GB 17015-1997 Diagnostic criteria and management principles for anthrax
Some standard content:
【GB170151997】
Diagnostic criteria and treatment principles for anthrax
Anthrax is a human-animal infectious disease caused by Bacillus anthracis. It mainly exists in herbivores and livestock communities and can cause extensive environmental pollution. Since anthrax spores have extremely strong resistance to the external environment, this kind of pollution often persists. Humans mainly contract the disease through contact with sick livestock, eating infected livestock meat, inhaling aerosols or dust containing the bacteria, and contacting contaminated fur and other livestock products. This standard specifies the diagnostic criteria for anthrax patients and the treatment principles that must be followed after diagnosis.
Appendix A and Appendix B of this standard are standard appendices; Appendix C and Appendix D are informative appendices.
This standard was proposed by the Ministry of Health of the People's Republic of China. This standard was drafted by the Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine.
The main drafters of this standard are Li Aifang, Dong Shulin, Liu Bingyang and Yu Dongzheng. The Chinese Academy of Preventive Medicine, the technical unit entrusted by the Ministry of Health, is responsible for the interpretation of this standard.
1 Scope
This standard specifies the diagnostic criteria for human anthrax and the principles for the treatment of human anthrax after it occurs. This standard is applicable to the diagnosis and treatment of human anthrax by medical care and epidemic prevention institutions at all levels and types.
2 Definitions
This standard adopts the following definitions.
2.1 Anthrax
All human infections caused by Bacillus anthracis, including mild cases with atypical symptoms. Animal anthrax is specifically specified in this standard, such as anthrax-sick animals. 2.2 On-site isolation
Isolate anthrax patients at the diagnosis site or at home. 2.3 Source of infection
The source from which anthrax patients acquire infection. This includes the source of infection, which refers to anthrax patients, sick animals or their households, as well as the environment and various objects contaminated by anthrax spores. 3 Diagnosis of anthrax patients
3.1 Diagnostic basis
3.1.1 Epidemiology
Patients living in areas where anthrax has been confirmed to exist, or have been to such areas within 14 days before the onset of the disease; engaged in occupations that are in close contact with animal products such as fur; have been in contact with suspected sick or dead animals or their remains, have eaten suspected sick or dead animal meat or their products; and have engaged in farming or digging in areas that may be contaminated by Bacillus anthracis should all be used as epidemiological clues.
3.1.2 Clinical manifestations
3.1.2.1 Superficial infection type (skin) anthrax: erythema, papules, and blisters appear on the skin of exposed parts such as the face, neck, hands or forearms, and the surrounding tissues swell and infiltrate, followed by a central ring of death to form an ulcerative black scorch, and the skin around the scorch is red, swollen, and pain is not significant. The lymph nodes draining the area are enlarged and often suppurative, accompanied by fever, headache, joint pain, etc. In a few severe cases, large areas of local edema and necrosis are present. 3.1.2.2 Oral infection type (intestinal) anthrax: acute onset, fever, abdominal distension, severe pain, diarrhea, usually bloody stool or bloody stool. There may be nausea and vomiting, and the vomitus contains blood and bile. It may affect systems other than the digestive tract. 3.1.2.3 Inhalation infection type (pulmonary) anthrax: high fever, dyspnea, chest pain and cough, mucus and bloody sputum. Pulmonary signs are often only scattered fine wet rales. The main manifestation of X-ray is widening of the mediastinal shadow. Pleural effusion is common. 3.1.2.4 Meningitis type anthrax: It can be secondary to 3.1.2.1~3.1.2.3 types, or it may occur directly. Severe headache, vomiting, stiff neck, followed by delirium, coma, respiratory failure, and cerebrospinal fluid is mostly bloody. 3.1.2.5 Anthrax septicemia: It can be secondary to the types 3.1.2.1~3.1.2.3, or it may occur directly. Severe systemic poisoning symptoms, high fever, chills, septic shock and disseminated intravascular coagulation (DIC), bleeding spots or large ecchymoses on the skin, active bleeding in the cavity, and rapid respiratory and circulatory failure. A large number of anthrax Bacillus can be detected in the circulating blood.
3.1.3 Laboratory test results
3.1.3.1 Bacillus anthrax can be found in the secretions of skin lesions, sputum, vomitus, excrement, or blood and cerebrospinal fluid specimens by microscopic examination. 3.13.2 Bacillus anthrax can be obtained by bacterial isolation and culture (see Appendix A for details). 3.1.3.3 The titer of serum anti-anthrax specific antibodies increases by 4 times or more (see Appendix B for details).
3.2 Diagnosis
3.2.1 Suspected diagnosis
Those with typical skin lesions in 3.1.2.1, or epidemiological clues in 3.1.1, and one of the clinical manifestations in 3.1.2.2 to 3.1.2.5. 3.2.2 Clinical diagnosis
Those with microscopic examination results in 3.1.3.1 and one of the clinical manifestations in 3.1.2.1 to 3.1.2.5.
3.2.3 Confirmed diagnosis
Obtain any of the laboratory results in 3.1.3.2 or 3.1.3.3. 4 Principles for the treatment of anthrax patients
4.1 Isolation
Anthrax patients should be isolated and treated from the time a suspected diagnosis is made. In principle, they should be isolated on the spot to avoid long-distance transportation of patients.
