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

Bii, C

Boga, H

Kangogo, M

Wanyoike, W

English

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August 2014 East african MEdical Journal    281East African Medical Journal Vol. 91 No. 8 August 2014ISOLATION AND CHARACTERISATION OF CRYPTOCOCCUS NEOFORMANS AND CRYPTOCOCCUS GATTII FROM ENVIRONMENTAL SOURCES IN NAIROBI, KENYAM. Kangogo, Department of Medical Microbiology, H. Boga, Department of Botany, W. Wanyoike, Department of Botany, Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000-00200, Nairobi and C. Bii, Medical Mycology Unit, Centre for Microbiology Research, Kenya Medical Research Institute, P. O. Box 54840-00202, Nairobi, KenyaISOLATION AND CHARACTERISATION OF CRYPTOCOCCUS NEOFORMANS AND CRYPTOCOCCUS GATTII FROM ENVIRONMENTAL SOURCES IN NAIROBI, KENYAM. KANGOGO, H. BOGA, W. WANYOIKE and C. BIIABSTRACTObjective: 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.INTRODUCTIONCryptococcus neoformans and Cryptococcus gattii are pathogenic basidiomycetous yeasts that cause cryptococcossis disease in immunocompromised and immunocompetent patients (1,2). With the emergence of the AIDS pandemic, the incidence of cryptococcosis is increasing and represents a major life threatening fungal infection in these patients (1,3). Globally, the risk of cryptococcal meningitis in HIV/AIDS is estimated between 0.04 to 12% in adults and about 1% in children. The highest population at risk of meningoencephalitis is in sub-Saharan Africa and South East Asia where the highest burden of HIV/AIDS exists (1,4). A low CD4 cell count is the main predictor of risk of cryptococcal meningoencephalitis; the vast majority of cases occur among AIDS patients with a CD4 cell count<100 cells/mm (5). Cryptococcus neoformans has historically been divided into three varieties of five serotypes based on antigenicity of the capsule: C. neoformans var. grubii (serotype A), C. neoformans var. gattii (serotypes B and C), C. neoformans var. neoformans (serotype D), and one hybrid (serotype AD) (6). In 2002, C. neoformans var. gattii (serotypes B and C) was awarded species status and renamed Cryptococcus gattii (7). The two varieties exhibit different sexual states; Fillobasidiella neoformans var. neoformans is the teleomorph of C. neoformans var. neoformans and F. neoformans var. bacilliospora is the teleomorph of C. neoformans var. gattii. The two varieties are morphologically similar except that basidiospores of var. neoformans are round and those of var. gattii are more elliptical in shape (7). The definitive identification of the two varieties is possible through biochemical tests such as resistance to canavanine and use of glycine as the sole carbon and nitrogen and resistance of their urease enzyme to EDTA (8). C. neoformans has a worldwide distribution and has been associated with a variety of environmental sources in particular, bird excreta and decaying wood (9,10). It is documented that 282 East african MEdical Journal August 2014 C. gattii is limited to tropics and sub-tropical regions and causes cryptococcosis in over 40 % of  non AIDS patients (2,11). Majority of the isolates from Europe belong to C. neoformans var. neoformans while only 15 % of the isolates from USA, Argentina and Canada belong to C. gattii (12). Such vast environmental reservoirs documented encourage the assumption of the existence of many other natural reservoirs of C. neoformans. The infectious particles of C. neoformans and C. gattii are presumed to be the dehydrated yeast cells, which enter the alveolar spaces of the lungs. Once inside the lungs, the yeast cells become rehydrated and acquire the polysaccharide capsule which is best visible in Indian ink preparations (13). The thickness of the capsule is strain related and varies with environmental conditions and is known to be responsible for virulence (13,14). Upon growth in 1% peptone solution, production of the capsule is enhanced. C. neoformans  can cause an asymptomatic pulmonary infection followed by the development of meningitis, which is often  the first indication of the disease limited to the lungs (13).  The study was aimed at establishing environmental reservoirs for C. neoformans and C. gattii in attempt to establishing the risk factors for the high prevalence of Cryptococcal infection in Kenya. The paucity of information on the natural reservoirs of C. neoformans in Kenya compared to other documented information is worthwhile.MATERIALS AND METHODSA total of 400 samples were collected from different locations in Nairobi, (1°17′S 36°49′E / 1.283°S 36.817°E),  Kenya. The city lies on the Nairobi River, in the south of the nation, and has an elevation of 1661 m (5450 ft) above sea level. Sampling was done from areas in the city with high concentrations of pigeons, birds, private chicken breeders’ homes and chicken selling markets. Others were from garbage damping sites within the city, (Table 1, Figure 1). Samples were treated as