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Genotyping of Paracoccidioides brasiliensis from formalin-fixed paraffin embedded (FFPE) tissue samples from patients treated in the Hospital of Medical School of Botucatu, São Paulo State University

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Author(s):
Luciana Bonome Zeminian de Oliveira
Total Authors: 1
Document type: Doctoral Thesis
Press: Botucatu. 2018-04-23.
Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina. Botucatu
Defense date:
Advisor: Luciane Alarcão Dias Melicio
Abstract

Paracoccidioidomycosis is a chronic granulomatous mycosis prevalent in Latin America, that until recently it was believed to be caused only by Paracoccidioides brasiliensis (P. brasiliensis). However, in 2006, researchers described cryptic species: S1, PS2, PS3, and PS4. In 2009, Paracoccidioides lutzii (Pb01-like) was described, and now, a new nomenclature was proposed for the other different agents: P. brasiliensis (S1), P. Americana (PS2), P. restrepiensis (PS3), and P. venezuelensis (PS4). All these agents are thermodimorphic fungi that develop as yeast in vivo, in host tissues or in vitro cultures at 37°C in culture media. It also grows as mycelium at room temperature ranging from 4 to 28°C. These species are not uniformly distributed throughout Latin America, some are more prominent in some regions than in others. The Hospital of Medical School of Botucatu - UNESP, which is a paracoccidioidomycosis study pole, is in São Paulo state midwest region, that is classified as an endemic area. Due to the existence of cryptic species of Paracoccidioides, further analyses of patient samples are needed for a better understanding the distribution and occurrence of these recently described species in Botucatu region, that could favor a possible correlation between genetic groups and mycological and clinical characteristics. Given the importance of this disease to the region, the aims of this study were to perform a retrospective epidemiological, geographical and clinical study gathering information available on medical records of patients treated in 10-year period from 2004 17 to 2014 in Botucatu and to genotype the clinical species present in the samples of our diagnosed patients. For the genotyping, it was used techniques for the removal of the paraffin from slices of the biopsies, followed by DNA extraction, PCR and sequencing of three target genes (ITS, CHS2 and ARF). It was that the most prevalent clinic form in Botucatu region is the chronic type (77,4%), most commonly presented in male individuals over 40 years old, smokers and alcoholics. It was also demonstrated that most of the patients, taking both clinical forms (acute and chronic) in consideration, are individuals self-declared as white. From the genotyping of species in the samples, we were able to find 44 positive samples. All the sequences were compared at with sequences deposited at GenBank to testify the presence of P. brasiliensis. The phylogenetic trees were constructed using the sequences of ITS, ARF and CHS2 genes and showed that 100% of our positive samples are from S1 cryptic species. This is an important data, demonstrating the possible absence of other species in our region. Moreover, the observation that white individuals are more prone to Paracoccidioidomycosis opens new perspectives of studies about a possible resistance of nonwhite individuals against the fungi. (AU)

FAPESP's process: 13/24877-9 - Genotyping of Paracoccidioides brasiliensis from different samples of paracoccidioidomycosis patients treated at Botucatu Medical School -UNESP
Grantee:Luciana Bonome Zeminian de Oliveira
Support Opportunities: Scholarships in Brazil - Doctorate