Methylene blue staining method for identification of Opisthorchis viverrini egg.

Methylene blue staining method was used to distinguish O. viverrini eggs from Haplorchis taichui and Prosthodendrium molenkampi eggs. All eggs were obtained from dissected adult worms, fixed in 10% formalin, and stained with methylene blue prior to light microscopy observation. The distinct musk-melon-like texture of the O. viverini eggshell surface and the thread-like texture of H. taichui eggshell surface were recognized, while P. molenkampi eggs showed a smooth eggshell. We also evaluated the sensitivity and specificity of the method by training investigators to differentiate surface textures. After training, the investigators were randomly tested with 10 slides containing fluke eggs. The sensitivity and specificity were 95% and 95%, respectively.

Studies on concomitant antigens of Bithynia funiculata for detection of antibody to Opisthorchis viverrini: effect of different centrifugal speeds on antigen preparation.

Antigens derived from somatic extracts of Bithynia funiculata, an intermediate snail host of O. viverrini, have been demonstrated to be highly heterogeneous in molecular weight (MW). These antigens have been suggested to be of potential use for serodiagnosis. In this study, B. funiculata somatic antigens were extracted using five different centrifugal speeds, namely 10,000 (C1); 20,000 (C2); 30,000 (C3); 40,000 (C4) and 50,000 (C5) rpm, with the aim of removing some non-specific antigens and determining the optimal centrifugal speed to obtain the highest efficiency of the test for which they will be used. The enzyme-linked immunosorbent assay technique was used to compare the reactivity of the five different centrifugal speed-prepared antigens. The sensitivity and specificity of all five antigens were compared by testing against sera from 81 opisthorchiasis patients, 30 parasite-free healthy individuals and 50 individuals infected with other helminthic infections, using mean + 4SD of all healthy individuals as the cut-off value. The sensitivity of these antigens was 69.1, 84.0, 80.2, 84.0 and 70.4, respectively; while the specificity was 66.2, 76.2, 82.5, 86.2 and 71.2, respectively. Immunoreactive components of each antigen were analyzed by SDS-PAGE and Western blot technique. The assay showed that three pairs of antigens with MW of 29 and 30, 47 and 50, and 86 and 90 kDa of all five antigens, which have previously been shown to be highly immunogenic, still reacted with a pooled serum from patients with opisthorchiasis. However, the C4 antigens gave more distinct components. Our results showed that 40,000 rpm is the optimal speed for antigen preparation for use in the serological diagnosis of opisthorchiasis, as demonstrated by the most satisfactory results of both sensitivity and specificity in the indirect ELISA and Western blot technique.

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda)

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.

The diagnosis of human opisthorchiasis.

Opisthorchiasis viverrini is a liver fluke infection causing a serious public health problem in Thailand, Lao PDR, Cambodia, and South Vietnam because it acts as a strong promoter of cholangiocarcinoma. The diagnosis of human opisthorchiasis is based on four approaches: clinical manifestations, parasitological, molecular biological, and immunological methods. These methods have advantages and disadvantages. Clinical manifestations of the patients are practically indistinguishable from those of other liver diseases. The features of the O. viverrini eggs are, by light microscopy, difficult to differentiate from those of other minute intestinal flukes' eggs. Polymerase chain reaction (PCR) is very complicated, needs special and expensive apparatus, and is time-consuming; it is, however, highly sensitive and specific. Immunological testing is the method of choice: the techniques are applicable to both routine laboratory work and field or epidemiological studies. Of these tests, enzyme-linked immunosorbent assay (ELISA) and immunoelectrotransfer blot assay are often used for the detection of O. viverrini-specific antigens (coproantigens) and antibodies (IgM, IgG, IgA, or IgE). Monoclonal antibodies are prepared to detect coproantigens, while the crude somatic and excretory-secretory antigens from the adult worms, metacercariae, eggs, and snail intermediate hosts are prepared in order to detect antibodies in sera. To eliminate the cross reactions between parasites, the appropriate amount, type, and efficacy of antigens or antibodies preparation should be considered. In this paper, the advantages and disadvantages of the four diagnostic methods are discussed.

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.

