Fungos isolados em travesseiros de crianças e adolescentes com rinite e/ou asma / Isolated fungi on pillows of children and teenagers with rhinitis and/or asthma

Introdução: Pouco se sabe sobre a distribuição de fungos dentro das casas e nos materiais onde ficam concentrados, como os travesseiros, objeto que passa maior tempo em contato próximo com o paciente, podendo ser um reservatório importante e facilitar a sensibilização e o desencadeamento de crises alérgicas. O conhecimento da ocorrência de fungos em locais pouco pesquisados, bem como sua taxonomia, torna-se fundamental. Objetivos: Avaliar ocorrência de fungos em travesseiros de crianças alérgicas, o ambiente e os aspectos relacionados. Métodos: Pacientes com rinite e/ou asma, e teste cutâneo positivo para fungos foram selecionados. Realizado questionário ambiental no domicílio e coletado travesseiros em uso, os quais foram aspirados na área interna e externa para obtenção de amostras de fungos. Travesseiros novos, comprados em comércio local, serviram de controle. Resultados: A prevalência de sensibilização dos pacientes a fungos foi de 5,46% (13 dos 238 avaliados). Nenhum ambiente revelou-se adequado para pacientes alérgicos. Todos os travesseiros, inclusive os controles, estavam contaminados, tanto na sua área externa como na interna; o número médio de unidades formadoras de colônias (UFC/m2) apresentou diferença significativa na parte externa, sendo maior naqueles com mais de 7 anos de uso. A diversidade e a quantidade de fungos encontrados nos travesseiros dos pacientes foi maior que nos controles. Das 39 espécies e/ou outro nível taxonômico identificados, 32 (82,0%) podem causar alergia do Tipo I - IgE mediada, e os mais frequentes foram Candida, Penicillium sp., Cladosporium sp., Mycelia sterilia, Fusarim sp., Aureobasidium pullulans e Aspergillus. Nenhum tipo de enchimento foi considerado ideal, e o que apresentou menor nível de contaminação foi o de viscoelástico. Conclusão: Travesseiros são fontes de fungos e seus alérgenos. A maioria dos fungos isolados pode causar sensibilização com resposta IgE mediada. O painel utilizado mostrou-se insuficiente para identificar sensibilização aos fungos isolados.

Introduction: Little is known about fungi distribution in houses and materials where they are concentrated. Pillows are objects that remain much time in close contact with people and can be an important fungal reservoir, thus facilitating sensitization and triggering allergic crises. The knowledge of occurrence of fungi in unfrequently reported places, as well as their taxonomy, has become fundamental. Objectives: To evaluate the occurrence of fungi in pillows of allergic children, including the environment and related aspects. Methods: Patients with rhinitis and/or asthma and positive skin test for fungi were selected. An environmental questionnaire was completed at home and pillows in use were collected and aspirated inside and outside to obtain fungal samples. New pillows, bought in local stores, served as controls. Results: The prevalence of sensitization of patients to fungi was 5.46% (13 of 238 patients evaluated). No environment proved to be suitable for allergic patients. All the pillows, including the controls, were contaminated, both inside and outside; the mean number of colony forming units (CFU/m2) showed a significant difference in the outside and was greater in pillows with more than 7 years of use. The diversity and amount of fungi found on patients' pillows was greater than in controls. Of the 39 species and/or other taxonomic level identified, 32 (82.0%) may cause type I IgE-mediated allergy, and the most frequent fungi were Candida, Penicillium sp., Cladosporium sp., Mycelia sterilia, Fusarim sp., Aureobasidium pullulans and Aspergillus. No type of filling material was considered ideal, and the one with lowest level of contamination was viscoelastic. Conclusion: Pillows are sources of fungi and their allergens. Most isolated fungi can cause sensitization with IgE-mediated response. The panel used was insufficient to identify sensitization to isolated fungi.

Use of fluorescent oligonucleotide probes for differentiation between Paracoccidioides brasiliensis and Paracoccidioides lutzii in yeast and mycelial phase

BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis.

Paracoccidioides brasiliensis AND Paracoccidioides lutzii, A SECRET LOVE AFFAIR / Paracoccidioides brasiliensis e Paracoccidioides lutzii, um caso secreto de amor

SUMMARYTo commemorate Prof. Carlos da Silva Lacaz's centennial anniversary, the authors have written a brief account of a few, out of hundreds, biological, ecological, molecular and phylogenetic studies that led to the arrival of Paracoccidioides lutzii, hidden for more than a century within Paracoccidioides brasiliensis. Lacaz's permanent interest in this fungus, and particularly his conviction on the benefits that research on paracoccidioidomycosis would bring to patients, were pivotal in the development of the field.

