Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in Clinical Stool Samples

American Society for Microbiology
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Journal Article
Journal Of Clinical Microbiology, 2011, 49 (1), pp. 257 - 262
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The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites; Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis from human clinical samples. A total of 472 faecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR assays (RT-PCR) and microscopy by a traditional modified iron haematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium spp. isolates. Detection and identification of the faecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results and when compared to RT-PCR the MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed faecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium ssp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica and 50% and 100% for G. intestinalis respectively. No cross reactivity was detected in 100 stool samples containing various other bacterial, viral and protozoan species. The MT-PCR assay was able to provide rapid, sensitive and specific simultaneous detection and identification of the four most important diarrhoea causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy and as such molecular methods such as MT-PCR must be considered the diagnostic method of choice for enteric protozoan parasites.
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