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

The enteric protozoan parasites spp., and 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 spp., and in clinical (stool) samples. The diagnostic performance of the assay was evaluated against a large panel of well-characterised DNA samples positive for spp. ( = 126), ( = 132) and ( = 49). The specificity of the test was assessed against a DNA panel from other intestinal or phylogenetically related parasites ( = 105) and faecal DNA from individuals without clinical manifestations ( = 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 (1 oocyst) and (5 × 10 cysts). The method allowed the detection of four species (, , and ) and five 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 , and in clinical settings.