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WS/T 566-2017 English PDF

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WS/T 566-2017: Diagnosis of fascioliasis
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Basic data

Standard ID	WS/T 566-2017 (WS/T566-2017)
Description (Translated English)	Diagnosis of fascioliasis
Sector / Industry	Health Industry Standard (Recommended)
Classification of Chinese Standard	C61
Word Count Estimation	13,169
Date of Issue	2017-08-01
Date of Implementation	2018-02-01
Regulation (derived from)	State-Health-Communication (2017) 11
Issuing agency(ies)	National Health and Family Planning Commission of the People's Republic of China

WS/T 566-2017: Diagnosis of fascioliasis

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Diagnosis of fascioliasis ICS 11.020 C 61 WS People's Republic of China Health Industry Standard Fascioliasis diagnosis 2017-08-01 released 2018-02-01 implementation Issued by the National Health and Family Planning Commission of the People's Republic of China

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009. Drafting organizations of this standard. Chinese Center for Disease Control and Prevention, Institute of Parasitic Disease Control, Anhui Institute of Parasitic Disease Control, China Shanghai Institute of Veterinary Medicine, Academy of Agricultural Sciences, Dali Prefecture Institute of Schistosomiasis Control, Yunnan Province, and Affiliated Hospital of Dali University. The main drafters of this standard. Xu Xuenian, Chen Jiaxu, Zhang Shiqing, Jiao Jianming, Liu Jinming, Fang Wen, Gu Wei, Liu Yuhua, Zhou Yan, Xiong Yan Hong. Fascioliasis diagnosis

1 Scope

This standard specifies the diagnostic basis, principles, diagnosis and differential diagnosis of Fasciolasis. This standard applies to the diagnosis of Fascioliasis by medical institutions and disease prevention and control institutions at all levels across the country.

2 Terms and definitions

The following terms and definitions apply to this document. 2.1 Fasciola spp. Fasciola hepatica (Fasciola hepatica) and giant fasciola Fasciola gigantica (see Appendix A). 2.2 Fascioliasis Diseases caused by Fasciola parasites in the human body, including acute phase caused by the migration of Fasciola juveniles in the abdominal cavity and liver parenchyma Damage, and chronic-phase damage mainly caused by bile duct epithelial hyperplasia, bile duct and gallbladder inflammation caused by adult parasites in the bile duct. 2.3 Asymptomatic case Those who have Fasciola parasitic in the body without obvious clinical symptoms and signs. 2.4 Fascioliasis endemic area There is an intermediate host of Fasciola (small soil snail and other freshwater snails of the family Vertebridae), and local people and/or ruminants such as cattle and sheep are infected Fasciola area.

3 Diagnosis basis

3.1 Epidemiological history Have a history of living, working, traveling in endemic areas, and have a history of eating aquatic plants or drinking raw water (see Appendix B, B.1). 3.2 Clinical manifestations 3.2.1 Acute phase Fever, abdominal pain, fatigue, or accompanied by anorexia, vomiting, abdominal distension, diarrhea and other symptoms; liver enlargement, liver area percussion pain and other signs (see attached Record B's B.2.1). 3.2.2 Chronic phase The clinical manifestations are relatively mild compared with the acute phase, mainly including abdominal pain, fatigue, anemia, anorexia, greasiness, jaundice and hepatomegaly, etc. Performance (see Appendix B, B.2.2). 3.3 Laboratory inspection 3.3.1 For examination of feces or duodenal drainage, see Fasciola eggs (see C.1 and C.2 of Appendix C). 3.3.2 For surgical or pathological biopsy, see Fasciola parasites (see C.3 of Appendix C). 3.3.3 Enzyme-linked immunosorbent test (ELISA) is positive (see C.4 of Appendix C). 3.3.4 The percentage and/or absolute value of peripheral blood eosinophils increased.

4 Principles of diagnosis

Diagnosis is based on epidemiological history, clinical manifestations and laboratory test results.

