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Diagnosis of Indolent Clonorchis sinensis and Opisthorchis viverrini Infections as Risk Factors for Cholangiocarcinoma: An Unmet Medical Need
Cholangiocarcinoma is a heterogeneous, highly aggressive cancer of the biliary tract epithelium with an overall 5-year relative survival rate of only 9%.1,2 Although surgical resection of localized, intrahepatic cholangiocarcinoma is associated with improved overall survival, most patients present with advanced disease not amenable to surgery due to a late onset of symptoms.2 Recently, an increased incidence of cholangiocarcinoma has been reported in the United States.3 Although relatively rare in the US, cholangiocarcinoma is prevalent across large parts of Asia, including China, Vietnam, Thailand, South Korea, and Taiwan.2
Risk Factors
To date, risk factors for developing cholangiocarcinoma have not been elucidated. 4,5 However, a growing body of literature suggests that chronic infection of genetically susceptible human subjects with Clonorchis sinensis ( C sinensis ) and Opisthorchis viverrini ( O viverrini ) plays a role. 6,7 The life cycle of these food-borne zoonotic trematodes involves eggs discharged in the stool of infected humans, the definitive host. 6,7 In nature, these eggs are ingested by freshwater snails, the intermediate host, where they undergo several developmental stages to form cercariae. Once released from snails into the water, free-swimming cercariae come in contact and penetrate freshwater fish where they encyst as metacercariae. Infection of humans occurs by ingesting undercooked, salted, pickled, or smoked freshwater fish infested with metacercariae. After ingestion, metacercariae excyst in the duodenum and ascend the biliary tract through the ampulla of Vater. They then mature into adult flukes that reside in small- and medium-sized intrahepatic biliary ducts. 6,7
Although most infected people remain asymptomatic, untreated indolent infections with C sinensis and O viverrini may persist in peripheral intrahepatic bile ducts for as long as 30 years, which is the lifespan of the trematodes.6,7 During this prolonged period, C sinensis and O viverrini feeding activities and their excretory-secretory products may damage bile duct epithelium and promote intense local inflammation.6,7 Conceivably, these pathological processes could then provoke the epithelial desquamation, adenomatous hyperplasia, goblet cell metaplasia, periductal fibrosis, and granuloma formation that are conducive to initiation and progression of cholangiocarcinoma in genetically susceptible people.8 Accordingly, the International Agency for Research on Cancer (IARC) has determined that there is sufficient evidence for the carcinogenicity of chronic infections with C sinensis and O viverrini in humans and that chronic infections with these trematodes cause cholangiocarcinoma.9 The IARC concluded that chronic infections with C sinensis and O viverrini are carcinogenic to humans (Group 1).9
Diagnosis
Presently, the diagnosis of C sinensis and O viverrini infection is based on microscopic identification and enumeration of the parasites’ eggs in weighted stool specimens using a formalin-ethyl acetate sedimentation concentration technique. 6,7 This approach requires a labor-intensive test that is conducted by an experienced technician. The test has low specificity and sensitivity because eggs could be confused with those of nonpathogenic intestinal flukes that are morphologically similar and because eggs are not present in feces during all stages of the infection. Although diffuse dilatation of intrahepatic bile ducts by screening sonography is used to diagnose clonorchiasis in endemic areas, it has low sensitivity, particularly in patients with low-level C sinensis and O viverrin i infections. 10
To address the current diagnostic gap, several enzyme-linked immunosorbent assays (ELISA) have been developed for the diagnosis of C sinensis, including monoclonal antibody-based (mAb) ELISA and indirect antibody ELISA.11,12 However, both have important limitations. The mAb ELISA detects only active infections while indirect antibody ELISA cross-reacts with other liver flukes.11,12 Taken together, these data illustrate the difficulties in diagnosing asymptomatic individuals with low-burden C sinensis or O viverrini infections by existing laboratory methods.
