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WS 283-2020 English PDF

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WS 283-2020: (Anthrax diagnosis)
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WS 283: Evolution and historical versions

Standard ID	Contents [version]	USD	STEP2	[PDF] delivered in	Standard Title (Description)	Status	PDF
WS 283-2020	English	349	Add to Cart	3 days [Need to translate]	(Anthrax diagnosis)	Valid	WS 283-2020
WS/T 283-2020	English	RFQ	ASK	3 days [Need to translate]	(Diagnosis of anthrax)		WS/T 283-2020
WS 283-2008	English	319	Add to Cart	3 days [Need to translate]	Diagnostic criteria for anthrax	Obsolete	WS 283-2008

PDF similar to WS 283-2020

Standard similar to WS 283-2020

GB 15979 GB/T 15981 WS/T 641 WS/T 293 WS 293

Basic data

Standard ID	WS 283-2020 (WS283-2020)
Description (Translated English)	(Anthrax diagnosis)
Sector / Industry	Health Industry Standard
Classification of Chinese Standard	C59
Word Count Estimation	14,192
Date of Issue	2020-04-21
Date of Implementation	2020-11-01
Older Standard (superseded by this standard)	WS 283-2008
Regulation (derived from)	State-health communication (2020) No. 6
Issuing agency(ies)	National Health Commission

WS 283-2020: (Anthrax diagnosis)

---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Diagnosis for anthrax ICS 11.020 C 59 WS People's Republic of China Health Industry Standard Replace WS 283-2008 Diagnosis of anthrax 2020-04-21 released 2020-11-01 implementation Issued by the National Health Commission of the People's Republic of China

Foreword

Chapter 6 of this standard is mandatory clauses, and the rest are recommended clauses. This standard was drafted in accordance with the rules given in GB/T 1.1-2009. This standard replaces WS 283-2008 "Diagnostic Criteria for Anthrax." Compared with WS 283-2008, the main technical indicators of this standard are as follows. -Added normative references (see Chapter 2); --Modified the laboratory inspection (see 4.3, 3.3 of the.2008 edition); -Modified the diagnosis of clinically diagnosed cases (see 6.2, 5.2 of the.2008 edition); -Modified the diagnosis of confirmed cases (see 6.3, 5.3 of the.2008 edition); --Added specimen storage and transportation requirements (see Appendix A); - Added biosafety requirements (see Appendix B); --- Added nucleic acid detection of Bacillus anthracis (see Appendix D); --Added Bacillus anthracis antigen detection (see Appendix E). Drafting organizations of this standard. Infectious Disease Prevention and Control Institute of China Center for Disease Control and Prevention, Liaoning Provincial Center for Disease Control and Prevention, Capital Medical Department Beijing Ditan Hospital affiliated to the University, Sichuan Provincial Center for Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Military Medicine of the Chinese People’s Liberation Army Institute of Military Veterinary Medicine, Academy of Sciences, Shaanxi Provincial Center for Disease Control and Prevention, Shenyang Sixth People's Hospital. The main drafters of this standard. Wei Jianchun, Li Wei, Yao Wenqing, Li Xingwang, Luo Longze, Yin Wenwu, Guo Xuejun, Liu Dongli, Mao Lingling, Zhang Mingxiang, Zhang Enmin, Zhang Huijuan. The previous editions of the standard replaced by this standard are as follows. --GB 17015-1997; --WS 283-2008. Diagnosis of anthrax

1 Scope

This standard specifies the diagnostic basis, principles, diagnosis and differential diagnosis of anthrax. This standard applies to the diagnosis of anthrax in various medical institutions and their medical staff at all levels across the country.

