Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or "internal proteins" are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

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.

Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas - FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

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%) were human immunodeficiency virus (HIV)-positive and 26 (41.3%) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7%) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5%), and 24/63 (38.1%) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8%). 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%) of the HIV-positive subjects and in 13/26 (50%) 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 fungemia: report of a case with lung and brain lesions mimicking radiological features of malignancy

A 64-year-old apparently immunocompetent white man developed lung and brain lesions of disseminated cryptococcosis. The radiologic features mimicked those of lung cancer metastatic to the central nervous system. C. gattii was recovered from cultures of bronchoalveolar lavage fluid, brain biopsy, and blood. The same fungus was recovered from pulmonary and brain specimens at autopsy. Serum and cerebrospinal fluid cryptococcal antigen tests were diagnostic in our case and should be included in the diagnostic evaluation of unexplained pulmonary and cerebral lesions. A literature search showed few reports of fungemia by this species of Cryptococcus, contrasting to C. neoformans.

FIRST REPORT ON Cryptococcus neoformans IN PIGEON EXCRETA FROM PUBLIC AND RESIDENTIAL LOCATIONS IN THE METROPOLITAN AREA OF CUIABÁ, STATE OF MATO GROSSO, BRAZIL

SUMMARYCryptococcosis is a severe systemic mycosis caused by two species of Cryptococcus that affect humans and animals: C. neoformans and C. gattii. Cosmopolitan and emergent, the mycosis results from the interaction between a susceptible host and the environment. The occurrence of C. neoformanswas evaluated in 122 samples of dried pigeon excreta collected in 49 locations in the City of Cuiabá, State of Mato Grosso, Brazil, including public squares (n = 5), churches (n = 4), educational institutions (n = 3), health units (n = 8), open areas covered with asbestos (n = 4), residences (n = 23), factory (n = 1) and a prison (n = 1). Samples collected from July to December of 2010 were seeded on Niger seed agar (NSA). Dark brown colonies were identified by urease test, carbon source assimilation tests and canavanine-glycine-bromothymol blue medium. Polymerase chain reaction primer pairs specific for C. neoformans were also used for identification. Cryptococcus neoformans associated to pigeon excreta was isolated from eight (6.6%) samples corresponding to six (12.2%) locations.Cryptococcus neoformans was isolated from urban areas, predominantly in residences, constituting a risk of acquiring the disease by immunocompromised and immunocompetent individuals.

ISOLATION OF Cryptococcus neoformans FROM ENVIRONMENTAL SAMPLES COLLECTED IN SOUTHEASTERN NIGERIA

SUMMARY Cryptococcosis caused by Cryptococcus neoformans is the second most common fungal opportunistic pathogen and a lifethreatening infection with serious clinical manifestations especially in HIV/AIDS and other immunocompromised patients. In Nigeria, HIV/AIDS infection has reached an alarming level. Despite this, information on the presence of this fungus in clinical and environmental samples is very scanty in Nigeria and many other parts of Africa. We set out to evaluate the presence of Cryptococcus neoformans or C. gattii in pigeon droppings obtained from Southeastern Nigeria. One hundred and seventy-seven samples of pigeon droppings from six sample types were collected. The area covered comprised of ten cities and other locations spanning across five States in Nigeria. Using established techniques, Cryptococcus neoformans was isolated from 39 of the 177 (22.0%) samples overall. No C. gattiiwas isolated. Most of the isolates (32.4%) were recovered from dovecotes (11 of 34) followed closely by samples taken from markets (31.8%; seven of 22) and least from the church (4.0%; one of 25). The highest isolation rate (38.9%) was found in samples from Enugu-Ezike(seven of 23) while the least came from Afikpoand the other locations each with 9.1% isolation rate. This is the first large-scale screening of Cryptococcus neoformans from pigeon droppings in Nigeria. The ecological and epidemiological significance of these findings are discussed.

Suscetibilidade a antifúngicos de variedades de Cryptococcus neoformans isoladas de pacientes em hospital universitário

Este trabalho identificou variedades de Cryptococcus neoformans e avaliou a suscetibilidade a antifúngicos pelo protocolo M27-A2 do National Committee for Clinical Laboratory Standards em isolados de 35 pacientes do Hospital Escola da Universidade Federal do Triângulo Mineiro. A variedade gatti foi identificada em 11.4% (nº = 4) dos casos. A concentração inibitória mínima (mg/ml) dos isolados de Cryptococcus neoformans neoformans variou de 0,062 - 2,000 (anfotericina B), 0,250 - 8,000 (fluconazol), 0,062 - 1,000 (itraconazol) e 0,125 - 1,000 (cetoconazol). A variedade gattii apresentou concentração inibitória mínima de 0,125 - 2,000 (anfotericina B), 0,250 - 16,00 (fluconazol), 0,062 - 1,000 (itraconazol) e 0,125 - 4,000 (cetoconazol). Detectaram-se 2 isolados resistentes ao itraconazol e 2 a anfotericina B (1 isolado de cada variedade por antifúngico). Os dados mostram a importância da determinação da variedade e da concentração inibitória mínima de isolados de Cryptococcus neoformans para monitorar o desenvolvimento de resistência e possibilitar uma terapia mais adequada na criptococose.