4. 2 Treatment
Before treatment begins, specimens should be taken to confirm the diagnosis. Based on antibiotic treatment, anti-shock and anti-DIC therapy should be adopted at the same time, and appropriate symptomatic treatment should be supplemented according to the situation (see Appendix C for details). 4.3 Disinfection of the patient's contaminated environment
The patient's discarded items must be incinerated, and all contaminated items should be incinerated as much as possible. The contaminated environment and items that cannot be destroyed should be disinfected using effective methods (see Appendix D for details). After the patient is discharged from the hospital or dies, the patient's environment should be disinfected. 4.4 Treatment of the patient's body
After the anthrax patient dies, the openings of the mouth, nose, anus and other cavities should be plugged with cotton or gauze soaked in chlorine-containing disinfectant, and the body should be wrapped with a sheet soaked in disinfectant and then cremated. 5 Determination and treatment of the source of infection
After the patient is diagnosed with anthrax, the source of infection should be determined as much as possible and treated appropriately to avoid further infection.
5.1 Collection of epidemiological data
When receiving patients suspected of anthrax, they should be asked about their contact history before the onset of the disease as much as possible to find the suspected source of infection.
5.2 Determine the source of infection
Sampling of suspected sources of infection should be conducted for bacteriological examination to determine whether they are indeed contaminated by Bacillus anthracis. In animal tissue specimens, Bacillus anthracis is found by microscopic examination; or Bacillus anthracis is isolated and cultured in specimens from various sources, which can be determined as the source of infection.
5.3 Principles for handling the source of infection
For the identified source of infection, the following treatment shall be carried out a) Isolate and treat patients;
b) Kill or isolate and treat sick animals;
c) Disinfect objects and environments contaminated by Bacillus anthracis; d) Vaccinate all livestock that are active in or around the contaminated area once every early spring.
A1 Collection of specimens
Appendix A
(Standard Appendix)
Anthrax bacteriological examination
A1.1 Two principles that must be followed when collecting specimensA1.1.1 Collect specimens before the start of antibiotic treatment as much as possible. A1.1.2 Specimens shall not be obtained by dissection unless necessary and in a laboratory equipped with the conditions for handling viruses and bacteria. The required blood and tissue specimens shall be obtained by puncture.
A1.2 Blood specimens
Blood specimens shall be collected from all suspected human cases and sick animals, and the specimen volume shall at least meet the needs of the following examinations:
a) Smear for microscopic examination;
b) Inoculation of culture medium for bacterial isolation and culture: c) Isolation of serum for antibody examination:
d) Routine blood examination.
A1.3 Stool and vomitus specimens
For suspected patients with digestive tract symptoms, collect stool or vomitus specimens, paying special attention to selecting the part mixed with blood and placing it in a sterile container. Al.4 Sputum and cough plate specimens
For suspected patients with respiratory tract symptoms, collect their sputum specimens. For those without sputum, take the culture medium for bacterial isolation and culture, open the blood plate cover and place it 10 cm from the patient's mouth and nose; ask the patient to cough into the plate, and then quickly cover it with blood. A1.5 Cerebrospinal fluid specimens
For patients with meningeal irritation symptoms, obtain cerebrospinal fluid by lumbar puncture. The specimen volume refers to the requirements of Al. 2.
A1.6 Body specimens
When herbivores die of anthrax, blood usually flows out of the mouth, nose, anus and other cavity openings. This blood should be the first specimen taken. If the blood has seeped into the soil, the soil mixed with blood should be collected as a specimen. When there is no blood outflow or it is impossible to obtain a blood sample, blood can be obtained by puncturing the heart or tissue samples can be obtained by puncturing solid organs such as the liver.
A1.7 Meat specimens
If the livestock suspected of anthrax has been slaughtered, or when the commercial meat is routinely inspected, small pieces of meat specimens can be cut. If possible, specimens rich in blood and lymphatic tissues such as liver and spleen should be cut.
A1.8 Fur or other suspected contaminated items specimens Cut small pieces of fur or other suspected items, cut them into pieces and place them in a sterile test tube, and soak them in an appropriate amount of sterile physiological saline.
A1.9 Water specimens
When checking water pollution, collect water samples in a wide-mouth bottle. If deep water samples need to be taken, put the wide-mouth bottle with a lid into the water, and then lift the bottle lid tied with a rope. A1.10 Soil specimens
At the place where livestock died or were slaughtered, soil specimens should be taken for inspection. A2 Microscopic examination
All specimens from patients, sick animals or households should be examined under a microscope first. A2.1 Compound negative staining method
Drop the specimen or suspension on the slide, immediately add an equal amount of carbon ink, mix well and slide. After drying, add a few drops of 95% ethanol, and then stain with 1% crystal violet solution for 1 minute after drying. Rinse with water and examine with an oil immersion lens after drying. If a clear and continuous interval is found between the bacteria and the carbon particles, it means that there is a capsule around the bacteria. It is recommended to use the compound negative staining method for liquid specimens (such as blood, cerebrospinal fluid) and suspension specimens (such as feces or soil mixed with blood) when it is possible to perform microscopic examination immediately after taking the specimen.
