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Cloning and expression of a 16-kDa recombinant protein from Angiostrongylus cantonensis for use in immunoblot diagnosis of human angiostrongyliasis

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Abstract

Angistrongylus cantonensis is a zoonotic nematode parasite and causative agent of human angiostrongyliasis, which clinically presents as eosinophilic meningitis or meningoencephalitis. Diagnosis of the disease is problematic since parasitologic findings are infrequent, and infection determinations must be based on the clinical symptoms and serological tests with limited specificities and sensitivities. The aim of the present study was to identify and generate a novel recombinant protein from A. cantonensis and evaluate its efficacy in the diagnosis of human angiostrongyliasis when incorporated into a Western blot serodiagnostic system. A cDNA protein expression library from adult A. cantonensis was constructed, followed by immunoscreening with serum from confirmed infected patients to identify and isolate immunoreactive clones. One clone, designated fAC40, possessed a partial sequence encoding a LisH protein domain with a predicted molecular weight of 16 kDa and containing four predicted antigenic peptides. By incorporating recombinant fAC40 in Western immunoblot tests using a serum panel consisting of confirmed and clinically diagnosed cases of human angiostrongyliasis and other helminthic infections, fAC40 exhibited a sensitivity and specificity of 91.8 and 100 %, respectively, and a positive and negative predictive value of 100 and 97.19 %, respectively, in the diagnosis of angiostrongyliasis. Importantly, it was not reactive with antibodies from serum of patients infected with Gnathostoma spinigerum and Cysticercus cellulosae, infections that clinically present neurological symptoms similar to angiostrongyliasis. These data demonstrate that the 16-kDa recombinant protein from A. cantonensis possesses high potential as a candidate antigen for a more sensitive and specific serodiagnosis of human angiostrongyliasis.

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Acknowledgments

This research was supported by the Commission on Higher Education, Ministry of Education, Thailand. We would like to thank the staff of the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Thailand, and the Department of Pathobiological Sciences, School of Veterinary Medicine, The University of Wisconsin-Madison, USA, for providing the laboratory facilities and assistance with the laboratory techniques.

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Authors and Affiliations

  1. Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, 65000, Thailand

    Apichat Vitta

  2. Center of Excellence in Medical Biotechnology (CEMB), Naresuan University, Phitsanulok, 65000, Thailand

    Apichat Vitta

  3. Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand

    Apichat Vitta, Paron Dekumyoy & Chalit Komalamisra

  4. Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand

    Thareerat Kalambaheti

  5. Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, 53706, USA

    Timothy P. Yoshino

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  1. Apichat Vitta
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  2. Paron Dekumyoy
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  3. Chalit Komalamisra
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  4. Thareerat Kalambaheti
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Corresponding authors

Correspondence to Paron Dekumyoy or Timothy P. Yoshino.

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Ethical statement

In the present study, we used laboratory-reared rats as experimental definitive hosts for A. cantonensis. The experiments involving the use of animals were approved by the Animal Care and Use Committee (project no. FTM-ACUC 009/2007) from the faculty of Tropical Medicine, Mahidol University, while those incorporating the use of human serum were approved by the Human Ethics Committee (project no. TMEC09-017).

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Vitta, A., Dekumyoy, P., Komalamisra, C. et al. Cloning and expression of a 16-kDa recombinant protein from Angiostrongylus cantonensis for use in immunoblot diagnosis of human angiostrongyliasis. Parasitol Res 115, 4115–4122 (2016). https://doi.org/10.1007/s00436-016-5184-1

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  • DOI: https://doi.org/10.1007/s00436-016-5184-1

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