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.

An Unusual Case of Gastric Gnathostomiasis Caused by Gnathostoma spinigerum Confirmed by Video Gastroscopy and Morphological and Molecular Identification.

Human gnathostomiasis is a harmful foodborne parasitic infection caused by nematodes of the genus Gnathostoma. Here, we report an unusual case of gastric gnathostomiasis seen in a hospital in Thailand along with the clinical characteristics, treatment, and outcome. A 39-year-old man presented with complaints of epigastric pain, dizziness, and history of passing dark, tarry stools for 2 days. The patient had a history of consuming raw freshwater fish. Supplementary differential diagnosis was performed via rapid serological testing, and presence of the causative agent was confirmed based on video gastroscopy, morphology of the removed parasite, and molecular identification. After its surgical removal from the stomach, the parasite was morphologically identified as Gnathostoma species. Molecular identification was performed via DNA extraction from the recovered worm, and amplification and sequencing of the second internal transcribed spacer (ITS2) region and partial cytochrome c oxidase subunit I (cox1) gene. The ITS2 and cox1 sequences were consistent with those of Gnathostoma spinigerum. Clinicians in endemic areas should therefore be aware of the rare clinical manifestations and use of supplementary serological tests to facilitate early diagnosis and treatment of gastric gnathostomiasis.

Effects of Gnathostoma spinigerum infective stage larva excretory-secretory products on NK cells in peripheral blood mononuclear cell culture: focused on expressions of IFN-γ and killer cell lectin-like receptors.

Human gnathostomiasis is mainly caused by third-stage larvae of Gnathostoma spinigerum (G. spinigerum L3). Excretory-secretory products (ES) released from infective helminthic larvae are associated with larval migration and host immunity modulation. Natural killer (NK) cells have important immune functions against helminth infection. Currently, the effects of ES from G. spinigerum L3 (G. spinigerum ES) on NK cell activity are unclear. This study investigated whether G. spinigerum ES affected human NK cells. Human normal peripheral blood mononuclear cell (PBMC) cultures were used to mimic immune cells within the circulation. PBMC were co-cultured with G. spinigerum ES (0.01-0.05 µg/ml) for 5 or 7 days. Levels of IFN-γ in cultured supernatants were measured by enzyme-linked immunosorbent assay. The expressions of mRNA encoding NK cell receptors, especially the C type killer cell lectin-like family (KLR; NKG2A, NKG2C, and NKG2D) and IFN-γ in ES induced PBMC were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). ES induced PBMC markedly decreased the levels of IFN-γ and increased the expressions of NKG2A and NKG2D on NK cells. In conclusion, low amounts of G. spinigerum ES modulated NK cells by downregulating the transcription of IFN-γ and upregulating the expressions of KLR (NKG2A and NKG2D receptors) during the 7-day observation period. These findings indicate more in-depth studies of NK cell function are required to better understand the mechanism involved in immune evasive strategies of human gnathostomiasis.

Proteomic analysis identification of antigenic proteins in Gnathostoma spinigerum larvae.

Gnathostoma spinigerum is the causative agent of human gnathostomiasis. The advanced third stage larva (AL3) of this nematode can migrate into the subcutaneous tissues, including vital organs, often producing severe pathological effects. This study performed immuno-proteomic analysis of antigenic spots, derived from G. spinigerum advanced third stage larva (GSAL3) and recognized by human gnathostomiasis sera, using two-dimensional (2-DE) gel electrophoresis based-liquid chromatography/tandem mass spectrometry (LC/MS-MS), and followed by the aid of a database search. The crude GSAL3 extract was fractionated using IPG strips (pH 3-11NL) and followed by SDS-PAGE in the second dimension. Each gel was stained with colloidal Coomassie blue or was electro-transferred onto a nitrocellulose membrane and probed with gnathostomiasis human sera by immunoblotting. Individual Coomassie-stained protein spots corresponding to the antigenic spots recognized by immunoblotting were excised and processed using LC/MS-MS. Of the 93 antigenic spots excised, 87 were identified by LC/MS-MS. Twenty-seven protein types were found, the most abundant being Ascaris suum37. Six spots showed good quality spectra, but could not be identified. This appears to be the first attempt to characterize antigenic proteins from GSAL3 using a proteomic approach. Immuno-proteomics shows promise to assist the search for candidate proteins for diagnosis and vaccine/drug design and may provide better understand of the host-parasite relationship in human gnathostomiasis.

Spontaneous cure after natural infection with Gnathostoma turgidum (Nematoda) in Virginia opossums (Didelphis virginiana).

Seasonality of the nematode Gnathostoma turgidum in Virginia opossums (Didelphis virginiana) in the wild has been reported; however, the mechanisms involved in deworming are unknown. We monitored the parasitologic and biologic changes in four Virginia opossums naturally infected with G. turgidum by coproparasitologic examination and abdominal ultrasonography. Eggs became detectable in the feces of opossums in May, peaked in July and August, and suddenly decreased in October. Adults of G. turgidum were expelled in the feces mainly in September. Ultrasonography of the liver showed slight damage during May. Lesions in the stomach appeared in April and persisted until September. The abnormalities of the liver and stomach were resolved in November. These data suggest that G. turgidum is likely expelled as a result of host immunologic mechanisms, although termination of a natural life span cannot be definitively excluded.

