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.

Evaluation of the Use of Singleplex and Duplex CerTest VIASURE Real-Time PCR Assays to Detect Common Intestinal Protist Parasites

“…The VIASURE D. fragilis assay achieved sensitivity and specificity values of 1 and 0.99 (respectively) in both singleplex and duplex versions. This diagnostic performance was in the higher range of what was previously documented in the use of other commercially available qPCR assays (sensitivity: 0.90-0.96; specificity: 1) [32,33].…”

“…Most previous similar studies that evaluated commercial qPCR assays used prospectively collected stool samples that were submitted for routine investigation in clinical settings [21,23,[25][26][27][28][29][30][31][32][33]. In contrast, this survey purposely used a DNA panel with a wide diversity of parasite species and genotypes, with the aim of providing an evidence-based answer to the question of if the evaluated qPCR assays were suitable for the detection of less common or rare species/genotypes and animal-adapted genetic variants with known zoonotic potential and diverse geographical origins.…”

A multiplex qPCR assay for transgenes detection: A novel approach for gene doping control in horseracing using conventional laboratory setup

“…Parker et al observed ~2‐fold to ~240‐fold drop in sensitivity when changing from multiple duplex assays in detecting respiratory viral pathogens to a multiplex platform 30 . Many publications are available to guide the optimisation of multiplex PCR or qPCR 35–37 . However, their primary focus on microbial pathogen detection represents a different context (e.g., microbial pathogen can be cultured to higher titre before extraction), and hence, not all of their considerations are specific to the context of doping control.…”

“…Parker et al observed $2-fold to $240-fold drop in sensitivity when changing from multiple duplex assays in detecting respiratory viral pathogens to a multiplex platform 30. Many publications are available to guide the optimisation of multiplex PCR or qPCR [35][36][37]. F I G U R E 1 The qPCR efficiency of each transgene in the multiplex qPCR assay.…”