2 Normative references

The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this article Pieces. For undated references, the latest version (including all amendments) applies to this document. GB 19489 Laboratory Biosafety General Requirements

3 Terms and definitions

The following terms and definitions apply to this document. 3.1 Anthrax anthrax An acute zoonotic disease caused by Bacillus anthracis. Mainly occurs in livestock houses, with herbivores such as cattle, sheep and horses being the most Susceptible. Humans get sick mainly through contact with animals suffering from anthrax or contaminated animal products, and environmental infections. The main clinical type is cutaneous anthrax, A few are pulmonary anthrax and intestinal anthrax, which can be secondary to sepsis and meningitis. Cutaneous anthrax has a low mortality rate, and other types of anthrax have a higher mortality rate. high.

4 Diagnosis basis

4.1 Epidemiological history Within 14 days before the onset of illness, have been in contact with a disease or dead animal or its remains suspected of anthrax, or have eaten meat of a disease or dead animal suspected of anthrax Or its products, or inhalation of suspected Bacillus anthracis contaminated dust, or engaged in close contact with animal products such as fur, and research with Bacillus anthracis Investigate the use of related occupations, or engage in activities such as breeding, grazing, cultivating or digging in areas that may be contaminated by anthrax spores. 4.2 Clinical manifestations and classification 4.2.1 Skin anthrax Unexplained macules, papules, blisters, swelling and infiltration of surrounding tissues appear on the local skin of exposed parts such as hands, forearms, face and neck. Then the central necrosis formed an ulcerative black eschar, and the skin around the eschar became red, swollen, painless, and slightly itchy. Typical skin damage It presents as a shallow ulcer with black scabs, small blisters around it, and non-pitting edema with extensive nearby tissues. In addition to skin lesions, patients often appear Symptoms and signs such as fever, headache, joint pain, general malaise, and local lymph node and splenomegaly. A few severe cases, with large patches of water locally Swelling and necrosis. 4.2.2 Intestinal anthrax Fever, bloating, severe abdominal pain, diarrhea, usually bloody or bloody stools. May have nausea, vomiting, and the vomit may contain blood Silk and bile. May be accompanied by symptoms and signs outside the digestive tract. 4.2.3 Pulmonary anthrax High fever, difficulty in breathing, chest pain and cough, and very thick bloody sputum. The lung signs often consist of only scattered rales. Chest X-ray The main manifestations are widened mediastinal shadows and pleural effusion. 4.2.4 Meningitis anthrax Severe headache, vomiting, stiff neck, delirium, coma, and respiratory failure. Cerebrospinal fluid is mostly bloody. Mostly secondary to 4.2.1~ 4.2.3, it may also happen directly. 4.2.5 Septic anthrax High fever, chills, septic shock and diffuse intravascular coagulation (DIC) manifestations, bleeding spots or large ecchymosis on the skin, cavities Blood, respiratory and circulatory failure quickly appeared. Often secondary to 4.2.1 ~ 4.2.3, may also occur directly. 4.3 Laboratory inspection 4.3.1 Clinical specimens of patients, bacterial isolation and culture to obtain Bacillus anthracis (see Appendix A, Appendix B). 4.3.2 In the patient's serum sample, the anti-anthrax specific antibody test was positive (see Appendix C). 4.3.3 Clinical specimens of the patient, microscopic examination revealed a large number of gram-positive large bacilli arranged in tandem with both ends flush. 4.3.4 In clinical specimens of patients, Bacillus anthracis-specific nucleic acid fragments were positive (see Appendix D). 4.3.5 The clinical specimen of the patient showed a positive Bacillus anthracis antigen test (see Appendix E). 4.3.6 Bacillus anthracis is obtained by separating and cultivating bacteria from exposed animal specimens or exposed environment specimens (see Appendix A and Appendix B).

5 Principles of diagnosis

Diagnosis is based on epidemiological history, clinical manifestations, laboratory tests, etc.

6 Diagnosis

6.1 Suspected cases Have 4.1, and have one of the clinical manifestations of 4.2.1 to 4.2.5. 6.2 Clinical diagnosis cases A clinically diagnosed case can be diagnosed if one of the following is met. a) Suspected cases, and those who have any of the items in 4.3.2 to 4.3.6; b) Those with a clear epidemiological history and typical skin lesions. 6.3 Confirmed cases A confirmed case can be diagnosed if one of the following is met. a) Suspected cases or clinically diagnosed cases, and those with 4.3.1; b) Suspected cases or clinically diagnosed cases, and have double serum anti-anthrax specific antibodies of the patients in 4.3.2 that have positive conversion or titer Those who have increased by 4 times or more; c) Suspected cases or clinically diagnosed cases, and those who have any 2 items in 4.3.2 ~ 4.3.6.

