Trematodes: Liver Flukes PDF

Trematodes: Liver Flukes Module 07: GI and Nutritional Pathologies Julius R. Migriño Jr, MD | Asynchronous TABLE OF CONTENTS B. DEMOGRAPHICS I. OVERVIEW....................................................................................... 1 II. FASCIOLA.................................................

Trematodes: Liver Flukes Module 07: GI and Nutritional Pathologies Julius R. Migriño Jr, MD | Asynchronous TABLE OF CONTENTS B. DEMOGRAPHICS I. OVERVIEW....................................................................................... 1 II. FASCIOLA.........................................................................................1 A. MORPHOLOGY............................................................................. 1 B. DEMOGRAPHICS.......................................................................... 1 C. LIFE CYCLE.................................................................................... 1 D. SYMPTOMS.................................................................................. 2 E. DIAGNOSIS................................................................................... 2 F. TREATMENT.................................................................................. 3 G. PREVENTION AND CONTROL....................................................... 3 III. CLONORCHIS AND OPISTHORCHIS...................................................3 A. MORPHOLOGY............................................................................. 3 B. DEMOGRAPHICS.......................................................................... 4 C. LIFE CYCLE.................................................................................... 4 D. SYMPTOMS.................................................................................. 5 E. DIAGNOSIS................................................................................... 6 F. TREATMENT.................................................................................. 6 G. PREVENTION AND CONTROL....................................................... 6 QUESTIONS......................................................................................... 6 ANSWER KEY.......................................................................................7 RATIONALE..........................................................................................7 I. OVERVIEW There are two major classifications of liver flukes: ○ Fasciola ○ Clonorchis, Opisthorchis Liver flukes are found worldwide ○ Usually found in countries where sheep and cattle are primarily raised ○ Increased incidence through the years ○ Fascioliasis: most important helminth infection in cattle but not in humans II. FASCIOLA Trematodes from genus Fasciola ○ F. hepatica: sheep liver fluke ○ F. gigantica: giant liver fluke Figure 2. Common geographic distribution of Fasciola. Top: F. hepatica. Bottom: F. gigantica F. hepatica: temperate liver fluke ○ Most common in Europe, Middle East, and Andean regions F. gigantica: tropical liver fluke ○ Most common in Asia, Africa, and Hawaii ▸ In Asia, it is commonly seen in China, Vietnam, Korea, India, and Thailand C. LIFE CYCLE A. MORPHOLOGY Figure 1. Morphology of Fasciola Larger compared to other flukes ○ Can grow up to 7 cm (70 mm) long and 1.5 cm (15 mm) wide Adult features: ○ Cone-shaped head (encircled in Figure 1) ○ Prominent shoulders (pointed in Figure 1) ▸ Delineate anterior portion from rest of the body YL6:07.32 Figure 3. Life cycle of Fasciola 1. Ingestion ○ Humans ingest metacercariae in contaminated freshwater plants ▸ Freshwater plant: second intermediate host – Common source of infection in Latin America, France, Algeria: watercress TG05: Artillaga, Avila, Cheng, Escalante, Lim, Marcos, Palencia, Quimson, Remitio, Salvador, Sandoval CG17: Angeles, Ayop, Basa, Dy, Layco, Lim, Medel, Ocampo, Regio, Salipsip, Simpao 1 2. 3. 4. 5. 6. 7. 