Eosinophilic meningitis caused by Angiostrongylus cantonensis: report of 17 cases.

PURPOSE: To describe two outbreaks of Angiostrongylus cantonensis infection that occurred in Kaohsiung, Taiwan, during 1998 and 1999, and to characterize the source of the outbreaks and the clinical manifestations of the disease. SUBJECTS AND METHODS: We performed a retrospective cohort study among Thai laborers with eosinophilic meningitis who ate raw snails (Ampullarium canaliculatus), as well as an environmental surveillance of larvae in snails. RESULTS: We enrolled 17 Thai laborers in whom severe headache and eosinophilia developed within 4 to 23 days after eating raw snails. Twelve (71%) developed eosinophilic meningitis. Third-stage larvae were found in the cerebrospinal fluids of 2 patients and in all 12 tested snails. Specific antibodies to A. cantonensis were detected in serum from 16 of the patients and in cerebrospinal fluid from 5 of the patients. Central nervous system manifestations included headache (n = 17 [100%]), fever (n = 11 [65%]), Brudzinski's sign/stiff neck (n = 11 [65%]), hyperesthesia (n = 3 [18%]), cranial nerve palsy (n = 2 [12%]), diplopia (n = 2 [12%]), and ataxia (n = 1 [6%]). Laboratory findings included peripheral eosinophilia (n = 15 [88%]) and cerebrospinal fluid eosinophilia (n = 12 [71%]); elevated immunoglobulin (Ig) E levels (n = 13 [100%]); and transient increases in white blood cell count (n = 7 [41%]) and in serum levels of creatine kinase (n = 7 [41%]), transaminase (n = 3 [18%]), and lactate dehydrogenase (n = 2 [12%]). The severity of illness and eosinophilia were correlated with the number of ingested snails. Meningeal and basal ganglion enhancement was noted on magnetic resonance imaging in several patients. Treatment with mebendazole combined with glucocorticosteroids appeared to shorten the course of the infection, but not the number of relapses. The eosinophil count fell to normal within 3 months, but IgE levels remained elevated for as long as 6 months. All patients recovered with minimal neurologic sequelae. CONCLUSION: Eosinophilic meningitis caused by A. cantonensis should be considered in patients who have headache or central nervous system manifestations after eating raw snails.

Detection of antibodies to Angiostrongylus cantonensis in serum and cerebrospinal fluid of patients with eosinophilic meningitis.

Adult and young adult antigens of Angiostrongylus cantonensis were purified by immuno-affinity chromatography and used to detect antibody in serum and cerebrospinal fluid (CSF), by enzyme-linked immunosorbent assay (ELISA), in cases of human eosinophilic meningitis or meningoencephalitis. The levels of IgG, IgA, IgM and IgE antibodies to A. cantonensis in these patients were higher than levels in control subjects. Antibodies in patients detected against adult and young adult worm antigens of A. cantonensis did not differ significantly. Significantly higher IgM and IgE antibody levels were observed in serum compared with CSF from infected patients (Student's t-test, P less than 0.05). Both adult and young adult A. cantonensis antigens proved to be highly sensitive in ELISA for serum antibodies; however, the sensitivity was significantly lower in tests on CSF.

Detection of circulating antigen by monoclonal antibodies for immunodiagnosis of angiostrongyliasis.

Two monoclonal antibodies, which recognize the epitope on an antigen with a molecular weight of 204 kD from the fifth-stage worm of Angiostrongylus cantonensis, were previously prepared and used to detect circulating antigens in patients with eosinophilic meningitis or meningoencephalitis and in mice experimentally infected with this parasite by a double-antibody, sandwich enzyme-linked immunosorbent assay (ELISA). The levels of this circulating antigen in experimentally infected mice were significantly higher three weeks after infection. The ELISA values in the detection of circulating antigens in cerebrospinal fluid (CSF) from patients were markedly higher than those in serum. Immunodiagnosis of patients with angiostrongyliasis by this technique proved to be highly specific for circulating antigens in serum and CSF specimens; however, the sensitivity in CSF was significantly higher than in serum.

Immunodiagnosis of human eosinophilic meningitis using an antigen of Angiostrongylus cantonensis L5 with molecular weight 204 kD.