Timely serodiagnosis of indolent C sinensis and O viverrini infections is important because these parasites have recently been raised as a risk factor for cholangiocarcinoma in veterans who served in Vietnam.13 The American War Library estimates that as of February 28, 2019, about 610,000 Americans who served on land in Vietnam or in the air over Vietnam between 1954 and 1975 are alive, and about 164,000 Americans who served at sea in Vietnam waters are alive.14 To that end, Psevdos and colleagues screened 97 US veterans who served in Vietnam and identified 50 who reported exposure to raw or undercooked fish while there.13 None had evidence of active C sinensis or O viverrini infection. Blood samples obtained from these veterans were analyzed for circulating C sinensis and O viverrini antibodies using an ELISA developed in South Korea and 12 blood samples tested positive for the trematodes. Imaging of extrahepatic and intrahepatic bile ducts was unyielding in all cases. One veteran diagnosed with cholangiocarcinoma had repeated negative tests. However, the results of this study were challenged by several experts in this field because the authors did not report the sensitivity and specificity of the ELISA assay used.15
Serologic testing of US veterans who served in C sinensis and O viverrini–endemic countries for indolent infections with these parasites is not recommended at present.15 Nevertheless, there is an urgent need to develop sensitive and specific serologic assays, such as ELISA tests with recombinant antigens, to detect both acute and indolent infections caused by each biliary liver fluke in the US, including in patients diagnosed with cholangiocarcinoma. We posit that testing and treatment of high-risk populations could lead to earlier detection and treatment of cholangiocarcinoma, leading to improved overall survival in the population at risk.
1. American Cancer Society. Survival rates for bile duct cancer. Updated March 1, 2023. Accessed March 17, 2023. https://www.cancer.org/cancer/bile-duct-cancer/detection-diagnosis-staging/survival-by-stage.html
2. Vij M, Puri Y, Rammohan A, et al. Pathological, molecular, and clinical characteristics of cholangiocarcinoma: A comprehensive review. World J Gastrointest Oncol. 2022;14(3):607-627. doi:10.4251/wjgo.v14.i3.607
3. Yao KJ, Jabbour S, Parekh N, Lin Y, Moss RA. Increasing mortality in the United States from cholangiocarcinoma: an analysis of the National Center for Health Statistics Database. BMC Gastroenterol. 2016;16(1):117. Published 2016 Sep 21. doi:10.1186/s12876-016-0527-z
4. Rustagi T, Dasanu CA. Risk factors for gallbladder cancer and cholangiocarcinoma: similarities, differences and updates. J Gastrointest Cancer. 2012;43(2):137-147. doi:10.1007/s12029-011-9284-y
5. Maemura K, Natsugoe S, Takao S. Molecular mechanism of cholangiocarcinoma carcinogenesis. J Hepatobiliary Pancreat Sci. 2014;21(10):754-760. doi:10.1002/jhbp.126
6. Steele JA, Richter CH, Echaubard P, et al. Thinking beyond Opisthorchis viverrini for risk of cholangiocarcinoma in the lower Mekong region: a systematic review and meta-analysis. Infect Dis Poverty. 2018;7(1):44. Published 2018 May 17. doi:10.1186/s40249-018-0434-3.
7. Kim TS, Pak JH, Kim JB, Bahk YY. Clonorchis sinensis, an oriental liver fluke, as a human biological agent of cholangiocarcinoma: a brief review. BMB Rep. 2016;49(11):590-597. doi:10.5483/bmbrep.2016.49.11.109
8. Murata M. Inflammation and cancer. Environ Health Prev Med. 2018;23(1):50. Published 2018 Oct 20. doi:10.1186/s12199-018-0740-1
9. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Biological agents. IARC Monogr Eval Carcinog Risks Hum. 2012;100(pt B):1-441.
