Conventional PCR for molecular diagnosis of human strongyloidiasis

Abstract

Strongyloidiasis is frequently asymptomatic and diagnosis of latent infection is difficult due to limitations of current parasitological and serological methods. This study aimed to verify the use of conventional polymerase chain reaction (PCR) assay for molecular diagnosis of Strongyloides stercoralis infection. Fresh stool samples were obtained from 103 individuals: 33 S. stercoralis positive, 30 positive for other parasites and 40 negative for parasitological methods. These samples were examined by the Lutz, Rugai and agar plate culture methods and conventional PCR assay. Two sets of primers (S. stercoralis species-specific and genus-specific sets), located in the 18S ribosomal RNA gene, were used for PCR. Of the 33 samples positive for S. stercoralis by parasitological methods, 28 (84.8%) were also detected by PCR assay using species-specific primers and 26 (78.8%) using genus-specific primers. Among the stool samples negative by parasitological methods, seven (17.5%) were positive by PCR using species-specific primers and two (5.0%) using genus-specific primers. In conclusion, the conventional PCR assay described in this study using a species-specific primer pair provided a molecular method for S. stercoralis diagnosis in human stool samples.

The laboratory diagnosis of Strongyloides stercoralis

“…the 18S rRNA small subunit (SSU); the internal transcribed spacer region 1 (ITS-1); the 28S rRNA gene; or the cyclooxygenase gene (cox1) 19,[23][24][25][26][27][28][29][30][32][33][34][35][36][37] . Published sensitivities and specificities for…”

“…Studies have also assessed the specificity of the PCR products by sequence analysis, with all finding 100% sequence homology with the target sequence of S. stercoralis. 24,25,27,29 . Sitta et al found a number of false positives, using published genus and species-specific primers, based on non-target sized bands on gel electrophoresis 19,25 .…”

“…24,25,27,29 . Sitta et al found a number of false positives, using published genus and species-specific primers, based on non-target sized bands on gel electrophoresis 19,25 . The genus-specific primers amplified sequences that generated nontarget bands on electrophoresis in specimens that contained…”

“…Blastocytis and other helminths on microscopy, and the speciesspecific primers amplified sequences that generated non-target bands on electrophoresis in specimens positive for hookworm on microscopy 25 . Similar accounts of cross-reactivity have not yet been reported, so further data will be useful to monitor the specificity of PCR in different populations.…”

Diagnóstico molecular da infecção por Strongyloides venezuelensis pela detecção específica de DNA em amostras de ratos experimentalmente infectados

“…A PCR tem sido mencionada como uma metodologia auxiliar, com alta sensibilidade e especificidade para o diagnóstico molecular de S. stercoralis (Repetto et al, 2013;Sitta et al, 2014). A detecção de DNA do parasito em amostras fecais através da amplificação por PCR em tempo real (qPCR) utilizando iniciadores referentes ao gene ribossomal, mostrou-se sensível e específico Kramme et al, 2011).…”