An antigen from Angiostrongylus cantonensis fifth-stage larvae was purified by immuno-affinity chromatography with a specific monoclonal antibody. The purified antigen showed only a single band with a molecular weight of 204 kD in SDS-PAGE, and no cross-reactivity to antibodies induced by several other species of helminths were observed in ELISA. When the purified antigen was used to examine serum and cerebrospinal fluid (CSF) specimens by ELISA, the antibody levels in patients with eosinophilic meningitis or meningoencephalitis (EME) were significantly higher than those of control subjects. The antibody levels in serum were slightly higher than those in CSF, and the levels in serum were positively correlated with the levels in CSF. The reliability in detection of antibodies in serum was slightly higher than that in the detection of antibodies in CSF specimens. The purification of a specific A. cantonensis antigen and its subsequent use in the development of an ELISA for detection of A. cantonensis specific antibodies in serum specimens constitute an important step towards improvement in the accuracy of diagnosis for A. cantonensis infections.

An outbreak of meningitis caused by Angiostrongylus cantonensis in Kaohsiung.

Eight Thai laborers developed meningitis after eating raw snails (Ampullarium canaliculatus) during the period from September 27 to October 6, 1998. The diagnosis of Angiostrongylus cantonensis infection was established in all patients by serologic studies of serum and cerebral spinal fluid (CSF). Clinical manifestations included meningitis, radiculitis and cranial nerve palsy. Symptoms included fever, headache, orbital pain, gastrointestinal upset, hyperesthesia, muscle weakness, skin rash and diplopia. Laboratory abnormalities included peripheral eosinophilia, CSF eosinophilia, transient elevation of liver enzymes and creatinine phosphokinase, elevation of IgE. No space occupying lesions were detected by magnetic resonance imaging of the brain. None of the patients developed severe sequelae during the 6-month follow-up except for occasional headache in one patient. This report also provides evidence that third stage larvae were present in the intermediate host, A. canaliculatus, which the laborers had eaten.

[Immunological screening of clonorchiasis].

Crude antigens of Clonorchis sinensis adult worms were purified by Sephadex G-200 column chromatography. A purified antigen was then applied to detect clonorchiasis patients and parasite-free subjects by an enzyme-linked immunosorbent assay (ELISA) and an intradermal test (IDT). The results of the ELISA showed that specific antibody levels of IgG, IgM, IgA, IgE and total IgE in 371 infected patients were significantly higher than those of 181 parasite-free subjects (Student's t-test, p less than 10(-6) or p less than 0.005). The sensitivity was 92.8% and the specificity was 89.0%. Significant correlations between the intensity of infection, expressed as egg per gram feces, and specific IgG, IgA and total IgE concentration were observed (linear regression, p less than 0.0005). Among the 309 subjects tested intradermally with Clonorchis antigen, 128 showed a positive reaction while all of the 60 parasite-free subjects showed negative results. By this test, the sensitivity was 49% and the specificity of IDT was 100%. The specific IgG, IgA and total IgE antibody levels of the IDT positive patients were significantly higher than those of the negative subjects (Student's t-test, p less than 0.0005). There was no correlation between the induced swelling diameter and the intensity of infection in IDT positive clonorchiasis patients. Cross reactions in ELISA were observed in patients infected with Capillaria philippinensis, Sparganum proliferum and Toxocara canis, when the antigens prepared in the present study were used.

Characterization of monoclonal antibodies to young-adult worms of Angiostrongylus cantonensis.

Three IgG1 and one IgG2a monoclonal antibodies were obtained by fusion of NS-1 cells with spleen cells of BALB/C mice immunized with young-adult worm antigen of A. cantonensis. These 4 monoclonal antibodies were specific to A. cantonensis without cross-reactivity to any antigens of Toxocara canis, Ascaris suum, Clonorchis sinensis, Dirofilaria immitis, Paragonimus westermani, Gnathostoma spinigerum, Strongyloides stercoralis and Anisakis spp. detected by ELISA. The affinity constants of monoclonal antibodies specific to young-adult worms antigen of A. cantonensis were in the range of 10(7) M-1 to 10(9) M-1. The immunoblots showed that the 4 monoclonal antibodies recognized epitope on molecular weight of 204 kiloDaltons. They will be utilized to purify their corresponding antigen by an immunoaffinity column for the specific immunodiagnosis.

Counterimmunoelectrophoresis test on human Clonorchis sinensis infection.

