Genetic differences among Haplorchis taichui populations in Indochina revealed by mitochondrial COX1 sequences.

Haplorchis taichui is an intestinal heterophyid fluke that is pathogenic to humans. It is widely distributed in Asia, with a particularly high prevalence in Indochina. Previous work revealed that the lack of gene flow between three distinct populations of Vietnamese H. taichui can be attributed to their geographic isolation with no interconnected river basins. To test the hypothesis that interconnected river basins allow gene flow between otherwise isolated populations of H. taichui, as previously demonstrated for another trematode, Opisthorchis viverrini, we compared the genetic structures of seven populations of H. taichui from various localities in the lower Mekong Basin, in Thailand and Laos, with those in Vietnam, using the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. To determine the gene flow between these H. taichui populations, we calculated their phylogenetic relationships, genetic distances and haplotype diversity. Each population showed very low nucleotide diversity at this locus. However, high levels of genetic differentiation between the populations indicated very little gene flow. A phylogenetic analysis divided the populations into four clusters that correlated with the country of origin. The negligible gene flow between the Thai and Laos populations, despite sharing the Mekong Basin, caused us to reject our hypothesis. Our data suggest that the distribution of H. taichui populations was incidentally associated with national borders.

Patterns of trematode infections of Opisthorchis viverrini (Opisthorchiidae) and Haplorchis taichui (Heterophyidae) in human populations from two villages in Savannakhet Province, Lao PDR.

The liver fluke, Opisthorchis viverrini, and the minute intestinal fluke, Haplorchis taichui, are prevalent in many Asian countries. This study analysed the patterns of infections of O. viverrini and H. taichui in Lahanam and Thakhamlien villages (Savannakhet Province, Lao PDR), in two cross-sectional investigations. Out of a total of 207 human participants, post-anthelmintic treatment positivity rates for expelled worms were 170 (82.1%) for H. taichui and 65 (31.4%) for O. viverrini. Both these species co-exist in the study villages. When each parasite was analysed separately, H. taichui infections reached a plateau among people aged >20 years. Opisthorchis viverrini infection rates were highest in the age group 21-30 years, with decreasing infection rates after the age of 30. Our findings indicated that fish-borne trematode infections were more prevalent among adults. Fish, common intermediate hosts, were acquired in the study area for analysis. The examination of 35 species of fish as intermediate hosts found O. viverrini metacercariae in only six species, and these were found mostly during the month of November. Many farmers who live on the rice fields obtain their food from their immediate environment, including these intermediate-host fish, potentially putting them at greater risk of O. viverrini infection. By contrast, H. taichui metacercariae were found in three species of fish obtained from the market, meaning that anyone could consume them and become infected. If people who work in rice fields limit the species of fish they consume, or avoid consuming raw fish during the month of November, they may reduce their risk of O. viverrini infection.

Recovery of Cyclospora cayetanensis among asymptomatic rural Thai schoolchildren.

OBJECTIVE: To obtain the prevalence with clinical symptoms of Cyclospora cayetanensis (C. cayetanensis), a coccidian protozoan parasite, in Thailand which is the cause of an intestinal infection characterized by sporadic-to-frequent explosive diarrhea. METHODS: In a field survey conducted by the Faculty of Tropical Medicine, Mahidol University, as part of the existing parasite-control program, a total of 2 540 faecal samples from villagers in Nan Province, Thailand, were collected and examined to determine the prevalence and clinical characteristics of parasitic infections. RESULTS: Twelve cases of C. cayetanensis infection were found during faecal examination of schoolchildren aged 5-12 years. None exhibited obvious clinical symptoms, especially evidence of diarrhea; 5 of 12 had loose faeces, one reported frequent symptoms of abdominal discomfort, and another had pale conjunctiva with low hematocrit. The children were generally asymptomatic. CONCLUSIONS: This finding confirms a public-health issue with potentially serious consequences whereby children can be exposed to an environment contaminated with food-and water-borne transmitted oocysts, and can hence become infected with C. cayetanensis.

Genetic differences among Vietnamese Haplorchis taichui populations using the COI genetic marker.

Adults of the fish-borne intestinal trematode species Haplorchis taichui were collected from humans in three provinces of Vietnam: Ha Giang, Thanh Hoa and Quang Tri. Genetic analysis revealed three groups of the parasite from clustering dendrograms, correlating with the localities in which they were collected. Measurements of evolutionary divergence over sequence pairs were greater between the different populations than within them, which indicated that the three populations were genetically different. The significance (Fst= 0.73; P value < 0.05) of the genetic variation of the three studied populations implied that genetic separation of the populations had already occurred, which may have been caused by a low gene flow among the different H. taichui populations. Factors contributing to the low gene flow may include isolation resulting from the intermediate-host fish rarely being sold outside of the rural commune where they are raised and the enclosed aquacultural areas themselves.

