The First Human Case of Thelazia callipaeda Infection in Vietnam

A 26-year-old man residing in a village of Thai Nguyen Province, North Vietnam, visited the Thai Nguyen Provincial Hospital in July 2008. He felt a bulge-sticking pain in his left eye and extracted 5 small nematode worms by himself half a day before visiting the hospital. Two more worms were extracted from his left eye by a medical doctor, and they were morphologically observed and genetically analyzed on the mitochondrial cytochrome c oxidase 1 gene. The worms were 1 male and 1 female, and genetically identical with those of Thelazia callipaeda. By the present study, the presence of human T. callipaeda infection is first reported in Vietnam.

Two Cases of Human Thelaziasis and Brief Review of Korean cases

The present study was performed to describe 2 cases of human thelaziasis (HT) which occurred in Gyeongsangnam- do and to briefly review the previously reported Korean cases. A 58-year old woman, residing in Hadong-gun, Gyeongsangnam-do, came to Gyeongsang National University Hospital (GNUH) complaining of foreign body sensation and itching of the right eye in March 2000. Total 6 adult nematodes of Thelazia callipaeda (2 males and 4 females) were detected in her right eye. A 80-year old man, residing in Jinju-si, Gyeongsangnam-do, came to GNUH complaining of foreign body sensation, itching, and pain of the right eye in December 2007. A total of 5 worms (4 females and 1 degenerated) were removed from his right eye. We analyzed characteristics of the total 39 Korean HT cases reported to date, including the present 2 cases. Most of the cases (71.8%) occurred in Seoul and Gyeonggi-do before 2000, and 21 cases (53.8%) were males and 18 (46.2%) were females. The prevalence was higher in younger ages below 30 years (48.7%) than 31-60 years (41.0%) and over 61 years (10.3%). The seasonal prevalence showed a higher incidence in autumn (43.6%) than in other seasons. Most of the cases (94.9%) were conjunctival sac infections and only 2 (5.1%) were intraocular cases. The present 2 HT cases are the first reported cases in Gyeongsangnam-do. Some characteristics of Korean HT cases were analyzed.

Incidence and genetic characterization of Gongylonema pulchrum in cattle slaughtered in Mazandaran Province, Northern Iran

The gullet worm, Gongylonema pulchrum Molin, 1857, is a thread-like spirurid nematode found in a variety of mammals worldwide. Its incidences in Iranian cattle of different breed or age have not been reported. The aims of the present study are to disclose the infection status of G. pulchrum in cattle slaughtered in northern region of Iran. Full-length esophagi of cattle of 97 native dairy breed and 41 Holstein-Friesian breed were collected at four local abattoirs in Mazandaran Province, northern Iran, from March 2006 to August 2007, and were examined parasitologically. Eight overlapping segments of the small-and large-subunits of rDNA were amplified by PCR, and the obtained nucleotide sequences were characterized. The incidences of G. pulchrum in female and male native dairy breed were 38.9% and 24.0%, respectively, whereas those in female and male Holstein-Friesian breed were 4.2% and 0%, respectively. The first internal transcribed spacer [ITS1] region of G. pulchrum rDNA showed an intra-individual variation in the sequence and length, and the variation was ascribed to some unstable repeats of "A" or "CA". Distinct incidences of G. pulchrum infection in native dairy breed and Holstein-Friesian breed might be ascribed to different animal husbandry manners for each breed in Iran; the former breed grazes freely in the pasture, but the latter breed is usually held in a pen. The rDNA sequence of Iranian G. pulchrum, obtained for the first time by us, might facilitate a reliable species identification of the parasite with a wide spectrum of morphological variations

Spinal gnathostomiasis resembling an intrinsic cord tumor/myelitis in a 4-year-old boy.