described by Staib, 1987 (15). Droppings, bark and soil samples were processed as follows: 5 g of the sample were suspended in 25 ml of phosphate-buffered saline (PBS) and allowed to settle for 30 min then the sample was filtered and 50 µl of antibiotic solution, (20 U/mL streptomycin and 40 U/mL penicillin) was added. Then 100 µL of the mixture was cultured on Niger-seed agar (Guizotia abyssinica). All plates were incubated at 37ºC for up to two weeks. The material collected with the swabs was directly plated onto the same medium. All brown colonies were cultured on Christensen’s urea agar and incubated at 37 ºC for 48 hours. Urease positive colonies were cultured on corn meal agar using slide culture technique. Each plate was divided into four parts and a single colony was seeded onto each part, it was covered with sterile cover slip and incubated at 37 ºC for 48 hours. The cover slip was removed aseptically and observed under microscope using X 40 resolution power. This was to distinguish different Cryptococcus neoformans from other yeasts. Cryptococcus neoformans do not produce pseudohyphae, are spherical, irregular in size and widely separated on corn meal agar. Observation for the presence of capsule which is a characteristic of Cryptococcus neoformans was done on Indian ink stain.The biovariety study was performed by culturing the isolates on Canavanine- Glycine-Bromthymol blue (CGB) medium to determine the use of glycine as a carbon source. A colour change from light yellow green to cobalt blue was considered a positive result for the CGB test, indicating presence of C. neoformans var. gattii (serotype B/C). The lack of colour change was considered a negative result, indicating absence of C. neoformans (serotype A/D). RESULTSThe environmental isolates of C. neoformans and C. gattii were recovered from different sources and sites as shown in Table 1and Figure 1. Cryptococcus neoformans was frequently isolated in all the droppings with the majority being from marabou stock (4/20; 20%) followed by chicken droppings (7/60; 11.7%) and 10% each from weaver birds (2/20), pigeon droppings (8/80), chicken cage (5/50) and pied crow droppings (2/20). In contrast, Cryptococcus gattii was less frequently isolated among the droppings with 3/80 (3.8 %) in pigeon droppings 1/20 (5%) from Marabou stock droppings and 1/60 (1.7%) from chicken droppings. Also, C. gattii was more frequently isolated among trees with 6/50 (12%) from Eucalyptus saligna and 1/10 (10%) from Acacia xanthophloea. C. neoformans 5/50 (10%) were recovered from Eucalyptus saligna. On the other hand C. gattii was not recovered from soil or garbage dumping sites.  C. neoformans was isolated from soil (2/30; 6.7%) and garbage damping sites (6/60; 10%) (Table 1).August 2014 East african MEdical Journal    283Table 1Environmental isolates of Cryptococcus neoformans and Cryptococcus gattii in Nairobi, KenyaSample Type  Positive Samples Total Number of Samples Cryptococcus neoformans Cryptococcus gattiiDroppingsMarabou stock (Leptoptilos crumeniferus) droppings20 4 (20%) 1 (5%)Weaver birds (Ploceus spekei) droppings20 2 (10%) 0Pigeon (Geopelia striata) droppings 80 8 (10%) 3 (3.8%)Chicken (Gallus gallus domesticus) droppings60 7 (11.7%) 1 (1.7%)Pied crow (Corvus albus ) droppings20 2 (10%) 0Tree swabsAcacia xanthophloea 10 0 1 (10%)Eucalyptus saligna 50 5 (10%) 6 (12%)Other sitesChicken (Gallus gallus domesticus) cage50 5 (10%) 0Garbage damping site 60 6 (10%) 0Contaminated Soil 30 2 (6.7%) 0Total 400 41 12Figure 1Map Showing Sampling Sites of Cryptococcus neoformans and C. gattii in Nairobi, Kenya284 East african MEdical Journal August 2014 DISCUSSIONWe were successful in recovering C. neoformans and C. gattii from different environmental sites in Nairobi, Kenya (Table 1 and Figure 1). This success was attributed to the fact that all the droppings analysed were dry and not wet at the time of collection. In a study conducted by Granados and Castañeda (16), it was noted that old excreta was more likely to harbour high numbers of C. neoformans than fresh excreta. We were also able to isolate C. gattii though in small percentages from pigeon droppings, marabou stock droppings and chicken droppings. C. gattii was not isolated from weaver bird and pied crow droppings. Limited studies have been carried out in Africa on the environmental isolation of C. neoformans (17-18). A study in Ethiopia on occurrence of C. neoformans in the environment showed the presence of C. neoformans in pigeon droppings and absence of C. neoformans from other bird droppings (18). The same study did not recover C. gattii from the pigeon droppings. In our study most of the C. neoformans isolates were recovered from pigeon (8/80; 10%) and chicken (7/60; 11.7 %) droppings. Although pigeon droppings has been documented as the most common source of C. neoformans in the environment, the presence of this yeast in many bird species other than pigeons, i.e., dove, psitaccines, budgerigars, canaries, parrots, cockatoos and Starlings had also been reported (19).  