Pila ampullacea and Pomacea canaliculata, as new paratenic hosts of Gnathostoma spinigerum.

Aquatic snails, Pila ampullacea and Pomacea canaliculata were experimentally found to be suitable paratenic hosts for advanced third-stage larvae (L3) of the nematode Gnathostoma spinigerum, the causative parasite of gnathostomiasis in humans. G. spinigerum (L3) were found to be encapsulated in the tissue of the snail's foot and its internal organs. The infection, intensity and survival of third-stage larvae of G. spinigerum in both species of aquatic snails are described. This is the first evidence to reveal that not only vertebrates but also invertebrates (snails) can serve as paratenic hosts to this parasite. Aquatic snails are one of several sources of human gnathostomiasis in Thailand.

Paragonimiasis prevalences in Saraburi Province, Thailand, measured 20 years apart.

Saraburi Province, Central Thailand has been a paragonimiasis-endemic area since 1956. This study compared the prevalences of human paragonimiasis in two villages near Chet Khot Waterfall, Kaeng Khoi District, investigated in 1984-1985 and 2005. The results from the 1980s showed 6.3% and 1% of villagers were positive for Paragonimus eggs in sputum and stool, respectively. In 2005, Paragonimus eggs were not found in feces or sputum. An IgG-ELISA for paragonimiasis was conducted on 33 serum samples collected in the 1980s, 23 collected in 2005 and 25 diagnosed with other parasitic infections. Ninety percent of the samples from the eighties were positive for paragoimiasis, and 43% from 2005 were positive, equivalent to 10.9% and 4.9% of the total population examined in the 1980s and 2005, respectively. Serodiagnosis is currently the best method for detecting paragonimiasis. The positive cases in the 1980s were age 10-60 years and in 2005 were age 34- 67-years-old. The prevalence and intensity of Paragonimus metacercariae in fresh Waterfall crabs collected from Chet Khot Waterfall were significantly lower in the 1980s than in 2005. The prevalence of paragonimiasis in this endemic area has decreased to the level that no egg-producing cases were detected. No infections were found in villagers age < 30 years, despite the high density of metacercariae in the crabs, indicating a change in the habit of eating raw food among the younger people.

Negative-ELISA using native and filtrated cystic fluid antigens to rule out cystic echinococcosis.

An increasing number of cases of echinococcosis in Thailand have been imported, probably native infections and medical transfers. Serodiagnosis is one diagnostic choice for interpreting infections before a further step is done. Due to limited antigen, indirect ELISA has been used as a negative screening test for IgG-detection to rule out echinococcosis. Native hydatid cystic fluid (HCF) antigen from Belgium was used for such testing, in which the ODs-ELISA of samples were compared with those of two positive controls. Subsequently, hydatid cyst fluid from a Thai patient was obtained and the filtrated cyst fluid antigen [(<30)-(>10) kDa, HCF30.10] was prepared to develop negative screening results for the serum samples. By using HCF, three of twenty-four samples resulted in higher ODs-ELISA than the controls. In an attempt to observe the cross-reactivity of this native antigen, IgG-antibodies from many helminthiases cross-reacted and showed high ODs-ELISA. The HCF30.10 Ag was used to develop the test and analyze IgG-antibodies from 5 positive controls (2 parasite-confirmed and 3 positive-serodiagnosed), 183 heterologous cases of 29 diseases and 50 healthy control sera. At a cut-off value of 0.484, the test had 100% sensitivity and 42% specificity. Only Malayan filariasis, onchocercosis, fascioliasis, amebiasis, giardiasis and blastocystosis gave true negatives. Antibodies from nematodiases strongly cross-reacted with HCF30.10 Ag. Nine of fifty (18%) healthy serum controls produced higher OD-values than the cut-off. The routine ELISA uses the HCF30.10 Ag to produce a negative result to echinococcosis, because limited cystic fluid antigen (Thai patient) for test improvement, a lot of cross-reactions and only two protoscolex-positive controls are available.

Host-finding behavior of Strongyloides stercoralis infective larvae to sodium cation, human serum, and sweat.

The host-finding behavior of Strongyloides stercoralis infective larvae was examined by in vitro agarose assay method. As human body fluid contains 0.85% (ca 0.15 molar) NaCl, various concentrations of sodium chloride, from 0.5M to 0.01M (7 steps), were examined. Many larvae were attracted at concentrations between 0.5 and 0.05M of sodium chloride. The concentration of 0.05M attracted the most larvae. The concentration of 0.02M of sodium chloride showed greatly reduced larval attraction compared with 0.05M. Therefore, the threshold concentration was determined as 0.05M. Then, 0.05M of chemicals were examined in a further experiment. Chloride compounds (NaCl, KCl, CaCl2, MgCl2) were investigated. These chemicals are components of human body fluids. Distilled water was used as the control in all experiments. Only sodium chloride attracted the larvae. Next, alkaline compounds were examined [NaOH, KOH, Ca(OH)2, and Mg(OH)2]. Larvae accumulated only at the NaOH site. The results suggested that the Na cation is important for larval attraction. A high pH value did not influence attraction at all. Next, human serum was tested. The human serum used was from normal serum to 1:32 diluted sera by distilled water (7 steps). Hierarchical attraction was seen according to serum concentration. Next, human sweat was collected from a limited zone of chest skin where only eccrine glands were distributed. Non-diluted sweat attracted the most larvae. Sweat might act as one of the most probable factors for infection by this skin-penetrating nematode.

