BackgroundNinh Binh province is known as an endemic area of fish-borne zoonotic trematode (FZT) transmission in Vietnam. A cross-sectional study was conducted in Gia Minh and Gia Thinh communes of Gia Vien district, Ninh Binh province to investigate the infections with different stages of FZT in various host species.MethodsFaecal samples from 1,857 humans were examined for trematode eggs using the Kato-Katz method, while faecal samples from 104 dogs, 100 cats, and 100 pigs were examined using the Formalin-ethyl acetate concentration technique (FECT). A total of 483 specimens of freshwater fish, representing 9 species, were examined for metacercariae using the artificial digestion method. Three methods of cercarial detection (shedding, crushing and cutting) were applied for examination of 3,972 specimens of freshwater snails, representing 7 species. All relevant data e.g. location, sex, age group, animal species, and habitat were recorded for statistical analyses.ResultsHelminth eggs were found in 65.5% of the human faecal samples, including 20.5% of faecal samples containing small trematode eggs. Infection with small trematodes differed among communes, age groups and sexes. Eggs of small trematodes were found in 32.7% of faecal samples from dogs, 49.0% from cats and 13.0% from pigs. The difference in prevalences and intensities were significant among species of animals but did not differ between the two communes. All fish species were infected with FZT, with an average prevalence of 56.1% and a mean intensity of 33.245 metacercariae per gram. Prevalence and intensity in fish differed significantly among cummunes and fish groups. Six species of zoonotic trematodes were identified. Metacercariae of the small liver fluke, Clonorchis sinensis, was only found in Hemiculter leucisculus. A total of 9 specimens from two snail species, Melanoides tuberculata and Bithynia fuchsiana, were infected with trematodes and four cercarial types were detected in the study sites.ConclusionsWe conclude that Gia Minh and Gia Thinh communes are continuing to be hot-spot endemic areas of FZT and other helminths infections where the habit of eating raw fish by the local people is still present.

We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.

Two members of the Paragonimus skrjabini complex, P. skrjabini and P. miyazakii, are now considered as two sub-species, P. skrjabini skrjabini and P. skrjabini miyazakii. They are well known as important pathogens for human paragonimiasis in China and Japan. Recently, members of this species complex have been reported from India. Here we report the first discovery of P. skrjabini from freshwater crab hosts in Thanh Hoa province, Vietnam. For morphological and molecular phylogenetic studies, adult worms were obtained by experimental infection in cats and dogs. Molecular analyses of metacercariae and adults revealed that the P. skrjabini population from Thanh Hoa, Vietnam was almost completely identical with that from Yunnan province, China. Those populations from Thanh Hoa, Vietnam and Yunnan, China and those from Manipur, India were significantly different from P. skrjabini populations reported from other localities of China in cytochrome oxidase subunit 1 (CO1) gene sequences, indicating considerable genetic variation within the P. skrjabini complex. Moreover, low bootstrap values in the CO1 tree suggested that more variant genotypes belonging to P. skrjabini complex may be found in other Asian countries in between Vietnam and India, such as Myanmar, Laos and Thailand. Since P. skrjabini is known as a pathogen for humans, paragonimiasis cases caused by P. skrjabini might be found in Vietnam and other Asian countries.

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