Considerable genetic diversity within Paragonimus heterotremus in Luang Prabang, northern Lao People's Democratic Republic

Paragonimiasis, caused by infection with lung flukes of the genus Paragonimus, remains a significant public health concern in Southeast Asia. In Lao People's Democratic Republic (Lao PDR), information on the distribution and genetic diversity of Paragonimus species is limited. This study investigated Paragonimus metacercariae in freshwater (mountain) crabs and analyzed their genetic diversity and phylogenetic relationships. Thirty-six crabs (Indochinamon sp.) were received from Xiang Ngeun and Pak Ou in Luang Prabang Province, northern Lao PDR. Partial mitochondrial 16S rRNA sequences obtained from four crabs indicated a moderately close relationship with Indochinamon ou. A total of 81 metacercariae identified morphologically as Paragonimus heterotremus were found among 13 out of the 32 crabs dissected (40.6 %). Molecular analyses targeting the ribosomal ITS2 region and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were conducted on these metacercariae. Phylogenetic analyses revealed that P. heterotremus sequences from Lao PDR clustered with those from neighboring countries-China, Myanmar, Vietnam and Thailand-suggesting potential genetic connectivity among eastern Asian populations. Haplotype-network analysis demonstrated significant genetic diversity within P. heterotremus populations from Lao PDR, separating into two distinct haplotype groups, one of which was unique to this study. This is the first report that Indochinamon sp. crabs serve as key intermediate host for a member of the P. heterotremus complex in Luang Prabang Province and highlights the parasite's genetic diversity in this region.