Immunoblot evaluation of the specificity of the 29-kDa antigen from young adult female worms Angiostrongylus cantonensis for immunodiagnosis of human angiostrongyliasis.

The antigenic components of Angiostrongylus cantonensis young adult female worm somatic extract (FSE) were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The sera tested were from patients with proven angiostrongyliasis, other parasitic diseases, and healthy adults. Both the sera and cerebrospinal fluid (CSF) were tested from patients with clinical angiostrongyliasis. The CSF from patients with other neurological diseases were also included. Using SDS-PAGE, we found that the FSE comprised more than 30 polypeptides. Immunoblot analysis revealed at least 12 or 13 antigenic bands in patients with proven or clinical angiostrongyliasis, respectively. The patterns of reactivity recognized by the serum and CSF antibodies against FSE were similar. These antigenic components had molecular masses ranging from less than 14.4 to more than 94 kDa. The prominent antigenic band of 29-kDa might serve as a reliable marker for the diagnosis of angiostrongyliasis. The sensitivity, specificity, positive and negative predictive values of immunoblot analysis in this antigenic band were 55.6%, 99.4%, 83.3% and 97.4%, respectively.

Affinity purified oval antigen for diagnosis of Opisthorchiasis viverrini.

Monoclonal antibodies (MAb) were raised against an oval antigen of the liver fluke Opisthorchis viverrini which is the causative agent of a parasitosis, i.e. opisthorchiasis in Thailand. The antibodies were used in an affinity column to purify the O. viverrini oval antigen from a crude extract of adult parasites by chromatography. The oval antigen was then used in a membrane (dot) ELISA for detecting antibodies in serum samples of parasitologically confirmed Opisthorchis viverrini infected individuals (adult parasites were found in stools after praziquantel treatment and salt purgation), as well as of individuals infected with other parasites and parasite-free controls. The MAb-based dot-ELISA using the affinity purified O. viverrini oval antigen revealed 100% sensitivity, specificity and accuracy for detecting O. viverrini infection. The test is simple, rapid and highly reproducible. Several samples can be tested at the same time without the requirement for special equipment or much increase in testing time; thus it is suitable for mass screening for O. viverrini exposure, especially in new endemic areas. Furthermore using serum specimens could increase patient and community compliance compared to the conventional parasitological survey which uses stool samples for the detection of O. viverrini ova, without treatment and subsequent salt purgation, this conventional method shows a low sensitivity and is also unpleasant to both the sample donors and the laboratory technicians which has historically shown a further negative impact on the final outcome.

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.

Gnathostoma spinigerum: analysis of protein patterns by two dimensional gel electrophoresis.

The protein extracts from male (MS) and female (FS) adults and advanced third-stage larvae (LS) of Gnathostoma spinigerum were separated by high resolution two-dimensional gel electrophoresis (2-DE). The polypeptide spots, as detected by silver staining, were subsequently identified. The spot patterns of LS, MS and FS were highly complex and consisted of more than 75, 44, 52 prominent spots, respectively. In addition, the stage-specific protein patterns were identified. This 2-DE database should provide an important reference for future biological and biochemical studies of G. spinigerum.

Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis.

Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.

Seroprevalence of specific total immunoglobulin (Ig), IgG and IgM antibodies to Toxoplasma gondii in blood donors from Loei Province, Northeast Thailand.

A total samples from 345 healthy blood donors from Loei Province, Northeast Thailand were examined for anti-Toxoplasma gondii antibodies by ELISA. The seroprevalence of the anti-Toxoplasma gondii total Ig, IgG and IgM antibodies was 4.9%, 4.1% and 4.3%, respectively. Overall seropositive rate was 33 out of 345 individuals (9.6%). Among the seropositve cases, 5 (15.2%), 2 (6.1%) and 13 (39.4%) of the samples were determined by using each type of anti-T. gondii total Ig, IgG and IgM antibodies, respectively. The seropositive sera was also determined by combining of anti-T. gondii antibodies (anti-T. gondii total Ig with IgG and anti-T. gondii total Ig with IgM antibodies). These results were 10 (30.3%) and 2 (6.1%) cases, respectively. Only one (3%) sample had all types of anti-T. gondii antibodies. In addition, the frequency distribution curves of ELISA optical densities of anti-T. gondii total Ig, IgG and IgM antibodies in blood donor presented "unimodal" curves. The negative results were found in the age group that less than 20 years old and more than 51. The highest seropositive results were found in two age groups (21-30 and 31-40 years old), and males were significantly higher than female (p < 0.05). These results demonstrated that when using anti-T. gondii total Ig, IgG and IgM antibodies for determining the seroprevalence, the sensitivity was twice that with the anti-T. gondii, total Ig antibody alone.

