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

• Published in: Journal of Clinical Microbiology 2011-01, Vol.49 (1), p.257-262
• Main Authors: Stark, D, Al-Qassab, S.E, Barratt, J.L.N, Stanley, K, Roberts, T, Marriott, D, Harkness, J, Ellis, J.T
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cryptosporidium; Cryptosporidium - genetics; Cryptosporidium - isolation & purification; Dientamoeba - genetics; Dientamoeba - isolation & purification; Dientamoeba fragilis; Entamoeba histolytica; Entamoeba histolytica - genetics; Entamoeba histolytica - isolation & purification; Feces - parasitology; Fundamental and applied biological sciences. Psychology; Giardia intestinalis; Giardia lamblia - genetics; Giardia lamblia - isolation & purification; Humans; Microbiology; Microscopy; Parasitology; Parasitology - methods; Polymerase Chain Reaction - methods; Protozoan Infections - diagnosis; Protozoan Infections - parasitology; Sensitivity and Specificity
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.01796-10

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 fecal 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 (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. 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 diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.