RESUMOPara comemorar o centenário de aniversário do Prof. Dr. Carlos da Silva Lacaz, os autores fazem um breve relato dos estudos sobre a biologia, ecologia e filogenia molecular que culminaram na revelação da espécie Paracoccidioides lutzii, que havia permanecido escondida por mais de um século ao lado de Paracoccidioides brasiliensis. O professor Lacaz exerceu papel central no desenvolvimento desta área do conhecimento, pois manteve interesse permanente nas pesquisas deste fungo e da paracoccidioidomicose, visando principalmente proporcionar benefícios aos pacientes acometidos por esta micose.

Candida species biofilm and Candida albicans ALS3 polymorphisms in clinical isolates

Over the last decades, there have been important changes in the epidemiology of Candida infections. In recent years, Candida species have emerged as important causes of invasive infections mainly among immunocompromised patients. This study analyzed Candida spp. isolates and compared the frequency and biofilm production of different species among the different sources of isolation: blood, urine, vulvovaginal secretions and peritoneal dialysis fluid. Biofilm production was quantified in 327 Candida isolates obtained from patients attended at a Brazilian tertiary public hospital (Botucatu, Sao Paulo). C. albicans ALS3 gene polymorphism was also evaluated by determining the number of repeated motifs in the central domain. Of the 198 total biofilm-positive isolates, 72 and 126 were considered as low and high biofilm producers, respectively. Biofilm production by C. albicans was significantly lower than that by non-albicans isolates and was most frequently observed in C. tropicalis. Biofilm production was more frequent among bloodstream isolates than other clinical sources,in urine, the isolates displayed a peculiar distribution by presenting two distinct peaks, one containing biofilm-negative isolates and the other containing isolates with intense biofilm production. The numbers of tandem-repeat copies per allele were not associated with biofilm production, suggesting the evolvement of other genetic determinants.

Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.

Evaluation of pathogenic fungi occurrence in traumatogenic structures of freshwater fish / Avaliação da ocorrência de fungos patogênicos em estruturas traumatogênicas de peixes fluviais

INTRODUCTION: Fungal infections in human skin, such as sporotrichosis, can occur after fish induced trauma. This work aimed to identify fungi in freshwater fish that are pathogenic to humans. METHODS: Extraction of dental arches from Serrassalmus maculatus (piranha) and Hoplias malabaricus (wolf fish), stings from Pimelodus maculatus (mandis catfish), dorsal fin rays from Plagioscion spp. (corvina) and Tilapia spp., for culture in Mycosel agar. Some cultures were submitted to DNA extraction for molecular identification by sequencing ITS-5.8S rDNA. RESULTS: Cultures identified most yeast as Candida spp., while sequencing also permitted the identification of Phoma spp. and Yarrowia lipolytica. CONCLUSIONS: While the search for S. schenckii was negative, the presence of fungus of the genera Phoma and Candida revealed the pathogenic potential of this infection route. The genus Phoma is involved in certain forms of phaeohyphomycosis, a subcutaneous mycosis caused by dematiaceous fungi, with reports of infections in human organs and systems. Traumatizing structures of some freshwater fish present pathogenic fungi and this may be an important infection route that must be considered in some regions of Brazil, since there are a large number of a fisherman in constant contact with traumatogenic fish.

INTRODUÇÃO: Infecções fúngicas na pele humana (como a esporotricose) podem se manifestar após traumatismos por peixes. O objetivo deste trabalho é procurar fungos patogênicos para o homem em peixes fluviais. MÉTODOS: Extração de arcadas dentárias Serrassalmus maculatus (piranha) e Hoplias malabaricus (traíra), ferrões de Pimelodus maculatus (mandis), raios da nadadeira dorsal de Plagioscion spp. (corvina) e Tilapia spp. para a realização do cultivo em agar Mycosel. Algumas culturas foram submetidas à extração de DNA para a identificação molecular pelo seqüenciamento da região ITS-5.8S do rDNA. RESULTADOS: As culturas mostraram que a maioria das leveduras era Candida spp. e o sequenciamento também permitiu a identificação de Phoma spp. e Yarrowia lipolytica. CONCLUSÕES: Embora a pesquisa para S. schenckii tenha sido negativa, a presença de fungos do gênero Phoma e Candida revela o potencial patogênico desta via de infecção. O gênero Phoma está envolvido em alguns casos de feohifomicoses, micoses subcutâneas causadas por fungos dematiáceos com relatos de infecções em órgãos e sistemas humanos. As estruturas traumatizantes de alguns peixes fluviais apresentam fungos patogênicos e esta pode ser uma importante via de infecção que deve ser considerada em algumas regiões do Brasil, uma vez que há um grande número de pescadores e peixes traumatogênicos.

