Elsevier

Experimental Parasitology

Volume 112, Issue 2, February 2006, Pages 109-114
Experimental Parasitology

Clonorchis sinensis and Opisthorchis viverrini: Development of a mitochondrial-based multiplex PCR for their identification and discrimination

https://doi.org/10.1016/j.exppara.2005.09.012Get rights and content

Abstract

We report a single, one-step PCR approach for detection and discrimination of Clonorchis sinensis and Opisthorchis viverrini in different life-stage forms (adults, metacercariae, and eggs) from fish intermediate hosts and from infected patients. Primers designed for species-specific PCR, amplifying portions of the mitochondrial (mt) genome, were also suitable for a multiplex PCR. The latter was a single, one-step reaction under high stringency conditions, using simultaneously 2 pairs of primers (1 pair for C. sinensis—product size 612 bp, and 1 pair for O. viverrini—product size 1357 bp). Assays using serially diluted templates demonstrated that as little as 0.78 ng of genomic DNA of either species could yield amplicons. Genomic DNA extracted from different life-stage forms including adult worms (of both species), eggs (of O. viverrini), eggs possibly of several trematode species (collected from patients infected with C. sinensis in Vietnam) and mixed metacercariae of common trematodes (collected from fishes in the C. sinensis endemic areas), yielded specific bands of the correct size and their identity was confirmed by sequence analysis. The multiplex PCR approach described here proved to be a species-specific, sensitive and fast tool for accurate diagnosis of clonorchiasis and/or opisthorchiasis, permitting the detection of their metacercariae in infected fishes or adult/eggs from patients in endemic areas.

Introduction

The two small liver flukes, Clonorchis sinensis and Opisthorchis viverrini, infect >30 million people in Asia including Korea, mainland China, Taiwan island, Thailand, and Indochina (Bruckner, 1999, Chai and Lee, 2002, King and Scholz, 2001, Sithithaworn and Haswell-Elkins, 2003). These parasites also infect a number of other mammals, including dogs, cats, pigs and rodents, that can serve as reservoirs of infection. In non-endemic areas (including the United States), the infection is sometimes found in Asian immigrants, or following ingestion of imported, undercooked or pickled freshwater fish containing metacercariae (Stauffer et al., 2004). The diseases caused by these zoonotic parasites can develop into a form of cholangiocarcinoma which is frequently fatal to humans (Choi et al., 2004, Mas-Coma and Bargues, 1997). Clonorchiasis and opisthorchiasis are thus important foodborne zoonotic parasitic diseases of much public concern (Fried et al., 2004, Lun et al., 2005).

Clonorchis sinensis is endemic in Vietnam, mainly in the Red River Delta provinces in the North. (De et al., 2003, Kino et al., 1998). Recently, we have used molecular techniques to identify O. viverrini from a range of provinces in the south of Vietnam (unpublished). Both species may overlap in Central Vietnam. Consequently, there is a need for accurate diagnosis/discrimination using eggs from patients or metacercariae from infected fishes.

Clonorchis sinensis and O. viverrini have similar life-cycles, location in the liver and pathogenicity and their morphologies, particularly in the egg and metacercariae forms, differ only slightly. These species have usually been diagnosed and distinguished from one another by detection of the eggs in feces or on examination of the reproductive organs of the adults if available (Bruckner, 1999, Fried et al., 2004). Identifying metacercariae from infected freshwater fish, or eggs from patients with mixed infections, is difficult. A single fish may host metacercariae of these liver flukes, and also of heterophyid trematodes such as Haplorchis spp. and Centrocestus spp. Consequently, humans eating such fish undercooked often host a number of trematode species producing similar eggs. Serological methods have been developed for diagnosis of clonorchiasis (Nagano et al., 2004) and common and specific antigens of Clonorchis and Opisthorchis have been used for immunoblot methods (Choi et al., 2003). In recent years, multiplex PCR approaches have been developed for rapid and accurate detection of organisms of particular interest, i.e., viruses (Mosquera Mdel et al., 2002), bacteria (Rivera et al., 2003), Plasmodium spp. (Patsoula et al., 2003), mosquitoes (Fettene and Temu, 2003), and free-living amoebae (Pelandakis and Pernin, 2002). A mitochondrial-based multiplex PCR probe has been designed for the large liver fluke, Fasciola hepatica in Brazil (Magalhães et al., 2004) and for the tapeworms, Taenia solium, T. asiatica, and T. saginata (Yamasaki et al., 2004).

Molecular diagnostic methods using PCR have also been introduced to detect O. viverrini in hamsters, human stool specimens, infected bithynid snails and cyprinoid fishes (Maleewong et al., 2003, Wongratanacheewin et al., 2001, Wongratanacheewin et al., 2002) and to distinguish O. felineus from other trematodes (Pauly et al., 2003). However, a multiplex PCR technique using a mix of specific primers, simultaneously in a single reaction under high stringency conditions, has not been developed for the discrimination of these two important species in every life-stage form.

In this paper, we present pairs of species-specific primers based on mitochondrial sequences for C. sinensis and O. viverrini and amplifying fragments of different lengths from the different species. The method is accurate and sensitive and could be used to detect and identify the particular parasite(s) when present in single or mixed infections.

Section snippets

Adults, metacercariae, and eggs

Adult worms of C. sinensis were collected from patients treated with praziquantel. Metacercariae of this species were digested from tissues of infected fishes. Eggs mixed with those of other trematodes (identified as Haplorchis spp., Centrocestus spp., Echinostoma spp., etc. by microscopy) were collected from patients in Nam Dinh province (Vietnam). Adult worms and eggs of O. viverrini were from Khon Kaen, Thailand. These worms were collected from golden hamsters 8 weeks after oral infection

Specificity of multiplex PCR

The species-specific primer pairs, wherever used together, always generated a clear band of the expected size for C. sinensis (612 bp) or O. viverrini (1357 bp), depending on the template present (Fig. 2). Specificity of amplification was confirmed by cloning and sequencing (data not shown).

Reaction sensitivity by diluted template

The reaction assays showed high and specific sensitivity (Figs. 3A for O. viverrini and B for C. sinensis, and C for the multiplex PCR of both species). Amplicons could be obtained in reactions contaning only

Discussion

Until now, microscopic examination has been the most appropriate method for detecting eggs in patients of late-stage clonorchiasis or opisthorchiasis, and for demonstrating metacercariae in fishes. Immunodiagnostic methods have also been proved to be sensitive and early techniques for early diagnosis using parasite egg antigen (Wongsaroj et al., 2001), crude specific antigen (common for both C. sinensis and O. viverrini—Choi et al., 2003); and recombinant antigens (Nagano et al., 2004). These

Acknowledgments

We thank our collaborators for their kind provision of materials used in this study. This investigation received financial support from Wellcome Trust, UK (Project No: 068762) to Thanh Hoa Le, Don McManus, and David Blair. The authors also thank Ms. B.N. Nguyen, Ms. T.T. Vu (Institute of Biotechnology, Hanoi, Vietnam) for their collaboration in the laboratory work.

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