A counterimmunoelectrophoresis test was used to detect antibodies against the adult worm antigen of Clonorchis sinensis in sera from 70 clonorchiasis patients, 20 uninfected healthy persons and 7 patients infected with other helminths. A constant voltage of 10 V/cm and a running time of 30 minutes was chosen in carrying out detection. Antibody titers of 1, 1:2 and 1:4 were obtained from 35, 21 and 14 clonorchiasis patients, respectively. Significant correlation was observed between worm burden in patients and antibody titer, the higher the antibody titer in patients, the more eggs per gram feces in their stool. Although cross reaction was observed with toxocariasis and angiostrongyliasis in this study, high (100%) sensitivity made it possible to screen the subjects in endemic areas to shorten the survey period.

Antibodies in serum of patients with clonorchiasis before and after treatment.

Sera of 31 patients infected with Clonorchis sinensis were examined using fraction 1 antigen by ELISA during a one-year observation. The results of ELISA with Igs, IgG and IgA demonstrated high sensitivity (100%, 100% and 90%) and specificity (100%, 100% and 87%). Sera specific Igs and IgG were significantly decreased in the 3rd month after treatment with praziquantel (25mg/kg body weight in one dose), and IgA was significantly decreased in the 1st month (paired t-test, p < 0.05). No eggs were found in the stool after treatment. Detection of sera specific Igs, IgG and IgA by ELISA was combined with stool examination to evaluate the effect of praziquantel and the completeness of the cure.

Preparation of monoclonal antibody against Angiostrongylus cantonensis antigen.

Monoclonal antibodies (MAb) against A cantonensis were produced through fusion of immunised spleen cells from BALB/c mice with NS-1 myeloma cells at a ratio of 10:1. The successful fusion rate on the 3rd day of fusion was 90.1%. Ten MAb were characterised, six of which were IgG1 and the remaining four were IgG2a, IgG2b, IgM and IgA respectively. Among 6 IgG1 MAb, four were A. cantonensis-specific, of which three reacted to adult worm antigen only and one reacted to both adult worm and juvenile worm antigens. Two other IgG1 MAb showed cross-reaction with other helminthic antigens of Toxocara canis. Ascaris suum. Paragonimus westermani, Dirofilaria immitis, Anisakis Spp, Gnatostoma Spinigerum and Clonorchis sinensis.

Humoral immune responses in mice infected with gamma-irradiated third-stage larvae of Angiostrongylus cantonensis.

Humoral immune responses in C57BL/6 strain mice infected with 1.0 kGy gamma-irradiated third-stage larvae (L3) and with non-gamma-irradiated L3 of Angiostrongylus cantonensis were assessed in this study. The young-adult worm (L5) antigen-specific antibodies in sera of both groups of mice elevated gradually and reached a peak in the third week after infection. The IgM antibodies of mice 1-week after infection with gamma-irradiated L3 and with non-gamma-irradiated L3 recognized several L5 antigens of the same molecular weight. However, only partially identical profiles were observed in the reaction of L5 antigens with IgM and IgG antibodies from mice 3 weeks after infection with gamma-irradiated L3 and with non-gamma-irradiated L3 respectively by western blot analysis. With regard to antibody-dependent cell-mediated cytotoxicity, antibodies from 3-week infected mice significantly increased the adherence of mouse eosinophils and neutrophils to L3 of A. cantonensis. The immunoresponses of splenic cells from mice in the third week of infection to sheep red blood cells were suppressed because the number of plaque forming cells obviously decreased more than the uninfected control.

Hepatic Schistosomiasis japonica in a patient with gallstones and bile duct stones--a case report.

Schistosomiasis, a common parasitic disease in many countries, is found as imported cases in Taiwan. Responsible for human infections are five species, one of which, Schistosoma japonicum, is currently endemic in China and South-east Asia. Chronic infection with S. japonicum may lead to the development of liver fibrosis, calcification and portal hypertension. Under investigation by sonography and computed tomography (CT) scan, a peculiar "turtle-back" appearance of liver fibrosis and calcification may be found. Herein, we report a case referred to our department due to jaundice. The sonography of liver showed typical "turtle-back" appearance. Gallstones and bile duct stones were also found in this case. Surgical interventions with percutaneous transhepatic biliary drainage (PTBD), cholecystectomy and choledocholithotomy were performed to relieve the obstructive jaundice and remove the stones. There were no parasitic eggs in the extracted stones or in drained bile juice. However, deposits of calcified S. japonicum eggs in liver parenchyma and portal tracts were identified in liver biopsy. No special treatment was given for the schistosomiasis japonica because the calcified parasitic eggs were the sequelae of past infection.

