) patients were found to be HIV positive (Moyale=42, Mandera=13, Turkana; Kakuma=8, Lopiding=31). Only 65 of 94 HIV positive patients provided saliva samples. Of these, 11 (17%) were TB smear positive and 19 (29.2%) were colonized by oral Candida species. The Candida isolates were as follows; Co-infection of Candida species and TB (n=4), C. albicans only (n=12), C. tropicalis only (n=1), C. albicans and C. glabarata (n=1) and C. albicans, C. glabarata and C. tropicalis. co-infection (n=1). CONCLUSION: The findings provides an important insight into the differences in mucosal susceptibility to bacteria (TB) infection and fungal (Candida species) colonization during HIV immunosuppression, based on collected blood, sputum and saliva specimens. Further studies are needed to elucidate the comparative transmission dynamics and pathogenetic mechanisms of these opportunistic infections-in different regions of Kenya. Such studies would improve the efficiency of directly observed preventive therapy programme (DOPT-P) whose implementation involves screening by tuberculin skin testing.

Pneumocystis carinii pneumonia in HIV/AIDS patients at an urban district hospital in Kenya.

BACKGROUND: Pneumocystis carinii pneumonia has generally been regarded to be an uncommon opportunistic infection in HIV infected individuals in sub-Saharan Africa. The reason for this has not been clear but postulates included a lack of suitable pathogenic types in the African environment, diagnostic difficulties and the more commonly held belief that African HIV infected individuals were dying early from common non-opportunistic pathogens before severe degrees of immunosuppression occured. Recently a trend has emerged at the Mbagathi district hospital whereby an increasing number of HIV infected patients are empirically treated for Pneumocystis carinii pneumonia (PCP) based on clinical and radiological features. OBJECTIVE: To determine the prevalence of PCP and clinical outcomes of HIV infected patients presenting at the Mbagathi District Hospital, Nairobi with the presumptive diagnosis of PCP. SETTING: Mbagathi District Hospital, a 169-bed public hospital in Nairobi, Kenya. METHODS: Patients presenting with a sub-acute onset of cough and dyspnoea were eligible for the study if they were found to have bilateral pulmonary shadows and had negative sputum smears for AFBS. Consenting patients who had no contraindication to fiberoptic bronchoscopy had a clinical evaluation which was followed with a fiberoptic bronchoscopy procedure where bronchoalveolar lavage fluid (BALF) was obtained. BALF was examined for cysts of P. carinii using toluidine blue stain and immunofluorescent antibody test (IFAT). BALF was also processed for fungi, bacteria and mycobacteria using routine procedures. Standard treatment with high dose cotrimoxazole was offered to all patients who were then followed up until discharge from hospital or death whichever came first. RESULTS: Between June 1999 and August 2000 a total of 63 patients were referred for bronchoscopy. Of these four declined to undergo the fiberoptic bronchoscopy procedure, four died before the procedure could be done, one was judged too sick to undergo the procedure and three had been on cotrimoxazole for longer than five days. Thus 51 patients underwent bronchoscopy. Pneumocystis carinii stain was positive in 19 (37.2%) while death occured in 16 (31.4%) of the 51 patients. There were more deaths in those without PCP but this difference was not statistically significant (odds ratio 0.68 (95% CI 0.35-1.32; P=0.2). CONCLUSION: PCP was found to be common in HIV infected patients presenting with clinical and radiological features of the disease. The mortality rate for patients with a presumptive diagnosis of PCP is high. This study suggests that cotrimoxazole preventive therapy may be a useful intervention in symptomatic HIV infected patients in Kenya for the prevention of PCP and may avert deaths from this disease.

Antifungal drug susceptibility of Candida albicans.

OBJECTIVE: To determine the susceptibility of clinical isolates of Candida albicans and to establish the minimum inhibitory concentrations (MIC) to commonly used antifungal drugs. DESIGN: Laboratory based experiment. SETTING: Mbagathi District Hospital, Nairobi, Kenya. SUBJECTS: Candida albicans isolated between 1998 and 2000 from the sputa of HIV/AIDS patients and throat swabs of children with acute respiratory infections (ARI). METHODS: Susceptibility to amphotericin B, clotrimazole, nystatin, and 5-fluorocytosine was done using agar dilution method (NCCLS 1997). RESULTS: Among the ARI isolates 29.3% and among HIV isolates 22.4% had MIC>0.5 microg/ml to amphotericin B. Over 80% of the ARI isolates had MICs > 1 microg/ml to clotrimazole. The MIC range of most isolates to nystatin was 4-16 microg/ml while most isolates were susceptible to 5-fluorocytosine. There were no significant differences in susceptibility between ARI and HIV isolates to commonly used antifungal drugs. CONCLUSION: Although fungal resistance has not been extensively studied, susceptibility tests showed some Candida albicans have increased MICs to commonly used antifungal drugs. The results call for further investigations on fungal resistance especially in the context of opportunistic infections in HIV/AIDS.