10. Mairiang E, Laha T, Bethony JM, et al. Ultrasonography assessment of hepatobiliary abnormalities in 3359 subjects with Opisthorchis viverrini infection in endemic areas of Thailand. Parasitol Int. 2012;61(1):208-211. doi:10.1016/j.parint.2011.07.009
11. Li HM, Qian MB, Yang YC, et al. Performance evaluation of existing immunoassays for Clonorchis sinensis infection in China. Parasit Vectors. 2018;11(1):35. Published 2018 Jan 15. doi:10.1186/s13071-018-2612-3
12. Hughes T, O’Connor T, Techasen A, et al. Opisthorchiasis and cholangiocarcinoma in Southeast Asia: an unresolved problem. Int J Gen Med. 2017;10:227-237. Published 2017 Aug 10. doi:10.2147/IJGM.S133292
13. Psevdos G, Ford FM, Hong ST. Screening US Vietnam veterans for liver fluke exposure 5 decades after the end of the war. Infect Dis Clin Pract (Baltim Md). 2018;26(4):208-210. doi:10.1097/IPC.0000000000000611
14. American War Library. In harm’s way... How many real Vietnam vets are alive today? Updated February 28, 2019. Accessed March 17, 2023. https://www.americanwarlibrary.com/personnel/vietvet.htm
15. Nash TE, Sullivan D, Mitre E, et al. Comments on “Screening US Vietnam veterans for liver fluke exposure 5 decades after the end of the war”. Infect Dis Clin Pract (Baltim Md). 2018;26(4):240-241. doi:10.1097/IPC.0000000000000659
Cholangiocarcinoma is a heterogeneous, highly aggressive cancer of the biliary tract epithelium with an overall 5-year relative survival rate of only 9%.1,2 Although surgical resection of localized, intrahepatic cholangiocarcinoma is associated with improved overall survival, most patients present with advanced disease not amenable to surgery due to a late onset of symptoms.2 Recently, an increased incidence of cholangiocarcinoma has been reported in the United States.3 Although relatively rare in the US, cholangiocarcinoma is prevalent across large parts of Asia, including China, Vietnam, Thailand, South Korea, and Taiwan.2
Risk Factors
To date, risk factors for developing cholangiocarcinoma have not been elucidated. 4,5 However, a growing body of literature suggests that chronic infection of genetically susceptible human subjects with Clonorchis sinensis ( C sinensis ) and Opisthorchis viverrini ( O viverrini ) plays a role. 6,7 The life cycle of these food-borne zoonotic trematodes involves eggs discharged in the stool of infected humans, the definitive host. 6,7 In nature, these eggs are ingested by freshwater snails, the intermediate host, where they undergo several developmental stages to form cercariae. Once released from snails into the water, free-swimming cercariae come in contact and penetrate freshwater fish where they encyst as metacercariae. Infection of humans occurs by ingesting undercooked, salted, pickled, or smoked freshwater fish infested with metacercariae. After ingestion, metacercariae excyst in the duodenum and ascend the biliary tract through the ampulla of Vater. They then mature into adult flukes that reside in small- and medium-sized intrahepatic biliary ducts. 6,7
Although most infected people remain asymptomatic, untreated indolent infections with C sinensis and O viverrini may persist in peripheral intrahepatic bile ducts for as long as 30 years, which is the lifespan of the trematodes.6,7 During this prolonged period, C sinensis and O viverrini feeding activities and their excretory-secretory products may damage bile duct epithelium and promote intense local inflammation.6,7 Conceivably, these pathological processes could then provoke the epithelial desquamation, adenomatous hyperplasia, goblet cell metaplasia, periductal fibrosis, and granuloma formation that are conducive to initiation and progression of cholangiocarcinoma in genetically susceptible people.8 Accordingly, the International Agency for Research on Cancer (IARC) has determined that there is sufficient evidence for the carcinogenicity of chronic infections with C sinensis and O viverrini in humans and that chronic infections with these trematodes cause cholangiocarcinoma.9 The IARC concluded that chronic infections with C sinensis and O viverrini are carcinogenic to humans (Group 1).9