8. – Common source of infection in South Korea: water parsley – Common sources of infection in Asia: water bamboo (jiao bai), water caltrop, morning glory ▸ Metacercariae: infective stage Excystation ○ Metacercariae excyst in the duodenum ○ Larvae are released in the GI tract Migration ○ Larvae penetrates the intestinal wall, enters the peritoneal cavity, then migrates to the liver parenchyma Sexual Maturation ○ Larvae migrate from the liver parenchyma to the biliary ducts, where they mature into adult flukes ▸ In humans, maturation from metacercariae into adult flukes usually takes 3-4 months after ingestion ▸ Lifespan of mature Fasciola: 9-13 years Reproduction ○ Adults produce and release unembryonated eggs into the biliary duct ○ Eggs eventually reach the stool and become excreted in feces Embryonization ○ Unembryonated eggs (diagnostic stage) in feces are embryonated in freshwater ▸ Takes around 9-15 days ▸ Eventually, embryonated eggs will hatch and release miracidia Invasion and Development ○ Miracidia invade the pond snail (infective stage for snails) ▸ Pond snail: first intermediate host ○ Miracidia undergo several developmental stages in the pond snail (sporocyst → rediae → cercariae) ▸ Takes a few weeks Encystation ○ Cercariae are released from the snail and attach to second intermediate host (freshwater plants) ○ Encyst as metacercariae in aquatic vegetation or other substances to complete the life cycle Take Note! Aside from humans, Fasciola also undergoes its complete life cycle in cattle and sheep, sometimes in camels For Fasciola: ○ Definitive host: herbivorous animals ○ Accidental host: humans ▸ Although humans are considered definitive hosts, it is more appropriate in this situation to call us accidental hosts ▸ Fasciola is considered a zoonotic disease ○ First intermediate host: pond snails ▸ Species of pond snails: – Galba truncata – Fossaria – Pseudosuccinae ○ Second intermediate host: freshwater plants ▸ Includes: – Watercress – Water lilies ○ Infective stage: metacercariae Due to the invasive nature of the larva, it elicits a high degree of immunologic response from the human body ○ Triad of diagnostic significance: ▸ High fever ▸ Hepatomegaly – Due to rampant migration inside the liver parenchyma ▸ Eosinophilia ○ RUQ pain (vague abdominal pain) ○ Diarrhea ○ Nausea/vomiting ○ Urticaria CHRONIC PHASE Adult stage ○ Parasite only eats and produces eggs Symptoms are usually more discreet ○ Fasciola adult resides very quietly inside the biliary tract ○ Does not elicit significant immune response from the human body If symptoms are present, they may be similar to acute intermittent biliary obstruction symptoms ○ Obstruction, inflammation, and fibrosis ▸ Rare: may lead to periductal cirrhosis ○ Cholangitis/cholecystitis ○ Lithiasis ▸ Adults and eggs become nidus for formation of stones ○ Pancreatitis ○ Malnutrition ○ Malabsorption of fats and amino acids ▸ Due to bile blockage ○ Rare: dissemination of eggs into the lungs, brain, and eyes [Clinical Correlation] Halzoun and Marrara Cases usually seen in the Middle East and parts of Africa ○ “Halzoun” (Lebanon) ○ “Marrara” (Sudan) Attachment of live adults to the posterior pharynx ○ Causes hemorrhagic nasopharyngitis and sometimes dysphagia Due to ingestion of raw infected liver containing adult worms ○ Not due to the ingestion of metacercariae E. DIAGNOSIS MICROSCOPY Usually through stool microscopy ○ May also look at duodenal and biliary drainage samples Detection of Fasciola eggs ○ Among the largest of the helminth eggs seen under the microscope ○ Ellipsoidal and operculated ○ Larger relative to an Ascaris egg Useful during the chronic phase Beware of false positives Active Recall Box 1. T/F: The adult features of Fasciola include a cone-shaped head. 2. T/F: The definitive hosts for Fasciola are herbivorous animals. Answers: 1T, 2T D. SYMPTOMS Symptoms associated with fascioliasis can be categorized into two major phases: acute and chronic ACUTE PHASE Revolves around larval migration YL6:07.32 Trematodes: Liver Flukes Figure 4. Fasciola egg under the microscope. Note the operculum at the upper left portion of the egg 2 ANTIBODY TESTS Used in diagnosing Fasciola infection Include enzyme immunoassay (EIA), Enzyme-Linked Immunoassay (ELISA), Western blot Primarily useful during the acute invasive phase ○ When the body has a lot of immune reactions Used to: ○ Diagnose ectopic fascioliasis ○ Rule-out false positives ○ Monitor therapy ○ Diagnose Halzoun and Marrara phenomena NOT useful in differentiating between past and present infections RADIOLOGIC TESTS CT scan ○ Multiple confluent nodules ○ Tunnel-like branching TRICLABENDAZOLE The current first-line treatment for human fascioliasis Only registered for human use in Egypt, Ecuador, Venezuela, and France 