Environmental isolation of Cryptococcus neoform ans var. gattii and C. neoformans var. neoformans in the city of São Paulo, Brazil

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1 Medical Mycology 2000, 38, Accepted 26 November 1999 Environmental isolation of Cryptococcus neoform ans var. gattii and C. neoformans var. neoformans in the city of São Paulo, Brazil H. MONTENEGRO* & C. R. PAULA *C entro de C ontrole de Zoonoses, Prefeitura Municipal de São Paulo, São Paulo, Brazil; Instituto de C iências Biomédicas, Universidade de São Paulo, São Paulo, Brazil Introduction Cryptococcus neoformans, an encapsulated yeast, is the etiological agent of human cryptococcosis. Infection begins by inhalation of fungal cells into the lungs but shows a remarkable propensity to spread hematogenously to the central nervous system; meningoencephalitis is the most common clinical manifestation [1]. Since the mid 1980s, cryptococcosis has emerged as a life-threatening infection to patients with acquired immune deciency syndrome (AIDS), where disseminated infection is commonly fatal even when treated with the most effective antifungal drugs [2,3]. Correspondence: Hildebrando Montenegro, Centro de Controle de Zoonoses, Rua Santa Eulalia, 86 Santana, CEP , São Paulo, SP, Brasil. Tel.: ; fax: ; hmontenegro@sti.com.br In order to determine the environmental occurrence of both varieties of Cryptococcus neoformans in the city of São Paulo, pigeon droppings and vegetable material from trees of the genus Eucalyptus were collected at typically crowded places. A total of 38 sites downtown where large heaps of pigeon droppings could be found were selected for sampling. Pigeon droppings from 10 (26 3%) of these sites were positive for C. neoformans var. neoformans in at least one sample. Twelve eucalyptus woods located within four municipal parks were also surveyed; vegetable material from Eucalyptus spp. trees were collected monthly over a 2-year period. C. neoformans var. gattii was recovered from a wood in Ibirapuera Park during the same season on two different occasions (November 1996 and November 1997); this park contained specimens of Eucalyptus camaldulensis, a natural habitat of C. n. var. gattii. C. n. var. neoformans was detected in a wood in Ibirapuera Park and Aclimação Park. The results show that both C. n. var. neoformans and C. n. var. gattii are present in the urban environment of São Paulo city at sites where large numbers of people normally gather. Keywords Cryptococcus neoformans, environmental isolation, Eucalyptus species, pigeon droppings On the basis of physiological, ecological and serological differences, C. neoformans has been subdivided into two varieties: C. neoformans var. neoformans (serotypes A, D and AD) and C. neoformans var. gattii (serotypes B and C). Subsequent to this study, Franzot et al. [4] proposed limiting the use of C. n. var. neoformans to serotype D isolates and described a new variety C. neoformans var. grubii for isolates of serotype A, based mainly on analysis of the URA5 gene sequences and DNA ngerprint patterns of the two serotypes. In the present study, C. n. var. neoformans was used for serotypes A and D. C. n. var. neoformans has a world-wide distribution and has been isolated from various sources in nature; it is known for its association with pigeon droppings but has also been found in soil, decaying wood, vegetables and fruits. C. n. var. neoformans is predominantly opportunis ISHAM

2 386 Montenegro & Paula tic, corresponding to almost all the clinical isolates from AIDS patients [1]. C. n. var. gattii shows a tropical and sub-tropical distribution and has been recovered from the environment in plant detritus of closely related eucalyptus species, such as Eucalyptus camaldulensis and E. tereticornis. Experiments conducted under the canopy of E. camaldulensis in bloom detected airborne propagules of the fungus, yet it was no longer possible to isolate the yeast from air or plant material after the owering period. It was suggested that the release of C. n. var. gattii propagules is seasonal and is associated with the blooming of these eucalyptus species [5]. C. n. var. gattii is predominantly pathogenic and can be found in patients with no underlying immunosuppression, but a few cases have been described in Brazilian patients with AIDS [6 8]. Studies conducted in São Paulo, Brazil, report the clinical isolation of both varieties of C. neoformans in the city [9 11]. The incidence of cryptococcosis caused by C. n. var. neoformans has been increasing steadily because of the AIDS epidemic [7] and a growing use of immunosuppressive drugs [12]. In the city of São Paulo, cryptococcosis due to C. n. var. neoformans is the presenting manifestation of AIDS in 6 7% of these patients [13]. The incidence of cryptococcosis indicates the presence of infective propagules in the urban area of São Paulo, where both varieties of the yeast must be encountering favorable environmental conditions for development. São Paulo city is the