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.

Increased cell proliferation of mouse fibroblast NIH-3T3 in vitro induced by excretory/secretory product(s) from Opisthorchis viverrini.

Infection by Opisthorchis viverrini is a strong risk factor for cholangiocarcinoma. However, the mechanism by which the parasite is involved in carcinogenesis is not clear. In addition to the direct damage of the bile duct epithelium via direct contact with O. viverrini, the excretory/secretory (ES) product(s) released from the parasites may play important roles in this process. We therefore investigated the responses of a fibroblast cell line, NIH-3T3, to ES product(s) released from O. viverrini by using a non-contact co-culture technique. In this culture system, the parasites in the upper chamber had no direct contact with the NIH-3T3 cells in the lower chamber of the culture plate. The results indicated a marked increase in NIH-3T3 cell proliferation in the non-contact co-culture condition with either 0% or 10% calf serum in the medium compared with that without parasites. ES product(s) increased cell proliferation by stimulating the expression of phosphorylated retinoblastoma (pRB) and cyclin D1, the key proteins in driving cells through the G1/S transition point of the cell cycle. This led to the induction of cells going into the S-phase of the cell cycle. ES product(s) also changed the morphology of NIH-3T3 cells to a refractive and narrow shape, which allowed the cells to proliferate in the limited culture area. For the first time, we have been able to demonstrate increased cell proliferation induced by the ES product(s) from O. viverrini; this finding may clarify how O. viverrini ES product(s) affect human bile duct epithelium during cholangiocarcinogenesis.

Localisation of parasite antigens and inflammatory responses in experimental opisthorchiasis.

The time course localisation of parasite antigens and related host pathology were studied in hamsters infected with 100 metacercariae of Opisthorchis viverrini for up to 6 months. Parasite antigens, as detected by immunofluorescence and/or immunoperoxidase-staining, were first observed in the flukes and the biliary epithelium of the intrahepatic and extrahepatic bile ducts as early as day 3 p.i. Antigens increased as the parasite matured, both in tissues in direct contact with the flukes and those surrounding the infection. Opisthorchis antigens were also observed in the first order bile ducts (small bile ducts) of the liver, which are not normally inhabited by flukes. In addition, they were found in damaged liver cells, Kupffer cells, macrophages, and within epithelioid and giant cells in the egg granuloma. The presence of the antigens was associated with heavy inflammatory cell infiltration, particularly with mononuclear cells. The results strongly support the role of fluke-associated antigens and local parasite-specific immune responses in the pathogenesis of opisthorchiasis.

Relationship between parasite-specific antibody responses and intensity of Opisthorchis viverrini infection in hamsters.

The kinetics of parasite-specific antibody responses in relation to worm burden and egg output were investigated in hamsters infected with 25, 50 and 100 Opisthorchis viverrini metacercariae (MC). Levels of antibody to egg, excretory-secretory (ES) and somatic antigens were examined by ELISA on days 1, 3, 7, 14 and month 1 postinfection (p.i.), and repeated monthly up to 6 months. The antibody responses were first detected as early as 14 days after infection. Hamsters that were infected with 100 MC and 50 MC showed higher antibody levels than those of 25 MC, during early infection until 1 month p.i. Then, the antibody levels were increased rapidly to a plateau at approximately month 2 p.i. and, subsequently, were relatively stable in all groups. The average antibody levels to egg and somatic, but not to ES antigens, were significantly higher in hamsters infected with 25 MC than those of 50 MC and 100 MC. These antibody responses, particularly to egg and ES antigens, were not correlated with worm burden or egg output. Overall, higher antibody responses were found in the order: ES, somatic and egg antigens. The significant lower antibody responses in chronic and heavy infections than those with mild infection may a result of immunosuppression.

Evaluation of a monoclonal antibody-based enzyme linked immunosorbent assay for the diagnosis of Opisthorchis viverrini infection in an endemic area.

