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This study investigated the relationship between immune responses to infection with the liver fluke. Opisthorchis viverrini, and the synthesis of the carcinogen, N-nitrosodimethylamine (NDMA) in humans. It also examined associations between synthesis of nitric oxide (NO) and nitrosation of amines, in vivo. Antibody and T cell responses to fluke antigens and post-alcohol urinary NDMA excretion were assessed among three groups of 40-50 men with no, moderate and heavy liver fluke infection. Markers of NO synthesis (nitrate, nitrite) and nitrosation (nitrosamino acids) were also measured in biological fluids. Assessments were carried out under controlled conditions which minimised intake of exogenous nitrate and nitrite and were carried out at two time points, namely before and 4 months after elimination of the infection with praziquantel treatment. No statistically significant variation was observed in the amount of NDMA excreted between the 3 groups. However, during active infection, a strong negative association was observed between in vitro lymphoproliferative responses to some liver fluke antigens and NDMA excretion. After treatment this association was reduced. Multivariate statistical models revealed a highly significant relationship between NDMA levels and urinary nitrate, stimulation indices for two T cell responses to two parasite antigens (MW 37 kDa and 110 kDa) and gall bladder dimensions. NDMA levels after treatment were best described by the ratio between parasite-specific IgG2 and IgE, background levels of T cell proliferation, a urinary marker of nitrosation (N-nitrosothioproline) and usual level of alcohol consumption. These results suggest that individual background immunologic activity, parasite-specific responses and/or parasite products and NO synthesis are important determinants of endogenous generation of nitrosamines in O.viverrini-infected humans.

The liver fluke, Opisthorchis viverrini, is both highly prevalent and closely associated with cholangiocarcinoma in northeast Thailand. This study measured associations between intensity of liver fluke infection and nonmalignant hepatobiliary disease diagnosed by ultrasonography among 1,807 largely asymptomatic adult residents drawn from endemic communities. Abnormalities significantly associated with intensity of infection included gallbladder enlargement in all dimensions, presence of sludge, irregular gallbladder wall, liver enlargement, and enhanced portal vein radicle echoes. While gallbladder enlargement was not sex-specific, the prevalence odds of the other abnormalities were 2-3 times higher among males compared with females. Those recently treated with the anthelmintic praziquantel had higher odds of these abnormalities compared with others with the same infection status who were untreated. The low prevalence of gallstones suggests that this impairment of gallbladder structure and function does not frequently stimulate gallstone formation. However, gallbladder disturbances, together with chronic inflammation and fibrosis of the bile ducts, which are visualized as enhanced portal vein radicle echoes, may contribute to the strikingly enhanced susceptibility to cholangiocarcinoma among people, especially males, with heavy liver fluke infection.

Infection with the liver fluke, Opisthorchis viverrini, is a causative agent of cholangiocarcinoma. One possible contributing factor in this carcinogenesis is the chronic, local generation of nitric oxide by inflammatory cells expressing inducible nitric oxide synthase and the production of N-nitroso compounds via the reaction between amines and nitrosating agents derived from nitric oxide. Our previous studies provided evidence that nitric oxide synthesis is elevated during human liver fluke infection. Here we present data on the same sample of men which definitively demonstrates increased nitrosation of proline and thioproline (thiazolidine-4-carboxylic acid) among infected men compared to uninfected control subjects on a low nitrate diet. This difference was specifically abolished by co-administration of ascorbic acid with proline and by elimination of parasites by praziquantel treatment. Multivariate statistical models demonstrate the importance of salivary thiocyanate levels to variation in the nitrosation of proline among uninfected individuals, but not among those with current fluke infection. This suggests that considerable generation of nitrosating agents (N2O3/N2O4) in infected people may be occurring via oxidation of arginine by nitric oxide synthase in inflamed tissue which is thiocyanate insensitive. Analyses revealed positive associations between N-nitrosoproline excretion and nitrate/nitrite levels in urine, plasma and saliva and with usual alcohol intake; with variation in these trends between groups. In conclusion, we have confirmed the relationship between O.viverrini infection and enhanced endogenous nitrosation, showing evidence of its extragastric site. New information is also provided on the determinants of N-nitrosamino acid excretion in men on a controlled low nitrate diet without smoking, conditions which reduce exogenous sources of nitrosating agents.