7 Differential diagnosis

7.1 Skin anthrax There is obvious edema in the early stage of the anthrax lesion, itching but not painful, and not purulent. It can be related to furuncle, cellulitis, scab of tsutsugamushi, sheep pox, melioidosis, Plague, tularemia, erysipelas, syphilis, chancre, and purulent ulcer are differentiated. The patient's occupation and history of contact with sick animals can be used for reference. 7.2 Intestinal anthrax Early intestinal anthrax should be differentiated from food poisoning, hemorrhagic necrotizing enteritis, dysentery, and acute abdomen. 7.3 Pulmonary anthrax Pulmonary anthrax viscous blood sputum is distinguished from foamy rust-colored sputum of lobar pneumonia, and it is distinguished from pulmonary plague and leptospirosis with diffuse lung hemorrhage Type identification. The effusion of pleurisy is bloody viscous fluid. AA

Appendix A

(Normative appendix) Collection, preservation and transportation of anthrax specimens A.1 Collection of patient specimens A.1.1 Principles to be followed when collecting specimens A.1.1.1 Collect specimens as far as possible before the start of antibiotic treatment. A.1.1.2 Except when necessary and in a laboratory with operating conditions, specimens shall not be obtained by dissection. Required blood and tissue mark This should be obtained by puncture. A.1.2 Blood specimen For all suspected cases, 5 mL blood samples should be collected aseptically for microscopic examination, culture, antigen and nucleic acid testing, and kept Take serum for antibody test. 5 mL of blood samples should be collected again during the recovery period, and serum should be collected for antibody testing. A.1.3 Skin lesion specimens In the blister phase, use two sterile cotton swabs to dip into the blister fluid; in the ulcer phase, use two sterile cotton swabs moistened with normal saline to apply on the skin lesions; During the eschar period, apply two sterile cotton swabs moistened with normal saline on the eschar repeatedly. Specimens are used for microscopy, culture, antigen and nuclear Acid detection. A.1.4 Stool and vomit specimens Suspected cases with gastrointestinal symptoms collect stool or vomit specimens, pay special attention to selecting the part mixed with blood, and place a sterile container A.1.5 Nasal (pharyngeal) swab or sputum specimen Suspected cases with respiratory symptoms are collected from nasal (pharyngeal) swabs or sputum samples for microscopic examination, culture, antigen and nucleic acid Detection. A.1.6 Cerebrospinal fluid specimen For suspected cases with meningeal irritation, the cerebrospinal fluid was obtained by lumbar puncture, and the sample volume was 1 mL to 2 mL. Used for microscopy, culture, Antigen and nucleic acid detection. A.1.7 Corpse specimens Death cases can be obtained by puncturing the heart to obtain blood or puncturing the liver and other solid organs to obtain tissue samples. Used for microscopy, culture, Antigen and nucleic acid detection. A.2 Collection of animal specimens and environmental specimens A.2.1 Animal tissue specimens Blood, meat, internal organs, etc., are collected according to the specific conditions and used for culture. A.2.2 Environmental specimens Collect soil, water, and other items contaminated by the blood of sick and dead animals for cultivation. A.3 Biosafety requirements for specimen collection When specimens are collected, secondary protection measures for infectious diseases should be taken, and medical protective masks, medical latex gloves, work caps, medical protective clothing, Protective shoes, wear protective goggles/face shields when necessary. A.4 Specimen storage requirements The specimens used for culture and nucleic acid detection should be stored in refrigerator at 2℃～8℃ and transported at low temperature. Serum samples used for serum antibody detection at -20°C The following freeze preservation, keep frozen for transportation. A.5 Transportation requirements The transportation of specimens or strains shall be carried out in accordance with the "Administrative Regulations on the Transportation of Highly Pathogenic Microorganisms (Virus) Species or Samples that Can Infect Humans". Bacillus anthracis strains or live cultures should be packaged and transported by air according to the requirements of UN2814, and other related samples should be packaged according to the requirements of UN3373 And air transport; transport by other means of transportation can refer to the above standard packaging. BB

