Marília Martins Nishikawa

Cryptococcus neoformans var. gattii — evidence for a natural habitat related to decaying wood in a pottery tree hollow

To study hollows of living trees as the natural habitat of Cryptococcus neoformans in an endemic area of cryptococcosis in the northeastern Brazilian region, samples of decaying wood were collected inside the hollows, plated on niger seed agar and inoculated into mice and hamsters. Identification of C. neoformans was based on morphological and physiological tests. Canavanine-glycine-bromothymol medium was used to screen the varieties and Crypto Check Iatron Kit to serotype the isolates. For a period of 29 months C. neoformans var. gattii serotype B was isolated repeatedly from the hollow of a pottery tree (Moquilea tomentosa), pointing to the natural occurrence of C. neoformans var. gatti in decaying wood forming hollows in living trees. Evidence for a natural habitat of the variety gattii other than that related to Eucalyptus camaldulensis are discussed.

Natural habitat of Cryptococcus neoformans var. neoformans in decaying wood forming hollows in living trees

Cryptococcus neoformans var. neoformans was repeatedly isolated from decaying wood forming hollows in living trees growing in urban areas of Rio de Janeiro, Brazil. A new natural habitat for C. neoformans var. neoformans has been found that is not associated with specific trees.

Serotyping of 467 Cryptococcus neoformans Isolates from Clinical and Environmental Sources in Brazil: Analysis of Host and Regional Patterns

ABSTRACT Cryptococcus neoformans is an important zoopathogen, and it is one of the most prevalent lethal mycotic agents. Its polysaccharide capsule, synthesized in vivo and in vitro, is a virulence factor, contains predominantly glucuronoxylomannan, and is responsible for the antigenic differentiation of serotypes A, B, C, D, and AD. A total of 467 isolates of C. neoformans obtained from clinical and environmental sources from Brazilian regions were studied serologically by using the Crypto Check Iatron RM 304-K kit. Serotyping of the clinical isolates showed the following prevalences of the serotypes: A (77.95%), followed by B (18.2%), AD (1.3%), D (0.4%), C (0.2%), and untypeable (1.93%). The epidemiology of serotype A in the Brazilian southern and southeastern regions reproduces the picture observed worldwide. In contrast, serotype B was the most frequent agent of cryptococcosis in the northeastern region, occurring nearly equally in male and female healthy hosts. Among the isolates from environmental sources, serotypes A and B were found to occur in the hollows of tropical trees of the genera Cassia, Ficus, and Moquillea. The few isolates from Eucalyptus camaldulensis debris were serotypes A and B and untypeable. Overall, no association with a specific host tree was identified for these serotypes, denoting a distinct ecoepidemiological regional pattern. The one serotype C isolate was recovered from a human immunodeficiency virus-negative host. Serotype AD predominated over serotype D among both clinical and environmental isolates.

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT alpha) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.

Isolation of both varieties of Cryptococcus neoformans from saprophytic sources in the city of Rio de Janeiro, Brazil

Using the method of intraperitoneal inoculation into mice, 11 (13·2%) out of 83 different samples from urban environments in the city of Rio de Janeiro, Brazil, were positive for Cryptococcus neoformans. Isolation of C. neoformans var. neoformans from wood and plant debris within a tree hollow of Syzygium jambolana (jambolana, Java plum) and the finding of C. neoformans var. gattii in bat guano collected in an old house are discussed with reference to our current knowledge on the ecology of C. neoformans.

Primary endemic Cryptococcosis gattii by molecular type VGII in the state of Pará, Brazil.

In order to study the infectious agents causing human disseminated cryptococcosis in the state of Pará, North Brazil, 56 isolates of Cryptococcusspp. (54 isolated from cerebral spinal fluid and two from blood cultures) from 43 cases diagnosed between 2003-2007 were analysed. The species were determined through morphological and physiological tests and genotypes were determined by URA5-RFLP and PCR-fingerprinting (wild-type phage M13). The following species and genotypes were identified: Cryptococcus neoformans VNI (28/56, 50%), Cryptococcus gattii VGII (25/56, 44.64%) and C. gattii VGI (3/56, 5.26%). The genotype VNI occurred in 12 out of 14 HIV-positive adults, whereas the genotype VGII occurred in 11 out of 21 HIV-negative adults (p < 0.02, OR = 6.6 IC95% 0.98-56.0). All patients less than 12 years old were HIV negative and six cases were caused by the VGII genotype, one by the VGI and one by VNI. Therefore, endemic primary mycosis in HIV-negative individuals, including an unexpectedly high number of children, caused by the VGII genotype deserves further study and suggests the need for surveillance on cryptococcal infection in the state of Pará, Eastern Amazon.

Cryptococcus neoformans isolated from human dwellings in Rio de Janeiro, Brazil: an analysis of the domestic environment of AIDS patients with and without cryptococcosis.