5 Diagnosis

5.1 Asymptomatic infection No obvious clinical symptoms and signs, conform to 3.1, and conform to any of 3.3.1 and 3.3.2 at the same time. 5.2 Acute Fascioliasis 5.2.1 Suspected cases It also complies with 3.1, 3.2.1 and 3.3.4. 5.2.2 Clinical diagnosis cases Suspected cases and also meet 3.3.3. 5.2.3 Confirmed cases Suspected cases and meet either 3.3.1 or 3.3.2 at the same time. 5.3 Chronic Fascioliasis 5.3.1 Suspected cases It also complies with 3.1, 3.2.2 and 3.3.4. 5.3.2 Clinical diagnosis cases Suspected cases and also meet 3.3.3. 5.3.3 Confirmed cases Suspected cases and meet either 3.3.1 or 3.3.2 at the same time.

6 Differential diagnosis

Should be combined with clonorchiasis, paragonimiasis, hepatic capillary worm disease, viral hepatitis, amoebic liver abscess, bacterial liver pus Differentiation of tumors and liver malignancies (see Appendix D). AA

Appendix A

(Informative appendix) Etiology A.1 Insect species Fasciola belongs to the genus Fasciola in the Facialidae of the order Polyclones. Its adults are mainly parasitic in the bile ducts of ruminants such as cattle and sheep, and can also be parasitic Inside the bile duct of the human body. Fasciola hepatica and Fasciola macrofasciatus (Veterinarians call Fasciola macrofasciola) insect). A.2 Form A.2.1 Adult The morphology of Fasciola hepatica and Fasciola giganteus is very similar. Adults have flat dorsal and abdomen, leaf-like, reddish-brown, hermaphrodite, covered with skin spine. There is a protruding cone-shaped nose cone at the front end of the insect body, and the rear cone becomes wider or forms a shoulder. The mouth sucker is located on the subventral surface of the nose The suction cup is located behind the base of the nose cone. There are genital holes between the mouth and abdominal suckers. The digestive system is composed of the pharynx, esophagus and two intestinal branches. The branch extends to the rear end of the worm body, and branches to the inner and outer sides. In the reproductive system, there are two testicles, which are highly branched, arranged one behind the other The middle of the body. One ovary, small, antler-shaped, located right behind the abdominal sucker and in front of the anterior testis. The uterus is short, coiled in the ovaries and Between the abdominal suckers, they are filled with eggs and open in the genital pores. The yolk glands are distributed on both sides of the worm, from the base of the cone to the back of the worm. Liver slices The main points for distinguishing the adult morphology of trematode and giant Fasciola are shown in Table A.1. A.2.2 Eggs The eggs are oblong, yellowish brown, with a small cover on one end. The egg shell is thin and divided into two layers. The egg is filled with multiple yolk cells, and one An egg cell that is not easily detected. The egg size of Fasciola hepatica is; Fasciola gigantea is. A.3 Life history The final hosts of Fasciola are mainly ruminants such as cattle and sheep. Humans are infected by metacercariae swallowed by raw aquatic plants and drinking raw water. its Adults parasitize in the bile ducts of the final host, and the produced eggs enter the intestines with bile, mix in feces and excrete from the body, and develop in water at a suitable temperature And hatched out mircaria, mircaria invaded the intermediate host (small soil snails and other freshwater snails of the family Vertebridae), and passed through the stages of larvae, mother rachis and daughter rachis After the development and asexual proliferation, it further develops to cercariae. Mature cercariae escape the snails and form metacercariae on aquatic plants or on water. End host Infection caused by accidental eating of live metacercaria. The young worm escapes from the metacercaria in the small intestine, passes through the intestinal wall, migrates in the abdominal cavity, and finally passes through the liver parenchyma Enter the bile duct and develop into an adult. The shortest time from swallowing metacercariae to finding eggs in feces is about 10 to 11 weeks. Adult lifespan is generally 4 Years to 5 years, it has been reported that it can be parasitic in human body for up to 12 years. BB