School-based health education for the control of soil-transmitted helminthiases in Kanchanaburi province, Thailand.

Soil-transmitted helminthiases are major parasitic diseases that cause health problems world-wide. School-based health education is one of several basic interventions currently recommended by the World Health Organization for the control of these infections. A 3-year programme of health education for the control of soil-transmitted helminths (STH) has recently been completed in four primary schools in the Hauykayeng subdistrict of Thong Pha Phum district, in the Kanchanaburi province of Thailand. Overall, the percentage of the schoolchildren found infected with STH increased between the start of year 1 of the intervention (16.6%) and the end of year 2 (23.8%) but showed signs of falling by the end of year 3 (19.4%). Although none of these year-on-year changes in overall prevalence was statistically significant, some significant trends were detected when the six school grades (i.e. age-groups) were considered separately. The grade showing the highest prevalence of STH infection changed, from grade 6 (representing the oldest children investigated) at the start of year 1 (when grade-1 children were excluded from the survey) to grade 1 (representing the youngest children) at the ends of year 2 and year 3. By the end of year 3, the children in grades 5 and 6 had significantly lower prevalences of infection than the grade-1 subjects. The prevalence of STH infection in the grade-1 children was significantly higher than that in any of the older grades at the end of year 2 and significantly higher than that in grades 3-6 at the end of year 3. These results indicate that the health education had a greater impact on the children in the higher grades (who, presumably had better levels of understanding and practised better, personal, infection prevention) than on the younger children. Although school-based interventions can serve as a useful entry point for parasite control, more effort, including anthelminthic treatment, may be required among the youngest children. The activities need to be sustainable and supported by appropriate school-health policies.

[Sequence analysis of ITS2 and CO1 genes of Paragonimus harinasutai].

OBJECTIVE: To identify Paragonimus harinasutai from Ninghai, Zhejiang Province, China. METHODS: Metacercariae were collected from the crabs Sinopotamon chekiangenes in Xixi village of Ninghai County for ITS2 sequence analysis, CO1 sequence analysis and endonuclease BsaHI and StuI analysis by PCR-RFLP. Results The fingerprintings of PCR-RFLP were virtually same to the isolate from Thailand (Nakorn-nayok). The ITS2 sequence with 366 bp and CO1 sequence with 390 bp of the metacercariae collected from Ninghai revealed a nucleotide identity 95.6% and 89.5% respectively to the Thai isolate. CONCLUSION: The study confirmed that Paragonimus harinasutai is present in Ninghai, China, with certain variation on molecular biology in comparison to the Thai isolate.

Mapping soil-transmitted helminths in Southeast Asia and implications for parasite control.

Establishing the current status and distribution of soil-transmitted helminths is essential for developing and implementing parasite control. Although Southeast Asia is known to have a high prevalence of infection, a precise estimate of the total disease burden has not been fully described. Here, we use Geographical Information Systems (GIS) to collate and map recent published surveys on soil-transmitted helminth epidemiology and distribution for this region. Distinct geographical variation was observed, which is suggested to reflect climatic variation, as well as behavioral differences. However, for much of the region few data are available, and therefore it proved necessary to generate predictions of the distribution of soil-transmitted helminths using remotely sensed (RS) satellite sensor environmental variables. A significant finding was the importance of land surface temperature in influencing the distribution of Ascaris lumbricoides and Trichuris trichiura. Spatial analyses using RS satellite sensor data were then used to generate predictive maps of infection risk. This information provided the basis for an estimate of the population at risk of infection and the numbers requiring treatment. These applications of GIS and remote sensing provide a good basis for developing control of soil-transmitted helminths in the region.

Social and behavioral factors associated with Clonorchis infection in one commune located in the Red River Delta of Vietnam.

Social behavioral factors associated with Clonorchis infection are needed for control measures. The population in Nga Tan commune were randomly sampled and questioned to determine knowledge, perception, and health behavioral factors associated with Clonorchis infection among heads of households. The cellophane thick smear method was applied to examine their stool samples. Seven hundred and seventy-one cases were examined, the positive rates were 17.2%, 66.9%, 78.7%, 15.9%, and 0.14% for Clonorchis sinensis, Ascaris lumbricoides, Trichuris trichiura, hookworm, and Dicrocoelium dendriticum respectively. There was no significant difference between the infection rate of clonorchiasis, education level, and family income groups (p > 0.05). But there was significance difference between the infection rate of clonorchiasis and people living in different family sizes (p < 0.01). Thirty-four clonorchiasis patients treated with praziquantel 25 mg/kg/day for three days showed a cure rate in 30 days of 97.1%.