In north-east Thailand a five-act drama is broadcast on the village sound system to catalyse involvement in planning and carrying out AIDS prevention activities. Each community's own suggestions for an effective strategy are presented to the relevant government and nongovernmental agencies for endorsement and support.

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.

Parasite-associated mortality: liver fluke infection and bile duct cancer in Northeast Thailand. International Journal for Parasitology 24: 833-843. Infection with the liver fluke, Opisthorchis viverrini, remains a major public health problem in Northeast Thailand, where approximately one-third of the population is infected. The northeast region is largely populated by Laos-descendent Thais who enjoy eating raw fish, which harbour the infective stage of the fluke. The parasite has maintained its presence in the population despite the widespread use of praziquantel and dissemination of health education material throughout the region by vigorous government-sponsored programs in recent years. The most severe consequence of liver fluke infection is cholangiocarcinoma, i.e. cancer of the bile duct epithelium. Although mortality due to the parasites alone appears to be uncommon, cholangiocarcinoma arising as a result of infection is one of the leading causes of death in the region.This paper reviews the pathogenesis of infection and the geographic, hospital-based and community studies which demonstrate the close relationship between infection and cancer. In addition, data from the Cancer Registry of Khon Kaen, Northeast Thailand and population-based studies using ultrasonography to visualize early tumours which illuminate the very high frequency of the cancer among heavily infected individuals and communities are discussed. Finally, the paper will close with a brief commentary on the prospects for control of the parasite and its likely impact on the frequency of cancer given the current epidemiological situation of liver fluke infection.

Cancers arising during bacterial, viral and parasitic infection provide useful models to investigate the link between inflammation and carcinogenesis. Because the inflammatory agent is known, relationships between immune responses, the production of DNA-damaging agents, such as nitric oxide radicles and N-nitroso compounds, and cancer risk can be explored. This paper first describes the close relationship between infection with the liver fluke, Opisthorchis viverrini, and cholangiocarcinoma in humans. Data are then presented which demonstrate an elevation in levels of salivary nitrite and urinary and plasma nitrate among men with moderate and heavy liver fluke infections compared to uninfected controls which was absent 4 months after the parasites were cleared with praziquantel. Because of the strict control over subject selection and dietary intake plus the absence of the increase following treatment, we conclude that the higher levels of nitrate and nitrite reflect endogenous generation of nitric oxide resulting from liver fluke infection. Excess nitric oxide generation in the inflamed tissue is likely to lead directly to the formation of N-nitroso compounds mediated by activated macrophages. Further work will attempt to demonstrate a link between this increase and both parasite-specific immune responses and the risk of cancer.

We describe an innovative strategy to quantify risk of cancer associated with varying levels of exposure to chronic parasitic infection through the identification of asymptomatic cases of cholangiocarcinoma within a population‐based survey of Opisthorchis viverrini infection. Stool samples from 12,311 adults over age 24 years from 85 villages in northeast Thailand were examined for intensity of liver fluke infection. People from varying egg count categories were selected for ultrasound examination to identify hepatobiliary disease. Fifteen preclinical cases of cholangiocarcinoma were diagnosed from a total of 1,807 people based on ultrasonographic evidence with confirmation by endoscopy where possible. The prevalence odds of the diagnosis of cholangiocarcinoma increased gradually within the light and moderate intensity groups. In contrast, sharply elevated prevalence odds [age‐, sex‐ and licality‐adjusted prevalence odds ratio (POR) 14.1, p < 0.05] were observed within the most heavily liver fluke‐infected group compared with the uninfected group. Males were more frequently affected than females (crude POR 4.5), but after controlling for intensity of infection, age and locality, the magnitude and significance of this measurement was reduced. Our data clearly demonstrate a significant relationship between intensity of liver fluke infection and cholangiocarcinoma and a strikingly high prevalence of the disease among heavily infected males. © 1994 Wiley‐Liss, Inc.