A monoclonal antibody-based enzyme-linked immunosorbent assay (MAb-ELISA) was evaluated for its potential in the diagnosis of opisthorchiasis in an area endemic for Opisthorchis viverrini infection. The method, based on the detection of the 89-kD O. viverrini metabolic antigen in the feces (coproantigen), was previously estimated to be sensitive enough to detect antigen excreted by a single mature fluke. In the present study, fecal specimens from 207 apparently healthy villagers in northeastern Thailand were analyzed in a double-blind test for the presence of O. viverrini eggs by microscopic examination and for antigen by MAb-ELISA. The microscopic examination was carefully done to minimize false-positive results due to eggs of Lecithodendriid trematodes. The specimens were divided into six groups based on the number of eggs per gram of feces, namely, egg negative, 1-500, 501-1,500, 1,501-3,000, 3,001-6,000, and more than 6,000. The results showed that the ELISA is sufficiently sensitive and specific for the diagnosis of O. viverrini infection. The slightly higher rate of coproantigen positive by the ELISA compared with microscopic examination may reflect lower specificity of the ELISA or its higher sensitivity over microscopic examination in detecting light infections. Different lines of evidence presented here support the latter explanation.

Comparative studies on the morphology of the eggs of Opisthorchis viverrini and lecithodendriid trematodes.

Iodine staining and scanning electron microscopy were used to determine the morphological features distinguishing Opisthorchis viverrini from lecithodendriid eggs in human feces. The embryonated eggs of lecithodendriid trematodes differ from O. viverrini by the presence of an iodophilic body, a large mass at the posterior end of miracidium that stains brown in 0.2% iodine solution and the curved miracidium of Phaneropsolus bonnei. All forms of lecithodendriid eggs can be differentiated from those of O. viverrini by a set of morphological features of the shell surface, the knob, the operculum, the shoulder, the shape and size. On the basis of these differences, it is possible to perform differential egg counts.

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.

Distribution patterns of Opisthorchis viverrini within a human community.

This paper describes patterns of infection with Opisthorchis viverrini within a human community assessed by egg count, parasite-specific antibody levels and worm burden determined by expulsion chemotherapy. All three measurements increased significantly with age, reaching a plateau in young adults. There were no significant sex-associated differences in the prevalence or intensity of infection, antibody levels, or in the frequency of heavy worm burdens. A striking 81.5% of the total Opisthorchis population and 74% of the total egg output were expelled by the most heavily infected 10% of the humans sampled. This extreme over-dispersion, together with recent findings that hepatobiliary disease frequently accompanies heavy infections, argues that the priority for community-based parasite control programmes should be to maximize treatment and health promotion efforts toward heavily infected individuals.

Phaneropsolus spinicirrus n. sp. (Digenea: Lecithodendriidae), a human parasite in Thailand.

Phaneropsolus spinicirrus n. sp., collected in a postpraziquantel treatment stool of a 44-yr-old woman from Kalasin Province in northeastern Thailand, is described. It is the second species of the genus Phaneropsolus Looss, 1899, found to parasitize a human host. It differs from the first species, Phaneropsolus bonnei Lie, 1951, in the presence of a short spinose cirrus and the structure and distribution of tegumental spines. The new species is most similar to Phaneropsolus perodictici Goodman and Panesar, 1986, isolated from the potto, Perodicticus potto, in Uganda because digeneans of both species have numerous spines on the cirrus. However, the new species differs from P. perodictici in having more conspicuous spines, the genital pore at the posterior border of the oral sucker or lateral to the pharynx, a body and internal organs of larger size, conical spines on the tegument, an ovoid to bilobed ovary and a large V-shaped excretory bladder. Phaneropsolus spinicirrus is distinguishable from other previously reported Phaneropsolus by possessing a spinose cirrus.

Immune responsiveness and parasite-specific antibody levels in human hepatobiliary disease associated with Opisthorchis viverrini infection.

