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 ...

Detection of Pyrazinamide Resisitance in Mycobacterium Tuberculosis by Sequencing of pncA Gene / 결핵

BACKGROUND: Examining the biological susceptibility of Mycobacterium tuberculosis to pyrazinamide (PZA) in vitro is very difficult as PZA is inactive under normal culture conditions. The susceptibility test, an enzyme assay for Pzase activity, and a genetic test for pncA gene mutations, were performed in order to predict PZA resistance. METHODS: 28 cultured clinical isolates of Mycobacterium tuberculosis were tested. The biological susceptibility was performed by the absolute concentration method using Lowenstein-Jensen media. The PZase activity was tested by means of Wayne's method. A 710-bp region includes the entire open reading frame of pncA was amplified and sequenced. RESULTS: All six strains with positive PZase activity exhibited no pncA mutations with one strain showing a false resistance in the biological susceptibility test. Among the 22 strains with no PZase activity, 21 exhibited showed pncA mutations. In the biological suscaptibility test, 20 strains were resistant, and one was susceptible, and the other failed to test. The mutation types varied with ten missense, one silent and one nonsense mutation 1 slipped-strand mispairing, and 6 frameshift mutations. Three strains had an adenine to guanine mutation at position - 11 upstream of the start codon. CONCLUSION: The mutation at the pncA promotor region is frequent at -11 upstream position. Automatic sequencing of pncA is a useful tool for rapid and accurate detection of PZA resistant M.tuberculosis, and for demonstrating the epidemiological relatedness of the PZA-resistant M.tubersulosis strains.