Clonorchis sinensis and Opisthorchis viverrini: Development of a mitochondrial-based multiplex PCR for their identification and discrimination
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Recently, we have used molecular techniques to identify O. viverrini from a range of provinces in the south of Vietnam (unpublished).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.
References (28)
Helminthic food-borne infections
Clinical Laboratory Medicine
(1999)- et al.
Food-borne intestinal trematode infections in the Republic of Korea
Parasitology International
(2002) - et al.
Mitochondrial genomes of parasitic flatworms
Trends in Parasitology
(2002) - et al.
Clonorchiasis: a key foodborne zoonosis in China
Lancet Infectious Disease
(2005) - et al.
Genomics of parasitic flatworms
International Journal for Parasitology
(2004) - et al.
Epidemiology of Opisthorchis viverrini
Acta Tropica
(2003) - et al.
Isolation and characterization of species-specific DNA probes from Taenia solium and Taenia saginata and their use in an egg detection assay
Journal of Clinical Microbiology
(1995) - et al.
Clonorchiasis and cholangiocarcinoma: etiologic relationship and imaging diagnosis
Clinical Microbiology Review
(2004) - et al.
Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda)
Korean Journal of Parasitology
(2003) - et al.
The food-borne trematode zoonoses of Vietnam
Southeast Asian Journal of Tropical Medicine and Public Health
(2003)
Species-specific primer for identification of Anopheles quadriannulatus sp. B (Diptera: Culicidae) from Ethiopia using a multiplex polymerase chain reaction assay
Journal of Medical Entomology
Food-borne intestinal trematodiases in humans
Parasitology Research
Trematodes of the family Opisthorchiidae: a minireview
Korean Journal of Parasitology
Epidemiology of clonorchiasis in Ninh Binh Province, Vietnam
Southeast Asian Journal of Tropical Medicine and Public Health
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