Application of multiplex pcr for species discrimination using individual metacercariae of Paragonimus occurring in Thailand.

A total of 6 lung fluke species have been documented in Thailand, of which P. heterotremus is the most important, since it affects humans. Although P. westermani is found as metacercariae in the same crab species as P. heterotremus in Thailand, human infections with P. westermani have not been confirmed. To accurately discriminate between the individual metacercariae of these two species, we established a multiplex PCR method. Using this method, two products each were amplified from the metacercarial DNA samples of P. heterotremus (ca. 310 and 520 bp) and P. westermani (ca. 140 and 520 bp). In contrast, 520-bp products alone were found to be generated from the DNA samples of P. siamensis, P. bangkokensis and P. harinasutai, 3 other species of lung flukes known to occur in Thailand. Digestion of these 520-bp products with the restriction enzyme ScrFI could unequivocally discriminate species by the number and size of the produced band(s): 3 bands (ca. 60, 210 and 250 bp) for P. harinasutai, 2 bands (ca. 250 and 270 bp) for P. bangkokensis, and an uncut band (520 bp) for P. siamensis. The established multiplex PCR used in combination with restriction enzyme digestion (PCR-RFLP with ScrFI) is effective for discriminating the 5 different species of lung flukes occurring in Thailand, even at the metacercarial stage.

Molecular discrimination between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand.

To accurately discriminate between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand, polymerase chain reaction (PCR)-based molecular methods were established and subjected to an evaluation. We first amplified and sequenced the second internal transcribed spacer (ITS2) region of the nuclear ribosomal DNA of the two species. Based on their nucleotide differences, P. heterotremus and P. westermani were unequivocally discriminated from each other. These nucleotide differences were further utilized to select the ApaL1 endonuclease site for PCR-restriction fragment length polymorphism (PCR-RFLP) analyses and to design species-specific primers for multiplex PCR reactions. Both PCR-RFLP and multiplex PCR methods allowed a more rapid and labor-effective species discrimination. Furthermore, the multiplex PCR method enabled the most efficient discrimination because species identification involved a single round of PCR in a single tube. In Thailand, P. heterotremus is the only species affecting humans. Thus, the methods established in the present study can be used as reliable tools to identify the lung fluke metacercariae that cause human disease.

Investigation on the epidemiological factors of Clonorchis sinensis infection in an area of south China.

To detect the epidemiological factors of Clonorchis sinensis infection in Hengxian County, one of counties in Guangxi Zhuang Autonomous Region where Schistosoma japonicum was endemic but eliminated in the late 1980s, a questionnaire was designed with 37 questions covering socioeconomic conditions and human behavior, contamination of the environment and fish ponds, inadequate farming/fishery practices; and the formalin-ether sedimentation technique was used to examine the feces of cats, dogs and pigs for the eggs of C. sinensis. Fresh fish was sliced and digested to detect the metacercariae of C. sinensis under a stereomicroscope. Of 1,521 people interviewed, 64% of the interviewees did not know about fluke disease or its transmission route, 46% of those who knew about the fluke believed that the infection caused no harm or only slight harm to their health. More than half of the interviewees (51%) ate raw fish at least 1-2 times per month, more among the middle-aged males. Eight percent of them used the same utensils for both raw fish and cooked food. When advice was given not to eat raw fish, 73% of the interviewees thought it was not feasible. In relation to pisciculture, 25% and 9% of the owners of fish ponds fed their fish with feces of domestic animals and human feces, respectively. The prevalences of C. sinensis infection in cats, dogs and pigs were 70, 50 and 27%, respectively, and the infection rate in fish was 40%. These results indicate the poor knowledge, and beliefs, and unhealthy behaviors in the human population. They also show poor environmental hygiene and inappropriate farming/fishery practices are important in the increase of C. sinensis prevalence in humans. Combined interventions, including health education, environmental modification, reform of traditional farming/fishery practices, mass screening and chemotherapy for humans, and the management of domestic animals are needed in developing control strategies to decrease C. sinensis infection in the human population.