Opisthorchis viverrini infection is associated with human hepatobiliary disease and cholangiocarcinoma, but the role of the immune response in the pathogenesis of infection is unclear. Here ultrasonography was used to examine the biliary tracts of residents from an endemic community. Delayed-type hypersensitivity responses to unrelated antigens, and fluke-specific IgG and IgA levels in serum of this group were also examined. Relationships between immunological parameters, intensity of infection and radiologically measured variables are reported. Immune responsiveness to unrelated antigens did not vary with intensity of parasite infection or disease status. Of all the variables, IgG levels were most markedly elevated in disease cases compared with normal subjects and were closely associated with gall bladder size and dysfunction. This is consistent with the hypothesis that an immunopathologic mechanism is involved in opisthorchiasis and suggests that antibody levels may be useful in screening populations for fluke-associated hepatobiliary disease.

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.

Opisthorchis viverrini: relationships between egg counts, worms recovered and antibody levels within an endemic community in northeast Thailand.

Three techniques for estimating the intensity of Opisthorchis viverrini infection in individuals from a Northeast Thai community are compared. Egg counts were determined using a quantitative formalin/ethyl acetate technique, worm burdens were estimated by expulsion chemotherapy and antibody levels were measured by ELISA. Log-transformed worm and egg counts were closely correlated (r = 0.80), suggesting that both measurements provide good assessments of relative intensity of infection. However, no Opisthorchis worms were recovered from 34 people with high egg counts; probably due to problems with the expulsion technique in some individuals. Examination of egg production per fluke indicated that each fluke contributed an average of 180 eggs per gram (epg) of faeces and fecundity was negatively associated with total worm burden. Serum IgG levels correlated significantly with Opisthorchis egg count (r = 0.61) at two independent assessments. Although significant associations were observed between antibody levels and echinostome infection, analysis suggested that these reflected independent associations between these two variables and Opisthorchis infection and age. We conclude that all three measurements are useful for epidemiological studies.

Influence of water current on the distribution of Opisthorchis viverrini infection in northeastern villages of Thailand.

The prevalences of Opisthorchis viverrini infection in the villages on the banks of rivers and those far from the rivers were studied in Loei and Nong Khai Provinces in Thailand. Most of the people examined in the present study were agriculturalists. The overall prevalence of O. viverrini infection was 41.3%. The prevalence of infection in males and in females in the villages far from the rivers were 52.6% and 51.7%, respectively, while the percent of people in the villages on the banks with infection were 27.9% and 21.7%, respectively. Prevalence of infection among the people residing far from the rivers was higher than those residing on the banks. This was observed despite the higher recording of raw fish consumption in villages on the banks. Infection level increased sharply in the age-group 6-10 years old among people residing far from the rivers. High prevalences of infection were observed in age groups from 11 to 50 years. Both types of villages had a similar proportion of houses with sanitary latrines.

A high frequency of hepatobiliary disease and suspected cholangiocarcinoma associated with heavy Opisthorchis viverrini infection in a small community in north-east Thailand.

A group of 87 adults from a small village in north-east Thailand was chosen to undergo ultrasound investigation based on their intensity of infection with the liver fluke, Opisthorchis viverrini, or clinical status (history of jaundice, current hepatomegaly). From this group, 8 cases of suspected early cholangiocarcinoma were found, and the diagnostic features of 6 of the 8 were confirmed by computerized tomography scan and endoscopic retrograde cholangiopancreatography. In addition, several cases of mild gall-bladder disease, chronic cholecystitis, cholelithiasis and parenchymal liver disease were detected. A highly significant positive relationship between the intensity of liver fluke worm burden and the severity of biliary tract disease within individuals is reported. These results indicate that Opisthorchis is associated with moderate to severe hepatobiliary disease in a considerable proportion of infected individuals.

Scanning electron microscopy of adult Echinostoma malayanum.

Scanning electron microscopy observations of E. malayanum adult obtained from small intestines of infected rats was made. The number of collar spines were 41. The features observed were a pair of corner spines (3 oral and 2 aboral) total 10; a pair of lateral collar spines (10 spines each side); total 20; dorsal collar spines (5 oral and 6 aboral) total 11. Sensory papillae were found more densely situated on the circumoral disc around the oral sucker and on the ventral sucker. Other sensory organs, dome shaped, found only on the circumoral disc. The scales appear mainly on the ventral surface. The microvilli are present on the tegument where the scales occur, while the other part of dorsal side had pitted tegument.