Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy

Abstract

Molecular detection of gastrointestinal protozoa is more sensitive and more specific than microscopy but, to date, has not routinely replaced time-consuming microscopic analysis. Two internally controlled real-time PCR assays for the combined detection of Entamoeba histolytica, Giardia lamblia, Cryptosporidium spp. and Dientamoeba fragilis in single faecal samples were compared with Triple Faeces Test (TFT) microscopy results from 397 patient samples. Additionally, an algorithm for complete parasitological diagnosis was created. Real-time PCR revealed 152 (38.3%) positive cases, 18 of which were double infections: one (0.3%) sample was positive for E. histolytica, 44 (11.1%) samples were positive for G. lamblia, 122 (30.7%) samples were positive for D. fragilis, and three (0.8%) samples were positive for Cryptosporidium. TFT microscopy yielded 96 (24.2%) positive cases, including five double infections: one sample was positive for E. histolytica/Entamoeba dispar, 29 (7.3%) samples were positive for G. lamblia, 69 (17.4%) samples were positive for D. fragilis, and two (0.5%) samples were positive for Cryptosporidium hominis/Cryptosporidium parvum. Retrospective analysis of the clinical patient information of 2887 TFT sets showed that eosinophilia, elevated IgE levels, adoption and travelling to (sub)tropical areas are predisposing factors for infection with non-protozoal gastrointestinal parasites. The proposed diagnostic algorithm includes application of real-time PCR to all samples, with the addition of microscopy on an unpreserved faecal sample in cases of a predisposing factor, or a repeat request for parasitological examination. Application of real-time PCR improved the diagnostic yield by 18%. A single stool sample is sufficient for complete parasitological diagnosis when an algorithm based on clinical information is applied.

Evaluation of a Rapid Enzyme-Linked Immunosorbent Assay for Diagnosis of Strongyloidiasis

“…The eventuality of undiagnosed strongyloidiasis, alone or as part of a polyparasitism, cannot be ruled out. Similarly, a molecular approach testing PCR amplification from stools underlined the lack of sensitivity of stool examination for strongyloidiasis diagnosis (2). In our experience, very few patients, including those with eosinophilia, are in fact investigated with 3 Baermann tests (personal data).…”

High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

“…These parent multiplex real-time PCR assays have been extensively validated against conventional techniques including enzyme-linked immunosorbent assay (ELISA) and microscopy and are being used in routine diagnostic settings and in epidemiological surveys. [6][7][8][9][10][11] In this study, these methods were adapted into a combined bead-based Luminex assay as an alternative molecular diagnostic platform to capture each of these parasites in a single protocol.…”

A Pentaplex Real-Time Polymerase Chain Reaction Assay for Detection of Four Species of Soil-Transmitted Helminths

“…26,[29][30][31] These diagnostic limitations can be avoided with real-time PCR because it allows simultaneous detection and quantification of different targets and has been shown to be highly sensitive and specific for detection of many intestinal parasites. 9,[12][13][14][15][32][33][34][35][36][37] In this study, a pentaplex real-time PCR was evaluated to detect infections with the human roundworm, two species of hookworm and threadworm. An additional fifth primer and probe set was used for the amplification and detection of an internal control for the detection of possible inhibition of the amplification reaction by fecal contaminants.…”