A2.2 Capsule staining method
Dry the specimen smear or print, properly package it and bring it back to the laboratory, fix it according to the conventional method, stain it with methylene blue dye for 3-5 minutes (if the membrane is not clear, the staining time can be slightly longer), wash it with water, dry it and then examine it under a microscope. The bacteria are blue and the capsule is red. Capsule staining method is recommended for tissue print specimens, or when microscopic examination cannot be performed immediately after preparation, or when a large area survey is conducted. A2.3 Determination of microscopic examination resultswww.bzxz.net
If thick rods surrounded by a wide capsule with flat ends are found in specimens from patients, sick animals or households, especially when a large number of pure and usually bamboo-shaped bacteria are found under the microscope, and there is no obvious contamination from other bacteria, the diagnosis of anthrax can be established. A3 Bacterial isolation and culture
All specimens listed in A1 should be subjected to bacterial isolation and culture. A3.1 Culture medium
The isolation and culture of Bacillus anthracis should be carried out with two culture media at the same time: conventional blood agar plate and selective plate. The latter is prepared according to the following prescription: Protein Chen
Sodium chloride
Alcohol mother extract powder
Thallium acetate
Salicin
0.2% bromothymol blue (BTB) 1.2mL agar powder
Add distilled water to 100mL, adjust pH to 7.6, sterilize at 121℃ for 20min, cool to 45-50℃, add: polymyxin B (3000u/mL aqueous solution) 0.1mL, lysozyme (30000u/mL aqueous solution) 0.1mL, mix well and pour into the plate. A3.2 Specimen processing
Specimens that should be sterile under normal circumstances, such as blood or cerebrospinal fluid, should be directly coated on the above two plates, 0.1mL per plate. Tissue specimens should first be cut with sterile scissors to make a section and imprint on the surface of the culture medium, and then coated with platinum ear. All contaminated and old specimens should first be made into suspensions. According to the amount of bacteria in the specimen, the suspension can be appropriately diluted, or after natural precipitation to remove coarse sediments, centrifuge at 3000r/min for 30min, and the enriched sediments can be taken. The resulting suspension is heated at 67℃ for 15min, and after cooling, it is coated on the above two plates, 0.1mL per plate. A3.3 Selection of suspicious colonies
After incubating the above plates at 37℃ overnight or 24h, check whether suspicious anthrax Bacillus colonies have grown.
The morphology of Bacillus anthracis on blood agar plates is: off-white, opaque, medium-sized, often irregular, with a frosted glass surface and no hemolytic ring around it. The colonies of Bacillus anthracis on selective plates are smaller than those on blood agar plates, with similar surface characteristics, and the colonies are blue-green. However, if the colonies are exactly the same as the above description but are yellow, the identification step A4 should also be performed. A4 Identification of Bacillus anthracis
Pick out the above suspicious colonies, streak them on ordinary nutrient agar plates, and paste paper strips soaked with diagnostic Bacillus anthracis phage and penicillin in the streaked areas. After incubation at 37C overnight or 24 hours, obvious antibacterial rings appear around both paper strips, and they can be determined to be Bacillus anthracis. The diagnosis of anthrax can be established by isolating Bacillus anthracis from patients, sick animals or households.
Appendix B
(Standard Appendix)
Anthrax serological examination
B1 Collection of specimens
When the conditions for bacteriological examination are not available, or when a suspected anthrax patient is found, and the patient has received antibiotic treatment, the patient's retrospective diagnosis can be determined based on the results of serological examination. Since the diagnosis must be made by two sera, it is necessary to emphasize the collection of acute-phase sera.
The first serum should be collected when the patient is first examined. Usually, blood specimens should be collected at one time for smear microscopy, bacterial isolation and culture, serological examination and routine blood examination. After the serum is separated, it is stored at 4℃. After the recovery serum is obtained, the antibody test is carried out together. The recovery serum should be collected about 15 days after the onset of the disease. B2 Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay is used to detect antibodies against the protective antigen of Bacillus anthracis in the patient's blood. The test method is as follows. B2.1 Titration plate coating
The protective antigen of Bacillus anthracis was diluted to 30-50 mg/mL with 0.05 mol/L carbonate buffer (pH 9.6), added to the wells of the titration plate, 0.1 mL per well, and incubated at 4°C for 18 hours. The antigen solution was removed the next day, and the plate was washed twice with 0.5 mol/L saline. B2.2 Serum to be tested
The serum to be tested was diluted in multiples with 0.01 mol/L PBS (pH 7.2) containing 0.05% bovine serum albumin, 0.1 mL per well, incubated at 37°C for 1 hour, and the serum was removed, and the plate was washed three times with 0.5 mol/L saline.
B2.3 Coupling enzyme labeling
Use horseradish peroxidase-labeled sheep (rabbit) anti-human IgG, or the enzyme-labeled Staphylococcus A protein to test the IgG antibody that has bound to the above antigen. Add 0.1mL to each well, incubate at 37℃ for 40min, pour off the labeling solution, and wash the plate 5 times with 0.5mol/L saline. B2.4 Color development
Add 0.1mL substrate solution to each well. The substrate solution should be prepared according to the following formula before use: 0.1mol/L citric acid
0.2mol/L disodium hydrogen phosphate
o-phenylenediamine
distilled water
30% hydrogen peroxide
After 20min at 37℃, add 1 drop of 2mol/L sulfuric acid to each well to terminate the reaction. B2.5 Result judgment
The experimental results can be read by an enzyme-labeled instrument. When the OD value of the tested well reaches 2.1 times the OD value of the negative serum control well, it can be judged as positive. In the absence of an enzyme-labeled instrument, it can also be judged by visual inspection. When the tested well shows a yellow-brown color that is significantly different from the negative control well, it is judged as positive.
Appendix C
(Suggested Appendix)
Treatment of Anthrax Patients
C1 Beginning of Treatment
From the time a suspected anthrax diagnosis is made, treatment should be given according to anthrax. The following two points should be achieved at the beginning of treatment.