Appendix B

(Informative appendix) Epidemiology and clinical manifestations B.1 Epidemiology Fasciola is distributed worldwide, with human cases reported in 51 countries on five continents. It is estimated that there are at least 2.4 million people worldwide Infected with Fasciola, 91.1 million people are threatened. The first survey on the distribution of human parasites in my country, the estimated number of people infected nationwide is 12 Million people. At present, 21 provinces (municipalities and autonomous regions) including Fujian, Jiangxi, Hubei, Inner Mongolia, Guangxi, and Yunnan have Fasciolasis Case report. B.2 Clinical manifestations B.2.1 Acute Fascioliasis Occurs from 2 weeks to 12 weeks after infection. It is caused by the migration of Fasciola juveniles in the abdominal cavity and liver parenchyma. The course of the disease is relatively slow. table Present. fever, abdominal pain, fatigue, or accompanied by anorexia, vomiting, abdominal distension, diarrhea and other symptoms; liver enlargement, liver percussion pain and other signs, some The patient developed signs of ascites and anemia. The percentage and/or absolute value of peripheral blood eosinophils increased. B.2.2 Chronic Fascioliasis Occurs after the acute phase, bile duct epithelial hyperplasia, bile duct hyperplasia, bile duct epithelial Inflammation of the duct and gallbladder, fibrosis, dilation and obstruction of the bile duct, etc. Manifestations. abdominal pain, fatigue, anemia, anorexia, greasiness, jaundice, The clinical manifestations of liver enlargement vary. The percentage and/or absolute value of peripheral blood eosinophils increased. B.2.3 Ectopic damage During the migration of the child worm in the abdominal cavity, the worm body can penetrate or be carried by the bloodstream to the organs and tissues other than the liver and cause ectopic damage, such as subcutaneous Ectopic parasites in tissues, abdominal wall muscles, peritoneum, lungs, eyes, brain and bladder are more common in subcutaneous tissues. Clinical manifestations of ectopic lesions It is more complicated and changeable, and the diagnosis is usually made by surgery. CC

Appendix C

(Normative appendix) Laboratory examination C.1 Stool examination C.1.1 Washing sedimentation method (or gravity sedimentation method) Put 20g-30g of feces in a beaker, add 10-12 times the volume of water, and stir thoroughly to form a slurry of feces. Through 250μm～425μm aperture (40 Mesh ～60 mesh) filter into a 500mL conical measuring cup, add water to rinse the fecal residue in the sieve until the fecal liquid in the measuring cup reaches 500mL. Stand still After 30 minutes, pour off the upper layer liquid, add water to 500 mL, and let it stand for 15-20 min. Repeat this for 3 to 5 times until the upper layer becomes clear. Finally, the upper layer was discarded, and the sediment smear was taken for microscopic examination of Fasciola eggs. C.1.2 Nylon silk bag egg collection method Take 20g～30g of feces and place them in a mesh sieve with 250μm～425μm aperture (40 mesh～60 mesh), and then connect with 55μm aperture (260 mesh). For dragon silk bags, clamp the bottom of the bag with iron clips. Sprinkle water, mash the feces, and filter the feces into nylon silk bags. Then remove the mesh screen and continue to shower Wash the fecal slurry in the bag, and use bamboo chopsticks to gently scrape the outside of the bag to aid filtration until the filtrate becomes clear. Remove the iron clamp and take a smear of the sediment for microscopic examination of Fasciola egg. C.2 Duodenal drainage fluid inspection Use the duodenal drainage tube to extract the duodenal drainage fluid and bile, and use the direct smear method to microscopically examine Fasciola eggs. In order to improve the effect of microscopy, The drainage fluid can be diluted with physiological saline and stirred, then divided into centrifuge tubes, centrifuged at 1500g for 5min to 10min, and the sediment smears are sucked for microscopic examination. C.3 Pathological biopsy The pathological section of the tissues taken through microscopic examination found that the microstructure of the fasciola body section can be used as a diagnostic basis, but it is not recommended It is used for further examination and confirmation of suspected cases. C.4 Enzyme-linked immunosorbent test (ELISA) C.4.1 Antigen preparation Collect fresh adult Fasciola parasitizing in the liver and bile ducts of cattle and sheep, and use 0.01mol/L pH7.4 phosphate buffered saline (PBS) After rinsing, add PBS and incubate at room temperature for 3h-6h. The culture solution was centrifuged at 3000g for 10 min at 4°C to remove the sediment (eggs, etc.). The supernatant is the excretion and secretion antigen of Fasciola, and the protein concentration is measured by the Coomassie brilliant blue method. C.4.2 Reagents Enzyme labeled conjugate. anti-human IgG horseradish peroxidase labeled conjugate; antigen coating diluent. 0.05mol/L pH9.6 carbonate buffer Washing solution; blocking solution. antigen-coated diluent containing 1% bovine serum albumin; washing solution. 0.01mol/L pH 7.4 containing 0.05% Tween-20 C.4.3 TMB substrate solution formulation Substrate color development solution A. sodium acetate 13.6g, citric acid 1.6g, 30% hydrogen peroxide 0.3mL, and distilled water to 500mL. Substrate color development liquid B. B 0.2g of disodium diaminetetraacetic acid, 0.95g of citric acid, 50mL of glycerol, 20mL of TMB solution (weigh 0.2g of TMB and dissolve it to 20mL of dimethyl sulfoxide Medium), distilled water is added to 500ml. Substrate color B solution should be stored in the dark. Before use, take the same amount of A and B liquids and mix them evenly before use. Or can Use commercially available TMB color developing solution directly. C.4.4 Operation steps The antigen is diluted to 5μg/mL～20μg/mL with the coating diluent, and 100μL is added to each well in a 96-well polystyrene plate, overnight at 4°C. Discard the solution in the well the next day and spin dry. Add.200μL/well of blocking solution and incubate at 37°C for 1h. Discard the blocking solution and wash 3 times with the washing solution, each time 5min, spin dry. Subject's serum or reference serum (2 negative, positive and blank controls are set for each batch, and each test sample is set for multiple hole testing) Dilute 1.200 to 1.400 with the diluent, add 100μL to each well, and incubate at 37°C for 1h. Pour out the serum sample and wash as before. Add enzyme label Record 100 µL/well of the conjugate (dilute with the diluent at the recommended concentration), and incubate at 37°C for 1 hour. Dump the enzyme-labeled conjugate, wash 4 times, 5min each time, Spin dry. Add 100µL of TMB substrate solution per well, and place it in the dark at room temperature for 5 minutes. Add stop solution 50μL/well. Use the blank control hole to zero, use Measure the optical density absorption (OD) value of each hole with a wavelength of 450nm with a microplate reader. C.4.5 Results judgment The average OD value of the tested sample/negative reference value (P/N) ≥ 2.1 is regarded as positive. DD