Diagnosis
Presently, the diagnosis of C sinensis and O viverrini infection is based on microscopic identification and enumeration of the parasites’ eggs in weighted stool specimens using a formalin-ethyl acetate sedimentation concentration technique. 6,7 This approach requires a labor-intensive test that is conducted by an experienced technician. The test has low specificity and sensitivity because eggs could be confused with those of nonpathogenic intestinal flukes that are morphologically similar and because eggs are not present in feces during all stages of the infection. Although diffuse dilatation of intrahepatic bile ducts by screening sonography is used to diagnose clonorchiasis in endemic areas, it has low sensitivity, particularly in patients with low-level C sinensis and O viverrin i infections. 10
To address the current diagnostic gap, several enzyme-linked immunosorbent assays (ELISA) have been developed for the diagnosis of C sinensis, including monoclonal antibody-based (mAb) ELISA and indirect antibody ELISA.11,12 However, both have important limitations. The mAb ELISA detects only active infections while indirect antibody ELISA cross-reacts with other liver flukes.11,12 Taken together, these data illustrate the difficulties in diagnosing asymptomatic individuals with low-burden C sinensis or O viverrini infections by existing laboratory methods.
Timely serodiagnosis of indolent C sinensis and O viverrini infections is important because these parasites have recently been raised as a risk factor for cholangiocarcinoma in veterans who served in Vietnam.13 The American War Library estimates that as of February 28, 2019, about 610,000 Americans who served on land in Vietnam or in the air over Vietnam between 1954 and 1975 are alive, and about 164,000 Americans who served at sea in Vietnam waters are alive.14 To that end, Psevdos and colleagues screened 97 US veterans who served in Vietnam and identified 50 who reported exposure to raw or undercooked fish while there.13 None had evidence of active C sinensis or O viverrini infection. Blood samples obtained from these veterans were analyzed for circulating C sinensis and O viverrini antibodies using an ELISA developed in South Korea and 12 blood samples tested positive for the trematodes. Imaging of extrahepatic and intrahepatic bile ducts was unyielding in all cases. One veteran diagnosed with cholangiocarcinoma had repeated negative tests. However, the results of this study were challenged by several experts in this field because the authors did not report the sensitivity and specificity of the ELISA assay used.15
Serologic testing of US veterans who served in C sinensis and O viverrini–endemic countries for indolent infections with these parasites is not recommended at present.15 Nevertheless, there is an urgent need to develop sensitive and specific serologic assays, such as ELISA tests with recombinant antigens, to detect both acute and indolent infections caused by each biliary liver fluke in the US, including in patients diagnosed with cholangiocarcinoma. We posit that testing and treatment of high-risk populations could lead to earlier detection and treatment of cholangiocarcinoma, leading to improved overall survival in the population at risk.