Appendix B

(Normative appendix) Bacteriological examination of anthrax B.1 Biosafety requirements It shall be implemented in accordance with the requirements of GB 19489 and the "List of Pathogenic Microorganisms Infected by Humans". B.2 Microscopic examination All specimens from patients or cadavers should be smeared, Gram stained, and examined under a microscope. If you find a lot of flat ends Qi's gram-positive large bacterium can be used as a diagnostic basis. B.3 Bacteria isolation and culture B.3.1 Specimen processing Skin lesion specimens, blood, cerebrospinal fluid, nasal (pharyngeal) swabs, sputum, autopsy specimens, animal tissues and other specimens are directly coated on the blood plate Or nutrient agar plates, blood samples can also be directly inoculated into blood culture bottles. Environmental specimens, stool and vomit specimens, use twice the amount of distilled water or Physiological saline is made into a suspension, after natural precipitation to remove coarse particles, the resulting suspension is taken 1 mL and heated at 65°C for 15 to 20 minutes. Take 100 µL～200 µL to coat the plate. B.3.2 Selection of suspicious colonies After incubating the above plate at 37°C for 8 h to 24 h, check whether there are suspicious Bacillus anthracis colonies. Bacillus anthracis colony The morphology is. off-white, opaque, round or irregular, the surface looks like ground glass, and the colony looks like curly hair under low power. No on blood plate Hemolysis or slight hemolysis. B.4 Identification of Bacillus anthracis Pick out the above suspicious colonies and inoculate them on a nutrient agar plate with a streak. Drop a drop of diagnostic anthrax phage in the streaked area. Stick a piece of penicillin sensitive paper. After incubating at 37°C for 8 hours to 24 hours, there is a clear plaque at the place where the phage drops, and the penicillin sensitive paper is around If there is an obvious inhibition loop, the inoculum can be determined to be Bacillus anthracis. Suspicious colonies can also be tested for nucleic acid, and Bacillus anthracis specific The heterosexual nucleic acid fragment can be judged as Bacillus anthracis (see Appendix D).