We were able to recover C. neoformans and C. gattii from two tree species namely Acacia and Eucalyptus. Most of the environmental C. gattii isolates were recovered from Eucalyptus trees (6/50; 12%) and only (1/10: 10%) was recovered from Acacia tree. Our findings agree with other studies that associates C. gattii with Eucalyptus and other non Eucalyptus trees as well (12,16). This was confirmed in a environmental surveillance study by  Granados Castaneda in Bogotá, Colombia whereby C. gattii was recovered from Eucalyptus trees as  well as other trees including Acacia, Cupresus, and Pinus (16). Similarly, C. neoformans was recovered also from Eucalyptus trees in this study. Many studies associate C. gattii isolation with Eucalyptus but our findings proved otherwise; the fact that these trees harbor different bird species could have contributed to the recovery of both isolates. Few reports exist of isolations of C. neoformans and C. gattii from the same habitats with the recognitions of natural hybrids between the two species. For instance, C. neoformans and C. gattii have been isolated from same sources, such as Eucalyptus spp. or Syzygium cumini trees or bird feces (20,21). C. neoformans was isolated from other sites including soil samples, chicken cage and swabs from garbage dumping sites. Contrary to some studies that have found soil to be a major reservoir of C. gattii (12,22), we did not recover C. gattii from soil in this study. The occurrence of C. neoformans and C. gattii in different environmental samples collected in the Nairobi, Kenya is significant. The isolation and identification of yeast from these environmental sources in Kenya might provide useful information for ecological and epidemiological studies of C. neoformans and C. gattii.ACKNOWLEDGEMENTSThis study was funded DAAD and JKUAT. We acknowledge the assistance rendered by Kenya Medical Research Institute in providing Laboratory space for sample collection and processing of the samples.REFERENCES1.  Park BJ, Wannemuehler K A, Marston BJ, Govender N, Pappas PG, Chiller TM. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS. 2009;23:525-530. 2.  Pappas PG. Cryptococcal infections in non-HIV-infected patients. Trans Am Clin Climatol Assoc. 2013;124:61-79.3.  Alemu AS, Kempker RR, Tenna A, et al. High prevalence of Cryptococcal antigenemia among HIV-infected patients receiving antiretroviral therapy in Ethiopia. PLoS One. 2013;8:e583774.  Tortorano AM, Viviani MA, Rigoni  AL, Cogliati M, Roverselli A, Pagano A. Prevalence of serotype D in Cryptococcus neoformans isolates from HIV positive and HIV negative patients in Italy. Mycoses. 1997;40:297-3025.  Jarvis JN, Harrison TS. HIV-associated cryptococcal meningitis. AIDS. 2007;21:2119-2129. 6.  Evans EE. The antigenic composition of Cryptococcus neoformans. I. A serologic classification by means of the capsular and agglutination reactions. J Immunol. 1950;64:423-430.7.  Kwon-chung KJ, Boekhout T, Fell JW, Diaz M. (1557) Proposal to conserve the name Cryptococcus gattii against C. hondurianus and C. basillisporus (Basidiomycota, Tremellomycetidae). Taxon. 2002;51:804-806.8.  Collier, L., Balows, A. & Sussman M. Topley & Wilson’s Microbiology and Microbial Infections.; 1998.9.  Kuroki M, Phichaichumpon C, Yasuoka A, et al. Environmental isolation of Cryptococcus neoformans from an endemic region of HIV-associated cryptococcosis in Thailand. Yeast. 2004;21:809-12. doi:10.1002/yea.1112.10.  Ferreira AS, Sampaio A, Maduro AP, et al. 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Zentralbl Bakteriol Mikrobiol Hyg. 1987;266:167-177.16.  Granados DP, Castañeda E. Isolation and characterization of Cryptococcus neoformans varieties recovered from natural sources in Bogotá, Colombia, and study of ecological conditions in the area. Microb Ecol. 2005;49:282-290. 17.  Litvintseva AP, Mitchell TG. Population genetic analyses reveal the African origin and strain variation of Cryptococcus neoformans var. grubii. PLoS Pathog. 2012;8: e1002495. 18.  Yimtubezenash W. Amanuel, Leykun Jemaneh DA. Isolation and characterization of Cryptococcus neoformans from environmental sources in Ethiopia. Ethiop J Heal Dev. 2001;15:45-49.19.  Kumlin U, Olsen B, Granlund M ELTA. Cryptococcosis and starling nests. Lancet. 1998;351:1181.20.  Lazera MS, Cavalcanti MA, Trilles L, Nishikawa MM WB. Cryptococcus neoformans var. gattii—evidence for a natural habitat related to decaying wood in a pottery tree hollow. Med Mycol. 1998;36:119-122.21.  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Isolation and characterisation of Cryptococcus neoformans and Cryptococcus gattii from environmental sources in Nairobi, Kenya

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Isolation and characterisation of Cryptococcus neoformans and Cryptococcus gattii from environmental sources in Nairobi, Kenya

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