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

Author:

Dashti Alejandro1ORCID,Alonso Henar2ORCID,Escolar-Miñana Cristina3ORCID,Köster Pamela C.145ORCID,Bailo Begoña1,Carmena David16ORCID,González-Barrio David1ORCID

Affiliation:

1. Parasitology Reference and Research Laboratory, National Centre for Microbiology, Health Institute Carlos III, 28220 Majadahonda, Spain

2. Department of Microbiology, Paediatrics, Radiology, and Public Health, Faculty of Medicine, University of Zaragoza, 50009 Saragossa, Spain

3. Department of Animal Production and Food Science, Faculty of Veterinary, University of Zaragoza, 50013 Saragossa, Spain

4. Faculty of Health Sciences, Alfonso X El Sabio University (UAX), 28691 Villanueva de la Cañada, Spain

5. Faculty of Medicine, Alfonso X El Sabio University (UAX), 28691 Villanueva de la Cañada, Spain

6. CIBER Infectious Diseases (CIBERINFEC), Health Institute Carlos III, 28008 Madrid, Spain

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.

Funder

Health Institute Carlos III

Spanish Ministry of Science, Innovation and Universities

Publisher

MDPI AG

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