largest urban center of South America and is ranked as the third largest world-wide. The population of pigeons is growing in this city and hence the amount of droppings in the environment is on the increase. Furthermore, the planting of eucalyptus in urban areas, including species associated with C. n. var. gattii, is a common practice in Brazil [14]. The aim of this study was to investigate the occurrence of C. n. var. gattii and C. n. var. neoformans in the city of São Paulo by analyzing samples of the most commonly reported substrates for both varieties of this fungus, that is, pigeon droppings and plant material from eucalyptus woods. Material and methods Study area The city of São Paulo (23 32Æ 36ÆÆ S latitude and 46 37Æ 59ÆÆ W longitude) is located 760 m above the sea level; it has a tropical climate that is inuenced by altitude, with an annual mean temperature of 19 C. This city is the largest urban and industrial center of Brazil, with almost 10 million inhabitants. Pigeo n excreta sampling Historically, the center of São Paulo is an ancient part of the city and comprises a great number of very old houses, churches and monuments that favor the settlement of pigeons. Within this area, we selected 38 sites highly contaminated with pigeon droppings for sampling of excreta. The places were separated by type into public and commercial buildings (n¾15), squares and plazas (n¾8), churches (n¾5), streets and avenues (n¾4) and others (n¾ 6); they were always close to crowded streets or a signicant ow of people. Those places were separated into protected and unprotected. Unprotected places were those which receive rain and solar radiation directly; protected places were those which in some way were protected from those factors. Every place was sampled twice at least from October 1995 until November 1997, but some of the places identied were followed more regularly; those places were sampled every 3 months during Each sample was collected at different locations within the area covered with pigeon excreta; sample weight varied g, with an average weight of 4 7 g. A positive place was considered one which was positive for C. n. var. neoformans in at least one sample. Eucalyptus woods sampling Four municipal parks located within the urban perimeter of the city were also selected. In Ibirapuera Park ( m 2 ), four eucalyptus woods were identied. This park is the biggest of the city and is located downtown; it is frequented by a large number of people every day. In Aclimação Park ( m 2 ), three eucalyptus woods were identied. This park is also located downtown, but is smaller, and frequented less. In Carmo Park ( m 2 ), four eucalyptus woods were identied. Carmo Park is one of the biggest parks of the city, but is located at the city s periphery and is frequented by large number of people on the weekends. In Piqueri Park ( m 2 ), only one eucalyptus wood was identied; this is the smallest park studied and is not frequented much. These eucalyptus woods identied in the parks have a mean area of m 2. They are typically reforested areas, composed only of eucalyptus species set out at regular intervals. One sample was collected per wood, every month, from January 1996 until December 1997; each sample was composed of soil containing mainly pieces of bark, leaves, fruits and owers as well as plant detritus from the ground. Collections were made randomly inside the wood, trying to cover the entire area. Sample weights varied g, with an average weight of 37 2 g.

3 Isolation of C. neoformans in São Paulo 387 Sample processing Samples were stored at 10 C and processed within 48 h. The material collected was suspended in sterile saline (1:10, w:v), shaken and allowed to stand for min. Next, 0 1 ml of the supernatant was streaked onto Sabouraud glucose agar (SGA) and on DOPA medium [15], both with chloramphenicol (0 2 g l ¼ 1 ), and incubated at 37 C for samples of pigeon droppings and 32 C for samples from the eucalyptus woods. Cultures were examined daily for 10 days. Isolation and identication of Cryptococcus species and varieties Brown colonies on DOPA medium or colonies with a suspected morphology on SGA were isolated for identication. Suspect colonies were identied using a battery of morphological and biochemical tests that included India ink preparation, urease production, assimilation tests and fermentation tests [16]. Colonies of the genus Cryptococcus were identied up to the species. The canavanine-glycine-bromthymol blue agar test was used to distinguish between of C. n. var. neoformans and C. n. var. gattii. Serotyping of C. neoformans isolates was carried out by the slide agglutination method [17]; as noted in the Introduction, the nomenclature C. n. var. neoformans is used for serotypes A and D isolates. The identication of C. n. var. gattii was conrmed by Dr Marcia Lazera with a commercial kit, Crypto Check RM 304-K (Iatron Lab. Inc., Tokyo, Japan). Results C. n. var. neoformans was isolated from 10 (26 3%) of the 38 sites selected in the centre of São