Development of a PCR Assay for Diagnosing Trematode (Opisthorchis and Haplorchis) Infections in Human Stools

Abstract

Abstract. We developed a combined conventional polymerase chain reaction (PCR) and real-time PCR (qPCR)-based assay for detecting and discriminating between Opisthorchis viverrini and Haplorchis taichui parasite infections. The first PCR amplifies the mitochondrial cytochrome c oxidase subunit I (COI) genes of parasites, and differential diagnosis is achieved by performing qPCR with specific primers and SYBR Green I. The detection limit of the assay was found to be 2.0 × 10 2 plasmid copies in a test in which a stool sample was spiked with a single egg, which is equivalent to 5 eggs per gram (EPG). The testing of 34 clinical stool samples that had been demonstrated to contain "Opisthorchis-like" eggs by microscopy showed that the novel assay exhibited a sensitivity of 100% for "Opisthorchis-like" parasitic infections, and 71% and 91% of these samples were found to be infected with O. viverrini and H. taichui, respectively. A further four parasitic infections were diagnosed in the 16 negative samples, and the microscopic findings of these samples were confirmed to be false negatives by sequencing analysis. The assay also displayed high specificity during the testing of 10 samples containing other common parasites. The fact that our qPCR SYBR Green I-based assay detected submicroscopic traces of parasitic DNA and was able to differentiate between parasites that produce eggs with similar morphologies indicates that it has a good potential for development of diagnostic application to use in areas where multiple parasites coexist.

Monsters in our food: Foodborne trematodiasis in the Philippines and beyond

“…A PCR assay that targeted the Internal Transcribed Spacer (ITS) regions in the ribosomal DNA of O. viverrini, C. sinensis, H. pumilio, and H. taichui was described by Sato et al (2009), and used by Li et al (2019) to diagnose a case of multiple foodborne trematode infections with systemic involvement in Shandong Province, China. To diagnose opisthorchiasis and haplorchiasis, a combined conventional PCR and real-time PCR assay that sequenced the cytochrome c oxidase 1 (cox-1) gene was developed by Lamaningao et al (2017). A simplex and a duplex PCR assays that targeted the second ITS (ITS-2) gene of F. hepatica and the NADH dehydrogenase 2 (nad2) gene of C. sinensis were found to have good diagnostic performance in detecting infections in sheep (Yang et al, 2015).…”

Newly developed SYBR Green-based quantitative real-time PCRs revealed coinfection evidence of Angiostrongylus cantonensis and A. malaysiensis in Achatina fulica existing in Bangkok Metropolitan, Thailand

“…The SYBR Green quantitative real-time PCR (qPCR) method was considered suitable for quantitative detection and species discrimination and has been used successfully in many studies to detect, quantify, and discriminate species. For example, Opisthorchis viverrini and Haplorchis taichui were detected from human stool samples, Leishmania was quantified in human samples, and Salmonella subspecies were all successfully discriminated ( Weirather et al, 2011 ; Barbau-Piednoir et al, 2013 ; Lamaningao et al, 2017 ).…”

SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

“…SYBR green qPCR has recently been used in many studies to detect, discriminate, and quantify species as, for example, in the discrimination and quanti cation of Leishmania in human samples and the discrimination of species and subspecies of Salmonella. The technique was also employed for the detection of Opisthorchis viverrini and Haplorchis taichui from human stool samples [28][29][30].…”