Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g-1)] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g-1, while the threshold for the ST was greater than 0.1.10³ CFU-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.

Cryptococcus gattii VGI and Cryptococcus neoformans VNI Associated with Wood Decay in Ficus Hollow Trees in Rio de Janeiro, Brazil.

Aim: Considering the geographic expansion of Cryptococcus gattii, the aim of this study was to investigate hollows of living trees as a reservoir of C. gattii in Rio de Janeiro, Brazil. Place and Duration of the Study: In an urban quarter of Rio de Janeiro city, 80 samples of decaying wood were collected. In addition, 85 decaying wood samples were collected in the wild rainforest. The samples were analyzed at the Mycology Laboratory, Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation, from 2008-2010. Methodology: Samples were collected by scraping the inner decaying wood of the hollows of the trunks of each tree. Pathogenic Cryptococcus species were identified by: brown colonies on niger seed agar (NSA) medium, thermotolerance at 35ºC, cycloheximide sensitivity, carbon and nitrogen assimilation tests performed by 32-Vitek System (Vitek ICB, bioMeriux, Durham, EUA). Canavanine-glycine-bromothymol blue medium (CGB) was used to determine the species of the isolates and the genotypes were determined by restriction fragment length polymorphism of URA5 gene. Results: After plating the samples on NSA, 584 colonies were obtained from the urban quarter. C. gattii VGI was identified in 98% of colonies, followed by C. neoformans VNI 2%. The positivity of the urban area was 7.8%. The concentrations of the fungi in hollows of ficus trees ranged from 50 to 56,250 colony-forming units per gram of sample (CFU/g). Conclusions: For the first time in Rio de Janeiro C. gattii VGI was isolated in a hollow of living tree.

Cryptococcus neoformans carried by Odontomachus bauri ants

Cryptococcus neoformans is the most common causative agent of cryptococcosis worldwide. Although this fungus has been isolated from a variety of organic substrates, several studies suggest that hollow trees constitute an important natural niche for C. neoformans. A previously surveyed hollow of a living pink shower tree (Cassia grandis) positive for C. neoformans in the city of Rio de Janeiro, Brazil, was chosen for further investigation. Odontomachus bauri ants (trap-jaw ants) found inside the hollow were collected for evaluation as possible carriers of Cryptococcus spp. Two out of 10 ants were found to carry phenoloxidase-positive colonies identified as C. neoformans molecular types VNI and VNII. The ants may have acted as a mechanical vector of C. neoformans and possibly contributed to the dispersal of the fungi from one substrate to another. To the best of our knowledge, this is the first report on the association of C. neoformans with ants of the genus Odontomachus.

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil)

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7 percent) were human immunodeficiency virus (HIV)-positive and 26 (41.3 percent) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7 percent) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5 percent), and 24/63 (38.1 percent) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8 percent). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6 percent) of the HIV-positive subjects and in 13/26 (50 percent) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.

Cryptococcus gattii molecular type VGII as agent of meningitis in a healthy child in Rio de Janeiro, Brazil: report of an autochthonous case / Cryptococcus gattii tipo molecular VGII como agente causador de meningoencefalite em criança saudável no Rio de Janeiro, Brasil: relato de um caso autóctone

Cryptococcus gattii causes meningoencephalitis in immunocompetent hosts, occurring endemically in some tropical and subtropical regions. Recently, this fungus was involved in an outbreak in Vancouver Island and British Columbia (Canada). In this temperate region, the VGII type is predominant. The paper describes an autochthonous case of meningoencephalitis by C. gattii VGII in a previously health child in Rio de Janeiro, considered nonendemic region of Brazil. The fungus was identified by biochemical tests and the molecular type was determined by URA5-RFLP. The present report highlights the need for clinical vigilance for primary cryptococcal meningitis in nonendemic areas.

Cryptococcus gattii é causa de meningoencefalite em hospedeiros imunocompetentes, ocorrendo endemicamente em regiões tropicais e subtropicais. Recentemente foi causador de surtos na Ilha de Vancouver e na Columbia Britânica (Canadá). Nesta região de clima temperado, o tipo VGII é predominante. Relatamos um caso de meningoencefalite pelo C.gattii tipo VGII acometendo criança previamente saudável autóctone do Rio de Janeiro, região não endêmica do Brasil. O agente foi identificado por testes bioquímicos e o tipo molecular determinado através de URA5-RFLP. O presente relato enfatiza a necessidade de vigilância clínica para a meningite criptocóccica primária em áreas não endêmicas.

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.

Efeito de diferentes técnicas de acabamento e polimento sobre a rugosidade superficial de resinas acrílicas utilizadas para restaurações provisórias / Effect of finishing and polishing techniques on the surface roughness of resin acrylic used on temporary restorations

Considerando a importância da lisura superficial das restaurações provisórias, o objetivo desse trabalho foi de avaliar o efeito de diferentes métodos de acabamento e polimento sobre a rugosidade superficial de 05 resinas acrílicas autopolimerizáveis (Dencor®, Dencrilay®, Duralay®, Instatemp® e Vipi Cor®). Um total de 200 corpos de prova foram confeccionados, sendo 40 corpos de prova para cada resina, que foram obtidos a partir de uma matriz bipartida com um molde cilíndrico de aço inoxidável de 10 mm de diâmetro e 3 mm de profundidade. Cada material foi proporcionado de acordo com a recomendação de seu fabricante, inseridos no molde na fase plástica e após polimerização, todos os espécimes foram armazenados em umidificador à 37ºC por 24 horas. Dez espécimes de cada resina foram submetidos a cada um dos seguintes procedimentos: a) sof-lex + branco de espanha; b) sof-lex + selante de superfície; c) pedra-pomes+ branco de espanha; d) pedra-pomes + selante de superfície. Em seguida, a rugosidade superficial média (Ra medidaem μm) foi aferida e registrada pelo rugosímetro (SJ-301, Mitutoyo – Japan) para posterior comparação entre os grupos. Os dados coletados foram submetidos à análise estatística através de testes paramétricos de “Kolmogorov-Smirnov” e análise de variância com nível de significância de 5%. Os resultados evidenciaram diferença estatisticamente significante em relação à rugosidade apenas entre a resina Instatemp® (menos rugosa) e a resina Vipi-Cor® (mais rugosa). No entanto, ao comparar os procedimentos de acabamento e polimento independente do tipo de resina acrílica, os dados evidenciaram diferenças numéricas, porém sem diferença estatisticamente significativa. Dentro das limitações desse estudo, concluiu-se que as resinas acrílicas apresentaram comportamento diferente em relação à rugosidade superficial média frente aos métodos de acabamento e polimento.

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 percent), Cryptococcus gattii VGII (25/56, 44.64 percent) and C. gattii VGI (3/56, 5.26 percent). 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 percent 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.

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 percent), followed by VGII (21 percent), VNII (5 percent), VGIII (4 percent), VGI and VNIV (3 percent each), and VNIII (< 1 percent). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.