One hundred and fifty-four human dwellings in the metropolitan area of Rio de Janeiro, Brazil were studied. A total of 824 samples of indoor dust, outdoor soil and avian droppings were collected. Cryptococcus neoformans var. neoformans was isolated from 20 (13%) dwellings, comprising five (15.6%) of 32 dwellings of patients with AIDS-associated cryptococcosis; four (8.9%) of 45 dwellings of patients with AIDS but without cryptococcosis; and 11 (14.3%) of 77 dwellings of apparently healthy individuals (P>0.05). The principal factor associated with domiciliar contamination by C. neoformans var. neoformans was the presence of avians in the domestic environment or nearby the home. Cryptococcosis was more frequent among AIDS patients residing in dwellings from which C. neoformans var. neoformans was isolated than among AIDS patients from whose domestic environment the fungus was not demonstrated by the methods used (odds ratio (OR)=2.05). These findings suggest that the distribution of C. neoformans var. neoformans in Rio de Janeiro is not restricted to the classically known biotopes as well as reinforce the possibility of exogenous infection in opportunistic cryptococcosis, including exogenous infection acquired in the domestic environment.

Decolorization of different textile dyes by Penicillium simplicissimum and toxicity evaluation after fungal treatment

The objective of this study was to investigate the capacity of decolorization and detoxification of the textile dyes Reactive Red 198 (RR198), Reactive Blue 214 (RB214), Reactive Blue 21 (RB21) and the mixture of the three dyes (MXD) by Penicillium simplicissimum INCQS 40211. The dye RB21, a phthalocyanine, was totally decolorized in 2 days, and the others, the monoazo RR198, the diazo RB214 and MXD were decolorized after 7 days by P. simplicissimum. Initially the dye decolorization involved dye adsorption by the biomass followed by degradation. The acute toxicity after fungal treatment was monitored with the microcrustacean Daphnia pulex and measured through Effective Concentration 50% (EC50). P. simplicissimum reduced efficiently the toxicity of RB21 from moderately acutely toxic to minor acutely toxic and it also reduced the toxicity of RB214 and MXD, which remained minor acutely toxic. Nevertheless, the fungus increased the toxicity of RR198 despite of the reduction of MXD toxicity, which included this dye. Thus, P. simplicissimum INCQS 40211 was efficient to decolorize different textile dyes and the mixture of them with a significant reduction of their toxicity. In addition this investigation also demonstrated the need of toxicological assays associated to decolorization experiments.

First isolation of Cryptococcus gattii molecular type VGII and Cryptococcus neoformans molecular type VNI from environmental sources in the city of Belém, Pará, Brazil

Cryptococcus neoformans and Cryptococcus gattii are important agents of meningoencephalitis in humans in the city of Belém. This clinical data suggests that the region may be a highly endemic area for the pathogenic Cryptococcus species within the state of Pará (PA), Northern Brazil. Preliminary analysis of 11 environmental samples from the city of Belém showed two positive locations, including a hollow of a kassod tree (Senna siamea) colonized simultaneously by C. gattii molecular type VGII and C. neoformans molecular type VNI, and a birdcage in a commercial aviary positive for C. neoformans, molecular type VNI. This is the first evidence of an environmental occurrence of molecular types VNI and VGII in PA.

Environmental isolation of Cryptococcus gattii VGII from indoor dust from typical wooden houses in the deep Amazonas of the Rio Negro basin.

Cryptococcosis is a human fungal infection of significant mortality and morbidity, especially in the meningoencephalitis form. Cryptococcosis is distributed worldwide and its agents, C. neoformans and C. gattii, present eight major molecular types—VNI-VNIV and VGI-VGIV respectively. The primary cryptococcosis caused by molecular type VGII (serotype B, MAT alpha) prevails in immunocompetent patients in the North and Northeast of Brazil, revealing an endemic regional pattern to this molecular type. Since 1999, C. gattii VGII has been involved in an ongoing outbreak in Canada, and is expanding to the Northwest of the United States, two temperate regions. Exposure to propagules dispersed in the environment, related to various organic substrates, mainly decomposing wood in and around dwellings, initiates the infection process. The present study investigated the presence of the agents of cryptococcosis in dust from dwellings in the upper Rio Negro, municipality of Santa Isabel do Rio Negro in Amazonas state. Indoor dust was collected from 51 houses, diluted and plated on bird seed agar. Dark brown colonies were identified phenotypically, and genotypically by URA5 restriction fragment length polymorphism analysis and multilocus sequence typing (MLST). The mating type was identified using pheromone-specific primers. Three of the 51 houses were positive for C. gattii molecular type VGII, MATα and MATa, showing a high prevalence of this agent. MLST studies identified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5 new subtypes unique to the region. For the first time in the state of Amazonas, C. gattii VGII MATα and MATa were isolated from the environment and correlates with endemic cryptococcosis in this state. This is the first description of MLST subtypes on environmental isolates in the Brazilian Amazon, indicating domiciliary dust as a potential source for human infection with different subtypes of C. gattii VGII MATα and MATa.