[Parasitic infections among wild rats from two areas of Kaohsiung].

Wild rats including four strains were captured for examination of parasitic infections. The numbers were 101 from areas of Kaohsiung harbor and 98 from areas of Kaohsiung airport. Most of the examined wild rats were Rattus norvegicus and second most were Suncus murinus. The infection rates of intestinal helminths in rats of both areas did not display any significant difference. The overall infection rate was 29.6%. The infection rate was highest in R. norvegicus and the most frequently found parasite in rats was Hymenolepis diminuta. Moniliformis moniliformis and Angiostrongylus cantonensis were revealed in two separated R. norvegicus rats. The infection rates of intestinal helminths in R. norvegicus were significantly correlated with the size of rats. There was no find in microscopic observation of blood smear from these rats stained with Giemsa's dye.

A survey on the intestinal parasites of the school children in Kaohsiung county.

The present study on the prevalence of intestinal parasites infection, from September to December 1999, was conducted among school children in Taoyuan Hsiang, Kaohsiung county. The investigated areas included three (Jiannshan, Shingjong and Taoyuan) primary schools. The overall infection rate in 305 children determined by Merthiolate-Iodine-Formaldehyde Concentration method of stools was 17%. Four confirmed species of helminthes (Ascaris lumbricoides, Hookworm, Trichuris trichiura and Hymenolepis nana) and three species of protozoa (Giardia lamblia, Entamoeba coli and Blastocystis hominis) were detected. Males and females had the infection rates of 24% and 11%. The infection rates of aboriginal and non-aboriginal children were 17% and 14%, respectively. Grade 1 and Grade 6 had the highest infection rate (21%). Following tape perianal examination of 302 children, the overall infection rate of Enterobius vermicularis was 25%. Males and females had the infection rates of 24% and 26%. The infection rates of aboriginal and non-aboriginal children were 27% and 11%, respectively. Grade 1 had the highest infection rate (37%). Based on these data, the infection rate of intestinal parasites among rural primary school children in Kaohsiung county remains high.

Topology of feather melanocyte progenitor niche allows complex pigment patterns to emerge.

Color patterns of bird plumage affect animal behavior and speciation. Diverse patterns are present in different species and within the individual. Here, we study the cellular and molecular basis of feather pigment pattern formation. Melanocyte progenitors are distributed as a horizontal ring in the proximal follicle, sending melanocytes vertically up into the epithelial cylinder, which gradually emerges as feathers grow. Different pigment patterns form by modulating the presence, arrangement, or differentiation of melanocytes. A layer of peripheral pulp further regulates pigmentation via patterned agouti expression. Lifetime feather cyclic regeneration resets pigment patterns for physiological needs. Thus, the evolution of stem cell niche topology allows complex pigment patterning through combinatorial co-option of simple regulatory mechanisms.

[Immune responses in rabbits infected with Clonorchis sinensis].

Experimental rabbits were divided into single infection group, reinfection group and treatment group. Both eggs per gram of feces and serum antibody were determined periodically by antiformin-ether concentration method and ELISA technic, respectively. Two rabbits were observed 72 weeks and 76 weeks after infection in single infection occasion. Antibody elevated to the maximum during 8 and 14 weeks after infection, and then gradually decreased but still kept in detectable level till the termination of observation. EPG kept in lower intensity and no apparent variation during the period of observation. In periodically re-infected groups, both antibody and EPG increased after every occasion of re-infection. But in rabbits re-infection every month, its antibody were maintained in a high level and kept this level even at the 5th to 7th re-infection. In general, EPG increased less rapidly after several times re-infection. Stool examinations showed negative results, but in antibody variations were not concordant in rabbits after treatment. In un-infected rabbits, antibody kept in negative reaction during the course of observation.

[Human clonorchiasis survey in Taiwan and its immunodiagnostic technics].

