WS/T 283: Evolution and historical versions
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(Diagnosis of anthrax)
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Diagnostic criteria for anthrax
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Basic data | Standard ID | WS/T 283-2020 (WS/T283-2020) | | Description (Translated English) | (Diagnosis of anthrax) | | Sector / Industry | Health Industry Standard (Recommended) | | Classification of Chinese Standard | C59 | | Date of Issue | 2020-11-01 | | Date of Implementation | 2020-11-01 | | Older Standard (superseded by this standard) | WS 283-2008 | | Regulation (derived from) | State-health communication (2020) No. 6 | | Issuing agency(ies) | National Health Commission |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/T 283-2020: (Diagnosis of anthrax)---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 of anthrax)
ICS 11.020
C 59
WS
People's Republic of China Health Industry Standard
Replace WS 283-2008
Diagnosis of anthrax
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 reference documents (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 the antigen test of Bacillus anthracis (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 versions 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 high mortality rate.
4 Diagnosis basis
4.1 Epidemiological history
Within 14 days before the onset of illness, have been in contact with suspected anthrax disease, dead animals or their remains, or have eaten suspected anthrax disease, dead animal meat
Or its products, or inhale dust suspected to be contaminated by Bacillus anthracis, or engage 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, farming, or excavation 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. In a few severe cases, large areas of edema and necrosis were present.
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 extremely thick bloody sputum. The lung signs often consist of scattered, fine rales. Chest X-ray
Mainly manifested as widening of the mediastinum and pleural effusion.
4.2.4 Meningitis anthrax
Severe headache, vomiting, stiff neck, then 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 patients, microscopic examination revealed a large number of gram-positive large bacilli arranged in series at both ends.
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 bacterial isolation and culture on exposed animal specimens or exposed environmental 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, with 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 anthrax lesions, 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 are available 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 pneumonic plague and leptospirosis with diffuse lung hemorrhage
Type phase identification. The effusion of pleurisy is bloody viscous fluid.
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 of blood samples should be collected aseptically for microscopic examination, culture, antigen and nucleic acid testing, and retained
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 stage, use two sterile cotton swabs to dip into the blister fluid; in the ulcer stage, 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. The specimens are used for microscopic examination, culture, antigen and nucleic acid detection.
A.1.4 Stool and vomit specimens
For 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 testing.
A.1.6 Cerebrospinal fluid specimen
For suspected cases with meningeal irritation, the cerebrospinal fluid was obtained by lumbar puncture, with a sample volume of 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 specimens. 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 objects 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 kept refrigerated at 2℃～8℃ and transported at low temperature. Serum samples used for serum antibody detection at -20℃
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 "Management Regulations on the Transportation of Highly Pathogenic 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 relevant 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.
Appendix B
(Normative appendix)
Bacteriological examination of anthrax
B.1 Biosafety requirements
It should 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, feces and vomit specimens, use twice the amount of distilled water or
Physiological saline is made into a suspension, after the coarse particles are removed by natural precipitation, 1 mL of the resulting suspension is 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 hours to 24 hours, 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 the low power lens. 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 drop of the phage, and the penicillin sensitive paper
If there is an obvious loop of inhibition, 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 an antibody test, and store the rest below -20°C. After the recovery serum is obtained, the two sera will be tested together
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.2.2.1 ELISA coating solution
0.01 mol/L phosphate buffered saline (PBS), pH 7.4.Weigh 8 g NaCl, 0.2 g KCl, 3.58 g Na2HPO4·12H2O,
0.3 g KH2PO4·2H2O, add about 900 mL of double distilled water, adjust the pH to 7.4 with HCl/NaOH, and finally dilute to 1000 mL, 15 psi (1.05
kg/cm
2) High pressure steam sterilization.
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, 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 that the content of Tween-20 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 Microplate 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, coat overnight at 4°C, and use within 7 days. Near
Before use, add 300 µL of washing solution to each well and wash 3 times for 3 minutes 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 samples were diluted with serum diluent to 1.50 and added to the ELISA plate. Two wells were tested in parallel, with 100 µL per well. Reagent and positive for each plate
Serum and negative serum control wells are 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 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 will be 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 diluted with ELISA diluent to each reaction well, and place it in a humidified chamber at 37°C
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 store in the dark for 20 min. 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 to measure the OD value of each well after zero adjustment with the reagent control well at a wavelength of 450 nm. 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 most
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 the specimen, and the test kit is properly diluted with normal 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 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.
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 a specimen, use Bacillus anthracis toxin genes and capsular synthesis-related genes 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 according to the amount of 10 µL/nmol to prepare a 100 µmol/L storage solution. Dilute 10 times when it is used.
As the concentration (10 µmol/L).
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