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WS/T 564-2017 English PDF

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WS/T 564-2017: Diagnosis of Babesiosis
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GBZ 57 GB/T 31989 WS/T 566 WS/T 568 WS/T 565

Basic data

Standard ID	WS/T 564-2017 (WS/T564-2017)
Description (Translated English)	Diagnosis of Babesiosis
Sector / Industry	Health Industry Standard (Recommended)
Classification of Chinese Standard	C61
Word Count Estimation	12,152
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 564-2017: Diagnosis of Babesiosis

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Diagnosis of Babesiosis ICS 11.020 C 61 WS People's Republic of China Health Industry Standard Babesia 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. Shanghai Society of Parasitology, Fudan University, Chinese Center for Disease Control and Prevention, Institute of Parasitic Disease Control, China Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences, Huashan Hospital Affiliated to Fudan University, Zhejiang Provincial Center for Disease Control and Prevention, Chinese Agricultural Sciences Shanghai Institute of Veterinary Medicine. The main drafters of this standard. Chen Jiaxu, Cheng Xunjia, Xu Xuenian, Qin Chuan, Zhang Wenhong, Yao Linong, Zhou Jinlin, Wei Qiang, Chen Shaohong, Zheng Bin, Chen Muxin. Babesia diagnosis

1 Scope

This standard specifies the basis, principles, diagnosis and differential diagnosis of Babesia. This standard applies to the diagnosis of Babesia in medical institutions and disease prevention and control institutions at all levels.

2 Terms and definitions

The following terms and definitions apply to this document. 2.1 Babesia Babesia.spp Protozoa that parasitize the red blood cells of humans and vertebrates. The main infections of humans are Babesia microti and Babesia diversiformis. (B.divergens), Duncan Babesia (B.duncani), Orion Babesia (B.venatorum), etc. (see Appendix A). 2.2 Babesia babesiosis/babesiasis A type of zoonotic parasitic disease caused by Babesia infection is mainly transmitted by ticks. 2.3 Asymptomatic case Babesia infection without clinical symptoms. 2.4 Severe babesiosis A confirmed case of Babesia, high fever, severe anemia, jaundice, hemoglobinuria, respiratory distress, renal failure, coma, etc. One or more clinical manifestations.

3 Diagnosis basis

3.1 Epidemiological history History of field activities, tick bites, blood transfusion or organ transplantation (see Appendix B). 3.2 Clinical manifestations 3.2.1 Common clinical manifestations. chills, fever, sweating, fatigue, nausea, loss of appetite, muscle pain, joint pain, headache, abdominal Pain, anemia, etc. (see C.1 of Appendix C). 3.2.2 Severe clinical manifestations. high fever, severe anemia, hemoglobinuria, jaundice, respiratory distress, renal failure, coma, etc. (see attached C.2) of C. 3.3 Laboratory inspection 3.3.1 See Babesia for blood smear microscopy (see D.1 of Appendix D). 3.3.2 Babesia nucleic acid test is positive (see Appendix D, D.2). 3.3.3 Babesia antibody test is positive (see D.3 of Appendix D). 3.3.4 Animals inoculated with Babesia are positive (see D.4 of Appendix D).

4 Principles of diagnosis

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

5 Diagnosis

5.1 Asymptomatic infection There are no obvious clinical symptoms and signs, and it meets any of 3.3.1, 3.3.2 and 3.3.4. 5.2 Suspected cases Comply with 3.1, and at the same time comply with any one of 3.2. 5.3 Clinically diagnosed cases Suspected cases and also meet 3.3.3. 5.4 Confirmed cases Clinically diagnosed cases or suspected cases, and meet any of 3.3.1, 3.3.2 and 3.3.4 at the same time. 5.5 Severe cases The confirmed cases are in compliance with 3.2.2.

