Detection of Gnathostoma spinigerum Antibodies in Sera of Non-Traumatic Subarachnoid Hemorrhage Patients in Thailand

Gnathostoma spinigerum can cause subarachnoid hemorrhage (SAH). The detection of specific antibodies in serum against G. spinigerum antigen is helpful for diagnosis of neurognathostomiasis. There is limited data on the frequency of G. spinigerum infection in non-traumatic SAH. A series of patients diagnosed as non-traumatic SAH at the Srinagarind Hospital, Khon Kaen University, Thailand between January 2011 and January 2013 were studied. CT or MR imaging of the brain was used for diagnosis of SAH. Patients were categorized as aneurysmal subarachnoid hemorrhage (A-SAH) or non-aneurysmal subarachnoid hemorrhage (NA-SAH) according to the results of cerebral angiograms. The presence of specific antibodies in serum against 21- or 24-kDa G. spinigerum antigen was determined using the immunoblot technique. The detection rate of antibodies was compared between the 2 groups. Of the 118 non-traumatic SAH patients for whom cerebral angiogram and immunoblot data were available, 80 (67.8%) patients had A-SAH, whereas 38 (32.2%) had NA-SAH. Overall, 23.7% were positive for specific antibodies against 21- and/or 24-kDa G. spinigerum antigen. No significant differences were found in the positive rate of specific antibodies against G. spinigerum in both groups (P-value=0.350).

Modulation of Antibody Responses against Gnathostoma spinigerum in Mice Immunized with Crude Antigen Formulated in CpG Oligonucleotide and Montanide ISA720

This study aimed to investigate the antibody responses in mice immunized with Gnathostoma spinigerum crude antigen (GsAg) incorporated with the combined adjuvant, a synthetic oligonucleotide containing unmethylated CpG motif (CpG ODN 1826) and a stable water in oil emulsion (Montanide ISA720). Mice immunized with GsAg and combined adjuvant produced all antibody classes and subclasses to GsAg except IgA. IgG2a/2b/3 but not IgG1 subclasses were enhanced by immunization with CpG ODN 1826 when compared with the control groups immunized with non-CpG ODN and Montanide ISA or only with Montanide ISA, suggesting a biased induction of a Th1-type response by CpG ODN. After challenge infection with live G. spinigerum larvae, the levels of IgG2a/2b/3 antibody subclasses decreased immediately and continuously, while the IgG1 subclass remained at high levels. This also corresponded to a continuous decrease of the IgG2a/IgG1 ratio after infection. Only IgM and IgG1 antibodies, but not IgG2a/2b/3, were significantly produced in adjuvant control groups after infection. These findings suggest that G. spinigerum infection potently induces a Th2-type biased response.

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).

Pineapple juice for digestion of swamp eel viscera for harvesting infective-stage larva of Gnathostoma spp.

Third-stage larvae were used as antigen in the diagnosis of gnathostomiasis in Western blot analysis. Normally, the larvae were obtained from digestion of eel's liver (Fluta alba) by the enzyme pepsin. We used pineapple juice (Ananus comosus) instead of enzyme pepsin in harvesting Gnathostoma spinigerum third-stage larvae. The difference in recovered larvae numbers, between pineapple juice and pepsin, were not statistically significantly different (p>0.05). The larvae from pepsin and pineapple juice digestion were cultivated on BME for 7 days; the survival rates were not significantly different (p>0.05). Thus, pineapple juice is another enzyme of choice for recovering Gnathostoma spinigerum third-stage larvae.

Preparation of Gnathostoma protein by ultrafiltration method using Nanosep membrane.

We report our experience with Gnathostoma protein preparation by the ultrafiltration method. Crude antigen was sonicated and ultrafiltrated using the Nanosep 100 K membrane. SDS-PAGE electrophoresis showed protein bands at 43, 41, 24, 22, 21, 19.5 kDa. Use of the ultrafiltration method can provide specific protein (24 kDa), similar to the non-ultrafiltration method, with the other 5 non-specific proteins. Using the non-ultrafiltration method, there were more (20) non-specific protein. The ultrafiltration method can be an alternative method for the preparation of protein, which can provide better results than non-ultrafiltration.

Specific IgE antibody responses to somatic and excretory-secretory antigens of third stage G. spinigerum larvae in human gnathostomiasis.

