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

## TL;DR

This study evaluates PCR tests for detecting intestinal parasites that cause diarrhea, finding them highly accurate and useful for clinical labs.

## Contribution

The study provides a clinical evaluation of singleplex and duplex PCR assays for detecting multiple intestinal protist parasites with high sensitivity and specificity.

## Key findings

- Singleplex assays showed high sensitivity (0.94–1) and specificity (0.96–1) for detecting Cryptosporidium, Giardia, and Entamoeba species.
- The duplex assay for Blastocystis sp. and Dientamoeba fragilis achieved 100% sensitivity and high specificity.
- The assays identified multiple species and subtypes, including six Cryptosporidium species and four Giardia assemblages.

## Abstract

Cryptosporidium spp., Giardia duodenalis and Entamoeba histolytica are species of protozoa- causing diarrhoea that are common worldwide, while Entamoeba dispar, Dientamoeba fragilis and Blastocystis sp. appear to be commensal parasites whose role in pathogenicity remains controversial. We conducted the clinical evaluation of five singleplex and one duplex CerTest VIASURE Real-Time PCR Assays against a large panel of positive DNA samples (n = 358), and specifically to Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25) E. dispar (n = 11), Blastocystis sp. (n = 42), D. fragilis (n = 37), and related parasitic phylum species such as Apicomplexa, Euglenozoa, Microsporidia and Nematoda. DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference centre. Estimated diagnostic sensitivity and specificity values were 0.94–1 for Cryptosporidium spp., 0.96–0.99 for G. duodenalis, 0.96–1 for E. histolytica, 1–1 for E. dispar, and 1–0.99 for D. fragilis in the evaluated singleplex assays. In the duplex assay for the simultaneous detection of Blastocystis sp. and D. fragilis these values were 1–0.98 and 1–0.99, respectively. Measures of diagnostic precision for repeatability and reproducibility were found to be under acceptable ranges. The assays identified six Cryptosporidium species (C. hominis, C. parvum, C. canis, C. felis, C. scrofarum, and C. ryanae), four G. duodenalis assemblages (A, B, C, and F), and six Blastocystis subtypes (ST1-ST5, and ST8). The evaluated singleplex and duplex VIASURE Real-Time PCR assays provide sensitive, practical, and cost-effective choices to the molecular diagnosis of the main diarrhoea-causing intestinal protists in clinical microbiology and research laboratories.