6 Differential diagnosis

The clinically diagnosed case should be compared with other diseases with fever as the main symptom, such as malaria, Lyme disease, tsutsugamushi, kala-azar, dengue fever, and abortion. Distinguish the blood and other phases (see Appendix E). AA

Appendix A

(Informative appendix) Etiology A.1 Classification Babesia belongs to Apicomplexa (Apicomplexa), Sporozoa (Sporozoa), Prioplasmasina (Prioplasmasina), Pear Piroplasmida (Piroplasmida), Babesiidae (Babesiidae), Babesia (Babesia). At present, more than 100 types of buses have been identified Shellworms can infect cows, horses, sheep, dogs and many other mammals and birds. The Babesia that can infect the human body is mainly the field mouse Babesia (Babesia microti), B.divergens, B.duncani, B.venatorum, etc. A.2 Form Babesia has various forms in red blood cells. Common worm body shapes are ring, round, rod, dot, pear, amoeba and so on. typical The form is pear-shaped, and there are often multiple parasites in one red blood cell, mostly with 1 to 4 parasites, which can form a triplet or quadruple type, namely Maltese cross shape; and can be worms of different developmental stages. After Wright or Gee's staining, the cytoplasm is blue and the nucleus is red. According to the body Size is divided into. large Babesia, body length 2.5 μm~5 μm, such as Babesia divergent; small Babesia, body length 1.0 μm~2.5 μm, such as field mouse Babesia. A.3 Life history The life history of Babesia mainly includes the developmental stages in the red blood cells of humans or vertebrates and the developmental stages in the body of the vector tick. Babesia Spores enter humans or vertebrates with saliva through tick bites, and after invading red blood cells, they develop through budding reproduction or two divisions. Merozoites. With the rupture of red blood cells, the merozoites are released and then invade new red blood cells, repeating division and proliferation. Part of the body is no longer split Proliferate and develop into male and female gametophytes. The gametophyte enters the body of the tick by sucking the blood of the host, develops into the gametes in the intestine, and then combines They become zygotes, then proliferate, and then migrate to various tissues in the tick through hemolymph. The zygote that migrates to the tick's salivary glands further develops into The sporozoite completes a life cycle. The transmission methods of Babesia among ticks are as follows. ①Transmission via eggs. After the female tick sucks blood, Babesia reproduces in the tick. After reproductive development, it enters the ovary of the tick and is passed to the next generation of ticks via the egg. ② Transmission during the period. after young ticks (or nymphs) suck blood containing Babesia Development, passing the worm to the next stage of development. BB

Appendix B

(Informative appendix) Epidemiology B.1 Source of infection Rodents such as humans and community rats, brown rats, yellow-breasted rats, and Apodemus agrarius, and animals such as cattle, deer, dogs, raccoons, and birds. B.2 Ways of transmission It is spread through tick bites, blood transfusions, or organ transplants. B.3 Susceptible population People are generally susceptible to Babesia. B.4 Regional distribution B.4.1 Distribution abroad Babesia has a worldwide distribution. Since 1888, Romanian scientist Babes first discovered Babesia dibromide in red blood cells of sick cattle. Since (Babesia bigemina), more than 100 species have been identified and reported, but the main ones that infect humans are Babesia voles, Babesia divergence and Duncan Babesia and Orion Babesia and other species. After Yugoslav scholars reported the first case of Babesia infection in humans in 1957, America, Europe, Cases of Babesia infection have been reported in Asia, Africa and Oceania, with most cases in the Americas and Europe. The United States has used Babesia as a Since it was a statutory infectious disease report, about 1,000 cases of infection have been reported every year. The main epidemic species is Babesia voles. And reported cases in Europe Slightly less, the main popular insect species are Babesia diversiformis, Babesia orion and Babesia voles. In recent years, Egypt, Mexico, South Africa, Mozambique Croatia, Australia, Brazil, Japan, South Korea, etc. have successively reported cases of human infection with Babesia. B.4.2 Distribution in my country The first report of Babesia infection in humans in my country dates back to 1944.Hong Shilu examined the blood of a suspected malaria parasite infection in Chongqing. During the smear examination, according to the analysis of the protozoan morphological characteristics, the patient was diagnosed with Babesia infection. So far, about 100 cases have been reported in my country. The reported cases include Heilongjiang, Yunnan, Chongqing, Guangxi, Shanghai, Xinjiang, Zhejiang, Inner Mongolia, Shandong and Taiwan. Most areas are Heilongjiang and Yunnan. The main species of insects distributed in my country are Babesia voles, Babesia orionata and Babesia divergence. Shellworms dominate, while the north is dominated by Orion Babesia. Most of the cases are concentrated in summer, probably because this season is the peak period of tick activity, Easy to cause human infection. In the literature reported in my country, a small number of patients have a clear history of tick bites and surgical blood transfusion, and some patients have no clear history Way of infection. CC