Cholangiocarcinoma is a heterogeneous, highly aggressive cancer of the biliary tract epithelium with an overall 5-year relative survival rate of only 9%.1,2 Although surgical resection of localized, intrahepatic cholangiocarcinoma is associated with improved overall survival, most patients present with advanced disease not amenable to surgery due to a late onset of symptoms.2 Recently, an increased incidence of cholangiocarcinoma has been reported in the United States.3 Although relatively rare in the US, cholangiocarcinoma is prevalent across large parts of Asia, including China, Vietnam, Thailand, South Korea, and Taiwan.2
Risk Factors
To date, risk factors for developing cholangiocarcinoma have not been elucidated. 4,5 However, a growing body of literature suggests that chronic infection of genetically susceptible human subjects with Clonorchis sinensis ( C sinensis ) and Opisthorchis viverrini ( O viverrini ) plays a role. 6,7 The life cycle of these food-borne zoonotic trematodes involves eggs discharged in the stool of infected humans, the definitive host. 6,7 In nature, these eggs are ingested by freshwater snails, the intermediate host, where they undergo several developmental stages to form cercariae. Once released from snails into the water, free-swimming cercariae come in contact and penetrate freshwater fish where they encyst as metacercariae. Infection of humans occurs by ingesting undercooked, salted, pickled, or smoked freshwater fish infested with metacercariae. After ingestion, metacercariae excyst in the duodenum and ascend the biliary tract through the ampulla of Vater. They then mature into adult flukes that reside in small- and medium-sized intrahepatic biliary ducts. 6,7
Although most infected people remain asymptomatic, untreated indolent infections with C sinensis and O viverrini may persist in peripheral intrahepatic bile ducts for as long as 30 years, which is the lifespan of the trematodes.6,7 During this prolonged period, C sinensis and O viverrini feeding activities and their excretory-secretory products may damage bile duct epithelium and promote intense local inflammation.6,7 Conceivably, these pathological processes could then provoke the epithelial desquamation, adenomatous hyperplasia, goblet cell metaplasia, periductal fibrosis, and granuloma formation that are conducive to initiation and progression of cholangiocarcinoma in genetically susceptible people.8 Accordingly, the International Agency for Research on Cancer (IARC) has determined that there is sufficient evidence for the carcinogenicity of chronic infections with C sinensis and O viverrini in humans and that chronic infections with these trematodes cause cholangiocarcinoma.9 The IARC concluded that chronic infections with C sinensis and O viverrini are carcinogenic to humans (Group 1).9
Diagnosis
Presently, the diagnosis of C sinensis and O viverrini infection is based on microscopic identification and enumeration of the parasites’ eggs in weighted stool specimens using a formalin-ethyl acetate sedimentation concentration technique. 6,7 This approach requires a labor-intensive test that is conducted by an experienced technician. The test has low specificity and sensitivity because eggs could be confused with those of nonpathogenic intestinal flukes that are morphologically similar and because eggs are not present in feces during all stages of the infection. Although diffuse dilatation of intrahepatic bile ducts by screening sonography is used to diagnose clonorchiasis in endemic areas, it has low sensitivity, particularly in patients with low-level C sinensis and O viverrin i infections. 10
To address the current diagnostic gap, several enzyme-linked immunosorbent assays (ELISA) have been developed for the diagnosis of C sinensis, including monoclonal antibody-based (mAb) ELISA and indirect antibody ELISA.11,12 However, both have important limitations. The mAb ELISA detects only active infections while indirect antibody ELISA cross-reacts with other liver flukes.11,12 Taken together, these data illustrate the difficulties in diagnosing asymptomatic individuals with low-burden C sinensis or O viverrini infections by existing laboratory methods.
Timely serodiagnosis of indolent C sinensis and O viverrini infections is important because these parasites have recently been raised as a risk factor for cholangiocarcinoma in veterans who served in Vietnam.13 The American War Library estimates that as of February 28, 2019, about 610,000 Americans who served on land in Vietnam or in the air over Vietnam between 1954 and 1975 are alive, and about 164,000 Americans who served at sea in Vietnam waters are alive.14 To that end, Psevdos and colleagues screened 97 US veterans who served in Vietnam and identified 50 who reported exposure to raw or undercooked fish while there.13 None had evidence of active C sinensis or O viverrini infection. Blood samples obtained from these veterans were analyzed for circulating C sinensis and O viverrini antibodies using an ELISA developed in South Korea and 12 blood samples tested positive for the trematodes. Imaging of extrahepatic and intrahepatic bile ducts was unyielding in all cases. One veteran diagnosed with cholangiocarcinoma had repeated negative tests. However, the results of this study were challenged by several experts in this field because the authors did not report the sensitivity and specificity of the ELISA assay used.15
Serologic testing of US veterans who served in C sinensis and O viverrini–endemic countries for indolent infections with these parasites is not recommended at present.15 Nevertheless, there is an urgent need to develop sensitive and specific serologic assays, such as ELISA tests with recombinant antigens, to detect both acute and indolent infections caused by each biliary liver fluke in the US, including in patients diagnosed with cholangiocarcinoma. We posit that testing and treatment of high-risk populations could lead to earlier detection and treatment of cholangiocarcinoma, leading to improved overall survival in the population at risk.