Specific IgE antibody levels in the serum of patients with proven gnathostomiasis and in those with intermittent cutaneous migratory swelling (CMS) were determined by the enzyme-linked immunosorbent assay (ELISA) using somatic extract and excretory-secretory (ES) products of Gnathostoma spinigerum infective larvae as antigens. The third stage larval used were obtained from naturally infected eels. There was an increase in specific IgE antibody to both antigens in these patients. The mean levels of these specific IgE antibodies were significantly higher than that of the healthy control (P<0.01). Comparison between using somatic extract and ES products in the test showed, a positive result in the group of suspected patients with gnathostomiasis or CMS was significantly higher when using ES products (81.81%) than somatic extract (59.09%) as the antigens (P<0.05). However, both somatic and ES antigens cross-reacted with other parasitic sera. The overall sensitivity of the ELISA for these IgE antibodies detection were 71.87 per cent and 87.50 per cent with somatic and ES antigens, respectively. The specificity was 57.53 per cent when somatic antigen was used and increased to 69.86 per cent when ES antigen was used. The positive and negative predictive values of the test were 42.59 per cent and 82.35 per cent by using somatic antigen. Both of these values, were also increased to 56.00 per cent and 92.72 per cent by using the ES antigen. It is obvious that more potential components may be present in ES products than those in the somatic extract. The ES antigen may have to be further purified and may be suitable for evaluation of the effectiveness of chemotherapy. As such, the antibody responses to secreted products are more closely related to active infection than the anti-whole worm antibody that may persist following the death of the parasites. However, in this disease, the effect of the IgE antibody on its pathophysiology it is still not known.

Antigenic components of Gnathostoma spinigerum recognized by infected human sera by two-dimensional polyacrylamide gel electrophoresis and immunoblotting.

Antigenic components of Gnathostoma spinigerum larval extract were revealed by two-dimensional gel electrophoresis (2-DE) and immunoblot analysis using sera from patients with 6 proven cases of gnathostomiasis, 5 presumptive cases of gnathostomiasis, 3 proven cases of angiostrongyliasis, 3 proven cases of cysticercosis, and pooled sera from healthy adults. By the 2-DE, the larval extract was highly complex and consisted of more than 75 polypeptides. Immunoblotting analysis of this larval extract after reaction with each of 6 proven gnathostomiasis sera revealed various numbers of antigenic spots ranging from 30 to 70 spots at the approximate molecular masses of less than 14.4 to more than 94 kDa with isoelectric points (pI) of less than 4.65 to 9.6. Antigenic spots at the approximate molecular mass of more than 30 kDa were recognized with the proven angiostrongyliasis, proven cysticercosis and healthy control sera but these sera did not react with the spots at approximate molecular masses of 23-25 kDa with pI of 8.3-8.5. The reacted spots, which consisted of at least 1 to 2 spots, were unique for the recognition of gnathostomiasis sera. Five out of 6 (83.3%) proven and 4 out of 5 (80%) presumptive gnathostomiasis sera reacted with these specific spots.

Immunohistochemical localization of Gnathostoma spinigerum larval antigens by monoclonal antibodies: II. Electron microscopy.

Immunocytochemical localization of antigens in advanced third-stage larvae of Gnathostoma spinigerum (GsAL3) was studied by immunogold labeling method using seven G. spinigerum specific monoclonal antibodies (MAbs), FS-3D11, SS-5H5, SK-6C4, SK-4E1, SK-7G6, SK-8D4 and SA-9B5. All these MAbs belong to the IgG1 subclass and only FS-3D11 and SS-5H5 recognize carbohydrate epitopes. The paraformaldehyde-fixed GsAL3 were embedded in Lowicryl K4M medium, and the gold colloidal particles used were 15 nm in size. When the worm sections were probed with FS-3D11, the gold particles appeared to concentrate specifically on the intestinal brush border. When SS-5H5 was applied, the particles were scattered densely over the brush border and in the cytoplasm of epithelial cells. The rest of the MAbs which recognize protein determinants exhibited a lack of labeling. The results suggested that the carbohydrate antigenic determinants recognized by the two MAbs are the most stable and most abundant particularly in the intestine of GsAL3. These results also confirmed the previous finding that the most antigenic site of GsAL3 is the intestine.

Purification of Gnathostoma spinigerum larval antigens by monoclonal antibody affinity chromatography.

An immunoaffinity column was prepared by coupling a partially purified Gnathostoma spinigerum-specific IgG1, MAb SK-6C4 (5 mg/ml) to CNBr-activated Sepharose 4B. Ten milliliters of approximately 0.3 mg/ml of crude soluble G. spinigerum larval antigens (GsAL3) were loaded onto the affinity column at a flow rate of about 5 ml/hour. Elution of the bound antigens was accomplished using 50 mM diethylamine-HCI containing 0.15 M NaCL, pH 11.5. The average amount of eluted antigens obtained by one passage of crude GsAL3 (1-4 mg) through 4 to 8 ml of column matrix was 143 micrograms (range, 67 - 414 micrograms). The minimal amount of purified GsAL3 detectable by ELISA using MAb SK - 6C4 (100 micrograms/ml) was 50 ng/ml. The SK - 6C4 affinity-purified GsAL3 was found to be relatively pure and immunologically specific as determined by SDS-PAGE and Western blotting, respectively.