C1.1 Take specimens to confirm the diagnosis. C1:2 Establish and maintain a patent intravenous access to prepare for effective rescue measures. C2
Antibacterial Treatment
C2.1 Penicillin G is the antibiotic of choice. The general dose for adults is 1.6 million to 3.2 million.9 Water specimens
When checking water pollution, collect water samples with a wide-mouth bottle. If deep water samples need to be taken, put the wide-mouth bottle with a cover into the water, and then lift the bottle cover tied with a rope. A1.10 Soil specimens
At the place where livestock died or were slaughtered, soil specimens should be taken for examination. A2 Microscope examination
All specimens from patients, sick animals or households should first be examined under a microscope. A2.1 Composite negative staining method
Drop the specimen or suspension on the slide, immediately add an equal amount of carbon ink, mix well and slide. After drying, add a few drops of 95% ethanol, and then stain with 1% crystal violet solution for 1 minute after drying, rinse with water, and examine with an oil microscope after drying. If a clear and continuous interval is found between the bacteria and the carbon particles, it means that there is a capsule around the bacteria. Liquid specimens (such as blood, cerebrospinal fluid) and suspension specimens (such as feces or soil mixed with blood) are recommended to use the composite negative staining method when the specimen can be examined under a microscope immediately after collection.
A2.2 Capsule staining method
Dry the specimen smear or print, properly package it and bring it back to the laboratory, fix it according to the conventional method, stain it with methylene blue dye for 3 to 5 minutes (if the membrane is not clear, the staining time can be slightly longer), wash it with water, dry it, and then examine it under a microscope. The bacteria are blue, and the capsule is red. The capsule staining method is recommended for tissue print specimens, or when microscopic examination cannot be performed immediately after preparation, or when a large-scale survey is conducted. A2.3 Determination of microscopic examination results
If large bacilli surrounded by a wide capsule and with flat ends are found in specimens from patients, sick animals or households, especially when large numbers of pure, usually bamboo-shaped bacteria are found under the microscope and there is no obvious contamination from other bacteria, the diagnosis of anthrax can be established. A3 Bacterial isolation and culture
All specimens listed in A1 should be subjected to bacterial isolation and culture. A3.1 Culture medium
The isolation and culture of anthrax Bacillus should be carried out using two culture media at the same time: conventional blood agar plates and selective plates. The latter is prepared according to the following prescription: protein Chen
sodium chloride
alcohol mother extract powder
thallium acetate
salicin
0.2% bromothymol blue (BTB) 1.2mL agar powder
add distilled water to 100mL, adjust pH to 7.6, sterilize at 121℃ for 20min, cool to 45-50℃, add: polymyxin B (3000u/mL aqueous solution) 0.1mL, lysozyme (30000u/mL aqueous solution) 0.1mL, mix well and pour into the plate. A3.2 Specimen processing
Specimens that should be sterile under normal circumstances, such as blood or cerebrospinal fluid, are directly coated on the above two plates, 0.1mL per plate. Tissue specimens should first be cut with sterile scissors to make a section and imprint on the surface of the culture medium, and then spread with platinum ear. All contaminated and old specimens should first be made into suspensions. Depending on the amount of bacteria in the specimen, the suspension can be appropriately diluted, or after natural sedimentation to remove coarse sediments, centrifuge at 3000r/min for 30min and take the enriched sediment. Heat the obtained suspension at 67℃ for 15min, and apply it to the above two plates after cooling, with 0.1mL per plate. A3.3 Select suspicious colonies
After incubating the above plates at 37℃ overnight or for 24h, check whether suspicious anthrax Bacillus colonies have grown.
The morphology of anthrax Bacillus on blood agar plates is: grayish white, opaque, medium size, often irregular, with a frosted glass surface, and no hemolytic ring around. The colonies of anthrax Bacillus on the selection plate are smaller than those on the blood agar plate, have similar surface characteristics, and are blue-green. However, if the colony morphology is exactly the same as the above description but is yellow, the identification step A4 should also be performed. A4 Identification of Bacillus anthracis
Pick out the above suspicious colonies, streak them on ordinary nutrient agar plates, and paste paper discs soaked with diagnostic Bacillus anthracis phage and penicillin in the streaked areas. After incubation at 37C overnight or for 24 hours, obvious antibacterial rings appear around the two paper discs, which can be identified as Bacillus anthracis. The diagnosis of anthrax can be established by isolating Bacillus anthracis from patients, sick animals or households.
Appendix B
(Standard Appendix)
Anthrax serological examination
B1 Collection of specimens
When the conditions for bacteriological examination are not available, or when a suspected anthrax patient is found, the patient has already received antibiotic treatment, the retrospective diagnosis of the patient can be determined based on the results of serological examinations. Since the diagnosis must be made by double sera, it is necessary to emphasize the collection of acute-phase sera.
The first serum should be collected when the patient is first examined. Usually, blood specimens should be collected at one time for smear microscopy, bacterial isolation and culture, serological examination and routine blood examination. After the serum is separated, it is stored at 4℃. After the recovery serum is obtained, the antibody test is carried out together. The recovery serum should be collected about 15 days after the onset of the disease. B2 Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay is used to detect antibodies against the protective antigen of Bacillus anthracis in the patient's blood. The test method is as follows. B2.1 Titration plate coating
The protective antigen of Bacillus anthracis is diluted to 30-50mg/mL with 0.05mol/L carbonate buffer (pH9.6), added to the titration plate wells, 0.1mL per well, 4℃18h. The antigen solution is poured off the next day, and the plate is washed twice with 0.5mol/L saline. B2.2 Serum to be tested
The serum to be tested was diluted in multiples with 0.01mol/LPBS (pH7.2) containing 0.05% bovine serum albumin, 0.1mL per well, incubated at 37℃ for 1h, the serum was removed, and the plate was washed 3 times with 0.5mol/L saline.
