title: Rapid and simple identification of human pathogenic heterophyid intestinal fluke metacercariae by PCR-RFLP.
abstract: Six species of heterophyid intestinal flukes (HIFs) constitute the major endemic zoonotic fish-borne pathogens in Asia: Haplorchis taichui, H. pumilio, H. yokogawai, Procerovum varium, Stellantchasmus falcatus, and Centrocestus formosanus. Several different species of these parasites are often found co-infecting the same second intermediate fish host. Because of their morphological similarities, differentiating between species of HIF metacercariae is difficult, time-consuming, and frequently results in misidentification. In this study, we aimed to develop an efficient and accurate method of identifying metacercariae of these 6 HIFs. Metacercariae were roughly grouped together, based on morphological characteristics seen under a stereomicroscope. Then, PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the exact species of each metacercaria, using the 28S ribosomal RNA gene as the genetic marker and MboII as the restriction enzyme. Using a combination of morphological and molecular methods eliminates the need for DNA sequencing and infecting animal subjects to obtain adult worms, increases accuracy, and decreases the need for laborious morphological identification. Because the method is simple, rapid, and relatively cheap compared with PCR-sequencing, it may be an effective tool for epidemiological studies of HIFs in endemic areas.
authors.affiliation: Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Thailand. tmurusa@mahidol.ac.th
authors.forename: Urusa
authors.initials: U
authors.lastname: Thaenkham
authors.forename: Orawan
authors.initials: O
authors.lastname: Phuphisut
authors.forename: Wallop
authors.initials: W
authors.lastname: Pakdee
authors.forename: Nirundorn
authors.initials: N
authors.lastname: Homsuwan
authors.forename: Surapol
authors.initials: S
authors.lastname: Sa-nguankiat
authors.forename: Jitra
authors.initials: J
authors.lastname: Waikagul
authors.forename: Yukifumi
authors.initials: Y
authors.lastname: Nawa
authors.forename: Do Trung
authors.initials: do T
authors.lastname: Dung
mesh_terms: D000818:Animals; D001208:Asia / Q000453:epidemiology; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D005393:Fish Diseases / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology; D005399:Fishes; D005618:Fresh Water / Q000469:parasitology; D006578:Heterophyidae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D006801:Humans; D007422:Intestines / Q000469:parasitology*; D058488:Metacercariae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D008853:Microscopy; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012339:RNA, Ribosomal, 28S / Q000032:analysis / Q000235:genetics; D014201:Trematode Infections / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*
chemical_list: D012339:RNA, Ribosomal, 28S; C024413:endodeoxyribonuclease MboII; D015252:Deoxyribonucleases, Type II Site-Specific