The molecular epidemiology of Dientamoeba fragilis isolates in an Australian population
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Dientamoeba fragilis is a trichomonad parasite that causes human gastrointestinal disease. It has been reported from most parts of the world in both rural and cosmopolitan areas and is a ‘neglected cause of diarrhoea’ and dysentry with chronic infections common. Current diagnosis of dientameobiasis is by microscopic identification of the trophozoite in stool. However this method is time-consuming and relatively insensitive while PCR technology offers an attractive alternative to conventional diagnosis. A conventional PCR assay based on the small-subunit ribosomal RNA gene of D. fragilis for the specific detection of D. fragilis DNA in fresh unpreserved stool samples was developed. The D. fragilis PCR was positive in 29/31 samples with positive microscopy and did not cross-react with other protozoan parasites. The PCR protocol showed a specificity of 100% and a sensitivity of 93.5% and the entire procedure can be performed in one day. A prospective study was also conducted over a 30 month period, in which 6,750 faecal samples were submitted to the Department of Microbiology at St. Vincent’s hospital Sydney, Australia. Trophozoites of Dientamoeba fragilis were detected in 60 (0.9%) patients by permanent staining and confirmation was performed by PCR. Gastrointestinal symptoms were present in all patients, with diarrhoea and abdominal pain the most common symptoms. Thirty-two percent of patients presented with chronic symptoms. The average age of infected patients was 39.8 years. No correlation was found between D. fragilis and Enterobius vermicularis, a proposed vector of transmission for D. fragilis. The genetic diversity of 50 D. fragilis isolates was examined by PCR and the PCR products were analysed for the presence of a restriction fragment length polymorphism. These results showed no variation in the small subunit rRNA gene and demonstrated a single genotype for all Australian isolates. This study indicates the potential pathogenic properties of D. fragilis, and the need for all laboratories to routinely test for this organism. I also developed a 5’ nuclease (TaqMan) based real-time PCR assay, targeting the small- subunit ribosomal RNA gene, for the detection of D. fragilis in human stool specimens and compared its sensitivity and specificity to the conventional PCR and microscopic examination by a traditional modified iron-haematoxylin staining procedure. Tests were performed using all three techniques on 200 stool specimens referred for screening on the basis of diarrhea. The real-time PCR assay exhibited 100% sensitivity and specificity compared with microscopy. The detection limit of both PCR tests was compared; real-time PCR was 100 times more sensitive than conventional PCR, with a detection limit of 0.01 trophozoites. In conclusion, all three methods for the detection of D. fragilis were highly specific, with real-time PCR being the most sensitive. The use of the real-time assay in a diagnostic laboratory provides a superior sensitive and specific method for the diagnosis of D. fragilis.
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