A 4-year-old boy presented with fever, myalgia followed by progressive quadriparesis and urinary retention. Spinal fluid from a lumbar puncture showed 42 WBC/microl with 100% lymphocytes, no RBC, a glucose of 54 mg/dl (blood glucose 107 mg/dl), and a protein of 39 mg/dl. The cerebrospinal fluid culture was negative. His white blood cell count was 10,860 cells/microl with a normal differential count. An MRI of the brain was negative. An MRI of the whole spine showed fusiform dilatation of the cervical cord from the cervicomedullary junction to the T4 level. The tentative diagnosis was acute hemorrhage of an intrinsic cord tumor versus acute myelitis. Intravenous dexamethasone was administered which resulted in a slight improvement in strength. One week later, he deteriorated precipitously and became flaccidly quadriplegic. Since the patient deteriorated rapidly and no definitive diagnosis was made, the patient underwent cervical cord biopsy. Intraoperatively, after the cervical cord had been opened, a living Gnathostoma spinigerum was found in the spinal cord parenchyma. The nematode was removed. Following the operation the patient was placed on albendazole 400 mg/d and metronidazole 250 mg three times per day for 3 weeks. He gradually improved over the next several weeks.

Ocular thelaziasis in Assam: a report of two cases.

Small, white, thread-like, motile worms were recovered from the conjunctival sac of a 13-year-old girl and a 50-year-old woman from Dibrugarh district, Assam, India. They were identified as thelazia species. These two cases have been reported due to their rarity in India and elsewhere.

Feasibility of using papaya skin extract for digestion of swamp eel viscera for harvesting infective stage larva of Gnathostoma spp.

In this study, we reported the feasibility of using papaya skin extract (Carica papaya L.) as an alternative to enzyme pepsin in harvesting Gnathostoma spinigerum third-stage larvae. From experimental digestion, we found that the different numbers of recovered larvae between papaya skin extract and pepsin were not statistically significant (p >0.05). When the derived larvae from pepsin and papaya skin extract digestion were cultivated in BME medium for 7 days, the survival rates were not significantly different either (p >0.05). Thus, papaya skin extract might be another choice for recovering Gnathostoma spinigerum third-stage larvae.

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.

Case report: intraocular gnathostomiasis in Vietnam.

This is the report of the first case of intraocular gnathostomiasis diagnosed in Vietnam. The disease progressed in two months in two distinct phases: in the first phase, the patient had swellings, which appeared at different times and in different locations, on his face--this phase lasted around one month; the second phase was the embedding of the parasite in the vitreous cavity of the right eye and uveitis. Surgical extraction of a living Gnathostoma larva was carried out. Based on morphological, histological criteria, the larva may have been an atypical third-stage of Gnathostoma spinigerum.

Various morphologic features of Gnathostoma spinigerum in histologic sections: report of 3 cases with reference to topographic study of the reference worm.

Gnathostomiasis is common in Southeast Asian countries and can be found sporadically in other parts of the world mainly due to human migration. The definitive diagnosis can be given either by identification of the parasite isolated from the patient or through histologic section of the lesion. It is therefore important for pathologists to be familiar with the morphology of parasitic larvae which varies according to the levels of section-cutting so that the diagnosis will not be misled. We present three cases of gnathostomiasis with different features of parasitic morphology and compare these with the reference adult worm.

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.

Intraocular gnathostomiasis.

We report a rare case of intraocular Gnathostomiasis, where a live worm, intracameral in location, was successfully removed. Its identity was confirmed by microscopy.

Immunohistochemical localization of Gnathostoma spinigerum larval antigens by monoclonal antibodies: I. Light microscopy.

Immunohistochemical localization of antigens in advanced third-stage larvae of Gnathostoma spinigerum (GsAL3) was studied by indirect enzyme immunostaining using 7 G. spinigerum specific monoclonal antibodies, FS-3D11, SS-5H5, SK-6C4, SK-4E1, SK-7G6, SD-8D4 and SA-9B5. All these MAb belong to the IgG1 subclass and only FS-3D11 and SS-5H5 recognize carbohydrate determinants. Each MAb exhibited a different reaction pattern and staining intensity in sectioned GsAL3. FS-3D11 bound primarily to the intestinal brush border whereas SS-5H5 reacted with various tissues of the parasite including intestinal epithelium and brush border, lateral cords, muscle, pseudocoel, and cuticle. SK-6C4 predominantly stained muscle, however, SK-4E1 and SK-7G6 exhibited a lack of labeling. SD-8D4 bound to the cuticle and the lateral cords whereas SA-9B5 reacted primarily with the pseudocoel. These results suggest that antigens sharing common epitopes are present in various structures of the larvae with the intestine being the most antigenic site. The present data also suggest that certain GsAL3 antigens recognized by the MAb obtained in this study are sensitive to formalin fixation and/or paraffin embedding since for 2 out of the 7 MAb staining was negative.