Appendix C

(Informative appendix) Clinical manifestations C.1 Common clinical manifestations The clinical manifestations of Babesia are related to the degree of destruction of red blood cells by Babesia during asexual reproduction and the immune status of the host. Its incubation period is 1 week to 4 week. People with normal immune function are mostly self-limiting, and symptoms can last for 2 to 4 weeks. Symptoms include chills, fever, sweating, fatigue, nausea, eating Loss of cravings, muscle pain, joint pain, headache, abdominal pain, anemia or hemoglobinuria, etc. C.2 Severe clinical manifestations Critically ill patients have a rapid onset, which usually occurs in patients with splenectomy, frail elderly and immunocompromised patients. The patient may have a high fever (body temperature up to 40 ℃), severe anemia, hemoglobinuria, soy sauce-colored urine, jaundice, respiratory distress, renal failure or coma, and even death. disease Human liver function is abnormal.

Appendix D

(Normative appendix) Laboratory examination D.1 Blood smear microscopy D.1.1 Preparation of blood smear A disposable lancet is used to collect blood from the earlobe or fingertip, and the baby can collect blood from the hallux or heel. Take blood on a clean, scratch-free glass slide Apply a thin blood film. Scrape 1.0 μL～1.5 μL of blood with the middle part of the slide, and push the lower edge of the slide flat against the midline of the slide. When the blood is on the slide When the two glass slides are extended to the width of about 2 cm from the push plate to the sides, keep the two glass slides at an angle of 25º～35º and quickly push them into a thin tongue-like blood film. D.1.2 Dyeing D.1.2.1 Gee's staining Fix the blood membrane with methanol before staining. For batch staining, insert the blood film into the staining tank in one direction, or the blood film of each pair of slides facing outward Insert it into the staining tank, pour the newly prepared 2% Ji's stain solution (2 mL of Ji's stock solution and 98 mL of distilled water or PBS buffer) to soak the thin blood After 30 minutes, pour tap water or PBS buffer into the dyeing tank to overflow, remove the scum on the surface of the dyeing solution, and remove the residual dye in the dyeing tank. Pour out the liquid, add new water, rinse repeatedly 2 to 3 times, then take out the glass slide, insert the blood film down on the drying plate to dry. Single blood film staining can be Take 2 mL of PBS buffer solution and add 1 to 2 drops of Ji's staining solution, mix them and drop them on the thin blood film. After 20 minutes to 30 minutes, wash with water and dry. D.1.2.2 Wright staining Add 5 to 8 drops of Wright's stain on the thin blood film, and fix the stain for 1 min to 2 min. Then add 5-8 drops of distilled water to the blood film, using a straw Mix the dye solution with distilled water evenly. After dyeing for 3 to 5 minutes, rinse off the dye solution gently with clean water and dry. D.1.3 Blood smear examination The stained blood film was examined with an optical microscope. After Wright or Gee's staining, the Babesia cytoplasm is blue and the nucleus is red. Common worms There are ring, round, rod, dot, pear, amoeba, Maltese cross and so on. The Babesia of the above-mentioned form was positive. D.2 Babesia nucleic acid detection D.2.1 Sample processing Use nucleic acid extraction kits or other genomic DNA extraction methods to extract Babesia DNA. D.2.2 Reagent composition Blood genomic DNA extraction kit and PCR amplification kit. D.2.3 Operation steps and interpretation of results D.2.3.1 Primer sequence Nested PCR method was used to detect Babesia 18S rRNA-specific genes from patients' red blood cells. The primer sequence is as follows. D.2.3.2 The first round of amplification D.2.3.3 The second round of amplification The second round of PCR products were subjected to electrophoresis to confirm a specific fragment with a size of approximately 400 bp. After the PCR product is recovered by tapping, Into the vector, clone and sequence analysis, confirm Babesia infection. D.3 Babesia antibody detection D.3.1 Method The indirect enzyme-linked immunosorbent assay (ELISA) method is used to detect Babesia IgG (or Babesia) in human serum, plasma or other body fluid samples. IgM) antibodies. D.3.2 Reagent composition Babesia diagnostic antigen (5 μg/mL~10 μg/mL) coated 96-well plate, staphylococcal protein A (SPA) or anti-human IgG (or IgM) Enzyme conjugate, washing solution (PBS buffer containing 0.05% Tween-20), diluent (washing solution containing 1% bovine serum albumin), Stop solution (the main component is 2 mol/L sulfuric acid solution), substrate A solution (the main component is hydrogen peroxide), substrate B solution (the main component is four Methylbenzidine), positive control and negative control. D.3.3 Operation steps In a 1.5 mL plastic centrifuge tube or dilution plate hole containing 0.5 mL diluent, add 5 μL of the sample to be tested and mix well. Babesia Add 100 μL of diluted sample to be tested to the original 96-well plate (replicate testing), set up 2 wells for positive control, 2 wells for negative control, and set up a blank pair Photograph 2 wells and incubate at 37 ℃ for 30 min. Then discard the liquid in the well, wash 3 times with washing liquid, each time interval of 1 min, and spin dry. Except blank pair Outside the wells, add 50 μL of enzyme label conjugate to each well and incubate at 37 ℃ for 30 min. Discard the liquid in the well and wash it 3 times with washing liquid, with 1 interval each time min, spin dry. Add 50 μL of Substrate A Solution and Substrate B Solution to each well, tap the microtiter plate to mix, and place it in the dark at 37 ℃ for 5 min to 10 min. Add 50 μL of stop solution to each well. D.3.4 Interpretation of results Adjust the blank hole to zero with a microplate reader at 450 nm wavelength, and measure the OD value of each test hole. Such as S/N (OD value of sample well/negative OD (average value of control wells) ≥ 2.1, the result is judged as positive. D.4 Animal inoculation Take the patient’s peripheral anticoagulant blood and inoculate it aseptically in BALB/c mice, SCID mice, NOD-SCID mice or hamsters, and each animal will be inoculated intraperitoneally 0.5 mL. One week after inoculation, blood was collected from the tail, blood smears were prepared and stained, and the red blood cell infection status was observed under microscope (see D.1). check Seeing Babesia is judged as pathogen positive. DE

