Development, Optimisation and Validation of a Novel Multiplex Real-Time PCR Method for the Simultaneous Detection of Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis

Abstract

The enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis are-to various extents-contributors to the burden of gastrointestinal illness in high-income countries. Detection of these pathogens by microscopy examination is challenging because of the limited sensitivity and need for specific staining procedures. We developed and optimised a new multiplex real-time PCR assay for the simultaneous detection of Cryptosporidium spp., G. duodenalis and D. fragilis in clinical (stool) samples. The diagnostic performance of the assay was evaluated against a large panel of well-characterised DNA samples positive for Cryptosporidium spp. (n = 126), G. duodenalis (n = 132) and D. fragilis (n = 49). The specificity of the test was assessed against a DNA panel from other intestinal or phylogenetically related parasites (n = 105) and faecal DNA from individuals without clinical manifestations (n = 12). The assay exhibited a diagnostic sensitivity of 0.90-0.97 and a diagnostic specificity of 1. The limit of detection was estimated for Cryptosporidium (1 oocyst) and G. duodenalis (5 × 10-4 cysts). The method allowed the detection of four Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. cuniculus) and five G. duodenalis assemblages (A-E) without cross-reacting with other parasites belonging to the phyla Amoebozoa, Apicomplexa, Euglenozoa, Microsporidia, Nematoda and Platyhelminthes. This newly developed multiplex real-time PCR assay represents a novel alternative for the rapid and accurate detection of Cryptosporidium, G. duodenalis and D. fragilis in clinical settings.