B2.3 Coupling enzyme labeling
Use horseradish peroxidase-labeled sheep (rabbit) anti-human IgG, or staphylococcal protein A labeled with the enzyme to test the IgG antibody that has bound to the above antigen. Add 0.1mL to each well, incubate at 37℃ for 40min, remove the labeling solution, and wash the plate 5 times with 0.5mol/L saline. B2.4 Color development
Add 0.1mL substrate solution to each well. The substrate solution should be prepared according to the following formula before use: 0.1mol/L citric acid
0.2mol/L disodium hydrogen phosphate
o-phenylenediamine
distilled water
30% hydrogen peroxide
After 20 minutes at 37℃, add 1 drop of 2mol/L sulfuric acid to each well to terminate the reaction. B2.5 Result judgment
The experimental results can be read by an enzyme-labeled instrument. When the OD value of the tested well reaches 2.1 times the OD value of the negative serum control well, it can be judged as positive. In the absence of an enzyme-labeled instrument, it can also be judged by visual inspection. When the tested well shows a yellow-brown color that is significantly different from the negative control well, it is judged as positive.
Appendix C
(Suggested Appendix)
Treatment of Anthrax Patients
C1 Beginning of Treatment
From the time a suspected anthrax diagnosis is made, treatment should be given according to anthrax. The following two points should be achieved at the beginning of treatment.
C1.1 Take specimens to confirm the diagnosis. C1:2 Establish and maintain a patent intravenous access to prepare for effective rescue measures. C2
Antibacterial Treatment
C2.1 Penicillin G is the antibiotic of choice. The general dose for adults is 1.6 million to 3.2 million.9 Water specimens
When checking water pollution, collect water samples with a wide-mouth bottle. If deep water samples need to be taken, put the wide-mouth bottle with a cover into the water, and then lift the bottle cover tied with a rope. A1.10 Soil specimens
At the place where livestock died or were slaughtered, soil specimens should be taken for examination. A2 Microscope examination
All specimens from patients, sick animals or households should first be examined under a microscope. A2.1 Composite negative staining method
Drop the specimen or suspension on the slide, immediately add an equal amount of carbon ink, mix well and slide. After drying, add a few drops of 95% ethanol, and then stain with 1% crystal violet solution for 1 minute after drying, rinse with water, and examine with an oil microscope after drying. If a clear and continuous interval is found between the bacteria and the carbon particles, it means that there is a capsule around the bacteria. Liquid specimens (such as blood, cerebrospinal fluid) and suspension specimens (such as feces or soil mixed with blood) are recommended to use the composite negative staining method when the specimen can be examined under a microscope immediately after collection.
A2.2 Capsule staining method
Dry the specimen smear or print, properly package it and bring it back to the laboratory, fix it according to the conventional method, stain it with methylene blue dye for 3 to 5 minutes (if the membrane is not clear, the staining time can be slightly longer), wash it with water, dry it, and then examine it under a microscope. The bacteria are blue, and the capsule is red. The capsule staining method is recommended for tissue print specimens, or when microscopic examination cannot be performed immediately after preparation, or when a large-scale survey is conducted. A2.3 Determination of microscopic examination results
If large bacilli surrounded by a wide capsule and with flat ends are found in specimens from patients, sick animals or households, especially when large numbers of pure, usually bamboo-shaped bacteria are found under the microscope and there is no obvious contamination from other bacteria, the diagnosis of anthrax can be established. A3 Bacterial isolation and culture
All specimens listed in A1 should be subjected to bacterial isolation and culture. A3.1 Culture medium
The isolation and culture of anthrax Bacillus should be carried out using two culture media at the same time: conventional blood agar plates and selective plates. The latter is prepared according to the following prescription: protein Chen
sodium chloride
alcohol mother extract powder
thallium acetate
salicin
0.2% bromothymol blue (BTB) 1.2mL agar powder
add distilled water to 100mL, adjust pH to 7.6, sterilize at 121℃ for 20min, cool to 45-50℃, add: polymyxin B (3000u/mL aqueous solution) 0.1mL, lysozyme (30000u/mL aqueous solution) 0.1mL, mix well and pour into the plate. A3.2 Specimen processing
Specimens that should be sterile under normal circumstances, such as blood or cerebrospinal fluid, are directly coated on the above two plates, 0.1mL per plate. Tissue specimens should first be cut with sterile scissors to make a section and imprint on the surface of the culture medium, and then spread with platinum ear. All contaminated and old specimens should first be made into suspensions. Depending on the amount of bacteria in the specimen, the suspension can be appropriately diluted, or after natural sedimentation to remove coarse sediments, centrifuge at 3000r/min for 30min and take the enriched sediment. Heat the obtained suspension at 67℃ for 15min, and apply it to the above two plates after cooling, with 0.1mL per plate. A3.3 Select suspicious colonies
After incubating the above plates at 37℃ overnight or for 24h, check whether suspicious anthrax Bacillus colonies have grown.
