Caracterização da diversidade genotípica de cepas de Candida parapsilosis isoladas de hemoculturas
Arquivos
Data
2007
Autores
Amorim, Cledja Soares de 
Orientadores
Colombo, Arnaldo Lopes
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A heterogeneidade genética de Candida parapsilosis tem sido reportada com
emprego de distintos métodos moleculares, identificando claramente três grupos (grupos I,
II e III). Recentemente, a análise filogenética baseada no polimorfismo de alguns genes
propôs uma reclassificação de C. parapsilosis grupo II e III em espécies distintas: Candida
orthopsilosis e Candida metapsilosis, respectivamente. Ainda há poucos estudos
epidemiológicos avaliando a prevalência e relevância clínica dessas espécies. Métodos
moleculares têm sido empregados para discriminar esses três grupos e também para
tipagem intra-específica dessas novas espécies. Entretanto, isolados de C. parapsilosis
não têm demonstrado grande diversidade genotípica por estes métodos. É necessário o
desenvolvimento de técnicas moleculares de baixo custo para viabilizar a identificação
dessas espécies em laboratório de rotina. Objetivos: 1) selecionar oligonucleotídeos para
utilização em RAPD que apresentem poder discriminatório para identificação de C.
parapsilosis, C. orthopsilosis e C. metapsilosis. 2) avaliar a prevalência dessas três
espécies em amostras de hemoculturas coletadas em hospitais da América Latina. 3)
analisar a diversidade genotípica entre isolados de C. parapsilosis oriundos de diferentes
áreas geográficas. Material e métodos: Para este estudo, foram selecionadas todas as
amostras de hemoculturas identificadas fenotipicamente como C. parapsilosis,
catalogadas no Banco de Microrganismos do Laboratório Especial de Micologia da
UNIFESP, que foram coletadas durante o período de 2003 a 2005, incluindo as cepas de
referência destas três espécies. Após identificação fenotípica, através do sistema de
ID32C e microcultivo, as colônias sugestivas de C. parapsilosis foram submetidas ao
método RAPD para identificação de espécie. Para interpretação dos resultados, os perfis
genotípicos gerados pelo RAPD foram submetidos à análise por dendrograma utilizando o
programa GEL COMPAR II versão 4.5. Todas as amostras identificadas como C.
orthopsilosis e C. metapsilosis neste estudo foram submetidas ao seqüenciamento da
região ITS para confirmação da identificação de espécie. Resultados: Inicialmente, foram
estudados 7 oligonucleotídeos para serem utilizados em RAPD que tivessem
reprodutibilidade e poder discriminatório em amostras de origem epidemiologicamente
conhecida. Desses, o iniciador 1281 apresentou melhor performance, portanto foi o escolhido para realização neste estudo. Do total das 166 amostras estudadas, 148 (89%)
eram C. parapsilosis, 13 (8%) C. orthopsilosis e 5 (3%) foram identificadas como C.
metapsilosis. A análise por dendrograma revelou que as cepas de C. orthopsilosis
apresentaram maior heterogeneidade genotípica entre si. Cepas de C. parapsilosis foram
geneticamente mais semelhantes entre si (Sab 80%) e formaram 6 subgrupos com uma
similaridade superior a 90%. Estes subgrupos eram constituídos, em sua maioria, por
cepas de mesma localidade geográfica demonstrando serem mais relacionadas entre si.
As análises dos isolados oriundos do estado e da cidade de São Paulo demonstraram
uma maior consistência do método, mostrando maior similaridade genética entre cepas de
cidades vizinhas e de mesmo centro médico, revelando disseminação de cepas de mesma
origem clonal que sofreram microevoluções nestes centros médicos. Conclusões: O
RAPD, com o emprego do iniciador 1281 demonstrou ser uma ferramenta de grande
utilidade na identificação das espécies do complexo C. parapsilosis. C. parapsilosis foi a
espécie de maior prevalência entre as amostras de hemocultura estudadas, seguida de C.
orthopsilosis e C. metapsilosis. Quanto à análise genotípica intra-específica, C.
orthopsilosis foi a espécie mais heterogênea do grupo. C. parapsilosis foi a espécie mais
homogênea geneticamente, provavelmente devido à sua maior adaptação ao homem.
Cepas dessa espécie podem tornar-se persistentes em cada cidade ou por centros
médicos.