Potassium permanganate staining for differentiation the surface morphology of Opisthorchis viverrini, Haplorchis taichui and Phaneropsolus bonnei eggs.

Potassium permanganate staining method was developed for differentiation Opisthorchis viverrini, Haplorchis taichui and Phaneropsolus bonnei eggs. The surfaces of O. viverrini, H. taichui and P. bonnei eggs stained permanently and temporarily were similar in appearance even the staining procedures were varied both in concentration and time. Determined under light microscope set at 400x, all of these eggs were oval-shaped, operculated at one pole and indistinct small knob at posterior end. O. viverrini eggs showed the distinct musk-melon-like prominent ridges on the surface. Haplorchis taichui eggs had a light striae pattern while P. bonnei eggs had a smooth egg shell. Length of these trematode eggs were significant different (chi2 test, p < 0.05). Mean +/- SD of O. viverrini, H. taichui and P. bonnei eggs were 26.34 +/- 1.65 microm, 29.03 +/- 1.48 microm and 23.00 +/- 1.49 microm, respectively. Regarding of their width, the mean +/- SD of O. viverrini, H. taichui and P. bonnei eggs were 15.54 +/- 0.69 microm. 14.94 +/- 0.91 microm and 12.25 +/- 1.02 microm, respectively. The means of width of O. viverrini and H. taichui eggs were not significantly different (chi2 test, p > 0.05), however, they were significantly different from those of P. bonnei (chi2 test, p < 0.05). Temporary staining using 1% w/v concentration and only 1 minute of time is useful in the mass fecal examination survey for the prevalence and intensity of truly Opisthorchis infection.

Evaluation of Bithynia funiculata snail antigens by ELISA-serodiagnosis of human opisthorchiasis.

Four batches of crude somatic antigens from: (1) Opisthorchis viverrini adult worms, (2) Bithynia funiculata-whole body, (3) B. funiculata-head-foot, and (4) B. funiculata-visceral mass were assayed against sera from 81 opisthorchiasis patients, 30 parasite-free healthy individuals, and 50 individuals infected with other helminthic infections, and their antibody levels determined. By IgG-ELISA, the antigenic reactive proteins were found in both the head-foot and the visceral mass of B. funiculata snails, but the whole snail antigens gave the best results. Furthermore, it was as good as when O. viverini antigens were used. Antibody levels of sera from patients with opisthorchiasis assayed against antigens from whole B. funiculata snails were significantly higher than those of the other two groups. The cut-off value for positivity at 0.228 gave 80.2% sensitivity and 81.2% specificity. Cross reactions were observed with sera from patients with paragonimiasis and strongyloidiasis. No cross reactions were found to occur with sera from healthy individuals.

Parasites elicited cross-reacting antibodies to Opisthorchis viverrini.

Two batches of crude antigens extracted from adult Opisthorchis viverrini worms were compared. One was derived from adult worms harvested from the livers of laboratory infected hamsters and another was obtained from worms sedimented from the faeces of opisthorchiasis patients following treatment with Praziquantel. SDS-PAGE and Coomassie brilliant blue staining revealed that the two preparations had similar protein components of which the predominant ones were the 17-18 kDa doublet. The antigens were used in an indirect ELISA for the detection of antibodies against O. viverrini in the sera of four groups of patients, ie. patients with opisthorchiasis (group 1), patients with mixed infections of O. viverrini and other parasites (group 2), patients with other parasitic infections (group 3), and normal-heathy, parasite-free individuals (group 4). The sensitivity of the test was high (91-92%), regardless of the batch of the antigen used. However, its specificity was relatively low (70-80%). Cross-reaction was observed with patients infected with Paragonimus heterotremus, Schistosoma spp.; Taenia spp.; Trichinella spiralis; Strongyloides stercoralis; hookworms; Plasmodium spp.; hookworms and Plasmodium spp.; S. stercoralis, Blastocystis hominis and yeast; and hookworms, Ascaris lumbricoides, Trichuris trichiura and P. falciparum. Western blot analysis revealed that sera of patients infected with these heterologous organisms contained antibodies reactive to O. viverrini antigenic components ranging from Mr 15.5 to 144.

Immunodiagnosis of opisthorchiasis.