The morphology of anthrax Bacillus on blood agar plates is: grayish white, opaque, medium size, often irregular, with a frosted glass surface, and no hemolytic ring around. The colonies of anthrax Bacillus on the selection plate are smaller than those on the blood agar plate, have similar surface characteristics, and are blue-green. However, if the colony morphology is exactly the same as the above description but is yellow, the identification step A4 should also be performed. A4 Identification of Bacillus anthracis
Pick out the above suspicious colonies, streak them on ordinary nutrient agar plates, and paste paper discs soaked with diagnostic Bacillus anthracis phage and penicillin in the streaked areas. After incubation at 37C overnight or for 24 hours, obvious antibacterial rings appear around the two paper discs, which can be identified as Bacillus anthracis. The diagnosis of anthrax can be established by isolating Bacillus anthracis from patients, sick animals or households.
Appendix B
(Standard Appendix)
Anthrax serological examination
B1 Collection of specimens
When the conditions for bacteriological examination are not available, or when a suspected anthrax patient is found, the patient has already received antibiotic treatment, the retrospective diagnosis of the patient can be determined based on the results of serological examinations. Since the diagnosis must be made by double sera, it is necessary to emphasize the collection of acute-phase sera.
The first serum should be collected when the patient is first examined. Usually, blood specimens should be collected at one time for smear microscopy, bacterial isolation and culture, serological examination and routine blood examination. After the serum is separated, it is stored at 4℃. After the recovery serum is obtained, the antibody test is carried out together. The recovery serum should be collected about 15 days after the onset of the disease. B2 Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay is used to detect antibodies against the protective antigen of Bacillus anthracis in the patient's blood. The test method is as follows. B2.1 Titration plate coating
The protective antigen of Bacillus anthracis is diluted to 30-50mg/mL with 0.05mol/L carbonate buffer (pH9.6), added to the titration plate wells, 0.1mL per well, 4℃18h. The antigen solution is poured off the next day, and the plate is washed twice with 0.5mol/L saline. B2.2 Serum to be tested
The serum to be tested was diluted in multiples with 0.01mol/LPBS (pH7.2) containing 0.05% bovine serum albumin, 0.1mL per well, incubated at 37℃ for 1h, the serum was removed, and the plate was washed 3 times with 0.5mol/L saline.
B2.3 Coupling enzyme labeling
Use horseradish peroxidase-labeled sheep (rabbit) anti-human IgG, or staphylococcal protein A labeled with the enzyme to test the IgG antibody that has bound to the above antigen. Add 0.1mL to each well, incubate at 37℃ for 40min, remove the labeling solution, and wash the plate 5 times with 0.5mol/L saline. B2.4 Color development
Add 0.1mL substrate solution to each well. The substrate solution should be prepared according to the following formula before use: 0.1mol/L citric acid
0.2mol/L disodium hydrogen phosphate
o-phenylenediamine
distilled water
30% hydrogen peroxide
After 20 minutes at 37℃, add 1 drop of 2mol/L sulfuric acid to each well to terminate the reaction. B2.5 Result judgment
The experimental results can be read by an enzyme-labeled instrument. When the OD value of the tested well reaches 2.1 times the OD value of the negative serum control well, it can be judged as positive. In the absence of an enzyme-labeled instrument, it can also be judged by visual inspection. When the tested well shows a yellow-brown color that is significantly different from the negative control well, it is judged as positive.
Appendix C
(Suggested Appendix)
Treatment of Anthrax Patients
C1 Beginning of Treatment
From the time a suspected anthrax diagnosis is made, treatment should be given according to anthrax. The following two points should be achieved at the beginning of treatment.
C1.1 Take specimens to confirm the diagnosis. C1:2 Establish and maintain a patent intravenous access to prepare for effective rescue measures. C2
Antibacterial Treatment
C2.1 Penicillin G is the antibiotic of choice. The general dose for adults is 1.6 million to 3.2 million.2mL agar powder
Add distilled water to 100mL, adjust pH to 7.6, sterilize at 121℃ for 20min, cool to 45-50℃, add: 0.1mL polymyxin B (3000u/mL aqueous solution), 0.1mL lysozyme (30000u/mL aqueous solution), mix well and pour into the plate. A3.2 Specimen processing
Specimens that should be sterile under normal circumstances, such as blood or cerebrospinal fluid, are directly coated on the above two plates, 0.1mL per plate. Tissue specimens should first be cut with sterile scissors to imprint on the surface of the culture medium, and then coated with platinum ear. All contaminated and old specimens should first be made into suspensions. According to the amount of bacteria in the specimen, the suspension can be appropriately diluted, or after natural sedimentation to remove coarse sediments, centrifuge at 3000r/min for 30min to take the enriched sediment. Heat the obtained suspension at 67℃ for 15min, and apply it to the above two plates after cooling, 0.1mL per plate. A3.3 Select suspicious colonies
After incubating the above plates at 37℃ overnight or for 24h, check whether suspicious anthrax Bacillus colonies have grown.
The morphology of anthrax Bacillus on blood agar plates is: grayish white, opaque, medium-sized, often irregular, with a frosted glass surface and no hemolytic ring around. The colonies of anthrax Bacillus on the selection plate are smaller than those on the blood agar plate, with similar surface characteristics, and the colonies are blue-green. However, if the colony morphology is exactly the same as the above description but is yellow, the identification step A4 should also be performed. A4 Identification of anthrax Bacillus
Pick out the above suspicious colonies, streak them on ordinary nutrient agar plates, and paste paper pieces soaked with diagnostic anthrax Bacillus phage and penicillin in the streaked area. After incubation at 37C overnight or for 24 hours, if obvious antibacterial rings appear around both paper strips, it can be determined to be Bacillus anthracis. The diagnosis of anthrax can be established by isolating Bacillus anthracis from patients, sick animals or households.