Introduction: The genetic heterogeneity of Candida parapsilosis clearly divided into three different groups (I, II and III) has been reported. Recently, phylogenetic analysis based on coding genes polymorphisms suggested the replacement of the old nomenclature C. parapsilosis groups II and III into two new species: Candida orthopsilosis and Candida metapsilosis, respectively. Only few epidemiological studies evaluate the prevalence and clinical relevance of these species. Molecular methods have been used to identify these three groups, as well as, for intra-specific typing of these new species. Nevertheless, C. parapsilosis isolates show low genetic diversity when these methods are used. Therefore, it is necessary to develop cheap molecular methods to make species identification in routine laboratories viable. Objectives: 1) To select primers to be used in RAPD that have discriminatory power to identify C. parapsilosis, C. orthopsilosis and C. metapsilosis. 2) To evaluate the prevalence of these tree species in blood culture samples from Latin America hospitals. 3) To analyze the genotypic diversity among C. parapsilosis isolates from different geographic areas. Materials and Methods: For this study, we selected the blood stream isolates phenotypically identified as C. parapsilosis collected between 2003 and 2005, and maintained at the yeast stock collection of the Special Mycology Laboratory, Universidade Federal de São Paulo, including the reference strains. After the phenotypic identification by the ID 32C system and micromorphology analysis, the colonies identified as C. parapsilosis were submitted to RAPD test. The RAPD genotypic profiles were analyzed by Gel Compar II program to generate dendrogram. All the strains identified as C. orthopsilosis and C. metapsilosis were submitted to ITS region sequencing to confirm species identification. Results: Seven different primers were tested on RAPD and we selected primer 1281 that showed high reproducibility and discriminatory power with strains from known geographical origin, to be used in this study. From the total of 166 strains tested, 148 (89%) were identified as C. parapsilosis, 13 (8%) as C. orthopsilosis and 5 (3%) as C. metapsilosis. The dendrogram analysis reveled that C. orthopsilosis strains had high genotypic heterogeneity among them. C. parapsilosis strains had the lowest genetic divergences between them (Sab 80%) and clustered in 6 subgroups with similarity higher than 90%. These subgroups clustered most strains from the same geographical locale showing to be highly related. The analysis, including isolates from São Paulo city and state, reveled greater genetic similarity among strains from the same medical center or cities geographically close. These data proved higher consistence of RAPD to analyze strains from close geographical locales and, also, showed the dissemination of strains with the same clonal origin that are undergoing microevolution in the sites studied. Conclusions: RAPD method using primer 1281, have been shown a useful tool for species identification of strains belonging to the C. parapsilosis complex. C. parapsilosis had the highest prevalence among all the blood stream samples studied, followed by C. orthopsilosis and C. metapsilosis. Concerning to intra-specific genotypic analysis, C. orthopsilosis was the most heterogeneous species, in contrast to C. parapsilosis that was the most homogeneous, probably due to its adaptation to the human host. Strains belonging to this species can become endemic in some cities or hospitals.
Introduction: The genetic heterogeneity of Candida parapsilosis clearly divided into three different groups (I, II and III) has been reported. Recently, phylogenetic analysis based on coding genes polymorphisms suggested the replacement of the old nomenclature C. parapsilosis groups II and III into two new species: Candida orthopsilosis and Candida metapsilosis, respectively. Only few epidemiological studies evaluate the prevalence and clinical relevance of these species. Molecular methods have been used to identify these three groups, as well as, for intra-specific typing of these new species. Nevertheless, C. parapsilosis isolates show low genetic diversity when these methods are used. Therefore, it is necessary to develop cheap molecular methods to make species identification in routine laboratories viable. Objectives: 1) To select primers to be used in RAPD that have discriminatory power to identify C. parapsilosis, C. orthopsilosis and C. metapsilosis. 2) To evaluate the prevalence of these tree species in blood culture samples from Latin America hospitals. 3) To analyze the genotypic diversity among C. parapsilosis isolates from different geographic areas. Materials and Methods: For this study, we selected the blood stream isolates phenotypically identified as C. parapsilosis collected between 2003 and 2005, and maintained at the yeast stock collection of the Special Mycology Laboratory, Universidade Federal de São Paulo, including the reference strains. After the phenotypic identification by the ID 32C system and micromorphology analysis, the colonies identified as C. parapsilosis were submitted to RAPD test. The RAPD genotypic profiles were analyzed by Gel Compar II program to generate dendrogram. All the strains identified as C. orthopsilosis and C. metapsilosis were submitted to ITS region sequencing to confirm species identification. Results: Seven different primers were tested on RAPD and we selected primer 1281 that showed high reproducibility and discriminatory power with strains from known geographical origin, to be used in this study. From the total of 166 strains tested, 148 (89%) were identified as C. parapsilosis, 13 (8%) as C. orthopsilosis and 5 (3%) as C. metapsilosis. The dendrogram analysis reveled that C. orthopsilosis strains had high genotypic heterogeneity among them. C. parapsilosis strains had the lowest genetic divergences between them (Sab 80%) and clustered in 6 subgroups with similarity higher than 90%. These subgroups clustered most strains from the same geographical locale showing to be highly related. The analysis, including isolates from São Paulo city and state, reveled greater genetic similarity among strains from the same medical center or cities geographically close. These data proved higher consistence of RAPD to analyze strains from close geographical locales and, also, showed the dissemination of strains with the same clonal origin that are undergoing microevolution in the sites studied. Conclusions: RAPD method using primer 1281, have been shown a useful tool for species identification of strains belonging to the C. parapsilosis complex. C. parapsilosis had the highest prevalence among all the blood stream samples studied, followed by C. orthopsilosis and C. metapsilosis. Concerning to intra-specific genotypic analysis, C. orthopsilosis was the most heterogeneous species, in contrast to C. parapsilosis that was the most homogeneous, probably due to its adaptation to the human host. Strains belonging to this species can become endemic in some cities or hospitals.
Descrição
Citação
AMORIM, Clédja Soares de. Caracterização da diversidade genotípica de cepas de Candida parapsilosis isoladas de hemoculturas. 2007. 133 f. Tese (Doutorado em Ciências) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2007.