Co-infection with Opisthorchis viverrini and Haplorchis taichui detected by human fecal examination in Chomtong district, Chiang Mai Province, Thailand.

Diseases caused by the liver fluke, Opisthorchis viverrini and the minute intestinal fluke, Haplorchis taichui, are clinically important, especially in the Northeast and North regions of Thailand. It is often difficult to distinguish between these trematode species using morphological methods due to the similarity of their eggs and larval stages both in mixed and co-infections. A sensitive, accurate, and specific detection method of these flukes is required for an effective epidemiological control program. This study aimed to determine the prevalence of O. viverrini and H. taichui infections in human feces by using formalin-ether sedimentation and high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR methods. Fecal specimens of people living along the Mae Ping River, Chomtong district were examined seasonally for trematode eggs using a compound microscope. Positive cases were analyzed in HAT-RAPD, DNA profiles were compared with adult stages to determine the actual species infected, and specific DNA markers of each fluke were also screened. Our results showed that out of 316 specimens, 62 were positive for fluke eggs which were pre-identified as O. viverrini and H. taichui. In addition, co-infection among these two fluke species was observed from only two specimens. The prevalence of H. taichui infections peaked in the hot-dry (19.62%), gradually decreased in the rainy (18.18%), and cool-dry seasons (14.54%), respectively. O. viverrini was found only in the hot-dry season (6.54%). For molecular studies, 5 arbitrary primers (Operon Technologies, USA) were individually performed in HAT-RAPD-PCR for the generation of polymorphic DNA profiles. The DNA profiles in all 62 positives cases were the same as those of the adult stage which confirmed our identifications. This study demonstrates the mixed infection of O. viverrini and H. taichui and confirms the extended distribution of O. viverrini in Northern Thailand.

Copro-diagnosis of Haplorchis taichui infection using sedimentation and PCR-based methods.

Fifty-one human fecal specimens were collected from villagers inhabiting along Mae Kuang River, Ban Sob Tha, Pa Sang District, Lamphun Province, Thailand. By the formalin-ether sedimentation technique (FEST) under a light microscope, eggs of 3 helminth species, Haplorchis taichui, Ascaris lumbricoides and unidentified hookworm species were detected with prevalences of 22, 14 and 4%, respectively. PCR amplification with H. taichui specific primers showed that H. taichui specific amplicon 260 bp was generated in all FEST-positive specimens, and also in some FEST negative specimens. This H. taichui specific PCR method can be used to detect this parasite in all developmental stages and in both definitive and intermediate hosts, which should be useful in prevention and control programs.

Molecular identification of larval trematode in intermediate hosts from Chiang Mai, Thailand.

Snail and fish intermediate hosts were collected from rice fields in 3 districts; Mueang, Mae Taeng and Mae Rim of Chiang Mai Province during April-July 2008. For identification of larval trematode infection, standard (cracked for snail and enzymatically digested for fish) and molecular methods were performed. The results showed that three types of cercariae were found, pleurolophocercus, cotylocercous, and echinostome among 4 species of snail with a prevalence of 29, 23 and 3% respectively. Melanoides tuberculata snail was the most susceptible host for cercariae infection. Four species of metacercariae, Haplorchis taichui, Stellantchasmus falcatus, Haplorchoides sp and Centrocestus caninus, were found with a prevalence of 67, 25, 60 and 20%, respectively. The Siamese mud carp (Henicorhynchus siamensis) was the most susceptible fish host for H. taichui, and half- beaked fish (Dermogenys pusillus) for S. falcatus metacercariae infection, whereas Haplorchoides sp and C. caninus were concomitantly found in Puntius brevis. HAT-RAPD profile confirmed that pleurolophocercus cercariae found in Melanoides tuberculata from Mae Taeng District belonged to H. taichui and in Tarebia granifera from Mueang District were S. falcatus.

Survival of Haplorchis taichui metacercariae in Lab-Pla, Thai traditional food preparation.

Lab-Pla is a famous kind of Thai traditional food in northern and northeasthern Thailand. Consumption of uncooked Lab-Pla can lead to trematode infection because cyprinoid fish serve as the 2nd intermediate hosts of trematodes. The preparation of Lab-Pla can be made in different ways, depending on seasonings used in different places. The effect of the seasonings used in Lab-Pla on the survival of Haplorchis taichui metacercariae were, therefore, determined. The methodology was done by the following: 100 g of chopped Siamese mud carp fish (Henicorhynchus siamensis) were used for each experiment of seasonings used for completely mixed Lab-Pla. Mixed materials were blended and digested by 1% pepsin solution to obtain metacercariae and then checked for the movement under a stereomicroscope. The results showed that metacercariae remained active in 5 experimental groups: fermented fish treated, fish sauce treated, chili powder treated, ground roasted rice treated, and complete mixed treated indicated by average as follows: 27, 38.7, 33.3, 42.7 and 21%, respectively. In conclusion, fermented fish and completely mixed methods have more effectiveness in killing metacercariae in Lab-Pla preparation than other seasonings (p < 0.05).