A cross-sectional study of intestinal parasitic infections among schoolchildren in Nan Province, Northern Thailand.

A cross-sectional study of the prevalence of intestinal parasitic infections at eight schools in Bo Klau district and four schools in Chalerm Prakiet district, Nan Province, in January and February, 2001. A total of 1,010 fecal samples were examined using the formalin-ether sedimentation technique. Results revealed that the rate of helminthic infection was 60.0%, while protozoa accounted for 36.2% of infections; mixed infections were common, resulting in a total prevalence of both parasites of 68.1%. Helminthic parasites, listed by frequency of infections, were Ascaris lumbricoides (21.7%), hookworm (18.5%), Trichuris trichiura (16.3%), Opisthorchis viverrini (1.7%), Strongyloides stercoralis (0.9%) and Enterobius vermicularis (0.9%). The protozoal infections were Entamoeba coli (25.8%), Giardia lamblia (5.3%), Endolimax nana (2.5%), Entamoeba histolytica (1.4%), Blastocystis hominis (0.8%), Chilomastix mesnili (0.3%) and Iodamoeba bütschlii (0.1%). This study emphasizes the need for improved environmental hygiene ie clean water supplies and enhanced sanitation, in affected communities. Health promotion, by means of a school-based educational approach is recommended; regular check-ups should be implemented, and a continuos program of treatment should be considered.

Epidemiology of trichinellosis in Asia and the Pacific Rim.

The epidemiology of trichinellosis, species of Trichinella present and the food and eating habits of people affected in Asia and the Pacific Rim are reviewed with emphasis on Japan, China and Thailand. Trichinella seems to be prevalent throughout this region although outbreaks of trichinellosis have not been reported in some areas. Major outbreaks of the disease have been reported primarily in China and Thailand. This is the result of three factors: (1) China and Thailand are highly endemic areas for this parasite; (2) the two countries are well-organized and there is a public health system that enables precise reporting of disease outbreaks and (3) culinary habits provide many opportunities to eat undercooked meats. Trichinella found in Asia and the Pacific Rim includes both encapsulated species (Trichinella spiralis, Trichinella britovi, Trichinella nativa) and noncapsulated species (Trichinella pseudospiralis, Trichinella papuae). T. britovi, isolated in Japan, is a different genotype from the European strain. Therefore, the Japanese strain of T. britovi is designated Trichinella T9. Human trichinellosis caused by T. pseudospiralis has occurred in New Zealand and Thailand. Tasmania has had animal cases of T. pseudospiralis infection and animals with T. papuae infection have been found in Papua New Guinea. Economic losses due to Trichinella infection are not negligible in China, where there have been more than 500 outbreaks of human trichinellosis, affecting more than 20,000 people and causing more than 200 deaths. In Thailand, over the past 27 years, 120 outbreaks were reported involving nearly 6700 patients and 97 deaths. Japan has had fewer outbreaks and some sporadic cases have been attributed to imported infection.

Efficacy of multiple dose mebendazole against trichuriasis in Thai and Karen patients.

Treatment of trichuriasis with mebendazole 500 mg for three days, and 100 mg twice daily for three days, yielded cure rates of 93.9 and 88.9% in Thai patients, while the cure rates in Karen patients were 96.2 and 95.5% respectively. The total number of Thai and Karen trichuriasis patients were 60 and 48, when tested by modified cellophane thick smear Kato-Katz technique. There were no significant differences among the two groups of patients and doses of treatment (p > 0.05).

Comparative studies on protein, isoenzyme and DNA restriction endonuclease profiles of Thai Paragonimus metacercariae.