Monoclonal antibody-based enzyme-linked immunosorbent assay and DNA dot blot hybridization techniques were developed and evaluated for their potential in the detection of Opisthorchis viverrini. A mixture of IgG monoclonal antibodies specific for the 89 kDa metabolic product of O. viverrini was captured on a microtiter plate by rabbit anti-mouse IgG and used in a sandwich ELISA for the detection of soluble parasite antigen in the feces of patients with opisthorchiasis. As little as 0.1 ng of the antigen could be detected. A specific O. viverrini DNA probe was used in a dot blot hybridization of parasite DNA. The labeled probe could detect DNA released from as few as five O. viverrini eggs. Both approaches were highly specific for O. viverrini and their sensitivity appeared to be comparable with that of the classical parasitological method. Preliminary data obtained from a field trial showed that these two methods have potential in the diagnosis of opisthorchiasis. Moreover, the limited data currently available showed that it is possible to use these methods to detect the presence of O. viverrini metacercariae in naturally infected fish.

Diagnosis of opisthorchiasis by enzyme-linked immunosorbent assay using partially purified antigens.

Opisthorchis viverrini antigens were partially purified from adult worms collected from liver and extrahepatic biliary system of infected hamsters. Tegument fraction was obtained by chemical extraction, whereas other fractions were purified by Sephadex G-200 gel filtration chromatography. Five fractions of O. viverrini antigens were obtained, namely tegument extract, somatic extract, fraction 1 (P1), fraction 2 (P2) and fraction 3 (P3), respectively. The enzyme-linked immunosorbent assay technique was used to compare the reactivity of the five partially purified antigens. The sensitivity and specificity of all five antigens were compared by testing against the sera of 78 O. viverrini-infected individuals from O. viverrini endemic areas and 70 individuals from non-endemic areas infected with hookworm, Trichuris and Ascaris including 49 individuals with negative stool examination. The assays performed with tegument extract, somatic extract and P1 fraction were found to have 100% sensitivity, whereas the sensitivities of those with P2 and P3 were 96.1% and 83.3%, respectively. The tegument extract had the highest specificity as demonstrated by the lowest cross-reactivity with other parasites. Our results indicated that surface tegument is the most suitable antigen for use in immunological diagnosis of opisthorchiasis.

Detection of antibodies against Opisthorchis viverrini in patients before and after treatment with praziquantel.

Levels of antibody in sera of 78 patients with opisthorchiasis, 30 patients with other liver diseases, 10 patients with schistosomiasis and 30 healthy individuals were compared using three serodiagnostic tests, namely indirect haemagglutination (IHA), enzyme-linked immunosorbent assay (ELISA) and lectin immuno test (LIT). The geometric mean reciprocal titer in sera of opisthorchiasis patients was significantly higher than patients with other diseases, patients with schistosomiasis and healthy individuals (p less than 0.00001). After treatment with praziquantel, the antibody titers were decreased and became lowest 120 days after treatment. A statistically significant decrease from the pre-treatment sample was observed only at 120 days after infection and not earlier and only with ELISA (p = 0.03) and not with IHA and LIT (p greater than 0.05). Even with ELISA, significant decrease in antibody titer was apparent only when the pre-treatment sera had high enough antibody titer. ELISA was therefore better than the other two tests for the assessment of cure provided that the titer of pre-treatment sera was high.

Analysis of Opisthorchis viverrini antigens: physicochemical characterization and antigen localization.

Antigens of Opisthorchis viverrini were identified and characterized by enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis following radioimmuno-precipitation, and indirect immunofluorescence. The immunoreactive protein with a relative molecular weight (Mr) of 89 kD was the predominating component of the parasite metabolic products. An immunofluorescence study showed it to be associated with parasite eggs, linings of the reproductive system and secretions therein. Protein of the surface tegument had Mr of greater than 116, 108, 64, 38, 34, 20 and 16-17 kD. The 16-17 kD surface molecule was the predominant protein, representing approximately 50% of the total protein in crude aqueous extracts of adult worms. However, it was poorly immunogenic when compared with the 89 kD molecule. Together with data presented previously, it appears that in addition to the 89 kD protein, several tegumental molecules are also specific for O. viverrini and have immuno-diagnostic potential.