10 mg/kg per orem once or twice ○ A benzimidazole ○ Prevents microtubule formation BITHIONOL A far second-line drug due to availability problems A study in Japan puts bithionol as an investigational new drug under the CDC 30-50 mg/kg on alternate days for 10-15 doses ○ A bisphenol ○ Inhibits oxidative phosphorylation POSSIBLE ALTERNATIVE TREATMENTS Albendazole Nitazoxanide Artesunate Mirazid® ○ Natural combination preparation of myrrh oleoresin ○ Currently being investigated as an anti-schistosomal drug G. PREVENTION AND CONTROL PUBLIC HEALTH STRATEGIES Figure 5. CT Scan showing presence of Fasciola Figure 5 Avoidance of contaminated water Proper washing and cooking of water plants Avoidance of contaminated liver (controversial) ○ Does eating processed liver products pose a public health problem regarding fascioliasis? Sanitation and education ○ Especially food handlers VETERINARY PUBLIC HEALTH MEASURES CT scan demonstrates multiple, round, clustered hypodense regions in the area Sonography ○ Small, clustered hypoechoic lesions Under the One Health initiative, control of Fasciola infections in humans heavily relies on cooperation of doctors and veterinarians Preventive chemotherapy of cattle ○ To counteract human Fasciola infections Do not use animal feces as fertilizers Cultivation of aquatic vegetables Veterinary capacity-building Vaccination in cattle against Fasciola ○ Currently being developed but still being studied with rat models ○ Fasciola hepatica cathepsin B2 ▸ Best vaccine candidate as of today Active Recall Box Figure 6. Ultrasound showing presence of Fasciola 3. Which phase of fascioliasis is characterized by larval migration? A. Chronic phase B. Acute phase C. Intermediate phase D. Latent phase 4. T/F: Halzoun and Marrara refer to the attachment of adult Fasciola worms to the posterior pharynx after ingestion of raw infected liver. 5. T/F: Fasciola infections can be treated with praziquantel. Figure 6 Answers: 3B, 4T, 5F Ultrasound demonstrates linear, echogenic material within the dilated common hepatic duct ○ Gives a clue as to the presence of dead adult Fasciola worms Rationale: 5F: Amongst all the flukes, Fasciola is the one which is NOT susceptible to praziquantel F. TREATMENT Amongst all the flukes, Fasciola is not susceptible to praziquantel ○ Fasciola is unresponsive/resistant to praziquantel ○ Therefore, alternative drugs have to be used YL6:07.32 Trematodes: Liver Flukes III. CLONORCHIS AND OPISTHORCHIS A. MORPHOLOGY Clonorchis and Opisthorchis are both trematodes Clonorchis sinensis: Oriental liver fluke ○ More common Clonorchis species 3 ○ Adult: around 1-2.5 cm long and around 0.5 cm wide ▸ Smaller than Fasciola adult Opisthorchis viverrini: Southeast Asian liver fluke ○ Adult: around over half the size of a typical Clonorchis adult Opisthorchis felineus: cat liver fluke Clonorchis and Opisthorchis look very similar to each other aside from their size difference ○ Key morphological difference: testes ▸ Clonorchis and Opisthorchis have two testes ▸ Clonorchis testes: arranged in tandem, very highly branched ▸ Opisthorchis testes: oriented diagonally, low Figure 7. Clonorchis sinesis (oriental liver fluke) with encircled testes Opisthorchis viverrini: seen in Thailand, Cambodia, Laos, and Vietnam ○ Thailand: highest prevalence of Clonorchis and Opisthorchis infections (particularly Opisthorchis) ▸ Prevalence: 9% ▸ Most common helminth infection in Thailand because of their food culture – Koi-pla (raw fish salad) – Pla-som (short-term fermented fish) – Pla-ra (long-term fermented fish) – Note: Each country and culture has versions of these delicacies ▸ Infections are declining due to successful public health strategies ○ Laos: prevalence remains high, mainly along the Mekong River ▸ Up to 67% in some villagers Opisthorchis felineus: seen in Europe and former Soviet Union C. LIFE CYCLE Almost the same for both Clonorchis and Opisthorchis Figure 8. Opisthorchis viverrini (southeast asian liver fluke) with encircled testes B. DEMOGRAPHICS Figure 10. Clonorchis sinensis life cycle Figure 9. Common geographic distribution of Clonorchis (top) and Opisthorchis (bottom). Note the countries highlighted in yellow Clonorchis sinensis (Oriental liver fluke): seen in China, Taiwan, North and South Korea, and Vietnam ○ China: about 12.5 million infected ○ Taiwan: about 1 million cases ○ Vietnam: about 1 million cases, with a lot of cases near Red River Delta ○ Russia: about 1 million cases YL6:07.32 Trematodes: Liver Flukes 1. Ingestion ○ Mammalian definitive host becomes infected by ingesting undercooked freshwater fish containing metacercariae ▸ Freshwater fish: second intermediate host – Family Cyprinidae: carps, minnows, goldfish 2. Excystation ○ Ingested metacercariae excyst in the duodenum 3. Migration ○ Metacercariae will ascend from duodenum to the liver through Ampulla of Vater (easier route) towards the biliary ducts, where it can reach the inside of the biliary ducts within the liver ▸ It does not penetrate the intestinal mucosa until it reaches the liver, unlike Fasciola ▸ Very important with regards to the symptomatology and pathogenesis of Clonorchis and Opisthorchis 4. Maturation ○ Occurs inside the biliary duct (habitat inside host) 5. Reproduction ○ Usually occurs 3-4 weeks after initial ingestion ○ Once the larva reaches sexual maturity inside the biliary ducts, they would continually release embryonated eggs ▸ It does not produce unembryonated eggs, unlike Fasciola 6. Embryonation ○ Occurs inside the adult before being released into the stool 7. Invasion and Development ○ Once the infected human defecates, and the embryonated eggs are released into the freshwater environment, the eggs are then ingested by snails ▸ Snail: first intermediate host ○ Eggs undergo several developmental stages inside the snail (miracidia → sporocyst → rediae → cercariae) ○ The cercariae will then be released from the snail ▸ This kills the first intermediate host snail 4 8. Encystation ○ The cercariae will swim around in freshwater until it finds its second intermediate host (freshwater fish) ○ Inside these fishes, they will migrate into the muscles and under the scales, and will encyst into metacercariae Take Note! For Clonorchis and Opisthorchis: ○ Definitive host: humans (accidental), carnivores ○ First intermediate host: snail (Parafossarulus spp.) ○ Second intermediate host: freshwater fishes (family Cyprinidae, carps and minnows) ▸ Vehicles for infection and what humans ingest ○ Reservoir host: domesticated omnivores (cats, dogs, pigs) ○ Infective stage: metacercariae Active Recall Box The highest rates of cholangiocarcinoma can be seen in northeastern Thailand (Khon Kaen province) ○ Corresponds to the highest prevalence of Opisthorchis viverrini infections and cholangiocarcinoma incidence in the world ▸ Due to the the province being landlocked – Their primary food source is agriculture and fishing in freshwater environments ▸ They are quite far from salt waters of Gulf of Thailand in the Indian Ocean Global incidence rates of cholangiocarcinoma, per 100,000 ○ North East Thailand ▸ Still has the highest among all other regions in the world – Second highest: North and Central Thailand – Third highest: South Thailand ○ Thailand generally has the highest global incidence rates of cholangiocarcinoma ○ Statistics are improving, but it is still a threat 6. The following countries are where you would find Opisthorchis viverrini EXCEPT A. Laos B. Korea C. Thailand D. Cambodia 7. T/F Release of the cercariae from the snail will end in the snail's death? 8. Where does Clonorchis undergo sexual maturation? A. Outside the human host B. Duodenum? C. Liver D. Biliary duct Answers: 6B, 7T, 8D Rationale: 7T: Release of Clonorchis cercariae from its intermediate host (snail) will lead to its death. D. SYMPTOMS ACUTE PHASE Larval migration phase Abdominal pain Diarrhea Nausea/vomiting Urticaria Eosinophilia In contrast to the acute phase of Fasciola, fever and hepatomegaly are not usually present ○ Due to the non-invasive nature of the larval migration of Clonorchis and Opisthorchis ○ Therefore, the human immune response is not that active during the acute phase CHRONIC PHASE Adult phase Symptoms are more discreet similar to fascioliasis If symptomatic, symptoms are similar to acute intermittent biliary obstruction symptoms ○ Hepatomegaly ▸ Usually seen during the chronic phase of Clonorchis and Opisthorchis infections rather than acute phase ○ Malnutrition or malabsorption ○ Severe cases ▸ Cholangitis, cholecystitis, pancreatitis O. felineus: more involved in the pancreatic duct and its associated symptoms Figure 11. Pathophysiology of liver fluke-induced