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.

A new record of Paragonimus other than P. westermani in southern Thailand.

Field surveys of Paragonimus in Surat Thani Province, southern Thailand, revealed a new record of a lung fluke species other than P. westermani. The metacercariae were obtained from the crab, Ranguna smalleyi. The cysts of the metacercariae were spherical in shape and the larval body in the cysts contained pinkish granules. Fully mature adult worms were obtained from experimental infections with a rat and a ferret. The adult worms from the two host animals resembled each other, except for size, and had the anatomical characteristics of P. bangkokensis, ie the cuticular spines were arranged mainly in groups, the ovaries were highly branched, while the testes were more simply divided. Chromosomal preparations of the testes showed a haploid number of 11. As no sequence data of P. bangkokensis has been deposited in the GenBank/EMBL/DDBJ nucleotide database, the ITS2 region was sequenced using the metacercariae as starting material. A similarity search of P. bangkokensis ITS2 sequence using the BLAST program revealed that there was only one base difference between this population and P. harinasutai occurring in central Thailand. The result may suggest a close relationship between P. bangkokensis and P. harinasutai. This is the first description of Paragonimus species other than P. westermani occurring in southern Thailand.

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.