Diagnosis of gnathostomiasis by skin testing using partially purified specific antigen and total IgE levels.

BACKGROUND: A finding of antibodies to Gnathostoma spinigerum 24-kDa antigen by immunoblot analysis is currently used to confirm a diagnosis of gnathostomiasis. A simple skin test for the diagnosis of gnathostomiasis was developed, and the results were evaluated and compared with the standard Western blot (WB) test. METHODS: This cross-sectional study was conducted at the Hospital for Tropical Diseases, Bangkok, Thailand, in 2008-2011. All eligible patients were tested with partially purified proteins of mAb-detected fractions pooled and sterilized by 0.2 µm diameter syringe filter, with a phenol saline solution of 1:10 w/v. RESULTS: A total of 69 cases, 39 gnathostomiasis cases and 30 controls, were enrolled into the study; the median age (IQR) was 40 (30.5-52.5) years. The most common presenting symptom was edema (56/69, 81%). Gnathostomiasis cases having strong cutaneous reactions to the intradermal test (81%) were also positive by immunoblot. A significant correlation between skin and immunoblot tests was detected (p<0.001). The difference in total IgE levels between cases and controls was not statistically significant (p=0.51). Logistic regression models showed that positive WB and skin-test results were significantly associated with gnathostomiasis (p=0.001 and p=0.007, respectively). CONCLUSION: Gnathostoma skin testing, using prepared fractionated antigen solution of Gnathostoma spinigerum, yields good reactivity and significantly correlates with the results of immunoblot testing.

Cutaneous gnathostomiasis with recurrent migratory nodule and persistent eosinophilia: a case report from China.

The present study reports a human case of cutaneous gnathostomiasis with recurrent migratory nodule and persistent eosinophilia in China. A 52-year-old woman from Henan Province, central China, presented with recurrent migratory reddish swelling and subcutaneous nodule in the left upper arm and on the back for 3 months. Blood examination showed eosinophila (21.2%), and anti-sparganum antibodies were positive. Skin biopsy of the lesion and histopathological examinations revealed dermal infiltrates of eosinophils but did not show any parasites. Thus, the patient was first diagnosed as sparganosis; however, new migratory swellings occurred after treatment with praziquantel for 3 days. On further inquiring, she recalled having eaten undercooked eels and specific antibodies to the larvae of Gnathostoma spinigerum were detected. The patient was definitely diagnosed as cutaneous gnathostomiasis caused by Gnathostoma sp. and treated with albendazole (1,000 mg/day) for 15 days, and the subsequent papule and blister developed after the treatment. After 1 month, laboratory findings indicated a reduced eosinophil count (3.3%). At her final follow-up 18 months later, the patient had no further symptoms and anti-Gnathostoma antibodies became negative. Conclusively, the present study is the first report on a human case of cutaneous gnathostomiasis in Henan Province, China, based on the past history (eating undercooked eels), clinical manifestations (migratory subcutaneous nodule and persistent eosinophilia), and a serological finding (positive for specific anti-Gnathostoma antibodies).

Gnathostoma spinigerum: immunodepression in experimental infected mice.

Mice were infected with 8- or 25-infective worms of advanced third stage Gnathostoma spinigerum larvae (L3) which were obtained from natural infected eels. On day 14, 60 and 200 post infections (PI), spleen cells of infected mice were tested for lymphoproliferative responses in vitro against the mitogen and specific L3 somatic antigen in order to clarify the cellular immune status of the host upon this nematode infection. Reduced responsiveness to Con A was observed in infected mice. These depressed responses were more pronounced in chronically infected mice (day 200, PI) than in day 14 and day 60, PI. There was no significant difference of lymphoproliferative response between groups of high (25 L3) and low (8 L3)-infective dose in the chronic readily stage. Regarding to the L3 somatic Ag stimulation, the depressed response was obviously detected in high dose and chronic infection. Our results demonstrated that in this G. spinigerum-mouse system T-cell response is defective. The depression could be reversible and was associated with active infection because it was abolished by anthelmintic (ivermectin) treatment. This study shows the involvement of Th-2 response to this nematode in regulating T cell proliferation.

Characterization of the humoral immune response against Gnathostoma binucleatum in patients clinically diagnosed with gnathostomiasis.

Gnathostomiasis is an emerging systemic parasitic disease acquired by consuming raw or uncooked fresh-water fish infected with the advanced third-stage larvae of Gnathostoma spp. This disease is endemic to the Pacific region of Mexico, and one of its etiologic agents has been identified as Gnathostoma binucleatum. We characterized the humoral immune response of patients clinically diagnosed with gnathostomiasis by detecting total IgM, IgE, and IgG class and subclasses against a crude extract of the parasite by Western blotting. Our results do not show differences in the antigens recognized by IgM and IgE. However, we found that the specific humoral immune response is caused mainly by IgG, specifically IgG4. We found that 43%, 65.2%, 54.1%, and 26.3% of the patients recognize the 37-kD, 33-kD, 31-kD, and 24-kDa antigens, suggesting that the 33-kD antigen is the immunodominant antigen of G. binucleatum.