Appendix C

(Normative appendix) Anthrax serology C.1 Specimen requirements Need to collect the patient's acute and convalescent serum for testing. Acute-phase serum should be collected during the first inspection of the patient, after serum separation First, take a small amount for one antibody test, and store the rest below -20°C. After the recovery period serum is obtained, the two sera will be tested together again. Body testing. Enzyme-linked immunosorbent assay, immunochromatography or other immunological methods can be used for detection. C.2 Enzyme-linked immunosorbent test (ELISA) C.2.1 Reagent selection You can use a commercial kit, follow the operating instructions, or follow the steps below. C.2.2 Reagent preparation C.2.2.2 Washing liquid Pipette 5 mL Tween-20 into 995 mL 0.01 mol/L PBS and mix well. C.2.2.3 Serum dilution Take 20 mL of 0.1 mol/L PBS, add 160 mL of double-distilled water, add 10 g of skimmed milk powder, add 1 mL of Tween-20, mix well, and adjust the pH to 7.4. Make the volume to.200 mL with double distilled water. Store at 4°C and use within two weeks. C.2.2.4 ELISA diluent Except for the content of Tween-20 which is 0.1%, the other ingredients and requirements are the same as the serum diluent. C.2.2.5 Chromogenic fluid 3,3',5,5'-Tetramethylbenzidine (TMB) solution is ready-to-use. C.2.2.6 Color development stop solution (0.5 mol/L H2SO4) Take 2.72 mL of 98% concentrated sulfuric acid, slowly add 90 mL of water, mix, add water to make the volume to 100 mL. C.2.3 ELISA plate coating The detection target of this method is the antibody against the protective antigen of Bacillus anthracis in the blood of the patient, and the protective antigen is used to coat the ELISA plate. Other specific antigen components of Bacillus anthracis can also be used to coat the ELISA plate, and the operation method can be adjusted according to the instructions of different kits. Antigen Dilute with ELISA coating solution to 1.0 µg/mL, add 100 µL to each reaction well used in the ELISA plate, cover overnight at 4℃, and use within 7 days. Near Before use, add 300 µL of washing solution to each well and wash 3 times, 3 min each time. C.2.4 Add serum to be tested C.2.4.1 Primary screening test of sample According to the actual situation, you can skip this step and proceed directly to C.2.4.2. The serum specimen was diluted 1.50 with serum diluent and added to the microtiter plate, and the two wells were tested in parallel, each well was 100 µL. Reagent and positive for each plate Serum and negative serum control wells were tested in parallel with 3 wells each. The ELISA plate was incubated in a humidified box at 37°C for 60 min. Spin dry the solution in the wells and wash each well Wash three times with 300 µL of washing solution, each for 3 minutes. C.2.4.2 Re-judgment experiment Serum specimens with positive initial screening tests were tested for re-judgment. The serum to be tested is first diluted with serum diluent to 1.50, and the diluted serum Add the first row of the ELISA plate, use two wells for parallel detection, and then dilute with the ELISA diluent to at least 1.3200, 100 µL per well. Make reagents, positive serum and negative serum control wells for each plate, and each 3 wells are tested in parallel. The ELISA plate was incubated in a humidified box at 37°C for 60 min. Spin hole Inner solution, add 300 µL of washing solution to each well and wash 3 times, 3 min each time. C.2.5 Enzyme-labeled antibody reaction Add 100 µL of horseradish peroxidase-labeled anti-human IgG at a working concentration diluted with ELISA diluent to each reaction well, and place in a 37℃ humidified box Incubate for 60 min. Wash 3 times with washing buffer, 300 µL per well, 3 min each time. C.2.6 Color development Add 100 µL of color developing solution to each reaction well, and keep it in the dark for 20 minutes. Add 100 µL of color development stop solution to each well to stop color development. C.2.7 Results judgment Within 30 minutes after the termination of color development, use a microplate reader at a wavelength of 450 nm to measure the OD value of each well after zero adjustment with the reagent control well. Negative serum and positive The OD value of sex serum should be within the range of quality control requirements. The sera whose OD value is greater than 2.5 times of the negative serum are defined as positive and the corresponding Large dilution is defined as positive titer. C.3 Colloidal gold immunochromatography C.3.1 Specimen preparation The detection target is generally Bacillus anthracis anti-capsular antibody. The patient’s serum is used as a specimen, and the test kit is appropriately diluted with saline release. C.3.2 Detection Unpack the Bacillus anthracis antibody detection reagent, and drip 150 µL～200 µL of the diluted patient serum into the sample hole. 2 min After that, the results were observed and the observation was terminated for 15 minutes. C.3.3 Interpretation results Two purple-red color bands appear, that is, the color of the quality control line (C) and the test line (T) is a positive result; only the color of the quality control line is a negative result; No band appears or only the detection line appears, indicating that the reagent is invalid, and the reagent should be replaced and tested again. CC