Primeiro isolamento ambiental de Cryptococcus gattii no Estado do Espírito Santo

A presença de Cryptococcus gattii foi investigada em diferentes regiões do Estado do Espírito Santo. A maioria (73) das amostras foi coletada de árvores localizadas em lugares públicos de Vitória; 47 amostras foram coletadas de áreas preservadas ou ainda com pouco impacto humano, situados nos arredores desta cidade, a altitudes entre 0 e 900m acima do nível do mar e 48 de árvores nativas das regiões norte e sul do estado. As amostras foram coletadas de ocos e troncos de árvores com auxílio de swab e resultaram em 2 (1,2%) isolados de Cryptococcus neoformans, 2 (1,2%) de Cryptococcus gattii e 1 (0,6%) de Cryptococcus laurentii. A espécie Cryptococcus gattii foi encontrada somente em árvores nativas da região norte, áreas que ainda apresentam resquícios de Floresta Atlântica, enquanto todas as amostras obtidas de vinte e duas espécies de árvores localizadas em área urbana não permitiram a detecção de Cryptococcus gattii. Esses resultados mostram uma possível relação entre ocorrência de Floresta Atlântica e Cryptococcus gattii e confirma que o meio ambiente é fonte de infecção desse fungo.

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.

Evidências epidemiológicas da ocorrência de escabiose, em humanos, causada pelo Sarcoptes scabiei (DeGeer, 1778) var. canis (Bourguignon, 1853)

A partir de 27 cães com sarna sarcóptica, envolvendo 143 pessoas expostas à infestação, observaram-se 58 (40,56%) com lesões cutâneas sugestivas de escabiose. Tais lesões mostraram-se mais incidentes nas mulheres do que nos homens e indivíduos de todas as faixas etárias foram acometidos, indistintamente. Foi demonstrada a presença do agente em 3 dos 12 casos humanos observados que mantiveram contato com animais escabiosos.

Susceptibility of Aedes aegypti larvae to temephos and Bacillus thuringiensis var israelensis in integrated control

The susceptibility of field collected Aedes aegypti larvae was evaluated in terms of median lethal time (LT50) and final mortality, when treated with temephos, Bacillus thuringiensis var israelensis as well as mixtures of these two agents. Third instar larvae were shown to be more susceptible than early and late fourth instar ones to the entomopathogen. Survival of some individuals when exposed to temephos suggest possible resistance. Temporal synergism in early fourth instar larvae was detected when they were exposed to mixtures of Bti-temephos. The possibility of this integrated treatment is commented on.

Susceptibilidade de populações de Simulium (Chirostilbia) pertinax Kollar, 1832 (Culicomorpha, Simuliidae) ao temephos e a um formulado à base de Bacillus thuringiensis var. israelensis

O uso de pequenas calhas de madeira no leito de riachos, manualmente colonizadas com borrachudos, é proposto para avaliações da eficiência de larvicidas 3 ou 4 horas após as aplicações. A susceptibilidade larval também foi avaliada pelo critério de TL50. Em três ensaios no litoral do Estado de São Paulo e Rio de Janeiro, populações de Simulium (C.) pertinax Kollar, 1832 mostraram-se resistentes ao temephos, mesmo em altas concentrações. Vectobac 12 AS, à base de Bacillus thuringiensis var. israelensis mostrou-se mais potente contra larvas dos últimos estádios e eficiente para controle em concentrações a partir de 7.200 UIP/1 (10 min.). O TL50para 3.744 UIP/1 (10 min.) foi de 70,9 minutos.

Esterilização por óxido de etileno: I. Influência do meio de esporulação na resistência dos esporos de Bacillus subtilis var. niger

Tendo por meta a padronização das variáveis influenciando a resistência de esporos empregados no controle do processo esterilizante por óxido de etileno, foram obtidos esporos de Bacillus subtilis var. niger, em meio sólido e líquido sintético de esporulação. Tais esporos, após padronização quantitativa dos 12 lotes obtidos, foram submetidos a exposições subletais como bioindicadores, tendo o papel como suporte. Construiu-se, então, a curva de letalidade característica de cada lote. A análise estatística empregada não evidenciou diferenças entre resistência dos 10 lotes obtidos em meio sólido e os 2 em meio líquido sintético, ressaltando-se a vantagem quanto ao rendimento que caracterizou a primeira metodologia.

O líquido cefalorraquiano inicial nas meningencefalites por Cryptococcus neoformans

A criptococose, conhecida desde 1905, é doença grave, causada pelo Cryptococcus neoformans. Nesta entidade o estudo do líquido cefalorraquiano é muito importante visto que o sistema nervoso está envolvido em 81% dos casos5. O propósito do presente trabalho é o de evidenciar as alterações do líquido cefalorraquiano inicial nas meningencefalites criptocócicas e demonstrar a importância de exame minucioso do líquido cefalorraquiano para o diagnóstico precoce desta doença. O material é constituído por 46 observações de pacientes com diagnóstico de meningencefalite criptocócica. O quadro inflamatório crônico, caracterizado por pleocitose predominantemente linfocitária, hiperproteinorraquia e glicose diminuída foi observado em 54% das amostras de líquido cefalorraquiano. Em 69% dos casos o líquido cefalorraquiano mostrava hipertensão. A glicose estava diminuída em 61% dos pacientes e a taxa de cloretos em 16%. Eosinófilos em baixa percentagem foram observados em 48% dos casos. O criptococo foi identificado no líquido cefalorraquiano de 98% dos pacientes; é salientada a importância da pesquisa a fresco do fungo pelo método da tinta da China, sempre que uma amostra de líquido cefalorraquiano revelar pleocitose. Esta é uma conduta básica para o diagnóstico precoce da criptococose do sistema nervoso.