In a series of control study on human clonorchiasis, surveys had been carried out for four years. During the fourth year of study, immunodiagnosis was also tried. Skin test and stool examination were performed as a routine epidemiological survey in this study. The result of skin test revealed that 7 out of 738 primary school children and 2 out of 274 inhabitants in Tong-ho Township, Taitung County showed positive reaction. Four out of 500 children and none of 9 teachers tested from the primary school of Nan-hua Township, Hualien County were positive. For stool examinations, only one was found with Clonorchis eggs among 34 children from Nan-hua school, Hualien County, but none of 549 children from Tong-ho school, Taitung County was found with eggs. When stool examination was performed to those who had history of eating raw fresh water fish, positive cases increased in numbers, such as in Pingtung County, 19 out of 96 children and in Meinung Township of Kaohsiung County, 52 out of 86 children and 14 out of 19 teachers were shown positive for Clonorchis eggs. Immunodiagnosis by means of ELISA had been carried out in 72 sera of clonorchiasis patients and CFT, CIE in 30 sera. Results showed that ELISA detectable antibody titer was between 1,600 and 25,600, CFT, between 16 and 128 and CIE, under 4. In 8 sera of non-infected and 6 infected with various intestinal helminths, negative reactions were obtained from each technic.

[Immunodiagnosis of rabbit clonorchiasis by enzyme-linked immunosorbent assay].

Enzyme-linked immunosorbent assay (ELISA) was successfully applied to the immunodiagnosis of parasitic disease in 1974 by Engvall and Perlmann. In our study, this technic was used to test rabbits infected with Chonorchis sinensis metacercariae experimentally. The optimal conditions for the test were determined and summarized as follows: 1. The optimal concentration of VBS antigen was 4 micrograms/ml, showing the highest O.D. in the detection of ELISA test. 2. Four hundred-fold diluted alkaline phosphatase conjugated goat antirabbit IgG (Sigma company) was selected for the test. 3. One hundred times diluted sera of 15 pre-infected rabbits were tested by ELISA and their mean and standard deviation of O.D. was 0.083 +/- 0.004, which was used as the cut-off value for testing post-infected rabbits. Fifteen rabbits were divided into two groups. Group A and B rabbits were infected one hundred and three hundred metacercariae respectively. Sera and stools were collected from all rabbits before and after infection. In serum examination, antibody was detectable by ELISA test during seven and twenty-one and twenty-eight days after infection. Optical density of fifteen infected rabbits detected by ELISA was significantly higher than before infection. ELISA test was successfully applied to the immunodiagnosis of Clonorchis sinensis infected rabbits.

A survey of Ampullarium canaliculatus for natural infection of Angiostrongylus cantonensis in south Taiwan.

Ampullarium canaliculatus were collected monthly from the field in five areas in Pintung County and examined for larvae of Angiostrongylus cantonensis. Snails from one area showed the highest positive rate (30.8%) and the highest number (31) of larvae per snail. Snails were placed in four groups according to size. The positive rates in snails increased from 10.0 to 29.4% and the average number of larvae per snail increased from 11 to 40 as the size of snails increased from 1.0 to 4.0 cm height or more. In the seasonal fluctuation of snails in the field, positive snails could be examined every month; however, the positive rate in snails (28.2%) and the average number of larvae per snail (33) in the dry season (November to April) were significantly higher than those (15.2% and 23 respectively) in the rainy season (May to October). Ampullarium canaliculatus were confirmed naturally infected with A. cantonensis in this investigation so more attention should be paid to the snails to prevent the infection in humans.

[Immunodiagnosis of human clonorchiasis by a micro-enzyme-linked immunosorbent assay].

A micro-enzyme-linked immunosorbent assay (ELISA) utilizing antigen from lyophilized Clonorchis sinensis adult worms extracted with triethanolamine buffered saline (TBS), veronal buffered saline (VBS) or phosphate buffered saline (PBS) were used to test 66, 31 and 9 sera from clonorchiasis patients, uninfected normal humen and patients with other parasitic infections, respectively. Four different combinations of incubation time between serum with antigen and serum with conjugate at either 37 degrees C or room temperature were tried. The combination of 6 hours and 16 hours reaction time processed at room temperature with shaking was chosen in this study because it revealed no pseudo-negative and cross reactions with high sensitivity, specificity and predictive value. There were no statistic difference among mean optical densities in micro-ELISA reactions of 66 sera (in 800 X dilution) from clonorchiasis patients with TBS, VBS or PBS antigen, respectively, but results obtained from TBS antigen seemed better than other two antigens. Good correlation was observed between the antibody titer or ELISA readings of sera form clonorchiasis patients and their worm burden expressed as eggs per gram of feces (EPG).