Efficiency of the Stool-PCR Test Targeting NADH Dehydrogenase (Nad) Subunits for Detection of Opisthorchis viverrini Eggs

Opisthorchis viverrini infection is the major parasitic infection problem in Southeast Asian countries, and long-term infection will lead to cholangiocarcinoma (CCA), the bile duct cancer. The early diagnosis of O. viverrini infection may interrupt the progression of the opisthorchiasis and other related illnesses, especially CCA. The current diagnostic procedure is stool examination by microscope-based methods such as direct smear and concentration techniques but it is limited by low parasite egg numbers. The molecular diagnosis prompts the chance to evaluate the light infection with low number of parasite eggs but is currently inconvenient for routine use due to special equipment requirement and unstable sensitivities. Our present study aims to establish the efficiency of OvNad subunits, the mitochondrial gene, for introducing as a potential diagnostic target by conventional PCR, the cheapest and easiest molecular procedure. A total of 166 stool samples were investigated microscopically by the PBS-ethyl acetate concentration technique (PECT); 75 samples were O. viverrini positive with 28 samples that were positive with single parasite (hookworm, A. lumbricoides, S. stercoralis, Taenia spp., and T. trichiura), 11 samples were with mixed infection, and 52 samples were without parasite detection. The detection limits of OvNad subunits were evaluated in artificially spiked samples containing 0, 1, 5, 10, 20, 50, and 100 Ov-eggs. The result suggested that the best detection efficacy was of OvNad5 that had exact detection limits at only 5 eggs. In the PCR amplification of OvNad subunits, there exist 100% specificities with varied sensitivities from 64%, 88%, 80%, and 100% of OvNad1, OvNad2, OvNad4, and OvNad5, respectively. OvNad subunits were amplified specifically without cross reactivity with the other collected parasites. Our study established that OvNad subunits, especially OvNad5, are the potent candidates for PCR amplification of stool containing Ov-eggs with high confidential sensitivity, specificity, PPV, and NPV even in the light infection that would be a benefit for developing as a routine diagnosis of O. viverrini infection.

Worm-free Cooking, Fish Safety: Recommendation to Strategy for Opisthorchiasis Prevention Program

Fish-borne parasitic zoonosis such as Opisthorchiasis caused by Opisthorchis viverrini remains a major public health problem in many parts of Southeast Asia and Me Kong Basin region including Thailand. The focal point of Opisthorchiasis is located in north-eastern part of Thailand, along with high prevalence coincidence of cholangiocarcinoma, a major primary carcinoma of the liver with a very poor prognosis. Opisthorchis viverrini infection caused by developed cholangiocarcinoma (CCA). O. viverrini infection is acquired by eating raw or partially cooked fish. In endemic areas, several food preparations contain uncooked or raw freshwater fish. The most common local freshwater fish used for this recipe are Koi Pla, Pla Som, Lab Pla and Pla Yang or Grill fish. Raw fish dishes are known to be associated with the risk of liver fluke infection due to the consumption of cyprinoid fish that contains metacercaria of O. viverrini. Grill fish and Lab-Pla are among the famous Thai traditional food in the northeastern and northern part of Thailand. The consumption of raw Grill fish can lead to O. viverrini infection because cyprinoid fish is a second intermediate host of O. viverrini. This study investigated the literature of previous working for prevention and control of Opisthorchiasis and confirm worm-free cooking safety of cooking with consideration of time for the preparation of freshwater fish-grill for the prevention and control of Opisthorchiasis and Cholangiocarcinoma infection. The unique experiment experimental designed. The freshwater fishes grill within the group of 0,1,2,3,4 and 5 minutes with 10 fresh water fishes from fisherman in Nakhon Phanom and Sakhon Nakorn provinces which reported high O. viverrini infection among risk people in 2009. The another experiment using frozen freshwater fish at -10 degree for 5 days. The results showed that metacercariae remained active in control and 1-2 minutes experimental groups. The groups 3-5 minutes of grill fish partially cooked showed inactive metacercariae. The excretory bladder spread with unclear form. The conclusion suggested that worm-free cooking under review, and unique experiment of cook safety is a crucial basic knowledge leading to bringing knowledge, practically for the prevention and control Liver fluke infection. Recommendation information also let people better understand the concerns during health literacy program to stop transmission of liver fluke and need to be discussed among head villagers, public health agencies and teachers in the public participation process and school health program.