Genotipagem do Paracoccidioides brasiliensis de diferentes amostras de pacientes atendidos no Hospital das Clínicas da Faculdade de Medicina de Botucatu
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Data
2018-02-27
Autores
Oliveira, Luciana Bonome Zeminian de
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Universidade Estadual Paulista (Unesp)
Resumo
A paracoccidioidomicose é uma micose granulomatosa sistêmica, prevalente na América Latina, e que até recentemente acreditava-se ser causada apenas por uma espécie de fungo, o Paracoccidioides brasiliensis (P. brasiliensis). No entanto, em 2006, pesquisadores descreveram três espécies crípticas: S1, PS2, PS3 e, posteriormente o PS4. Em 2009, o Paracoccidioides lutzii (Pb01-like) foi descrito, e ano passado, em 2017, uma nova nomenclatura foi proposta para esses agentes etiológicos distintos: P. brasiliensis (S1), P. Americana (PS2), P. restrepiensis (PS3) e P. venezuelensis (PS4). Todos esses agentes são fungos termodimórficos que crescem como levedura in vivo, no hospedeiro, em tecidos ou em culturas in vitro a 37°C e como micélio à temperatura ambiente (4 a 28°C). As espécies não são uniformemente distribuídas pela América Latina, sendo algumas mais proeminentes em algumas regiões do que em outras. O Hospital das Clínicas da Faculdade de Medicina de Botucatu é polo de estudo da paracoccidioidomicose e situado na região centro-oeste do estado de São Paulo, considerada uma área endêmica. Devido à existência de espécies crípticas de Paracoccidioides, análises mais detalhadas nas amostras de pacientes tornaram-se necessárias para uma melhor compreensão de distribuição e ocorrência das espécies recentemente descritas nessa região, favorecendo uma possível correlação entre os grupos genéticos e características micológicas e clínicas. Os objetivos foram obtenção de dados epidemiológicos, geográficos e clínicos dos pacientes diagnosticados e tratados em um período de 10 anos, de 2004 até 2014 em Botucatu, a partir da análise dos prontuários médicos e genotipagem as espécies clínicas presentes nas amostras desses pacientes que foram aqui diagnosticados. Para a genotipagem, usamos técnicas de extração de DNA, PCR e sequenciamento de três genes alvo (ITS, CHS2 e ARF). Identificamos que em Botucatu a forma clínica prevalente foi a forma crônica (77,4%), mais comumente apresentada por indivíduos do sexo masculino acima dos 40 anos de idade, fumantes e etilistas. Identificamos que a maior parte dos pacientes, tanto da forma aguda, bem como da forma crônica, eram indivíduos auto declarados brancos. Em relação à genotipagem das espécies presentes nessas amostras, conseguimos identificar 44 amostras positivas. As sequências dos genes alvo foram avaliadas no GenBank para certificação da presença de Paracoccidioides. As árvores filogenéticas foram construídas usando as sequências de genes ITS, ARF e CHS2 e mostraram que 100% de nossas amostras positivas pertenciam à espécie críptica S1. Esse é um dado importante, demonstrando a possível ausência de outras espécies em nossa região. Além disso, a observação de que indivíduos brancos são mais acometidos pela doença abre novas perspectivas de estudos sobre uma possível resistência de indivíduos pardos e negros contra o fungo.
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.
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.
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Palavras-chave
Paracoccidioicomicose, Paracoccidioides brasiliensis, Espécies Crípticas, Sequenciamento, Paracoccidioidomycosis, Cryptic species, Sequencing