Specificity of antibodies in cerebrospinal fluid of human cerebral gnathostomiasis cases.

Specificity of antibodies in cerebrospinal fluid (CSF) of human cerebral gnathostomiasis cases were examined by indirect fluorescent antibody technique against paraffin sections of Gnathostoma spinigerum larva. Specific greenish fluorescence was observed at cuticle, esophagus, muscle cells, intestinal cell cytoplasm and microvilli. CSF of confirmed cerebral cysticercosis cases gave fluorescence mostly at the cuticle. It is suggested that parasite-specific antigen may be present on intestinal cell microvilli and CSF would be a good source of antibodies in studying specificity of antibodies to gnathostome infections.

Gnathostoma larva migrans among guests of a New Year party.

An outbreak of Gnathostoma larva migrans occurred among guests of a New Year's party in Chachoengsao, Thailand. Nine people who consumed a raw fish dish called 'Hu-sae' contracted the disease. Five of them developed gastro-intestinal symptoms consisting of nausea, vomiting, abdominal cramps and diarrhea as early as within the first 24 hours, while in the other four, symptoms started on the following day. After the initial symptoms pertaining to the gut, malaise, chest discomfort, cough, myalgia, weakness, itching and migratory swellings were experienced. Eosinophilia was demonstrated in every patient with a mean (+/- SE) count of 5,516 +/- 1,010 cells/cu mm. Detection of antibody against aqueous extracts of G. spinigerum adult antigen using an enzyme-linked immunosorbent assay showed a titer of 1:1,600 or greater in every patients except one who had a titer of 1:400 (positive greater than or equal to 1:400). This outbreak illustrates the high attack rate when heavily infected fish are consumed.

Practicable IFAT slide antigen preparations for detection of Gnathostoma spinigerum antibodies in rabbits.

This study was designed to determine which stage of Gnathostoma spinigerum and which method of the preparation of test antigens are the most suitable for the detection of antibodies in serum of rabbits infected with advanced third stage larvae (AL3) of G. spinigerum by the indirect fluorescent antibody test (IFAT). Antigens from parasite ova and first stage larvae (L1) were obtained from freshly preserved specimens and affixed to glass slides with egg albumin. AL3 antigens consisted of paraffin sections, cryostat sections and pellets of crude worm soluble extract. Slides of adult male and female worms were prepared in cryostat sections. Pellets of crude worm soluble extract (AL3) smeared onto slides gave the best positive reaction followed by AL3 cryostat sections and L1.

Antigens, antibodies and immune complexes in cerebrospinal fluid of patients with cerebral gnathostomiasis.

Methods for the detection of antigens, antibodies and immune complexes in the cerebrospinal fluid (CSF) of patients with neurological manifestations suggestive of cerebral gnathostomiasis were developed, in the hope that they may be useful in confirming the diagnosis of Gnathostoma spinigerum infection. Gnathostoma antigens were determined by a sandwich enzyme linked immunosorbent assay (ELISA) using antibodies from rabbits immunized with the excretory/secretory (ES) antigens obtained from the in vitro supernatant fluid in which the third-stage G. spinigerum larvae were maintained. With a biotin streptavidin procedure, the presence of G. spinigerum antigens as low as 2 ng in one ml of CSF could be detected. An indirect ELISA was used for the quantitation of IgG antibodies in the paired serum and CSF of these patients. A complement consumption method was used for the detection of immune complexes in the concentrated CSF specimens. Of the 11 patients with clinical signs and symptoms suggestive of having G. spinigerum infection involving the central nervous system, only one patient had antigens detected in the CSF and in this one patient no antibody could be demonstrated. One other patient had immune complexes in her CSF. All remaining patients had IgG antibodies demonstrable in the CSF specimens. These data suggest that the detection of IgG antibodies in CSF is more reliable than the other two methods in confirming the diagnosis of cerebral gnathostomiasis.

ELISA for diagnosis of gnathostomiasis using antigens from Gnathostoma doloresi and G. spinigerum.

ELISA was developed for the detection of IgG antibody in sera obtained from patients in Japan and in a foreign country. Gnathostoma doloresi adult antigen was less specific than G. spinigerum advanced third-stage larval antigen but their sensitivity were similar. Cross reactivity was observed in Toxocara canis-, Anisakis-, Paragonimus westermani- and Fasciola-infected sera when G. doloresi adult worms but not G. spinigerum advanced third-stage larvae were used as antigens.

