Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil.

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Avaliação da heterogeneidade molecular de Cryptosporidium sp. em amostras clínicas / Evaluation of the molecular heterogeneity of Cryptosporidium sp. in clinical specimens

O Cryptosporidium é um parasito coccídeo reconhecido por causar diarréia em humanos e animais em todo o mundo. O gênero compreende pelo menos 20 espécies confirmadas, sendo o C. hominis e C. parvum as principais espécies causadoras de criptosporidiose em humanos. Ferramentas moleculares têm sido desenvolvidas para detectar e diferenciar espécies/genótipos e subgenótipos de Cryptosporidium. O objetivo do trabalho foi avaliar a heterogeneidade molecular de Cryptosporidium sp. obtidos de amostras clínicas provenientes dos municípios do Rio de Janeiro e de Salvador, através da PCR em tempo real e seqüenciamento automático. Foram analisadas 85 amostras, distribuídas em 3 grupos distitntos, sendo 45 delas do município do Rio de Janeiro e 40 amostras provenientes de Salvador, Bahia. Todas as amostras foram positivas para Cryptosporidium sp. pelo método de coloração de Kinyoun a frio. O ensaio da PCR em tempo real combinou uma reação multiplex para a detecção do gênero Cryptosporidium e da espécie C. parvum e uma reação simples para a detecção de C. hominis. Na detecção do gênero Cryptosporidium foram utilizados par de primers e uma sonda TaqMan desenhados a partir do alinhamento de seqüências conservadas do gene 18S rRNA de várias espécies de Cryptosporidium disponíveis no GenBank. Para a detecção das espécies C. parvum e C. hominis foram utilizados primers e sondas específicos obtidos a partir de seqüências de cada espécie disponíveis no GenBank. A detecção do gênero Cryptosporidium através da sonda 18S rRNA, na reação duplex, foi visualizada em 63 de 85 amostras totais. Destas, a sonda TaqMan específica para C. parvum detectou 6 amostras e a sonda Taqman específica para C. hominis detectou 42 amostras. Quinze amostras não puderam ser detectadas pelas sondas C. hominis ou C. parvum. Nos ensaios da PCR para o gene 18S, 31 amostras foram positivas e 27 delas sequenciadas. As análises filogenéticas confirmaram a presença de C. hominis, C. parvum e C. felis ...

Cryptosporidium is a coccidia parasite known to cause diarrhea in humans and animals worldwide. The genus comprises at least 20 confirmed species, with C. hominis and C. parvum major species that cause cryptosporidiosis in humans. Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotypes and subtype levels. The objective of this study was to evaluate the molecular heterogeneity of Cryptosporidium sp. clinical samples obtained from Rio de Janeiro and Salvador (Bahia), through real-time PCR and automatic sequencing. We analyzed 85 samples, distributed in three distinct groups, 45 of them in the city of Rio de Janeiro and 40 samples from Salvador. All samples were positive for Cryptosporidium sp. by modified Kinyoun acid-fast staining technique. The real-time PCR procedure combined a duplex reaction for the detection of Cryptosporidium species and C. parvum and a simple reaction for the detection of C. hominis. The detection of the genus Cryptosporidium have been used a pair of primers and TaqMan probe designed from the alignment of conserved sequences of the 18S rRNA gene of several species of Cryptosporidium available in GenBank. For the detection of species C. parvum and C. hominis were used specific primers and probes derived from sequences of each species available in the GenBank. Detection of Cryptosporidium sp. by 18S rRNA probe, in the duplex reaction, was visualized in 63 of 85 total samples. Of these, the C. parvum TaqMan probe detected 6 samples and the C. hominis TaqMan probe detected 42 samples. Fifteen samples could not be detected by C. hominis or C. parvum probes. In the 18S PCR assays, 31 samples were positive and 27 of them sequenced. Phylogenetic analysis confirmed the presence of C. hominis, C. parvum and C. felis. The analysis of the phylogenetic tree obtained by Neighbor Joining showed that the sequences obtained in this study were grouped with Cryptosporidium species described ...