1. American Cancer Society. Survival rates for bile duct cancer. Updated March 1, 2023. Accessed March 17, 2023. https://www.cancer.org/cancer/bile-duct-cancer/detection-diagnosis-staging/survival-by-stage.html
2. Vij M, Puri Y, Rammohan A, et al. Pathological, molecular, and clinical characteristics of cholangiocarcinoma: A comprehensive review. World J Gastrointest Oncol. 2022;14(3):607-627. doi:10.4251/wjgo.v14.i3.607
3. Yao KJ, Jabbour S, Parekh N, Lin Y, Moss RA. Increasing mortality in the United States from cholangiocarcinoma: an analysis of the National Center for Health Statistics Database. BMC Gastroenterol. 2016;16(1):117. Published 2016 Sep 21. doi:10.1186/s12876-016-0527-z
4. Rustagi T, Dasanu CA. Risk factors for gallbladder cancer and cholangiocarcinoma: similarities, differences and updates. J Gastrointest Cancer. 2012;43(2):137-147. doi:10.1007/s12029-011-9284-y
5. Maemura K, Natsugoe S, Takao S. Molecular mechanism of cholangiocarcinoma carcinogenesis. J Hepatobiliary Pancreat Sci. 2014;21(10):754-760. doi:10.1002/jhbp.126
6. Steele JA, Richter CH, Echaubard P, et al. Thinking beyond Opisthorchis viverrini for risk of cholangiocarcinoma in the lower Mekong region: a systematic review and meta-analysis. Infect Dis Poverty. 2018;7(1):44. Published 2018 May 17. doi:10.1186/s40249-018-0434-3.
7. Kim TS, Pak JH, Kim JB, Bahk YY. Clonorchis sinensis, an oriental liver fluke, as a human biological agent of cholangiocarcinoma: a brief review. BMB Rep. 2016;49(11):590-597. doi:10.5483/bmbrep.2016.49.11.109
8. Murata M. Inflammation and cancer. Environ Health Prev Med. 2018;23(1):50. Published 2018 Oct 20. doi:10.1186/s12199-018-0740-1
9. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Biological agents. IARC Monogr Eval Carcinog Risks Hum. 2012;100(pt B):1-441.
10. Mairiang E, Laha T, Bethony JM, et al. Ultrasonography assessment of hepatobiliary abnormalities in 3359 subjects with Opisthorchis viverrini infection in endemic areas of Thailand. Parasitol Int. 2012;61(1):208-211. doi:10.1016/j.parint.2011.07.009
11. Li HM, Qian MB, Yang YC, et al. Performance evaluation of existing immunoassays for Clonorchis sinensis infection in China. Parasit Vectors. 2018;11(1):35. Published 2018 Jan 15. doi:10.1186/s13071-018-2612-3
12. Hughes T, O’Connor T, Techasen A, et al. Opisthorchiasis and cholangiocarcinoma in Southeast Asia: an unresolved problem. Int J Gen Med. 2017;10:227-237. Published 2017 Aug 10. doi:10.2147/IJGM.S133292
13. Psevdos G, Ford FM, Hong ST. Screening US Vietnam veterans for liver fluke exposure 5 decades after the end of the war. Infect Dis Clin Pract (Baltim Md). 2018;26(4):208-210. doi:10.1097/IPC.0000000000000611
14. American War Library. In harm’s way... How many real Vietnam vets are alive today? Updated February 28, 2019. Accessed March 17, 2023. https://www.americanwarlibrary.com/personnel/vietvet.htm
15. Nash TE, Sullivan D, Mitre E, et al. Comments on “Screening US Vietnam veterans for liver fluke exposure 5 decades after the end of the war”. Infect Dis Clin Pract (Baltim Md). 2018;26(4):240-241. doi:10.1097/IPC.0000000000000659