Appendix D

(Normative appendix) Nucleic acid detection of Bacillus anthracis D.1 Polymerase chain reaction (PCR) detection of Bacillus anthracis specific genes D.1.1 Target gene When detecting Bacillus anthracis nucleic acid from specimens, B. anthracis toxin genes and capsular synthesis-related genes are used as target genes. Other specific genes can also be selected. D.1.2 Reagent selection You can use a commercial kit, follow the operating instructions, or follow the steps below. D.1.3 Reference primer The reference primer sequence is shown in Table D.1.The synthesized primers are usually freeze-dried products. They should be centrifuged briefly before opening the lid. They need to be dissolved and Dilute into storage solution. Add pure water in the amount of 10 µL/nmol to prepare a 100 µmol/L storage solution. Dilute it by 10 times when it is used. As the concentration (10 µmol/L). D.1.4 Template preparation D.1.4.1 Kit method Commercial genomic DNA extraction kits were used for the original specimens, and the specific operation methods were carried out according to the kit instructions. D.1.4.2 Boiling method The bacterial colonies that have been isolated and cultured are prepared using the boiling method for simple template preparation. Bacteria were inoculated on nutrient agar plates and cultured at 37°C for 18 h～24 h; scrape 1 inoculation loop (approximately 2 µL～5 µL) of the lawn and put it into a 1.5 mL centrifuge tube containing 800 µL of pure water or TE, and mix; 100°C Heating for 10 min; centrifugation at 12 000 ×g for 10 min; aspirate the supernatant and filter with a 0.22 µm filter; the filtrate is the PCR template. D.1.5 PCR reaction system A reaction system with a total amount of 25 µL at a time contains. 12.5 µL of enzyme and buffer (2×), upstream primer (10 µmol/L) and downstream primer The sample (10 µmol/L) is 1 µL each, the template to be tested is 1 µL～5 µL, and the volume is made up to 25 µL with pure water. Establish quality control parameters for the reaction. use pure water as a negative control; use a known Bacillus anthracis template as a positive control. Adding order. First add the negative control, then add the template to be tested, and finally add the positive control. D.1.6 PCR amplification D.1.7 Detection and analysis of PCR amplification products Take 5 µL of each PCR product and mix it with 1 µL of loading buffer, add it to the sample well of 1% agarose gel, add DNA molecular weight to each piece of gel Standard 1-3 wells, 5 µL per well, 6 V/cm electrophoresis for 40 min. PCR products can also be sequenced and analyzed. D.1.8 Interpretation of results Observe the results in a UV transilluminator or gel imaging system. When the negative control and the positive control are established, such as the specificity of Bacillus anthracis A positive gene amplification indicates a positive specimen. When the negative control and the positive control are not established, the reagents need to be replaced and the test should be performed again. Sequencing knot The fruit matches the Bacillus anthracis-specific gene sequence, indicating that the specimen is positive. D.2 Real-time fluorescent quantitative polymerase chain reaction (Real-Time PCR) detection of Bacillus anthracis specific genes D.2.1 Target gene Same as D.1.1. D.2.2 Reagent selection You can use a commercial kit, follow the operating instructions, or follow the steps below. D.2.3 Reference primer and probe sequence The reference primer and probe sequence are shown in Table D.2, and the primer and probe dilution method is the same as D.1.3. D.2.4 Template preparation Same as D.1.4. D.2.5 Real-Time PCR reaction system A 20 µL reaction system contains. enzyme and buffer (2×) 10 µL, upstream primer (10 µmol/L) and downstream primer (10 µmol/L) 0.4 µL each, 0.4 µL of the probe solution (10 µmol/L), 1 µL～5 µL of the template to be tested, and make up the volume to 20 µL with pure water. Establish quality control parameters for the reaction. use pure water as a negative control; use a known Bacillus anthracis template as a positive control. Adding order. First add the negative control, then add the template to be tested, and finally add the positive control. D.2.6 Real-Time PCR amplification A two-step method is generally used for amplification. The reference procedure is as follows. 95℃ pre-denaturation for 5 min; amplification reaction at 95℃ for 10 s, 58℃ for 45 s, 40 cycles. The amplification procedures of different fluorescent quantitative PCR instruments will have some differences, and the reaction procedures can be appropriately adjusted according to the instrument used. Adjustment. D.2.7 Results judgment The result is judged when the negative control and the positive control are established. When the Ct value is less than 35, it is judged to be positive, and the reaction can be adjusted appropriately when the Ct value is less than 38. The system is re-tested, and it is judged as negative ...

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