Genotipagem de multilocus de Cryptosporidium hominis associado a surto diarréico em creche de São Paulo

A number of species of Cryptosporidium are associated with diarrhea worldwide. Little data exists regarding the genotypes and species of Cryptosporidium associated with cases of infections in Brazil. PURPOSE: In the present study, we ascertained by molecular methods the species and the genotype of Cryptosporidium sp from a diarrhea outbreak diagnosed in a day care at the Hospital Clínicas, São Paulo University Medical School. MATERIALS AND METHODS: Specific identification and typing of the isolates associated with the outbreak was done by DNA sequencing analysis of fragments amplified by polymerase chain reaction (PCR) from 3 different Cryptosporidium loci: the SSUrRNA coding region, the Cryptosporidium oocyst wall protein (COWP) gene, and the microsatellite locus 1 (ML1), a tandem GAG-trinucleotide repeat containing substitutions that differentiate the genotypes of Cryptosporidium parvum and Cryptosporidium hominis. RESULTS: A total of 29 positive samples from the outbreak were studied by the molecular methods described. Our study revealed the presence of a single genotype of Cryptosporidium hominis in all samples. CONCLUSION: The molecular analysis reinforced the hypothesis that the transmission of Cryptosporidium hominis during the period the samples were collected occurred in an outbreak pattern, possibly by person-to-person contact through the fecal-oral route. As far as we know, this is the first time that molecular tools have been used to identify the species and the genotype of isolates showing the presence of the ML1 genotype in samples from Brazilian patients.Mundialmente, diferentes espécies de Cryptosporidium estão relacionadas com doenças diarréicas. No Brasil há poucos dados sobre os genótipos das espécies de Cryptosporidium associadas a infecções. OBJETIVO: No presente estudo, caracterizamos, por métodos moleculares, a espécie e o genótipo de Cryptosporidium sp diagnosticado em surto diarréico ocorrido na creche do Hospital das Clínicas, São Paulo, Brasil. MATERIAL E MÉTODOS: Identificação específica e tipagem dos isolados associados ao surto foram feitos a partir do seqüenciamento de fragmentos de DNA amplificados por PCR dos seguintes loci: a região que codifica o SSUrRNA, o gene que codifica uma proteína do envoltório dos oocistos de Cryptosporidium (COWP), e o locus de microsatélite ML1, representado por seqüências repetitiva de três nucleotídeos GAG contendo substituições que diferem entre os genótipos de Cryptosporidium parvum e Cryptosporidium hominis. RESULTADOS: Um total de 29 amostras positivas para Cryptosporidium associadas ao surto diarréico foi analisado com base nos métodos moleculares acima descritos. O estudo revelou a presença do genótipo ML1 de Cryptosporidium hominis. DISCUSSÃO: A análise molecular reforçou a hipótese de que a transmissão de Cryptosporidium hominis durante o surto diarréico ocorreu de pessoa a pessoa através da rota fecal oral. Esta é a primeira vez que ferramentas moleculares são utilizadas para identificação de espécies e genótipos de isolados acusando a presença do genótipo ML1 em pacientes brasileiros

Multilocus genotyping of Cryptosporidium hominis associated with diarrhea outbreak in a day care unit in São Paulo

A number of species of Cryptosporidium are associated with diarrhea worldwide. Little data exists regarding the genotypes and species of Cryptosporidium associated with cases of infections in Brazil. PURPOSE: In the present study, we ascertained by molecular methods the species and the genotype of Cryptosporidium sp from a diarrhea outbreak diagnosed in a day care at the Hospital Clínicas, São Paulo University Medical School. MATERIALS AND METHODS: Specific identification and typing of the isolates associated with the outbreak was done by DNA sequencing analysis of fragments amplified by polymerase chain reaction (PCR) from 3 different Cryptosporidium loci: the SSUrRNA coding region, the Cryptosporidium oocyst wall protein (COWP) gene, and the microsatellite locus 1 (ML1), a tandem GAG-trinucleotide repeat containing substitutions that differentiate the genotypes of Cryptosporidium parvum and Cryptosporidium hominis. RESULTS: A total of 29 positive samples from the outbreak were studied by the molecular methods described. Our study revealed the presence of a single genotype of Cryptosporidium hominis in all samples. CONCLUSION: The molecular analysis reinforced the hypothesis that the transmission of Cryptosporidium hominis during the period the samples were collected occurred in an outbreak pattern, possibly by person-to-person contact through the fecal-oral route. As far as we know, this is the first time that molecular tools have been used to identify the species and the genotype of isolates showing the presence of the ML1 genotype in samples from Brazilian patients