RESUMO
Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes.
Assuntos
Pareamento de Bases/genética , Criptosporidiose/parasitologia , Cryptosporidium parvum/classificação , Cryptosporidium/classificação , DNA de Protozoário , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/parasitologia , Criptosporidiose/veterinária , Cryptosporidium/genética , Cryptosporidium parvum/genética , DNA de Protozoário/genética , DNA Ribossômico/análise , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 18S/genéticaRESUMO
Previous studies have provided histological evidence of an association between primary Pneumocystis infection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clustered Pneumocystis organisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derived PNEUMOCYSTIS: Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of the Pneumocystis species using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding to Pneumocystis carinii were found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims. Pneumocystis jirovecii was successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that the Pneumocystis species found in the lungs of Chilean SID victims was P. jirovecii.