Appendix B
(Standard Appendix)
Anthrax serological examination
B1 Collection of specimens
When the conditions for bacteriological examination are not available, or when a suspected anthrax patient is found, the patient has already received antibiotic treatment, the retrospective diagnosis of the patient can be determined based on the results of serological examination. Since the diagnosis must be made by two sera, it is necessary to emphasize the collection of acute-phase sera.
The first serum should be collected when the patient is first examined. Usually, blood specimens should be collected at one time for smear microscopy, bacterial isolation and culture, serological examination and routine blood examination. After the serum is separated, it is stored at 4°C. After the recovery serum is obtained, the antibody test is carried out together. The recovery serum should be collected about 15 days after the onset of the disease. B2 Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay is used to detect antibodies against the protective antigen of Bacillus anthracis in the patient's blood. The test method is as follows. B2.1 Titration plate coating
The protective antigen of Bacillus anthracis is diluted to 30-50 mg/mL with 0.05 mol/L carbonate buffer (pH 9.6) and added to the titration plate wells, 0.1 mL per well, 4°C for 18 hours. The antigen solution is poured off the next day and the plate is washed twice with 0.5 mol/L saline. B2.2 Serum to be tested
The serum to be tested was diluted in multiples with 0.01mol/LPBS (pH7.2) containing 0.05% bovine serum albumin, 0.1mL per well, incubated at 37℃ for 1h, the serum was removed, and the plate was washed 3 times with 0.5mol/L saline.
B2.3 Coupling enzyme labeling
Use horseradish peroxidase-labeled sheep (rabbit) anti-human IgG, or staphylococcal protein A labeled with the enzyme to test the IgG antibody that has bound to the above antigen. Add 0.1mL to each well, incubate at 37℃ for 40min, remove the labeling solution, and wash the plate 5 times with 0.5mol/L saline. B2.4 Color development
Add 0.1mL substrate solution to each well. The substrate solution should be prepared according to the following formula before use: 0.1mol/L citric acid
0.2mol/L disodium hydrogen phosphate
o-phenylenediamine
distilled water
30% hydrogen peroxide
After 20 minutes at 37℃, add 1 drop of 2mol/L sulfuric acid to each well to terminate the reaction. B2.5 Result judgment
The experimental results can be read by an enzyme-labeled instrument. When the OD value of the tested well reaches 2.1 times the OD value of the negative serum control well, it can be judged as positive. In the absence of an enzyme-labeled instrument, it can also be judged by visual inspection. When the tested well shows a yellow-brown color that is significantly different from the negative control well, it is judged as positive.
Appendix C
(Suggested Appendix)
Treatment of Anthrax Patients
C1 Beginning of Treatment
From the time a suspected anthrax diagnosis is made, treatment should be given according to anthrax. The following two points should be achieved at the beginning of treatment.
C1.1 Take specimens to confirm the diagnosis. C1:2 Establish and maintain a patent intravenous access to prepare for effective rescue measures. C2
Antibacterial Treatment
C2.1 Penicillin G is the antibiotic of choice. The general dose for adults is 1.6 million to 3.2 million.2mL agar powder
Add distilled water to 100mL, adjust pH to 7.6, sterilize at 121℃ for 20min, cool to 45-50℃, add: 0.1mL polymyxin B (3000u/mL aqueous solution), 0.1mL lysozyme (30000u/mL aqueous solution), mix well and pour into the plate. A3.2 Specimen processing
Specimens that should be sterile under normal circumstances, such as blood or cerebrospinal fluid, are directly coated on the above two plates, 0.1mL per plate. Tissue specimens should first be cut with sterile scissors to imprint on the surface of the culture medium, and then coated with platinum ear. All contaminated and old specimens should first be made into suspensions. According to the amount of bacteria in the specimen, the suspension can be appropriately diluted, or after natural sedimentation to remove coarse sediments, centrifuge at 3000r/min for 30min to take the enriched sediment. Heat the obtained suspension at 67℃ for 15min, and apply it to the above two plates after cooling, 0.1mL per plate. A3.3 Select suspicious colonies
After incubating the above plates at 37℃ overnight or for 24h, check whether suspicious anthrax Bacillus colonies have grown.
The morphology of anthrax Bacillus on blood agar plates is: grayish white, opaque, medium-sized, often irregular, with a frosted glass surface and no hemolytic ring around. The colonies of anthrax Bacillus on the selection plate are smaller than those on the blood agar plate, with similar surface characteristics, and the colonies are blue-green. However, if the colony morphology is exactly the same as the above description but is yellow, the identification step A4 should also be performed. A4 Identification of anthrax Bacillus
Pick out the above suspicious colonies, streak them on ordinary nutrient agar plates, and paste paper pieces soaked with diagnostic anthrax Bacillus phage and penicillin in the streaked area. After incubation at 37C overnight or for 24 hours, if obvious antibacterial rings appear around both paper strips, it can be determined to be Bacillus anthracis. The diagnosis of anthrax can be established by isolating Bacillus anthracis from patients, sick animals or households.
Appendix B
(Standard Appendix)
Anthrax serological examination
B1 Collection of specimens
When the conditions for bacteriological examination are not available, or when a suspected anthrax patient is found, the patient has already received antibiotic treatment, the retrospective diagnosis of the patient can be determined based on the results of serological examination. Since the diagnosis must be made by two sera, it is necessary to emphasize the collection of acute-phase sera.