Demonstration of species-specific antigens of Gnathostoma spinigerum, a preliminary attempt.

An attempt was made to demonstrate the presence of species-specific antigens for Gnathostoma spinigerum advanced third-stage larvae (GsAL3) in a rabbit receiving weekly immunization with GsAL3 for seven weeks. The homologous and heterologous antibodies against GsAL3 and G. doloresi adult worm (Gd) antigens were initially detected by immunoelectrophoresis (IEP) and ELISA after the second immunization, and their levels were gradually increased with the number of immunizations. Though cross-reactivity between GsAL3 and Gd were shown with both tests, species-specific antibodies for the homologous antigens were demonstrated. After cross-absorption of rabbit hyperimmune serum was collected after the seventh immunization, seven 'putative' species-specific precipitin bands of GsAL3 were identified. The ELISA values of the rabbit hyperimmune serum showed 50% inhibition after absorption with 0.7 micrograms/ml of homologous GsAL3 antigens as opposed to 1.0 micrograms/ml of the heterologous Gd antigens.

Antibody responses in human gnathostomiasis.

Sera from 4 patients with parasitologically confirmed gnathostomiasis and from 18 healthy individuals were studied by SDS-PAGE and Western blot analysis using radioiodinated protein A to detect antibody responses against crude aqueous somatic extract of advanced third stage larvae of Gnathostoma spinigerum (L3G). It was found that the L3G extract was highly complex, comprising of more than 40 polypeptides among which more than 20 components were antigenic in human. The relative M.W. of the proteins ranged from 13 kd to 150 kd with the major antigenic bands at 150, 135, 120, 94, 84, 82, 72, 55, 54, 49, 43, 38, 35, 32 and 28 kd. All 4 sera from gnathostomiasis patients gave almost an identical pattern of reactivities against the L3G antigens whereas sera from the normal individuals gave much lower reactivities against the L3G antigen of M.W. 38 kd and, in certain individuals, those of 49 and 43 kd. The present findings suggest that the serum antibody response against the parasite is specific and may be useful in a specific or a confirmed immunodiagnosis of human gnathostomiasis.

Serodiagnosis of human gnathostomiasis.

An enzyme-linked immunosorbent assay (ELISA) was evaluated for serodiagnosis of human ocular and visceral gnathostomiasis in comparison to an indirect haemagglutination (IHA) and precipitin (PPT) tests. The ELISA antibody titers were found to range from 1:400 to 1:51,200 against somatic and 1:200 to 1:25,600 against excretory-secretory (ES) antigens. When sera were tested at single dilutions, the ELISA was positive in 7 of 8 gnathostomiasis cases while only 5 and 3 were positive by IHA and PPT respectively. The overall specificity of the ELISA was 96.7% and 97.4% with somatic and ES antigens respectively. Since somatic and ES antigens produced similar ELISA results, either can be used for diagnostic purpose. It was suggested that the ELISA was a reliable serodiagnostic test for human gnathostomiasis.

A study on immunodiagnosis of gnathostomiasis by ELISA and double diffusion with special reference to the antigenicity of Gnathostoma doloresi.

In order to diagnose gnathostomiasis immunologically, Gnathostoma doloresi was evaluated for the antigenicity in comparison with G. hispidum which was recently reported in Japan by using micro-ELISA. The study revealed that G. doloresi can be used as the alternate source of antigen in the test. A significant increase of specific IgG antibodies was seen in 22 (73.3%) out of 30 gnathostomiasis cases. Although double diffusion was slightly less sensitive than ELISA, it was considered more specific than the latter method.

Detection of humoral immune response to Gnathostoma spinigerum in mice.

The humoral immune response to early third stage larvae (EL3) and advanced third stage larvae (AL3) of Gnathostoma spinigerum infection was studied in mice by Ouchterlony gel diffusion technique. The antibodies was detected at week 3 in mice infected with EL3 and remained up to week 10 after infection. Highest positive sample of sera were demonstrated at week 4 to week 7. Similar results were obtained from AL3 infected sera except the antibodies was found and disappeared earlier (week 2 to week 6). G. spinigerum larvae recovery from mice in both groups showed that the number of advanced third stage larvae located in muscle correlated to the peak of positive sera. No cross reaction was observed on positive sera of G. spinigerum and antigens of A. cantonensis, P. siamensis, T. spiralis, O. viverrini and A. ceylanicum. Cross reaction was shown on the G. spinigerum antigen against rat sera with angiostrongyliasis and bandicoot sera with paragonimiasis.