General proteins and 14 enzymes from metacercariae of Paragonimus heterotremus, P. siamensis and P. westermani were determined by vertical polyacrylamide gel electrophoresis. The isoenzyme profiles showed considerable interspecific polymorphism for general protein (PT), malate dehydrogenase (MDH), malic enzyme (ME) and tetrazolium oxidase (TO) while those of glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) showed similarity. The Pt-6 and To-I loci can be used as identification markers for these three species. The preliminary study of the molecular biology of Paragonimus heterotremus P. siamensis and P. westermani was based on analysis of metacercarial genomic DNA with restriction endonuclease Pst I. Agarose gel electrophoresis revealed restriction fragment length differences among the three species studied. The DNA restriction fragments were approximately 4-6 fragments, ranging from 5.35 to 14.67 kb. Among these. P westermani shared two homologous fragments with P. siamensis, ie, 5.35 and 7.22 kh, none with P. heterotremus, while P. heterotremus shared only one with P. siamensis, ie, 8.16 kb. Thus, the DNA restriction fragment length differences can be used to differentiate among these three species.

IgG- and IgG4-detected antigens of Dirofilaria immitis adult worms for bancroftian filariasis by enzyme-linked immunoelectrotransfer blot.

In Thailand, Wuchereria bancrofti filariasis has persisted along the border between Thailand and Myanmar, its dynamic distribution caused by the infected transmigrants between neighboring countries, and the availability of susceptible mosquito vectors. Dirofilaria immitis adult worm was used as a source of antigens, excretory-secretory (ES) and partial surface extracts, to detect human filariasis. ES products showed several stained bands with Coomassie brilliant blue ranging from 14.5-93 kDa and mostly being glycoproteins as shown by concurrent reaction with Concanavalin A, except those at 18, 16 and 14.5 kDa which stained only with Coomassie brilliant blue. Surface proteins of 33.5-91.5 kDa were stained with Coomassie brilliant blue and showed smear bands with Concanavalin A. By enzyme-linked immunoelectrotransfer blot, Bancroftian filariasis sera gave specific reactions with glycoprotein ES antigens at MW 20.5 kDa against anti-human IgG. A prominent band of 18 kDa appeared consistently with the IgG4-ES antigen system. Surface extracts reacting with IgG and IgG4 were considered to be unsuitable as antibodies from all cases of filariasis could not detect any bands.

Clinostomum trematode from human eye.

The first human case of Clinostomum lacramalitis in Thailand is reported. A 38-year-old man visited an ophthalmology clinic after having itchy pain in the right eye for two days. Ophthalmological examination revealed a living worm adhered to the lacramal opening and after removal, it was identified as Clinostomum sp. The patient admitted that he used to eat raw freshwater fish, he caught in Prachin Buri Province where he lives.

A molecular perspective on the genera Paragonimus Braun, Euparagonimus Chen and Pagumogonimus Chen.

The status of the genera Euparagonimus Chen, 1963 and Pagumogonimus Chen, 1963 relative to Paragonimus Braun, 1899 was investigated using DNA sequences from the mitochondrial cytochrome c oxidase subunit I (CO1) gene (partial) and the nuclear ribosomal DNA second internal transcribed spacer (ITS2). In the phylogenetic trees constructed, the genus Pagumogonimus is clearly not monophyletic and therefore not a natural taxon. Indeed, the type species of Pagumogonimus, P. skrjabini from China, is very closely related to Paragonimus miyazakii from Japan. The status of Euparagonimus is less obvious. Euparagonimus cenocopiosus lies distant from other lungflukes included in the analysis. It can be placed as sister to Paragonimus in some analyses and falls within the genus in others. A recently published morphological study placed E. cenocopiosus within the genus Paragonimus and probably this is where it should remain.

Gnathostome infection in swamp eels, Fluta alba, in central Thailand.

To investigate the distribution of gnathostome worms in central Thailand, the infective larvae of Gnathostoma spp were examined from the flesh and liver of swamp eels, Fluta alba. Seven hundred and eighty-eight eels were purchased from markets in 11 provinces; Ang Thong (30), Ayutthaya (36), Chachoengsao (30), Lop Buri (30), Nakhon Nayok (437), Pathum Thani (30), Prachin Buri (48), Ratchaburi (53), Saraburi (30), Samut Prakan (30) and Suphan Buri (34). The highest rate of gnathostome infection was observed in swamp eels from Nakhon Nayok (68.7%). The infection rates in Ayutthaya, Ang Thong, Prachin Buri, Ratchaburi, Saraburi and Lop Buri were 33.3%, 26.7%, 25.0%, 18.9%, 13.3% and 10.0% respectively. Gnathostome larvae were not found in swamp eels from Chachoengsao, Pathum Thani, Samut Prakan and Suphan Buri. Among the 9,573 larvae recovered, almost all were the advanced third stage larvae of G. spinigerum, except one larva from Nakhon Nayok and two larvae from Ratchaburi which were identified as the advanced third stage larvae of G. vietnamicum and G. hispidum respectively. This study is the first report of swamp eels as natural intermediate hosts of G. vietnamicum and G. hispidum.