Paulo for their marked contamination by pigeon droppings (Table 1). All the isolates belonged to serotype A. These positive places corresponded mainly to public buildings, like the Main Post Ofce, the Municipal Theatre, The City s Court of Justice, the Central Railroad Station and a Technical Highschool, but C. n. var. neoformans was also isolated in a square, in a street, in two house and in a shoeshine box. At the Central Railroad station, samples were taken from inside the main building; at the other places, samples were collected from the outer surfaces of the buildings. C. n. var. neoformans was isolated in eight (72 7%) of the 11 protected places sampled and in two (7 4%) of the 27 unprotected places sampled. A total of 112 pigeon droppings specimens were collected at those 38 places, 43 from protected and 69 from unprotected places (Table 2). C. n. var. neoformans was isolated in 30 specimens (69 8%) collected in protected places and in two specimens (2 9%) collected in unprotected places. At nine places followed regularly during 1997, a total of 36 pigeon droppings samples were collected (Table 3). C. n. var. neoformans was isolated in 13 (65 0%) of the 20 samples collected in ve protected places and in two (12 5%) of the 16 samples collected in four unprotected places. With respect to the samples collected in eucalyptus woods, C. n. var. gattii, C. n. var. neoformans, Cryptococcus albidus and Cryptococcus laurentii were isolated from eucalyptus debris (Table 4). In Aclimação Park, C. n. var. neoformans was isolated in wood # 1 (April 1996); C. albidus was isolated in wood # 1 and # 3 (both in August 96); C. laurentii was isolated in wood # 1 (April and September 1996) and # 3 (April, August and September 1996). In Carmo Park, C. laurentii was isolated in wood # 1 (August 1997), # 2 (April 1996, June and August 1997) and # 4 (May 1997). In Piqueri Park, no Cryptococcus species was recovered from eucalyptus debris. In Ibirapuera Park (Fig. 1), C. n. var. gattii was isolated in wood # 1 (November 1996 and 1997), C. n. var. neoformans was isolated in wood # 1 (July 1996) and C. laurentii was isolated in wood # 1 (April 1996) and # 4 (August and September 1996 and September 1997). C. n. var. gattii isolates from eucalyptus debris belonged to serotype B and C. n. var. neoformans isolates belonged to serotype A. Discussion The incidence of cryptococcosis in the city of São Paulo has been documented since before the AIDS epidemic. Table 1 C. neoformans var. neoformans isolation at different types of places Positive Place Type n n % Public and commercial buildings P UP Squares and plazas P UP Streets and avenues P UP Churches P UP Others P UP TOTAL P, protected place; UP, unprotected place.

4 388 Montenegro & Paula Table 2 Number of samples collected at different types of places Positive Place Type n n % Public and commercial buildings P UP Squares and plazas P UP Streets and avenues P UP Churches P UP Others P UP Total P, protected place; UP, unprotected place. However, an interest in C. neoformans in clinical settings has increased considerably due to its importance in immunosuppressed patients. Less attention has been paid to environmental sources of the fungus within the urban perimeter of this city, despite the epidemiological relevance of such data. In São Paulo, cryptococcosis due to both C. n. var. neoformans and C. n. var. gattii is known to occur [9,10]. The present study focused on the most important substrates of each variety in order to identify natural sources of infective propagules within the city s urban environment. São Paulo s population of pigeons has increased considerably due to greater availability of both shelter and food, to the point of becoming a public health problem. Pigeon droppings usually accumulate near the birds dwellings and nests, which are usually of difcult access and hence impose a certain limitation to signicant sampling. The sites surveyed in this study were Fig. 1 Ibirapuera Park with the locations of eucalyptus woods sampled. chosen after an extensive search for places close to the ow of people in the centre of the city. The percentage of positive samples of C. n. var. neoformans in pigeon droppings from heavily contaminated downtown locations (26 3%), is similar to available data for other Brazilian cities and other countries [18]. The samples were collected in an area of high population density and this implies that many of the inhabitants are frequently exposed to C. n. var. neoformans. Indeed, the incidence of human cryptococcosis due to this variety in São Paulo, before and Table 3 C. neoformans var. neoformans isolation at places sampled every 3 months during the year of 1997 Place Type February May August November Central Railroad Station P Governor s Wife Cabinet P Technology Highschool P Central Mail Ofce P Municipal Theatre P Marechal Deodoro Square UP Santa Cecõ lia Plaza UP Fishery Institute UP Roosevelt Square UP P, protected place; UP, unprotected place., Positive sample;, Negative sample.