Search for Mycobacterium leprae in wild mammals

Leprosy is still a worldwide public health problem. Brazil and India show the highest prevalence rates of the disease. Natural infection of armadillos Dasypus novemcinctus with Mycobacterium leprae has been reported in some regions of the United States. Identification of bacilli is difficult, particularly due to its inability to grow in vitro. The use of molecular tools represents a fast and sensitive alternative method for diagnosis of mycobacteriosis. In the present study, the diagnostic methods used were bacilloscopy, histopathology, microbiology, and PCR using specific primers for M. leprae repetitive sequences. PCR were performed using genomic DNA extracted from 138 samples of liver, spleen, lymph nodes, and skin of 44 D. novemcinctus, Euphractus sexcinctus, Cabassous unicinctus, and C. tatouay armadillos from the Middle Western region of the state of São Paulo and from the experimental station of Embrapa Pantanal, located in Pantanal da Nhecolândia of Mato Grosso do Sul state. Also, the molecular analysis of 19 samples from internal organs of other road killed species of wild animals, such as Nasua nasua (ring-tailed coati), Procyon cancrivoros (hand-skinned), Cerdocyon thous (dog-pity-bush), Cavia aperea (restless cavy), Didelphis albiventris (skunk), Sphigurrus spinosus (hedgehog), and Gallictis vittata (ferret) showed PCR negative data. None of the 157 analyzed samples had shown natural mycobacterial infection. Only the armadillo inoculated with material collected from untreated multibacillary leprosy patient presented PCR positive and its genomic sequencing revealed 100 percent identity with M. leprae. According to these preliminary studies, based on the used methodology, it is possible to conclude that wild mammals seem not to play an important role in the epidemiology of leprosy in the Middle Western region of the São Paulo state and in the Pantanal of Mato Grosso do Sul state.

Molecular approaches for eco-epidemiological studies of Paracoccidioides brasiliensis

Medical mycology has greatly benefited from the introduction of molecular techniques. New knowledge on molecular genetics has provided both theoretical and practical frameworks, permitting important advances in our understanding of several aspects of pathogenic fungi. Considering Paracoccidioides brasiliensis in particular, important eco-epidemiological aspects, such as environmental distribution and new hosts were clarified through molecular approaches. These methodologies also contributed to a better understanding about the genetic variability of this pathogen; thus, P. brasiliensis is now assumed to represent a species complex. The present review focuses on some recent findings about the current taxonomic status of P. brasiliensis, its phylogenetic and speciation processes, as well as on some practical applications for the molecular detection of this pathogen in environmental and clinical materials.

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.

Experimental infections with Paracoccidioides brasiliensis obtained from armadillos: comparison to clinical isolates

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM) that is one of the most prevalent systemic human mycoses in Latin America. Armadillos show a high incidence of PCM infection and could, therefore, be a natural reservoir for this fungus. In this study were compared the virulence profiles of isolates obtained from nine-banded armadillos (Dasypus novemcinctus) (PbT1 and PbT4) and isolates from PCM patients (Pb265 and Bt83). Pathogenicity was evaluated by fungal load and analysis of colony morphology. Immunity against the fungus was tested by delayed type hypersensitivity test (DTH) and antibody quantification by ELISA. The higher virulence of PbT1 and PbT4 was suggested by higher fungal load in spleen and lungs. Armadillo isolates and Bt83 presented a cotton-like surface contrasting with the cerebriform appearance of Pb265. All isolates induced cellular and humoral immune responses in infected BALB/c mice. DTH reactions were similarly induced by the four isolates, however, a great variability was observed in specific antibody levels, being the highest ones induced by Bt83 and PbT4. The present work confirms that armadillos harbor P. brasiliensis, whose multiplication and induced immunity in experimentally infected mice are heterogeneous, resembling the behavior of isolates from human PCM. This study reinforces the possibility that armadillos play an important role in the biological cycle of this pathogen.