1. American Cancer Society. Survival rates for bile duct cancer. Updated March 1, 2023. Accessed March 17, 2023. https://www.cancer.org/cancer/bile-duct-cancer/detection-diagnosis-staging/survival-by-stage.html
2. Vij M, Puri Y, Rammohan A, et al. Pathological, molecular, and clinical characteristics of cholangiocarcinoma: A comprehensive review. World J Gastrointest Oncol. 2022;14(3):607-627. doi:10.4251/wjgo.v14.i3.607
3. Yao KJ, Jabbour S, Parekh N, Lin Y, Moss RA. Increasing mortality in the United States from cholangiocarcinoma: an analysis of the National Center for Health Statistics Database. BMC Gastroenterol. 2016;16(1):117. Published 2016 Sep 21. doi:10.1186/s12876-016-0527-z
4. Rustagi T, Dasanu CA. Risk factors for gallbladder cancer and cholangiocarcinoma: similarities, differences and updates. J Gastrointest Cancer. 2012;43(2):137-147. doi:10.1007/s12029-011-9284-y
5. Maemura K, Natsugoe S, Takao S. Molecular mechanism of cholangiocarcinoma carcinogenesis. J Hepatobiliary Pancreat Sci. 2014;21(10):754-760. doi:10.1002/jhbp.126
6. Steele JA, Richter CH, Echaubard P, et al. Thinking beyond Opisthorchis viverrini for risk of cholangiocarcinoma in the lower Mekong region: a systematic review and meta-analysis. Infect Dis Poverty. 2018;7(1):44. Published 2018 May 17. doi:10.1186/s40249-018-0434-3.
7. Kim TS, Pak JH, Kim JB, Bahk YY. Clonorchis sinensis, an oriental liver fluke, as a human biological agent of cholangiocarcinoma: a brief review. BMB Rep. 2016;49(11):590-597. doi:10.5483/bmbrep.2016.49.11.109
8. Murata M. Inflammation and cancer. Environ Health Prev Med. 2018;23(1):50. Published 2018 Oct 20. doi:10.1186/s12199-018-0740-1
9. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Biological agents. IARC Monogr Eval Carcinog Risks Hum. 2012;100(pt B):1-441.
10. Mairiang E, Laha T, Bethony JM, et al. Ultrasonography assessment of hepatobiliary abnormalities in 3359 subjects with Opisthorchis viverrini infection in endemic areas of Thailand. Parasitol Int. 2012;61(1):208-211. doi:10.1016/j.parint.2011.07.009
11. Li HM, Qian MB, Yang YC, et al. Performance evaluation of existing immunoassays for Clonorchis sinensis infection in China. Parasit Vectors. 2018;11(1):35. Published 2018 Jan 15. doi:10.1186/s13071-018-2612-3
12. Hughes T, O’Connor T, Techasen A, et al. Opisthorchiasis and cholangiocarcinoma in Southeast Asia: an unresolved problem. Int J Gen Med. 2017;10:227-237. Published 2017 Aug 10. doi:10.2147/IJGM.S133292
13. Psevdos G, Ford FM, Hong ST. Screening US Vietnam veterans for liver fluke exposure 5 decades after the end of the war. Infect Dis Clin Pract (Baltim Md). 2018;26(4):208-210. doi:10.1097/IPC.0000000000000611
14. American War Library. In harm’s way... How many real Vietnam vets are alive today? Updated February 28, 2019. Accessed March 17, 2023. https://www.americanwarlibrary.com/personnel/vietvet.htm
15. Nash TE, Sullivan D, Mitre E, et al. Comments on “Screening US Vietnam veterans for liver fluke exposure 5 decades after the end of the war”. Infect Dis Clin Pract (Baltim Md). 2018;26(4):240-241. doi:10.1097/IPC.0000000000000659