Mycoplasma pneumoniae in children with pneumonia at Mbagathi District Hospital, Nairobi.

OBJECTIVE: To determine the prevalence of mycoplasma pneumoniae in nasopharyngeal aspirates of children under five years of age suffering from pneumonia. DESIGN: Cross-sectional survey. METHODS: Two primer sets targeting the genes coding for adhesion protein and 16S rRNA were used in PCR and M. pneumoniae specific antibodies were detected using commercial article agglutination kit. Microbiological investigations to isolate common acute respiratory infectious pathogens were also carried out. RESULTS: M. pneumoniae was detected in nasopharyngeal aspirates of 33.7% of the cases by PCR while serology was positive in only 4.1%. M. pneumoniae alone (single detection) was detected in 25% of the cases while 75% occurred with other acute respiratory infectious (ARI) pathogens. However, the results did not correlate with clinical outcome or the severity of pneumonia. No significant aetiology was found in 28% of the cases investigated, however microbiological investigations by culture revealed the presence of other aetiological agents as follows: Streptococcus pneumoniae (26%), Klebsiella pneumoniae (1%), Staphylococcus aureus (3%), E. coli (2%), parainfluenza viruses (5%), Salmonella(1%), adenovirus (4%), RSV (22%) and Candida spp(13%), Mycoplasma pneumoniae was more prevalent in children aged between six months and three years. Cases of M. pneumoniae PCR positive and S. pneumoniae exhibited similar seasonal distribution with peaks in May and September. However, there was no relationship between M. pneumoniae PCR positive and the severity of pneumonia. CONCLUSION: More investigation is required to establish the significance of atypical pathogens in respiratory infections in Kenya.

Plant essential oils with promising antifungal activity.

OBJECTIVE: To establish therapeutic efficacy of Tagetes minuta extracts. DESIGN: Laboratory-based experiment. METHOD: In vitro fungitoxicity test of essential oils was studied on Trichophyton mentagrophytes, Microsportum gypsum, Candida albicans ATCC 10231, Cryptococcus neoformans, Aspergillus niger and Penicillium species. The effect of oil on sporulation, budding and germination of fungal conidia was studied. The lowest active concentration (lac) was determined and compared with that of one per cent clotrimazole and one per cent nystatin. RESULTS: T. minuta essential oils were more effective on filamentous fungi than the standard antifungal drugs (p<0.05) but was less active on the yeast. It was fungicidal on filamentous forms of fungi and fungistatic on the yeast. The activity of the flower oil was significantly higher than that of the leaves (p=0.015). The oil significantly inhibited conidia germination but had no effects on budding of yeast. Gas chromatograph analysis of the oil showed a total of 30 compounds with five major peaks. CONCLUSION: The fungitoxicity of T. minuta oil can be exploited for its antifungal properties if toxicity and the active compound is studied further.

The role of airborne fungal spores from garbage dumps in respiratory diseases.

In recent years, there has been a growing global, regional and local concern about the potential health hazards associated with air pollution. Great attention has so far been paid to pollution associated with oxides of sulphur, CO, nitrogen oxides, O(3), trace organics, selected trace metals like lead, and miscelleneous suspended particulate matter. But, apart from these much talked about pollutants, urban air pollution associated with fungal spores from garbage pose a major environmental human health problem in most urban centres in Africa. Garbage probably accounts for most of the fungal spores in air, and, therefore, people, particularly those living close to heaps of garbage are exposed to very high concentrations of fungal spores. There is a possibility that a high correlation exists between the current incidence of respiratory infections and levels of fungal spores in the air. Air pollution by fungal spores has not been a matter of great concern in the past, and therefore, it is time we drew the environmentalists' attention and public awareness on this real and potentially hazardous issue. In this article, potential human health hazards resulting from moulds growing on crude garbage dumps in the vicinity of human habitation are discussed.