Scanning electron microscopy of newly excysted metacercariae of Paragonimus westermani-like, Nakhon Nayok, Thailand.

Scanning electron microscope (SEM) observation of surface structure was done on newly excysted metacercariae of Paragonimus westermani-like (Nakhon Nayok, Thailand). The surface of the body was covered with numerous single-pointed tegumentary spines, large dome-shaped papillae, small one with a pit, and small one with a smooth surface were situated around the suckers. There were 27 to 30 of the small dome-shaped papillae with a pit around the oral sucker and 10 to 13 of the small ones with a smooth surface around the ventral sucker. The present report is the new record of excysted metacercariae of P. westermani-like (Nakhon Nayok, Thailand) by SEM.

Excretory-secretory antigenic components of adult Fasciola gigantica recognized by infected human sera.

The immunogenic components of Fasciola gigantica excretory-secretory (ES) products were revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting technic using sera from patients with F. gigantica infection, from patients with clinical suspected fascioliasis, from patients with other illness and from healthy adults. By SDS-PAGE, it was found that the ES products comprised more than 6 polypeptides. Immunoblotting analysis revealed 12 components which were strongly recognized by fascioliasis antisera. These antigenic components had a molecular mass ranging from less than 14.4 to 38 kDa. One antigenic band of 27 kDa was found to give a consistent reaction with fascioliasis antisera (100% sensitivity and 98% specificity). The present findings suggest that the 27 kDa components are sensitive and specific for the diagnosis of human F. gigantica infection.

Antigenic components of somatic extract from adult Fasciola gigantica recognized by infected human sera.

The antigenic components of Fasciola gigantica somatic extract were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting technique using sera from patients with F. gigantica infection, patients with clinically diagnosed fascioliasis, patients with other infections/illness and healthy adults. By SDS-PAGE, it was found that the somatic product comprised more than 22 polypeptides. Immunoblotting analysis revealed at least 13 components which were strongly recognized by sera of patients with fascioliasis. These antigenic components had molecular weights ranging from less than 14.4 to more than 94 kDa. One antigenic component, i.e. 38 kDa was found to give a consistent reaction with sera of patients with fascioliasis (100% sensitivity and 96.7% specificity). The finding suggests that the 38 kDa components may be a potential diagnostic antigen for fascioliasis.

A dot-ELISA test using monoclonal antibody-purified antigens for the diagnosis of paragonimiasis caused by Paragonimus heterotremus.

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody-affinity chromatography was developed for detecting antibodies to Paragonimus heterotremus in four groups of subjects. They consisted of 30 patients with P. heterotremus infection, 93 patients with other parasitic infections, 18 patients with pulmonary tuberculosis and 30 normal, healthy controls. Sensitivity, specificity, as well as positive and negative predictive values of the test were 100, 97, 88, and 100%, respectively.

Recent advances in diagnosis of paragonimiasis.

Paragonimiasis in endemic areas can be diagnosed by clinical symptoms. However, the diagnosis should always be confirmed by microscopic examination of the sputum or stool in order to find Paragonimus eggs. Within recent years marked advances in diagnosis of paragonimiasis have been made. Two new approaches comprising a genetic probe and immunological tests have been developed with claims to be as good or better than microscopic examinations. This report reviews these two areas, especially in paragonimiasis caused by Paragonimus heterotremus and P. westermani. In addition, problem areas in assay development are discussed.

Specific monoclonal antibodies to Fasciola gigantica.

