MaxEnt modeling of soil-transmitted helminth infection distributions in Thailand.

Infections due to soil-transmitted helminths (STHs), i.e. Ascaris lumbricoides, Trichuris trichiura, hookworms, and Strongyloides stercoralis, are widely distributed in tropical and subtropical areas in which approximately 1.5 billion people are infected. A clear understanding of the epidemiology and distribution of diseases is an important aid for control and prevention. The aim of our study was to identify the effects of environmental and climatic factors on distribution patterns of STHs and to develop a risk map for STH infections under current environmental and climate regimes in Thailand. Geographical information systems (GIS), remote sensing, and Maximum Entropy (MaxEnt) algorithm software were used to determine the significant factors and to create predictive risk maps for STH infections in Thailand. The disease data from Thailand covered the years from 1969 to 2014, while environmental and climatic data were compiled from the Worldclim database, MODIS satellite imagery, Soilgrids and ISCGM. The models predicted that STHs occur mainly in southern Thailand. Mean annual precipitation was the factor most affecting the current distribution of A. lumbricoides, T. trichiura, and S. stercoralis. Land cover class was the main predictor for distribution of S. stercoralis and important for hookworms. Altitude was the dominant factor affecting the distribution of hookworms, and mean temperature of the wettest quarter was significantly associated with A. lumbricoides distribution. A predicted distribution map of STHs to identify environmental risk factors in Thailand is presented. This work provides a model for use in STH monitoring and health planning not only in Thailand but also in other countries with similar disease conditions.

Egg-Hatching Mechanism of Human Liver Fluke, Opisthorchis viverrini: A Role For Leucine Aminopeptidases From the Snail Host, Bithynia siamensis goniomphalos.

The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24-28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.

Activity of tribendimidine and praziquantel combination therapy against the liver fluke Opisthorchis viverrini in vitro and in vivo.

Opisthorchiasis, caused by the liver fluke Opisthorchis viverrini, a food-borne trematode, is an important public health problem; however, only a single drug, praziquantel is available. We investigated tribendimidine-praziquantel combinations against O. viverrini in vitro and in vivo. The IC50 values of 0.16 µg/ml and 0.05 µg/ml were determined for praziquantel and tribendimidine, respectively, against adult O. viverrini in vitro. When O. viverrini was exposed to both drugs simultaneously (using a drug ratio based on the IC50 (1:3.2)) a synergistic effect was calculated (combination index (CI) at the IC50= 0.7). A similar result was observed when drug addition in vitro was spaced by the respective half-lives of the drugs (a CI of 0.78 at the IC50 for tribendimidine followed by praziquantel and a CI of 0.47 at the IC50 for praziquantel followed by tribendimidine). In vivo median-effect dose (ED50) values of 191 mg/kg and 147 mg/kg were calculated for praziquantel and tribendimidine, respectively. Low to moderate worm burden reductions (38-62%) were observed in O. viverrini infected hamsters when both drugs were administered simultaneously or on subsequent days, pointing to antagonistic effects in vivo. Further studies are necessary to understand the striking differences between the in vitro and in vivo observations using combinations of praziquantel and tribendimidine on O. viverrini.

Effects of Bayluscide on Bithynia siamensis goniomphalos, the first intermediate host of the human liver fluke, Opisthorchis viverrini, in laboratory and field trials.

The molluscicidal effects of Bayluscide (niclosamide) were investigated on Bithynia siamensis goniomphalos, the first intermediate host of human liver fluke, Opisthorchis viverrini. Lethal concentrations of 50% (LC(50)) and 95% (LC(95)) against young and adult males were 0.38 and 0.80, 0.42 and 0.86 ppm, respectively. The LC(50) and LC(95) against young and adult females were 0.42 and 0.86, 0.46 and 0.97 ppm, respectively. No significant differences in mortality rate between sexes or snail size (p > 0.05) was detected. Bayluscide-related tissue damage in B. siamensis goniomphalos included detachment of cilia of the epithelial layer of the digestive tract and decreased number of calcium cells. In tests of lethal concentrations of Bayluscide on non-target animals, no lethal effect was observed on Filopaludina martensi martensi (Viviparous snail) but high mortality rates were recorded in Puntius gonionotus fingerling, Ricefish (Oryzias mekongensis) and shrimp (Macrobrachium lanchesteri), but lower in guppy fish (Poecilia reticulata) after 24h exposure. For field trials, sufficient Bayluscide was sprayed in 3 roadside ditches to result in final concentrations of 5, 10 or 20 ppm, with mortality rates on B. siamensis goniomphalos of 10.94, 20.00 and 31.25%, respectively. Non-target snails died in small numbers but no effect was observed in other aquatic vertebrate animals. Field trials of Bayluscide on B. siamensis goniomphalos revealed low mortality rates, suggesting the need for application methods of higher efficacy or that Bayluscide is not suitable for application to operculate snails or snails which are able to escape by burying in mud.

