- INTRODUCTION
- MATERIALS AND METHODS
- Ethical approval.
- Parasites.
- Identification of Fasciola worms by morphology and molecular techniques.
- Human sera.
- Assessment of the Fasciola IgG ELISA kit produced in the United States and selection of positive control sera.
- Crude worm extract.
- Cystatin capture ELISA with CWE.
- Dot-blot ELISA using recombinant F. gigantica proteins.
- Synthetic peptides design based on FgCB5 sequence.
- Indirect ELISA with synthetic peptide antigen based on FgCB5.
- Statistical analysis.
- RESULTS AND DISCUSSION
- Identification of Fasciola species.
- Assessment of the Fasciola IgG ELISA kit from the United States and selection of positive control sera for subsequent experiments.
- Diagnostic efficacy of CC ELISA using crude worm extract.
- Selection of recombinant proteins using dot-blot analysis.
- Peptide selection and efficacy of synthetic peptide-based ELISA in comparison with other ELISA systems.
- Supplementary Files
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Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Kit and In-House Fasciola gigantica Cysteine Proteinases-Based Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Fascioliasis
Fascioliasis, caused by Fasciola hepatica and Fasciola gigantica infection, is a major food-borne trematodiasis in many places of the world, with the central region of Vietnam being reported as a highly endemic area. Stool examination for Fasciola eggs is not a sensitive method, and immunodiagnostic methods are preferable. We investigated various enzyme-linked immunosorbent assays (ELISAs) to evaluate their efficacy for fascioliasis diagnosis. Test sera used are primarily screened using an ELISA kit produced in Vietnam (VN kit; Viet Sinh Chemical Producing & Trading Co. Ltd., Ho Chi Minh City, Vietnam): Seropositive individuals having symptoms compatible with fascioliasis were regarded as clinically diagnosed fascioliasis cases. A commercial Fasciola IgG ELISA kit from Diagnostic Automation/Cortez Diagnostics, Inc. (USA kit; Woodland Hills, CA), which has been commonly used in Vietnam, was assessed and compared with in-house ELISA systems, including a cystatin-capture (CC) ELISA using crude worm extract (CWE) and an indirect ELISA using a synthetic peptide Ac-TPTCHWECQVGYNKTYDEE-NHMe designed from the F. gigantica cathepsin B (FgCB5) molecule. The USA kit was suitable for routine diagnosis after recalibration of the manufacturer’s suggested cutoff point. Cystatin-capture ELISA with CWE provided good sensitivity and specificity with perfect agreement to the results of the USA kit. In dot-blot ELISA, recombinant FgCB5 reacted more strongly with human antisera than did other F. gigantica antigens tested. Enzyme-linked immunosorbent assay using the synthetic peptide fragment of the FgCB5 exhibited nearly 80% sensitivity and specificity, but the test results showed low agreement with CC-ELISA or the USA kit. In conclusion, the commercially available Fasciola IgG ELISA kit from the United States and the in-house CC ELISA using CWE are suitable for practical diagnosis for fascioliasis.
Author Notes
Financial support: This research was supported by Invitation Research, Faculty of Medicine (IN61241), and the Postgraduate Scholarship for International Students, Faculty of Medicine, Khon Kaen University.
Authors’ addresses: Na T. D. Tran, Kitti Intuyod, and Somchai Pinlaor, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mails: t.tranthidiemna@kkumail.com, kitti.i@kkumail.com, and psomec@kku.ac.th. Phuong Anh Ton Nu, Department of Parasitology, Hue University of Medicine and Pharmacy, Hue, Vietnam, E-mail: tnpanh@huemed-univ.edu.vn. Ly T. K. Dao, Institute of Malariology, Parasitology and Entomology Quy Nhon, Quy Nhon, Vietnam, E-mail: khanhly75@yahoo.com. Porntip Pinlaor, Faculty of Associated Medical Sciences, Centre for Research and Development of Medical Diagnostic Laboratories, Khon Kaen University, Khon Kaen, Thailand, E-mail: porawa@kku.ac.th. Yukifumi Nawa, Faculty of Medicine, Tropical Diseases Research Centre, Khon Kaen University, Thailand, E-mail: yukinawa@kku.ac.th. Kiattawee Choowongkomon, Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand, E-mail: kiattawee.c@ku.th. Amornrat Geadkaew-Krenc, Nanthawat Kosa, and Rudi Grams, Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand, E-mails: amornrut_gead@hotmail.com, nantha.ko@hotmail.com, and rgrams@tu.ac.th.