This paper presents a case study of the development, implementation and evaluation of an HIV prevention strategy for rural, married women in the northeastern, Isan region of Thailand. The strategy is based on established principles of behaviour change, social learning, and community health promotion; grounded in baseline research, that identifies relevant contextual factors; community-based; evaluated; and applicable elsewhere. The methodology of the project includes four phases: data collection, strategy design, implementation, and evaluation. Baseline data were collected from an age-stratified sample of married women from 24 randomly chosen villages in Khon Kaen province in two waves. A total of 654 women participated in structured face-to-face interviews and 150 also participated in focus groups. Using the baseline data, a health promotion strategy including three components was designed: meetings with and training of village leaders and health workers; a five-part week long audio-drama, together with posters and other publicity conducted in villages; and village meetings to discuss the potential impact of HIV/AIDS on the village and to establish a village strategy to address this. This paper reports the results of the baseline data collection, how the data were used to produce an HIV prevention initiative for rural villages, results of the process evaluation and future development in HIV/AIDS prevention initiatives in this region.

We recently demonstrated a number of biliary tract abnormalities associated with moderate to heavy Opisthorchis viverrini infection among 95 selected village residents in north-east Thailand, who were treated with praziquantel immediately after initial examination and re-examined by abdominal ultrasonography 10 months later. Dramatic improvement in the gall-bladder status of previously infected individuals was observed, including reduction of gall-bladder length and width and regained contractibility of the organ following fatty meal consumption. The frequency of severe enhanced portal vein radicle echoes and gall-bladder sludge and wall irregularities also decreased following treatment, but persisted in many individuals. Two masses were observed in the right hepatic lobe of one individual who was previously considered normal. The findings suggest that gall-bladder abnormalities are reversible following elimination of liver fluke infection, but malignancies, once initiated, are not likely to be affected by treatment.

Twenty‐four locality‐, age‐ and sex‐matched groups of village residents with no light, moderate and heavy Opisthorchis viverrini infection were examined by ultransonography. Highly significant differences were observed between the groups in the relative size of the left lobe of the liver and the fasting and post‐meal size of the gall‐bladder. In addition, indistinct gall‐bladder wall, the presence of gall‐bladder sludge and strongly enhanced portal vein radicle echoes were most frequently observed in the heavily infected group. Two suspected cases of cholangiocarcinoma were identified from the heavy group. The results highlight the importance of intensity of infection on the frequency and severity of fluke‐associated hepatobiliary disease.

Free to read 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.

Free to read 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 0. 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 0. 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.

A group of 87 adults from a small village in north-east Thailand was chosen to undergo ultra-sound 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.

Summary A semi–quantitative analysis of individual human antibody responses to larval Ascaris excretory and secretory (ES) antigens using radioimmunoprecipi–tation and SDS–PAGE is presented. A significant relationship was observed between the intensity of antibody–precipitated radiolabeled ES antigens and host age. The antibody response profile followed a similar age–related pattern to that of intensity of infection, with blood samples from 5–9–year–old children showing the strongest banding patterns and the heaviest infections. These findings support the hypothesis that the degree of exposure to infective stages of Ascaris is a major determinant of the convex age–intensity profile observed in the community. Considerable heterogeneity was observed in the antibody profiles of individuals, particularly in the recognition of a 14 kD molecule. Positive correlations were observed between the strength of banding at several mol. wts and the worm burdens of individuals. However, the sample size was too small to determine whether these relationships simply reflect age–related profiles or represent independent associations between antibody levels and worm burden. When the data were stratified by age, negative associations between the strength of recognition of some bands and the intensity of infection were suggested and require further study.