The ELISA-based detection of anti-Opisthorchis viverrini IgG and IgG4 in samples of human urine and serum from an endemic area of north-eastern Thailand.

The levels of correlation between the number of Opisthorchis viverrini eggs excreted in the faeces and levels of anti-Opisthorchis IgG and IgG(4) in the serum and urine (as indicated by absorbances in ELISA) have recently been evaluated in north-eastern Thailand. The 225 subjects investigated in detail, all of whom came from an endemic village in Chaiyaphum province, were selected on the basis of the numbers of O. viverrini eggs that they were excreting. ELISA based on a crude antigen extract of the trematode were then used to determine the levels of specific IgG and IgG(4) in serum and urine samples. Compared with the egg-negative, the villagers who were found to be egg-positive for O. viverrini had significantly higher levels of specific IgG in their urine and serum and significantly higher levels of specific IgG(4) in their serum. The serum levels of specific IgG and IgG(4) and the urine levels of specific IgG all correlated with the numbers of O. viverrini eggs/g faeces [with correlation coefficients (r) of 0.251, 0.121 and 0.142, respectively]. Although the serum levels of IgG were positively correlated with the urine levels of IgG (r=0.098), there was no significant relationship between the serum and urine levels of specific IgG(4) (r=0.051). When the 225 subjects investigated in the ELISA were divided according to whether they had no detectable Opisthorchis eggs in their faeces (N=57), or 1-100 (N=154), 101-1000 (N=5), 1001-1500 (N=5) or >1501 (N=4) eggs/g faeces, the serum and urine levels of specific IgG and the serum (but not urine) levels of specific IgG4 were also found to correlate significantly with the infection-intensity categories (with r-values of 0.550, 0.146 and 0.578, respectively). When the results of the faecal examinations were treated as the 'gold standard', the ELISA for the detection of (Opisthorchis-specific) serum IgG, serum IgG(4), urine IgG and urine IgG(4) had sensitivities of 99.2%, 23.1%, 43.0% and 45.9% and specificities of 93.0%, 29.6%, 45.9% and 67.2%, respectively. Although the study was limited by the small number of subjects with intense infections, it appears worth investigating urine samples for subclasses of specific IgG other than IgG(4).

Granulomatous, hepatolithiasis and hepatomegaly caused by Capillaria hepatica infection: first case report of Thailand.

This is the first case report in Thailand of a Capillaria hepatica infection causing a granulomatous hepatic lesion, bile duct dilatation, hepatolithiasis and hepatomegaly. The patient's chief complaint was abdominal pain with fever and chills. Imaging of the liver revealed a 3-cm mass in the postero-inferior sub-segment of the right lobe of the liver with bile duct dilatation. Right hepatectomy and cholecystectomy were performed. Gross pathology of the right hepatectomy revealed focal intrahepatic duct dilatation with prominent periductal fibrosis. The histopathological section revealed chronic inflammation and some granuloma formation surrounding the bile ducts, generalized portal infiltration, prominence of eosinophils and hepatolithiasis. Histopathotogical section revealed oblique sections of C. hepatica egg (size 35.4+/-6.38 microm in width) and brown amorphous pigment.

Early stage biliary and intrahepatic migration of Opisthorchis viverrini in the golden hamster.

In Thailand, infection with the liver fluke Opisthorchis viverrini is a serious health problem, with over 8.6 million human infections each year. Early stage biliary intrahepatic migration and infection of O. viverrini in the Syrian golden hamster were used to study the growth and development of this fluke. Fifty metacercariae of O. viverrini were introduced into each hamster by gastric intubation. Worms were found to migrate rapidly from the stomach to the gall bladder and hepatic duct, where they remained in relatively constant numbers until the end of week 8. Sexual development of worms was rapid, with full development of the uterus and testes by one and one half weeks and the appearance of eggs in the uterus by the beginning of the third week of infection. Worm growth as indicated by body length had ceased by week 8. Hamsters demonstrated development of a full reproductive cycle with in three to four weeks, and this generation continued until 8 weeks.

Comparative study on DNA sequences of ribosomal DNA and cytochrome c oxidase subunit 1 of mitochondrial DNA among five species of gnathostomes.