CCA Figure 11 Pathophysiology of Liver Fluke-Induced CCA ○ Liver fluke CCA is only associated with Clonorchis and Opisthorchis infection ○ Liver fluke CCA is caused by epithelial hyperplasia brought about by the following: ▸ Chronic inflammation ▸ Nitroso compounds ▸ Nitric oxide from inflammatory cells ▸ Mechanical damage ▸ Indirect parasite products ○ Chronic inflammation: chronic damage by adult worms → chronic inflammation → fibrosis → DNA damage and potential carcinogenesis ○ Nitroso compounds: fermentation and addition of preservatives (e.g., nitrosamines in food) → DNA damage and potential carcinogenesis ○ Mechanical damage in the biliary tract by adult worms via parasite suckers → chronic lesions → morphological changes in epithelial cells → potential carcinogenesis ○ Excretory and secretory parasite products from the fluke may contribute directly and indirectly in producing NO and oxygen radicals → tissue and DNA damage [Clinical Correlation] Cholangiocarcinoma (CCA) Clonorchis and Opisthorchis infections have a direct relationship with the prevalence of cholangiocarcinoma YL6:07.32 Trematodes: Liver Flukes 5 E. DIAGNOSIS Active Recall Box MICROSCOPY 9. What are the regions with the second highest rates of incidence of cholangiocarcinoma, secondary to Opisthorchis infections, in Thailand? 10. What adult worm structure causes mechanical damage to the biliary tract? 11. What shape does Clonorchis/Opisthorchis look like under the microscope? Answers: 9 North and Central Thailand, 10 Suckers, 11 Sesame seed-like Figure 12. Clonorchis/Opisthorchis egg under the microscope Figure 12 QUICK REVIEW Left image ○ Encircled: abopercular knob ○ Lined with red: operculated end Right image shows sesame seed-like shape Visualization of the egg/ova of Clonorchis/Opisthorchis: ○ Shaped like sesame seeds ○ Similar to Fasciola, the ova are operculated and has a very small abopercular knob ○ Amongst the smallest helminth ova Usually of stools, but also of duodenal and biliary drainage Most practical diagnostic OTHER DIAGNOSTICS Radiography Serologic ○ ELISA ○ PCR ○ Immunochromatographic point-of-care-test or rapid tests Figure 12. immunochromatographic device for the diagnosis of opisthorchiasis and clonorchiasis (Sadaow et al,(2019) F. TREATMENT Drug of choice: Praziquantel Second line treatment: Albendazole G. PREVENTION AND CONTROL Avoidance of contaminated water Ecological control of snails Proper washing, pre-treatment, and cooking of freshwater fishes Education and sanitation: proper human waste disposal Question from the lecture Which strategy do you think is more effective to change eating habits to curb cholangiocarcinoma (CCA) cases in Thailand? A. Banning the sale of freshwater fishes caught from known hotspots B. Talking to village elders about the dangers of liver flukes C. Changing the school curriculum of the younger generation YL6:07.32 Rationale: 11T: Clonorchis/Opisthorchis ova look like sesame seeds under the microscope and are amongst the smallest helminth ova. Trematodes: Liver Flukes QUESTIONS 1. Which of the following statements about Fasciola hepatica is false? A. F. hepatica is also known as the sheep liver fluke. B. F. hepatica is common in tropical areas such as Asia and Africa. C. F. hepatica is found in areas where sheep and cattle are raised. D. The infective stage of F. hepatica is the metacercariae. 2. Which of the following is the correct order for the life cycle of Fasciola? A. Excystation → Migration → Maturation → Encystation B. Excystation → Invasion → Maturation → Encystation C. Ingestion → Embryonization → Excystation → Development D. Ingestion → Embryonization → Encystation → Development 3. Which of the following is not part of the triad of diagnostic significance in the acute phase? A. High fever B. Eosinophilia C. Bradycardia D. Hepatomegaly 4. T/F: Diagnosis of Fasciola through microscopy is useful during the chronic phase while diagnosis through antibody tests is useful during the acute phase. 