Application of Recombinant Gnathostoma spinigerum Matrix Metalloproteinase-Like Protein for Serodiagnosis of Human Gnathostomiasis by Immunoblotting

Penchom Janwan Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Pewpan M. Intapan Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Hiroshi Yamasaki Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Porntip Laummaunwai Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Kittisak Sawanyawisuth Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Chaisiri Wongkham Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Chatchai Tayapiwatana Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Amnat Kitkhuandee Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Viraphong Lulitanond Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Yukifumi Nawa Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Wanchai Maleewong Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; National Institute of Infectious Diseases, Tokyo, Japan; Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

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Matrix metalloproteinase (MMPs) is the extracellular zinc-dependent endopeptidase and is secreted for degrading extracellular matrix molecules of host tissues. A cDNA encoding MMP-like protein of Gnathostoma spinigerum larvae was amplified by reverse transcription-polymerase chain reaction, and was cloned into a prokaryotic expression vector, and expressed in Escherichia coli. Total immunoglobulin G class (total IgG) antibody responses to the recombinant MMP-like protein were analyzed by immunoblot diagnosis of human gnathostomiasis. Serum samples from proven and clinically suspected cases of gnathostomiasis, other parasitic diseases patients, and from healthy volunteers were tested. The immunoblotting gave high sensitivity (100%) and specificity (94.7%). Positive and negative predictive values were 85.4% and 100%, respectively. Recombinant MMP-like protein can be used as a diagnostic antigen and potentially replace native parasite antigens to develop a gnathostomiasis diagnostic kit.

Author Notes

* Address correspondence to Wanchai Maleewong, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand. E-mail: wanch_ma@kku.ac.th

Financial support: This research was supported by grants from the Office of the Higher Education Commission, Thailand for supporting by grant fund under the program Strategic Scholarships for Frontier Research Network for the Ph.D. Program Thai Doctoral degree (CHE PhD scholarship); the Higher Education Research Promotion and National Research University Project of Thailand, and Faculty of Medicine, Khon Kaen University. PJ was supported by a CHE PhD scholarship. WM was supported by TRF Senior Research Scholar Grant, Thailand Research Fund grant no. RTA5580004. This research was also funded by a grant from the Association for Preventive Medicine of Japan in 2011 and 2012 to Wanchai Maleewong and Hiroshi Yamasaki.

Authors' addresses: Penchom Janwan, Pewpan M. Intapan, Porntip Laummaunwai, and Wanchai Maleewong, Department of Parasitology, Faculty of Medicine and Research, and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand, E-mails: pair_wu@yahoo.com, pewpan@kku.ac.th, porlau@kku.ac.th, and wanch_ma@kku.ac.th. Hiroshi Yamasaki, Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan, E-mail: hyamasak@nih.go.jp. Kittisak Sawanyawisuth, Department of Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mail: kittisak@kku.ac.th. Chaisiri Wongkham, Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mail: chaisiri@kku.ac.th. Chatchai Tayapiwatana, Biomedical Technology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at the Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand, E-mail: asimi002@chiangmai.ac.th. Amnat Kitkhuandee, Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mail: amnaki@kku.ac.th. Viraphong Lulitanond, Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mail: viraphng@kku.ac.th. Yukifumi Nawa, Research Division, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mail: yukinawa@kku.ac.th.

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