The nucleotide sequences of partial 18S, complete internal transcribed spacer region 1 (ITS1), complete 5.8S, complete ITS2 and partial 28S of ribosomal DNA (rDNA) and cytochrome c oxidase subunit 1 of mitochondrial DNA (MCOI) from five species of gnathostomes (G. spinigerum, G. doloresi, G. nipponicum, G. hispidum and G. binucleatum with the former four species being distributed in Japan and Asia) that cause human gnathostomiasis were compared by direct polymerase chain reaction cycle-sequencing. The nucleotide sequences of each region of the18S (613 bp), 5.8S (158 bp) and 28S (598 bp) rDNA from the five species were almost identical. The ITS1 region was different in length for the five species. The nucleotide sequences of each region of ITS2 and partial MCO1 regions were different among the five species. Therefore, these two regions can be used as genetic markers for identification of worms.

Expression of tenascin in bile duct cancer of hamster liver by combined treatment of dimethylnitrosamine with Opisthorchis viverrini infections.

Tenascin is an extracellular matrix glycoprotein known to be an essential factor for the modulation of reciprocal interactions between the epithelium and mesenchyme during embryogenesis and tumourigenesis. The interactions between the expression of tenascin in the liver of Syrian golden hamster and the development of bile duct cancer in an Opisthorchis viverrini-associated cholangiocarcinoma model were investigated. The tenascin was expressed in connective tissues surrounding the dilated ducts, ductal rims and the stroma of cancers, and strongly in the stroma flame of necrotic cancer nodules. The mRNA signal for tenascin was also recognized in the stroma cells. The potential roles of tenascin as prognostic tumour markers are discussed.

Sensitive and specific enzyme-linked immunosorbent assay for the diagnosis of Wuchereria bancrofti infection in urine samples.

We developed an enzyme-linked immunosorbent assay (ELISA) that detects filaria-specific immunoglobulin G4 antibodies in unconcentrated urine. The ELISA was positive in 87 of 91 (95.6%) urine samples collected from people with Wuchereria bancrofti microfilariae, antigen, or both. Of 298 urine samples collected in Thailand, Lao People's Democratic Republic, and Japan, where no human filariasis is known, 295 (99.0%) were negative by ELISA. Various intestinal nematode and fluke infections did not interfere with the ELISA. Urine samples with sodium azide could be kept at 37 degrees C for 4 weeks, and the time of urine collection did not influence ELISA results. This ELISA can be used to identify endemic foci of filariasis.

The antiulcerative effect of Thai Musa species in rats.

Bananas are reported to have an antipeptic ulcer effect, however, the beneficial action can be affected by many factors, including the variety. Our study was undertaken to investigate the antipeptic ulcer effect of the Palo and Horn varieties of banana, grown and consumed in the northeast of Thailand. Indomethacin and acetic acid-induced gastric lesions in rats were employed as models of peptic ulcer disease. The lengths of gastric lesions in the glandular part of the stomach were measured for the assessment of the protective effect of bananas. The healing effect was studied by histological examination of the ulcerated area. The lesions in rats treated with the extract of banana were significantly less dominant than those of the control. The average length of total lesions of rats treated with an extract of Palo or Horn bananas at a dose of 1.0 g/kg/d for 3 days prior to indomethacin administration were 4.47+/-1.2 and 1.87+/-0.44 mm, respectively, whereas those observed in the control rats were 14.56+/-2.43 mm. In the ulcer-healing model, only the Hom-banana-extract-treated group showed a beneficial effect which manifested as a milder degree of histological change than that of the indomethacin-induced-chronic-ulcer control group. However, in acetic acid-induced ulcers, the histological changes of every group were similar. The present findings indicate that bananas of different varieties have varying antipeptic ulcer effects. The extracts of Palo and Hom bananas have a prominent gastroprotective effect, whereas only the extract of Hom banana had an observed ulcer-healing effect.

Nucleotide sequence of mitochondrial CO I and ribosomal ITS II genes of Opisthorchis viverrini in northeast Thailand.

The mitochondrial cytochrome c oxidase subunit I (CO I) gene and the second internal transcribed spacer region (ITS II) gene of Opisthorchis viverrini were compared among O. viverrini from various areas in northeast Thailand. The nucleotide sequences of partial CO I gene (417 bp) of O. viverrini differed among O. viverrini originated from Ubon Ratana, Leongpleuy, Ban Phai, Maha Sarakham, and Chatturat. These intraspecific variations were classified into 5 patterns but no area-specific pattem was observed. Amino acid sequence deduced from the nucleotide sequences of these genes was identical. Nucleotide sequences of a region of the O. viverrini ITS II gene (296 bp) from different areas were identical. However, they were different from those of Clonorchis sinensis, Haplorchis taichui, H. pumilio, Fasciola gigantica, Echinostoma malayanum and Centrocestus sp..