Anatomia do encéfalo de Dasypus novemcinctus (xenarthra, dasypodidade) / Brain anatomy of Dasypus novemcinctus (Nine-band armadillo) (xenarthra, dasypodidade) / Anatomía del Encéfalo de Dasypus novemcinctus (XENARTHRA, DASYPODIDAE)

Os tatus são animals pertencentes à família Dasypodidae (Ordem Xenarthra). Possuem uma carapaça dorsal bem desenvolvida, pela qual são facilmente identificados. Este trabalho teve por objetivo descrever as estruturas mais relevantes do encéfalo do tatu-galinha (tatu-de nove-bandas) (Dasypus novemcinctus), a fim de contribuir para o conhecimento da anatomia dessa espécie e, assim, entender algumas de suas funções biológicas. Foram utilizados 5 animals, cujos crânios foram separados do restante do corpo e fixados em solução aquosa de paraformaldeído (formol) a 20%, na qual os espécimes foram mergulhados durante 30 dias. Os encéfalos foram conservados em nova solução de formalina a 20% durante tempo suficiente, até que se fizessem as análises morfológicas. O encéfalo dos tatus contém todas as estruturas encontradas nos mamíferos. O telencéfalo é liso, porém apresenta rudimentos de sulcos. O cerebelo é grande, em relação aos hemisférios cerebrais. O bulbo olfatório é bastante evidente nesses animals, sugerindo uma orientação espacial pelo olfato.

The armadillos belong to the Dasypodidae famiIy (Xenarthra order); they are diggers and nocturnal. This article had aimed at describing the most evident structures of the brain of the Dasypus novemcinctus, in order to contribute to the knowledge of such species, thus understanding some of its biological functions. Five animals were used for this study; they were beheaded, and their brains were put into 20% formalin aqueous solution where the specimens were kept for 30 days. The brains were kept in a new 20% formalin solution for enough time until we did the morphological analysis. The brains of armadillos present all the structures found in mammals. The brain hemispheres are smooth, but with some circumlocutions. The cerebellum is prominent in these animals, which suggests a spatial orientation through olfact.

Pythium insidiosum: relato do primeiro caso de infecção humana no Brasil / Pythium insidiosum: report of the first case of human infection in Brazil

A pitiose é causada por microorganismo aquático, fungo-símile, o Pythium insidiosum, patógeno de homens e animais. Observou-se um paciente com úlcera fagedênica no membro inferior, com exame anatomopatológico sugestivo de zigomicose, pouco sensível à terapêutica antifúngica, obtendo-se cura por meio de ampla exérese. A comprovação etiológica resultou de métodos moleculares, com amplificação e seqüenciamento de DNA de organismo isolado em ágar Sabouraud, observando-se 100 por cento de analogia com seqüências de P. insidiosum depositadas no GenBank.

Pythiosis is caused by an aquatic fungus-like organism, Pythium insidiosum, pathogenic to men and animals. A patient with a phagedenic ulcer on the leg is reported. Histopathological examination was suggestive of zygomycosis, response to antifungal drugs was poor and cure was obtained by means of wide surgical excision. Etiologic diagnosis was confirmed by molecular amplification and DNA sequencing of colonies isolated in Sabouraud agar. After BLAST analysis, the sequence showed 100 percent identity with those of P. insidiosum deposited on the GenBank.

Isolation of Paracoccidoides brasiliensis from armadillos (Dasypus novemcinctus) captured in an endemic area of paracoccidioidomycosis

Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), was first isolated from the Amazonian gerion where the mycosis is uncommon. In the present study, we report on the high incidence of PCM infection in armadillos from a hyperendemic region of the disease. Four nine-banded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, Sao Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions. Fragments of lung, spleen, liver and mesenteric lymph nodes were precessed for histology, cultured on Mycosel agar at 37ºC, and homogenized for inoculation into the testis and peritoneum of hamster. The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37ºC. Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph node hamster. In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node. The three aramdillos isolates (Pb-A1, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticulary inoculation hamster. The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients. Taken together, the present data confirm that armadillos are a natural reservoir of P. brasiliensis and demonstrate that the animal is a sylvan host to the fungus