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Twenty provinces in northeastern Thailand were investigated for fluke metacercariae contamination in pickled fish, or pla-som, during January –June 2016. A total of 129 pickled fish shops were randomly chosen. Samples were digested with acid-pepsin and those found to be infected with metacercariae were fed to hamsters to test for metacercariae infectivity. The results demonstrated that only 20.2% of the pla-som samples were infected with fluke metacercariae (mc), at various levels (1 to 268 mc/kg). All recovered fluke metacercariae were inactive, degenerated and could not develop to adults in the animal model. In conclusion, the fluke mc infection status in pla-som was correlated with the prevalence of fluke infection in this region known for high O.viverrini and cholangiocarcinoma development. Clearly, systematic control of the fluke life cycle is needed. Whether pickling is an effective preventive measure needs further assessment.

Background: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective; thus innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. This study aimed to investigate the synergistic effects of forbesione combined with 5-fluorouracil (5-FU) in hamster cholangiocarcinoma (Ham-1) cells both in vitro and in vivo. The anti-tumor effects of 5-FU combined with forbesione in vitro were determined using the Sulforhodamine B (SRB) assay and the effects in vivo were assessed in transplanted Ham-1 allograph models. Using ethidium bromide/acridine orange (EB/AO) staining, the morphological changes of apoptotic cells was investigated. The expressions of apoptosis-related molecules after combined treatment with forbesione and 5-FU were determined using real-time RT-PCR and western blot analysis. Forbesione or 5-FU alone inhibited proliferation of Ham-1 cells in a dose-dependent manner and their combination showed a synergistic proliferation inhibitory effect in vitro. In vivo studies, forbesione in combination with 5-FU exhibited greater inhibition of the tumor in the hamster model compared with treatment using either drug alone. Forbesione combined with 5-FU exerted stronger apoptotic induction in Ham-1 cells than did single drug treatment. The combination of drugs strongly suppressed the expression of B-cell lymphoma 2 (Bcl-2) and procaspase-3 while enhancing the expression of p53, Bcl-2-associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1), caspase-9 and caspase-3, compared with single drug treatments. These results explained the decreased expression of cytokeratin 19 (CK19) positive cells and proliferation cell nuclear antigen (PCNA) positive cells in Ham-1 cell tumor tissues of the treated hamsters. There was no apparent systemic toxicity observed in the treated animals compared with the control groups. Forbesione combined with 5-FU strongly induced apoptosis in Ham-1 cells. The growth inhibitory effect of combined treatment using these two drugs was much greater than treatment with either drug alone, both in vitro and in vivo.

Consumption of fluke-free fish is the most important factor in controlling Opisthorchis viverrini (OV) infection in endemic areas such as northeast Thailand and thereby reducing the risk of cholangiocarcinoma. Cooking fish is the best way to avoid infection; however, the cultural practice of eating raw or fermented fish is difficult to change. We investigated the food preparation process, using freezing, heating and fermentation to kill OV metacercariae in fish. Uncooked cyprinid fish infected with OV were divided into three groups: refrigerated at 4 oC for 24, 48 or 72 h (control group); frozen at -20 oC for 24, 48 or 72 h; or heated by microwaving (at 400 or 800 W) or boiling at 90 oC for 1, 5 or 10 min. Moreover, pickled (fermented) fish were divided into two groups: refrigerated at 4 oC (control) or frozen at -20 oC for 24 or 48 h. The infectivity of recovered metacercariae was confirmed by infecting hamsters with OV and then evaluating the recovery of adult worms after 1 month. We found that a heating process, by boiling or microwaving at 400 or 800 W for at least 5 min, could kill OV metacercariae, and freezing pickled fish at -20 oC for 48 h could kill OV metacercariae in all sizes of fish. The present study found that heating and freezing processes, as well as the fermentation process under optimal conditions, could kill OV metacercariae in a timely manner. This knowledge is valuable for implementation in endemic areas to control OV infection and cholangiocarcinoma.

1) Expression of apoptosis-related factors in muscles infected with Trichinella spiralis, 2) Differences and similarities of nurse cells in cysts of Trichinella spiralis and T. pseudospiralis