Detection of IgG antibodies of Brugian filariasis with crude male and female antigens of Dirofilaria immitis.

Crude antigens from male and female Dirofilaria immitis were used to detect antibody to Brugian filariasis in humans by indirect ELISA. Both antigens were tested with 42 cases of Brugian filariasis, 131 cases of 20 heterologous infections and 35 healthy controls. The results--using male and female antigens--showed sensitivity of 88.1% and 88.1%, and specificities of 64.1% and 51.8%, respectively. Cross-reaction from other helminthic infections using crude male antigen gave false-positives with 48 sera from 13 heterologous diseases at the threshold value of 0.180, while the female antigen gave 63 sera from 15 diseases, at 0.309. Serum antibodies from patients with other helminthic infections--gnathostomiasis, strongyloidiasis, hookworm infections, trichinellosis, capillariasis, angiostrongyliasis, ascariasis, trichuriasis, toxocariasis, neurocysticercosis, cystic echinococcosis, taeniasis and opisthorchiasis--resulted in false-positives with both male and female antigens. One each of sparganosis and paragonimiasis heterotremus sera cross-reacted with only crude female antigen and their OD values were close to the threshold value. Although crude male antigen showed better specificity than crude female antigen, both female and male worms are sources of antigens needed for further purification. This study provides baseline data for further serodiagnosis of Brugian filariasis using dirofilaria antigen.

Paragonimiasis in Nan Province, northern Thailand.

Two cases of paragonimiasis were identified within the hill-tribe population living on the Thai-Laotian border of Nan Province, northern Thailand, where information on Paragonimus was then still limited. The patients were in the habit of eating improperly cooked crabs and freshwater prawns. A survey for natural intermediate hosts to complete the life cycle was in progress at that time, and the detection of paragonimiasis cases indicated that there was persistence of paragonimiasis in the endemic area of Nan Province.

Double-dose ivermectin vs albendazole for the treatment of gnathostomiasis.

A comparative study was performed for the treatment of gnathostomiasis patients with ivermectin 0.2 mg/kg for 2 days in 15 patients vs albendazole 400 mg twice daily for 21 days in 14 patients. The ivermectin and albendazole gave cure rates of 100% and 78.5%, respectively, however the difference was not statistically significant between the two drugs (Fisher's exact, p=0.0996). One year after treatment, the patients who had no migratory swellings and a drop in ELISA titers or a negative immunoblot test were considered to be cured. The side effect of ivermectin for two days was dizziness. The side effects of albendazole were nausea, dizziness, and an increased alkaline phosphatase.

Cysticercosis: IgG-ELISA evaluations of peak1 antigen and <30 kDa antigen of delipidized extract of Taenia solium metacestodes.

The antigenicity of ether-delipidized Taenia solium metacestode extract (DLPAg) was investigated by IgG-ELISA. The antigen showed higher antigenicity than that of non-delipidized antigen (NDLPAg). Then the DLPAg was subjected to Sephacryl S-200 gel chromatography and a partially purified antigen (DLPP1Ag) was identified as the promised antigen by IgG-ELISA using 25 sera from cysticercosis cases, 177 cases of 24 heterologous infections, and healthy controls. Sensitivity was 52% and specificity was 91.8% at the cut-off value (X + 7SD), 0.399. Cross-reactivity occurred with 17 cases of eight diseases: cystic echinococcosis (7/11), taeniasis (1/16), gnathostomiasis (2/8), strongyloidiasis (1/12), angiostrongyliasis (1/12), paragonimiasis heterotremus (2/15), opisthorchiasis (1/9) and fascioliasis (2/7). When DLPP1Ag was fractionated through Ultra free centrifugal tube (retained 30 kDa) and Amicon (PM10), MWCOP1Ag (<30-10> kDa) was obtained; the antigen showed better results than DLPP1Ag with 88% sensitivity and 95.6% specificity at the cut-off value (X + 4SD), 0.264. Nine cases of six diseases cross-reacted with this antigen: cystic echinococcosis (2/11), gnathostomiasis (2/8), trichinellosis (2/12), toxocariasis (1/5), schistosomiasis (1/6), and fascioliasis (1/7). MWCOP1Ag gave higher sensitivity than that of DLPP1Ag but some cross-reactivity occurred.

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.

Improved antigens for IgG-ELISA diagnosis of strongyloidiasis.

Two preparations of antigens for the diagnosis of strongyloidiasis were prepared from an extract of the infective larvae of Strongyloides stercoralis: a crude antigen (CA) and a molecular weight cut-off antigen (MWCOA). Both antigens were analysed by indirect ELISA against the sera of strongyloidiasis (26 cases), other helminthiases (167) and normal controls (30). The larvae were obtained from fecal culture by a modified polyethylene tube technique after screening tests by triple simple smears per case. The larvae were extracted with distilled water and further sonicated to obtain a supernatant, the CA. A part of the CA was separated for an antigen containing molecules of lower than 30 kDa by an ultrafree-MC centrifugal filter tube (PLTK): this was designed as the MWCOA. The CA gave 96.15% sensitivity and 40.12% (67/167) specificity at a cut-off value of 0.980 (5SD); false positives were produced by 19 of 20 different helminthiases. The MWCOA produced 96.15% sensitivity at cut-off value of 0.71 (4SD); the specificity of the test was 78.44% (131/167), higher than that of CA. False positives also appeared with 15 other helminthic infections. This study suggests that MWCOA is more specific than CA. A purified MWCOA will be necessary in order to reduce cross-reactivity and provide the suitable diagnosis of strongyloidiasis.