Appendix E

(Informative appendix) Differential diagnosis E.1 Malaria Have traveled or worked in malaria endemic areas, and have a history of mosquito bites. The typical malaria episodes include chills, fever, sweating, and fever. Periodic symptoms, positive for Plasmodium microscopy or positive for rapid diagnosis of malaria. Microscopic examination of the difference between Babesia and Plasmodium after staining blood slices. Red blood cells infected by Plasmodium vivax were significantly larger than normal red blood cells, and the size of red blood cells infected by Babesia did not change significantly; Plasmodium falciparum infected red blood cells There is no change in cell size, ring-shaped body is common, and Babesia has various forms. At the same time, pay attention to the differential diagnosis of Plasmodium vivax and Plasmodium ovale. E.2 Lyme disease Lyme disease is also an infectious disease transmitted by ticks. It is a natural foci disease caused by Borrelia burgdorferi. Patients often Symptoms such as migratory erythema, fatigue, chills and fever, headache, nausea, vomiting, joint pain or muscle pain. E.3 tsutsugamushi There is a history of chigger bites. The patient has eschar or soybean-large ulcer on the genitals or delicate skin, and the superficial lymph nodes all over the body are enlarged, which disappears in several months Lost, 4 to 6 days after the illness, there were red macules on the chest and abdomen. The thermal type is a missed or relaxed type. Waifei test is positive. E.4 Kala-azar Have been to the endemic area of kala-azar and have a history of sandfly bites. Usually there are symptoms such as irregular fever, hepatosplenomegaly, lymphadenopathy, and anemia. Leishmania can be found on bone marrow smears. E.5 Dengue fever Have been to dengue fever endemic areas and have a history of mosquito bites. The onset is sudden, the clinical manifestations are complex and diverse, including high fever, headache, eyeball pain, muscle Symptoms such as pain in meat and joints, epistaxis, swollen lymph nodes, rashes, etc. Generally, rashes appear when fever is 4 to 5 days, which are distributed on the trunk and face And limbs, the rash disappeared with the decrease in body temperature. The serum dengue virus specific IgM antibody was positive. Serum IgG antibody in recovery phase is higher than in acute phase 4 Times more. E.6 Sepsis There are symptoms of chills, high fever, sweating, etc. The heat type is mostly relaxation fever, without periodicity, the total number of white blood cells increases with neutrophils, blood Cultivation shows pathogenic bacteria, with a history of primary lesions, skin abscesses, and extruded boils.

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