Seasonal variation in the intensity of Gnathostoma larvae in swamp eels (Fluta alba) sold in a local market in Bangkok.

The viscera of swamp eels were obtained from a local market in Bangkok twice a month from June 1996 to May 1997. The livers were separated, weighed and counted. Gnathostome larvae were recovered from the livers by the digestion technic, examined, identified, and counted. A total of 12,278 Gnathostoma larvae were obtained from 18,561.1 g (15,264 pieces) of eel livers. The overall average number of larvae/g liver and the overall average number of larvae/liver are 0.91 and 0.94, respectively. The greatest number of larvae/g liver (on average) was in December (high levels of infection during the months of October to December) whereas the lowest was in April (lowest levels of infection during the months of March to April). Thus there was a marked decrease in the average number of larvae/g liver during January to April, which then started to rise in May. This finding suggests that the level of infection abruptly decreases soon after the completion of the rainy season, starts to rise when the rain has come, and reaches its peak when the amount of rainfall is highest. More than 99% of the total gnathostome larvae recovered were identified to be G. spinigerum, and 25.4% of the entire larvae recovered bore variant or abnormal cephalic hooklets. The most common unusual feature was that there were extra rudimentary hooklets above row one, below row four and in between the four rows of hooklets which comprised 21.4%. In addition, the body size and the number of cephalic hooklets of G. spinigerum are also discussed.

Human lung fluke Paragonimus heterotremus: differential diagnosis between Paragonimus heterotremus and Paragonimus westermani infections by EITB.

The specificity of three major polypeptides (35, 33 and 32.5 kD) from Paragonimus heterotremus antigens prepared from ether-extracted adult worms was tested against sera from heterologous infections as well as against P. westermani-infected sera. Only the 35 kD polypeptide was not present, its antigenic determinant being bound to the antibodies from all P. westermani-infected cases. Its cross-reactivity against various sera from heterologous helminthiases and other lung infections showed that it is not bound to these antigenic polypeptides. These major bands cannot be detected by Concanavalin A detector. Our research encourages the pattern (35, 33 and 32.5 kD) of immunoblot reactions for the diagnosis of P. heterotremus infections; the 35 kD antigen is specific for corresponding species and able to differentiate infections between both species of Paragonimus.

Opisthorchis viverrini metacercaria in Thai freshwater fish.

Examination for metacercaria in freshwater fish, the common intermediate hosts of Opisthorchis viverrini was carried out during 1992-1996. The 4-year survey of fish from markets in 14 provinces revealed that metacercariae of O. viverrini were found in fish from Udon Thani, Sa Kaeo and Prachin Buri Provinces; fish from Aranyaprathet district had the highest positive rates (25-28%). Fish from 12 provinces were found to be positive with heterophyid metacercariae, namely: Haplorchis pumilio, H. taichui, H. yokogawai, Stellantchasmus falcatus, Centrocestus formosanus and Haplorchoides cahirinus. It was also observed that the prevalence of O. viverrini metacercaria in fish decreased markedly during the last 10 years.

Use of delipidized antigens of Taenia solium metacestodes in IgG-ELISA for detection of neurocysticercosis.

The development of IgG-ELISA for detecting neurocysticercosis is aimed at the routine laboratory, and requires a particular antigen preparation, an acceptable number of serum samples to be tested (both homologous and heterologous) and patients with a diversity of helminthic infections to rule out cross-reactions. This study characterizes IgG-antibodies from cases of neurocysticercosis by assaying the sera against ether-delipidized antigens (5 microg/ml) prepared from metacestodes of Taenia solium. The test had a sensitivity of 90% and a specificity of 83%. IgG-antibodies from heterologous serum samples elicited a number of false positives (25/147) from six different helminthic infections, ie paragonimiasis, echinococcosis, opisthorchiasis, ascariasis, taeniasis and fascioliasis. In additional tests to detect antibody levels to these stage-related antigens, one of three serum samples from T. solium-infected cases gave negative at OD value of 0.187 while the others yielded 0.472 and 0.576. Conversely, assays of all serum samples from neurocysticercosis cases reacted against antigens from Echinococcus granulosus cystic fluid, Paragonimus heterotremus and Opisthorchis viverrini adult worms. In comparison, the antigens from these three species yielded higher mean OD values when assayed against the corresponding infected serum samples. Furthermore, neurocysticercosis cases yielded OD values that are separate and distinct from those of paragonimiasis cases.