5 Isolation of C. neoformans in São Paulo 389 Table 4 Cryptococcus spp. isolation in eucalyptus woods located at municipal parks Location Wood J F M A M J J A S O N D J F M A M J J A S O N D Aclimação Park # : # # : Carmo Park # # # # Ibirapuera Park # # # # Piqueri Park # , C. neoformans var. gattii; 2, C. neoformans var. neoformans; 3, C. albidus; 4, C. laurentii. after the AIDS epidemic, conrms such exposure to the fungus. With respect to protected and unprotected places, our data suggest a correlation between protected places and heaped pigeon droppings positive for C. n. var. neoformans. The fungus was more frequently isolated from pigeon droppings sampled in protected places (72 7%) than in unprotected places (7 4%). Protected places favor desiccation of the excreta and it is known that C. n. var. neoformans is highly resistant to desiccation. This characteristic favors the survival of the fungus when competing with other microbial populations present in pigeon droppings [1]. Since the dry yeast is easily aerosolized [2], dry pigeon droppings positive for C. n. var. neoformans must act as a focal source of fungal dissemination. Conversely, open and exposed places were rarely positive for C. n. var. neoformans, despite the large amounts of accumulated pigeon droppings found. This variety is very susceptible to solar radiation [19] and has a low thermotolerance [1]. Such features probably limit the development of this yeast in unsheltered droppings. In fact, a closer examination of some of the places identied reinforces this observation (Table 3). C. n. var. neoformans was isolated in 65 0% of the samples collected in protected places, whereas in unprotected places the fungus was recovered from 12 5% of the samples. It is noteworthy that in one of the protected places, the Municipal Theatre, heaps of pigeon droppings were removed during the rebuilding of the theatre. Samples of pigeon droppings were negative for C. n. var. neoformans in the rst two collections after removal (February and May), but the fungus was newly recovered in subsequent collections (August and November). The high prevalence of cryptococcosis due to C. n. var. gattii in Brazil [1] reects its typical tropical and subtropical distribution. Lacaz & Rodrigues [9] reported a frequency of 36% of this variety among Brazilian isolates recovered from clinical sources during Paula et al. [10] studied 40 clinical isolates obtained from non- AIDS patients in 1988 and found a frequency of 32 5%of C. n. var. gattii. The prevalence of C. n. var. gattii in Brazil, before and after the AIDS epidemic, is considered high even among South American countries [20]. Despite this, there are few studies about environmental sources of C. n. var. gattii in this country. In a study carried out at an E. camaldulensis plantation in the state of Sergipe, only C. laurentii was recovered from plant material [21]. C. laurentii was also recovered in seven of the 12 eucalyptus woods sampled at this study (Table 4). A recent study in the city of Teresina provides evidence of another natural habitat of C. n. var. gattii than that related to E. camaldulensis and E. tereticornis trees; the authors repeatedly isolated this variety in decaying wood forming the hollow in a pottery tree (Moquilea tomentosa) [22]. According to Ellis & Pfeiffer [5], the high frequency of cryptococcosis due to this variety in endemic areas is related to the presence of E. camaldulensis and E. tereticornis. Brazil has the largest plantation area of eucalyptus in the world; the planting of this type of tree in the urban environment is very common in the country, and E. camaldulensis and E. tereticornis are among the 10 pre-

6 390 Montenegro & Paula ferred species [14]. In our study, C. n. var. gattii was isolated from the same wood in Ibirapuera Park during the month of November, on two consecutive years. It is worth noting that the presence of E. camaldulensis has already been described in a taxonomic study performed in this park [23]. Ellis & Pfeiffer [5] also reported the isolation of C. n. var. gattii in plant samples derived from E. camaldulensis during the month of November in Australia. In view of these considerations, our ndings lend support to the hypothesis of a seasonal dispersal of this fungus in São Paulo associated with eucalyptus in bloom. Ibirapuera Park is one of the largest and most visited parks in the city of São Paulo and, considering our ndings, presents an increased risk of the exposure of individuals to C. n. var. gattii propagules when the corresponding eucalyptus host species are in bloom. To conclude, our data indicate that both C. n. var. neoformans and C. n. var. gattii are present in the urban environment of the city of São Paulo, at sites where large crowds of people normally gather. Considering the incidence of human cryptococcosis in this Brazilian city, our results emphasize the need for further studies on the distribution of C. neoformans in urban areas, in order to better understand the epidemiology of this fungus. Acknowledgements We wish to express our thanks to Dr Carlos S. Lacaz, Laboratório de Micologia Médica, Instituto de Medicina Tropical, Universidade de São Paulo, for his helpful advice during the study, to Dr Claudia Maffei, Departamento de Microbiologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, for serotyping our strains and to Dr Marcia Lazera, Laboratório de Micologia Médica, Hospital Evandro Chagas, Instituto Oswaldo Cruz, for conrming our C. neoformans var. gattii strains. References 1 Kwon-Chung KJ, Bennett JE. 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