The first serum should be collected when the patient is first examined. Usually, blood specimens should be collected at one time for smear microscopy, bacterial isolation and culture, serological examination and routine blood examination. After the serum is separated, it is stored at 4°C. After the recovery serum is obtained, the antibody test is carried out together. The recovery serum should be collected about 15 days after the onset of the disease. B2 Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay is used to detect antibodies against the protective antigen of Bacillus anthracis in the patient's blood. The test method is as follows. B2.1 Titration plate coating
The protective antigen of Bacillus anthracis is diluted to 30-50 mg/mL with 0.05 mol/L carbonate buffer (pH 9.6) and added to the titration plate wells, 0.1 mL per well, 4°C for 18 hours. The antigen solution is poured off the next day and the plate is washed twice with 0.5 mol/L saline. B2.2 Serum to be tested
The serum to be tested was diluted in multiples with 0.01mol/LPBS (pH7.2) containing 0.05% bovine serum albumin, 0.1mL per well, incubated at 37℃ for 1h, the serum was removed, and the plate was washed 3 times with 0.5mol/L saline.
B2.3 Coupling enzyme labeling
Use horseradish peroxidase-labeled sheep (rabbit) anti-human IgG, or staphylococcal protein A labeled with the enzyme to test the IgG antibody that has bound to the above antigen. Add 0.1mL to each well, incubate at 37℃ for 40min, remove the labeling solution, and wash the plate 5 times with 0.5mol/L saline. B2.4 Color development
Add 0.1mL substrate solution to each well. The substrate solution should be prepared according to the following formula before use: 0.1mol/L citric acid
0.2mol/L disodium hydrogen phosphate
o-phenylenediamine
distilled water
30% hydrogen peroxide
After 20 minutes at 37℃, add 1 drop of 2mol/L sulfuric acid to each well to terminate the reaction. B2.5 Result judgment
The experimental results can be read by an enzyme-labeled instrument. When the OD value of the tested well reaches 2.1 times the OD value of the negative serum control well, it can be judged as positive. In the absence of an enzyme-labeled instrument, it can also be judged by visual inspection. When the tested well shows a yellow-brown color that is significantly different from the negative control well, it is judged as positive.
Appendix C
(Suggested Appendix)
Treatment of Anthrax Patients
C1 Beginning of Treatment
From the time a suspected anthrax diagnosis is made, treatment should be given according to anthrax. The following two points should be achieved at the beginning of treatment.
C1.1 Take specimens to confirm the diagnosis. C1:2 Establish and maintain a patent intravenous access to prepare for effective rescue measures. C2
Antibacterial Treatment
C2.1 Penicillin G is the antibiotic of choice. The general dose for adults is 1.6 million to 3.2 million.1mL, incubate at 37℃ for 40min, pour off the labeling solution, and wash the plate 5 times with 0.5mol/L saline. B2.4 Color development
Add 0.1mL substrate solution to each well. The substrate solution should be prepared according to the following formula before use: 0.1mol/L citric acid
0.2mol/L disodium hydrogen phosphate
o-phenylenediamine
distilled water
30% hydrogen peroxide
After 20min at 37℃, add 1 drop of 2mol/L sulfuric acid to each well to terminate the reaction. B2.5 Result judgment
The experimental results can be read by an enzyme-labeled instrument. When the OD value of the tested well reaches 2.1 times the OD value of the negative serum control well, it can be judged as positive. In the absence of an enzyme-labeled instrument, it can also be judged by visual inspection. When the tested well shows a yellow-brown color that is significantly different from the negative control well, it is judged as positive.
Appendix C
(Suggested Appendix)
Treatment of Anthrax Patients
C1 Beginning of Treatment
From the time a suspected anthrax diagnosis is made, treatment should be given according to anthrax. The following two points should be achieved at the beginning of treatment.
C1.1 Take specimens to confirm the diagnosis. C1:2 Establish and maintain a patent intravenous access to prepare for effective rescue measures. C2
Antibacterial Treatment
C2.1 Penicillin G is the antibiotic of choice. The general dose for adults is 1.6 million to 3.2 million.1mL, incubate at 37℃ for 40min, pour off the labeling solution, and wash the plate 5 times with 0.5mol/L saline. B2.4 Color development
Add 0.1mL substrate solution to each well. The substrate solution should be prepared according to the following formula before use: 0.1mol/L citric acid
0.2mol/L disodium hydrogen phosphate
o-phenylenediamine
distilled water
30% hydrogen peroxide
After 20min at 37℃, add 1 drop of 2mol/L sulfuric acid to each well to terminate the reaction. B2.5 Result judgment
The experimental results can be read by an enzyme-labeled instrument. When the OD value of the tested well reaches 2.1 times the OD value of the negative serum control well, it can be judged as positive. In the absence of an enzyme-labeled instrument, it can also be judged by visual inspection. When the tested well shows a yellow-brown color that is significantly different from the negative control well, it is judged as positive.
Appendix C
(Suggested Appendix)
Treatment of Anthrax Patients
C1 Beginning of Treatment
From the time a suspected anthrax diagnosis is made, treatment should be given according to anthrax. The following two points should be achieved at the beginning of treatment.
C1.1 Take specimens to confirm the diagnosis. C1:2 Establish and maintain a patent intravenous access to prepare for effective rescue measures. C2
Antibacterial Treatment
C2.1 Penicillin G is the antibiotic of choice. The general dose for adults is 1.6 million to 3.2 million.
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