Appendix D

(Informative appendix) Differential diagnosis D.1 Clonorchiasis sinensis It is a relatively common food-borne parasitic disease caused by raw freshwater fish and shrimp containing Clonorchis sinensis metacercariae. Born in the hepatobiliary duct of the host, the clinical manifestations are similar to that of Fascioliasis, but most patients have mild symptoms. Stool examination can detect clonorchis sinensis Eggs, the size is (27～35)µm×(10～20)µm. D.2 Paragonimiasis of the liver type Humans are infected with freshwater crabs and crickets containing Paragonimus metacercaria from raw or semi-raw food, causing liver damage. Flukeworm disease is mainly manifested as liver enlargement, pain in the liver area, and abnormal liver function. The serum immunological test of Paragonimus-specific antibodies was positive. D.3 Hepatic capillary worm disease Capillaria hepatica is a parasite of rodents and a variety of mammals. Adults parasitize the liver and occasionally infect humans, causing capillariasis of the liver. People get infected by eating food or water contaminated with eggs during the infection period. Clinical manifestations include fever, hepatosplenomegaly, and a significant increase in eosinophils. through The pathogen was confirmed by liver biopsy. D.4 Viral hepatitis Patients with acute hepatitis and chronic hepatitis often have obvious abnormalities in liver function, and the antigen or antibody test of viral hepatitis is positive. Fasciola The immunological and etiological examinations of the disease were negative. D.5 Amoebic liver abscess Some patients have a history of dysentery or diarrhea. Patients often have low-grade fever, liver area pain, and tenderness. The histolytic endoamidine can be found in the stool Bar trophozoites or cysts. B-ultrasound examination revealed an echoless area at the liquefied area, and a typical chocolate-like pus can be obtained from liver biopsy. The trophozoites of solubilized endoamoeba can be detected in the pus. D.6 Bacterial liver abscess Patients often have chills, high fever, severe infection and poisoning symptoms, pain in the liver area, elevated white blood cells, elevated neutrophils, and acidophilia The percentage and/or absolute value of neutrophils is normal. Multiple abscesses are common in B-ultrasound examination, and pus from liver biopsy is often present Yellowish white, smelly, smear or culture with bacteria, often metastatic abscess, anti-amebic treatment is ineffective. D.7 Malignant tumor of liver Common clinical manifestations include liver pain, abdominal distension, anorexia, fatigue, weight loss, progressive hepatomegaly or upper abdominal masses, etc.; usually liver Visceral enlargement, jaundice, ascites and other signs. Positive for alpha-fetoprotein. Imaging examination revealed space-occupying lesions.

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