Ultrastructural and immunohistochemical analysis of cholangiocarcinoma in immunized Syrian golden hamsters infected with Opisthorchis viverrini and administered with dimethylnitrosamine.

Utilizing the experimental model in Syrian golden hamsters, we explored the role of immunization in carcinogenesis. The animals, which were infected with liver flukes (Opisthorchis viverrini), and administered a subcarcinogenic dose of dimethylnitrosamine, developed cancer. Pre-immunizing with a crude somatic antigen did not reduce cancer development, but accelerated carcinogenesis. Histopathological analysis of the cancer tissues was done once at week 30 and again at week 39 using H and E staining, immunostaining for the p53 tumor suppressor phosphoprotein, and electron microscopy. Thirty weeks after immunization, the immunized hamsters developed tubular adenocarcinoma at a higher rate (71.43%) than the non-immunized group (20.00%). This rate (20.00%) increased to 63.64% by week 39. The small foci cancer in the non-immunized group decreased in frequency from 80.00% (at week 30) to 36.36% (by week 39), suggesting the small foci cancer progressed to tubular adenocarcinoma during the 9-week interval. Most of the observed tubular adenocarcinoma was well differentiated. Nearly all hamsters that tested positive for cancer also tested positive for p53 immunostaining in the epithelia of the small bile ducts. The positive reaction for p53-immunostaining was localized in the rough endoplasmic reticulum, Golgi apparatus and perinuclear membranes. The electron micrographs of these positive p53-immunostained cells showed characteristics of early cancer. The detection of p53 in early cancer development makes it a candidate as a tumor marker.

Natural infection of trematodes in Lymnaea (Radix) auricularia rubiginosa in water reservoirs in Amphoe Muang, Khon Kaen Province.

Lymnaea (Radix) auricularia rubiginosa (Michelin, 1831) was surveyed in 54 reservoirs of 18 districts in Amphoe Muang, Khon Kaen Province during February to May 1994. Lymnaeid snails were found in the water of 20 reservoirs, of which 16 reservoirs contained clear water and 4 turbid water. Two of the four turbid water reservoirs received drainage water from Khon Kaen Town. Two thousand four hundred and eight L. auricularia rubiginosa were collected and examined by shedding and crushing. Trematode infection occurred in 163 (6.77%) of 2,408 L. auricularia rubiginosa and some snails were infected with more than one cercarial species. Ninety-nine snails (4.11%) were infected with echinostomes, while mixed infection of echinostomes with Fasciola gigantica and with schistosomes was found in 5 snails (0.21%) and 2 snails (0.08%), respectively. Only 1 snail (0.04%), 19 snails (0.79%) and 37 snails (1.54%) were infected with F. gigantica, schistosomes and unidentified species, respectively. The mean size of infected snails was 6.89 +/- 2.02 mm (6.20-22.36) while the mean of sampled snails was 13.46 +/- 3.64 mm (4.00-26.55). The water plants which were found in reservoirs and presented with snails, were creeping water primose (Jusstaea repens), water lily (Nymphaea sp), water hyacinths (Eichornia crassipes) and grass.

Metacercariae isolated from cyprinoid fishes in Khon Kaen District by digestion technic.

Several kinds of fresh-water fishes collected from natural water resources in Khon Kaen District were digested with 0.25% pepsin A to obtain the infective stage or metacercariae of fish-borne trematodes. Most of them harbored at least 3 species of trematodes. Two families of medical trematodes, i.e., Family heterophyidae and Family Opisthorchiidae, were isolated. The heterophyid flukes consisted of Haplorchis taichui, H. pumilio and Centrocestus formosanus. Opisthorchis viverrini was the only parasite of the Family Opisthorchiidae found in these fish. Among these four species, H. taichui was predominant and found in all kinds of fish examined, especially Hampala dispar.

Field trials in the control of Opisthorchis viverrini with an integrated programme in endemic areas of northeast Thailand.