Monoclonal antibodies (mAbs) were produced against soluble metabolic products (excretory/secretory; ES) of the liver fluke, Fasciola gigantica. The ES antigen was obtained from spent culture medium in which the adult flukes had been maintained in vitro. From two cell fusions, the mAbs produces were exclusively associated with either IgG or IgM isotypes. When screened against a panel of homologous and heterologous parasite antigens by indirect ELISA, two mAbs were highly specific for F. gigantica. The remainder cross-reacted extensively with other parasites. Results from immunoblotting and immunofluorescence exhibited two patterns of reactivity. The first group of mAbs which recognized the multiple bands between > 14.4-27.5 kDa gave extremely bright fluorescence over all major muscular systems, vitelline gland, testes and intestinal caeca. The second group which reacted with the 185 kDa band showed a bright fluorescence over a thinner muscular layer underlying the tegument, intestinal caeca and testes.

Comparison of adult somatic and excretory-secretory antigens in enzyme-linked immunosorbent assay for serodiagnosis of human infection with Fasciola gigantica.

Adult somatic antigen extract of Fasciola gigantica was compared with excretory-secretory (ES) antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of human fascioliasis gigantica. The absorbance values in ELISA using the adult somatic antigen were not significantly different from the values obtaining using ES antigen (p > 0.05). The diagnostic sensitivity, specificity and positive and negative predictive values of the test using adult somatic extract as antigen were 100%, 98%, 70% and 100%, respectively. On the other hand, these values of the test using adult ES antigen were 100%, 99.3%, 87.5% and 100%, respectively. It appears that both somatic and ES antigens are effective antigens for use in the serodiagnosis of human fascioliasis gigantica.

Partially purified antigens of Paragonimus heterotremus for serodiagnosis of human paragonimiasis.

The preparative crude extract of Paragonimus heterotremus was fractionated by isoelectric focusing. Fractions at pH 5 which contained a specific antigen with a relative molecular weight of 31.5 kDa were pooled and used in an indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis for diagnosis of human paragonimiasis. The sensitivity and specificity of ELISA were found to be 100% and 99% respectively. The band of 31.5 kDa antigenic component was found to give consistent reaction with paragonimiasis sera. The sensitivity, specificity and predictive value (positive and negative) of immunoblot analysis for the 31.5 kDa band were all 100%.

Analysis of antibody levels before and after praziquantel treatment in human paragonimiasis heterotremus.

Enzyme immunoassays (ELISA) and Western blot analysis were used to determine IgG antibody levels in patients infected with Paragonimus heterotremus from Thailand before and after treatment with praziquantel. An IgG antibody ELISA showed that a substantial reduction of antibody levels occurred after one year of treatment. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed that P. heterotremus adult extract is highly complex, consisting of more than 9 antigenic bands with molecular size ranging from 123 kDa to less than 12.3 kDa. Two prominent bands of 31.5 kDa and 18.5 kDa were found to show consistent reactions with all serum samples from the pretreatment group. There was a marked reduction in the intensity of the reaction of the 31.5 kDa band with each serum sample from post-treatment patients but the other bands disappeared during the one year interval.

Prevalence and intensity of Opisthorchis viverrini in rural community near the Mekong River on the Thai-Laos border in northeast Thailand.

The prevalence and intensity of Opisthorchis viverrini in fourteen villages in Nakhon-Phanom province, Northeast, Thailand have been investigated. Overall prevalence of O. viverrini infection was 66.4 per cent in a total population of 2,412 individuals. The prevalence was 18.5 per cent in children under 5 years, 38.9 per cent in those aged 5-9 years, and ranged from 64.9 per cent to 82.2 per cent in the age group above 10 years. The intensity of O. viverrini infection increased with age. The mean faecal egg output was highest in the 30-34 year age group and remained relatively constant through older ages. In all age groups the prevalence and intensity of infection in both men and women were similar. The population was divided according to the presence and intensity of infection as follow, 33 per cent were uninfected, 59 per cent had light infections (less than 1,000 eggs per g of faeces; EPG), 7 per cent had moderate infections (1,000-10,000 EPG), and 1 per cent had heavy (greater than 10,000 EPG). Other important intestinal infections found in this community are hookworm, Taenia spp. and Trichuris trichiura with the prevalence of 17.9 per cent, 1.1 per cent and 1.1 per cent 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.