5. The following are prevention and control strategies to combat Fasciola infections EXCEPT? A. Sanitation and education, especially among food handlers B. Preventive chemotherapy C. Avoid contaminated soil D. Vet capacity building 6. Choose the correct pairing. A. O. viverrini: tandem and highly branched testes B. C. sinensis: diagonal and low testes C. O. viverrini: diagonal and low testes D. C. sinensis: tandem and highly branched testes E. Both A and B F. Both C and D 7. Where does maturation occur in the Clonorchis life cycle? A. Duodenum B. Biliary duct C. Ileum D. Stomach 8. A patient reported feeling sick after eating copious amounts of koi-pla from their recent trip abroad. Which of the following symptoms are NOT likely to be present? A. Diarrhea B. Fever C. Urticaria D. Abdominal pain 9. What organism has more involvement in the pancreatic duct and its associated symptoms? A. O. sinensis B. O. felineus C. O. viverrini D. P. westermani 6 10. Which of the following is/are FALSE regarding the pathogenesis of liver fluked-induced cholangiocarcinoma (CCA)? A. Flukes may release oxygen radicals that can cause DNA damage B. Nitrosamines in food preservatives have no effect on cholangiocytes and do not play a role in causing CCA C. Parasite suckers cause mechanical damage to the biliary tract which may lead to CCA D. Chronic inflammation can induce nitric oxide synthase and release oxygen radicals that can cause DNA damage 11. What is the most practical diagnostic for Clonorchis/Opisthorchis? A. ELISA B. PCR C. Microscopy D. Radiographs ANSWER KEY 1B, 2A, 3C, 4T, 5C, 6F, 7B, 8B, 9B, 10B, 11C Concerns and Feedback form: http://bit.ly/YL6CFF2027 How’s My Transing? form: https://bit.ly/2027YL6HMT Mid-Semester Evaluation form: https://bit.ly/2027YL6MidSem End-of-Semester Evaluation form: https://bit.ly/2027YL6EndofSem Errata Points Trackers: https://bit.ly/YL62027EPT YL6 TransMap: https://bit.ly/2027YL6TransMap RATIONALE 1. B. F. hepatica is common in tropical areas such as Asia and Africa. This is descriptive of F. gigantica, also known as the tropical liver fluke. In contrast, F. hepatica is common in Europe, Middle East, and the Andean regions and is thus known as the temperate liver fluke. 2. A. Excystation → Migration → Maturation → Encystation. Metacercariae excyst in the duodenum then migrate to the liver parenchyma and to the biliary ducts where they mature into adult flukes. Eventually the embryonated eggs will hatch and release miracidia, which undergoes invasion and development, and encystation. In encystation, cercariae are released and encyst as metacercariae. 3. C. Bradycardia. While bradycardia might occur in some medical conditions, it is NOT part of the triad of diagnostic significance for fascioliasis in its acute phase. 4. True. Diagnosis of Fasciola through microscopy is useful during the chronic phase while diagnosis through antibody tests is useful during the acute invasive phase. 5. C. Avoid contaminated soil. To prevent and control Fasciola infections, it is best to avoid contaminated water (not soil). Recall metacercariae-contaminated freshwater or water plants are the second intermediate host. 6. F. Both C and D. The testes of C. sinensis are usually found in tandem and are highly branched while the testes of O. viverrini are usually arranged diagonally and low. This is how you would be able to distinguish between these two trematodes. 7. B. Biliary duct. Maturation of Clonorchis occurs inside the biliary ducts (habitat inside host). 8. B. Fever. Fever and hepatomegaly are usually not present in the acute phase of Clonorchis and Opisthorchis infections because the human immune response is not that active during the acute phase. 9. B. O. felineus. It has more involvement in the pancreatic duct and its associated symptoms. 10. B. Nitrosamines in food preservatives have no effect on cholangiocytes and do not play a role in causing cholangiocarcinoma. Nitrosamines from food preservatives may cause carcinogenesis via DNA damage 11. C. Microscopy. Visualization of the ova of Clonorchis/Opisthorchis through microscopy is considered the most practical diagnostic test, although rapid tests and other serologic tests are available. FREEDOM SPACE REFERENCES 💻 Migriño, J. R., MD (2020, September 25). Liver Flukes (Part 1 of 2): Fasciola [Video]. YouTube. https://youtu.be/wtnsCABwq70 💻 Migriño, J. R., MD (2020, September 25). Liver Flukes (Part 2 of 2): Clonorchis and Opisthorchis [Video]. YouTube. REQUIRED https://youtu.be/hyso9G1c6xE 📖 Belizario Jr., V. Y. & De Leon, W. U. (2013). Medical Parasitology in the Philippines. The University of the Philippines Press. SUPPLEMENTARY YL6:07.32 Trematodes: Liver Flukes 7