A control programme of opisthorchiasis was carried out for three years in three villages of northeast Thailand in order to compare the effectiveness of two intervention measures. The intervention measures employed were to give praziquantel treatment to all infected people either once (village I) or twice (village II) per year with the integration of regular health education and sanitation improvement. A control village (village III) received no intervention during the study. Sampling of the population in these villages to estimate prevalence, intensity, reinfection and incidence of infection as well as knowledge of opisthorchiasis and number of latrines was performed at 6 monthly intervals throughout the duration of the study. At the initial assessment, there were no significant differences in the prevalence and intensity of infection of villages I and II. Both values were greatly reduced when assessed 6 months after the initiation of treatment, and the decrease in prevalence and intensity of infection did not differ between village I and II. The control village (village III), during the first 2 years, exhibited slight variation in the prevalence and intensity, and these decreased considerably in the third year of the study. The reinfection rate per 6 months in villages I and II was nearly equal. The incidence rate per 6 months in the two treated villages was lower than that of the control village. Marked improvement in knowledge of opisthorchiasis, behavioural changes of eating raw fish and increased numbers of latrines was evident in all of the villages during this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Seasonal variation in the prevalence and intensity of canine Gnathostoma spinigerum infection in northeastern Thailand.

Gnathostoma spinigerum was found in gastric nodules in 4.1% of 2940 dogs surveyed in northeastern Thailand. The prevalence and worm burden of G. spinigerum exhibited a seasonal fluctuation. The parasites were more abundant in the rainy season and the early winter (August-December) than in the summer (April-March). Most parasites were sexually mature between August and December while immature worms were observed during March and April. The distribution of gnathostomes within the sampled dogs was highly dispersed and few animals were found to harbour more than five worms.

Eggshell morphology of the small eggs of human trematodes in Thailand.

Light and scanning electron micrographs of Opisthorchis viverrini, Haplorchis taichui, H. pumilio, A Phaneropsolus bonnei, and Prosthodendrium molenkampi eggs were studied. Under light microscopy. O. viverrini eggs had rough eggshells and prominent shoulders. H. taichui, H. pumilio were similar in shape and had smooth eggshells and prominent shoulders. H. pumilio eggs were bigger than H. taichui eggs. P. bonnei and P. molenkampi eggs had smooth eggshells and indistinct shoulders. P. bonnei eggs were thinner and bigger than P. molenkampi eggs. Some deformed eggs of O. viverrini and Haplorchis sp. were found and they had no embryos and indistinct opercula. Under scanning electron microscopy, O. viverrini eggs looked like musk-melon skin; they had prominent shoulders and long knobs. H. taichui eggs had curly, thread-like ridges and prominent shoulders and knobs. H. pumilio eggs had streched ridges, like Chinese bitter mormodica, and prominent shoulders. P. bonnei and P. molenkampi eggs had smooth eggshells and small shoulders and knobs.

Quantitative post-mortem study of Opisthorchis viverrini in man in north-east Thailand.

The results of an autopsy study of Opisthorchis viverrini infection in 181 accident subjects in northeast Thailand are presented. The age-prevalence profiles were similar whether determined by faecal examination for parasite eggs or by recovery of worms from the liver. The prevalence increased rapidly with age and reached a plateau at 70-80% in adults. The overall prevalence estimated by faecal examination was 69.2%, while that measured by worm recovery was 79.2%. The average intensity of infection, as indicated by eggs per gram of faeces, rose with age and became relatively stable in groups over 20 years of age. Worm burden also increased rapidly with age, reaching a peak in the 31-40 year groups and slightly declining in the 2 oldest age groups. Fluke distribution between individual subjects was highly dispersed; most people harboured no or low worm burdens (0-200 worms) while a small group of subjects (16.5%) carried 66% of the total worms recovered. The distribution of flukes within the liver appeared to be homogeneous. The results of the present study were similar to those from field studies, and possible mechanisms that generate these observed patterns of infection are discussed.

Relationship between faecal egg count and worm burden of Opisthorchis viverrini in human autopsy cases.

The relationship between faecal examination for egg output and worm burden of Opisthorchis viverrini in man of 181 autopsy cases from Northeast Thailand is described. Diagnosis of the parasite infection by stool examination for the presence of eggs was less sensitive than the worm recovery technique. Using Stoll's dilution and formalin-ether technique, no eggs were detected in the faeces of 20 cases harbouring low worm burdens (less than 20 worms). The quantitative faecal egg count by Stoll's dilution technique showed a strikingly close positive correlation with the number of worms recovered (r = 0.96, P less than 0.001) indicating a strong linear association between eggs per gram of faeces (epg) and worm burden. The number of epg per worm was inversely correlated to the worm burden (P less than 0.001), suggesting that density-dependent constraints on fecundity could operate to restrict egg output in heavy infections. The accuracy of egg counts for estimating worm burden and its relevance to parasite epidemiological research are discussed.