Article ; Online: Development, Optimisation and Validation of a Novel Multiplex Real-Time PCR Method for the Simultaneous Detection of Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis
Pathogens, Vol 11, Iss 1277, p
2022 Volume 1277
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 ... ...
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. |
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Keywords | diagnosis ; enteric parasites ; diarrhoea ; clinical setting ; Medicine ; R |
Subject code | 610 |
Language | English |
Publishing date | 2022-10-01T00:00:00Z |
Publisher | MDPI AG |
Document type | Article ; Online |
Database | BASE - Bielefeld Academic Search Engine (life sciences selection) |
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