Evaluation of immunodiagnostic tests for human gnathostomiasis using different antigen preparations of Gnathostoma spinigerum larvae against IgE, IgM, IgG, IgG1-4 and IgG1 patterns of post-treated patients.

OBJECTIVES: The aims of the study were two-fold: (1) antigen (Ag) preparation and evaluation of three antigens of Gnathostoma spinigerum infective larvae (GsL3), crude somatic antigen (CSAg), excretory-secretory antigen (ESAg) and partially purified antigens (namely P1Ag, P2Ag and P3Ag) to differentiate IgE, IgG, IgG1-4 and IgM for human gnathostomiasis diagnosis; and (2) application of the selected ELISA for following up stored sera of patients treated with ivermectin (IVM) and albendazole (ABZ). METHODS: Different antigens were analysed by antibodies of gnathostomiasis cases, other parasite infections and healthy controls using indirect ELISA to differentiate IgE, IgG, IgG1-4 and IgM. Then, prominent antigen and immunoglobulin were used in antibody predictions of gnathostomiasis cases treated with albendazole or ivermectin. RESULTS: Sensitivity of all evaluated ELISAs: IgM-, IgG-, IgG1- and IgG4-ELISA, was 100%. IgM-ELISA with CSAg and P3Ag exhibited the highest specificity of 99%. IgG-ELISA with P2Ag resulted in the highest specificity of 92.3%. IgG1-ELISA with P2Ag and P3Ag showed excellent results with 100% specificity. Finally, P2Ag evaluated IgG1 of the followed-up cases with ABZ and IVM. Decreasing antibody IgG1 levels were mostly found in both treatments at Month 9 and long follow-up was over 12 months. A Gnathostoma worm was extracted from each two treated patients. CONCLUSIONS: Using IgG1-ELISA against P2Ag and P3Ag gave excellent results with 100% sensitivity and specificity. These tests can be an alternative to immunoblotting for gnathostomiasis. IgG1 decreased at least 9 months in most cases, so long-term treatment should be performed over 1 year.

Molecular cloning and characterization of serine protease inhibitor from food-borne nematode, Gnathostoma spinigerum.

Gnathostoma spinigerum is a causative agent of human gnathostomiasis and infects people residing in endemic areas as well as travelers. Cutaneous and visceral larval migrants cause clinical manifestations, resulting in severe morbidity and mortality. To survive in hosts, these parasites have evolved various immune evasion mechanisms, including the release of regulatory molecules. Serine protease inhibitors (serpins) that are present in many parasitic helminths are proteins suspected of suppressing host serine protease-related digestion and immune responses. In this study, the serpin secreted by G. spinigerum (GsSerp) was characterized using bioinformatics and molecular biology techniques. The bioinformatics revealed that GsSerp contains 9 helices, 3 ß-sheets, and a reactive central loop, which are conserved structures of the serpin superfamily. Recombinant GsSerp (rGsSerp) was expressed in E. coli (molecular weight, 39 kDa) and could inhibit chymotrypsin. Mouse polyclonal antibody against GsSerp could detect the native GsSerp in crude worm antigen but not the excretory-secretory product (ES) of infective-stage larva (aL3Gs). Moreover, the expression of GsSerp in the aL3Gs tissue was located in the hemolymph and intestinal tissue, indicating its role in parasite homeostasis. Our findings may help develop effective strategies for preventing and controlling gnathostomiasis.