GB 15988-1995 Diagnostic criteria and treatment principles for brucellosis
Some standard content:
National Standard of the People's Republic of China
Diagnostic criteria and principles of management of brucellosis1 Subject content and scope of applicationWww.bzxZ.net
This standard specifies the diagnosis of brucellosis (abbreviated as brucellosis) in the human population and the principles of management of epidemic areas. This standard is applicable to all types of medical and health and epidemic prevention institutions. 2 Terminology
Skin allergy test:
GB15988-1995
Delayed allergic reaction of the skin after being infected with Brucella and then stimulated by Brucella allergens. Brucellosis epidemic area;
Any natural village (village) or livestock farm (team) where new patients occur or infected livestock are detected is considered a brucellosis epidemic area. Quarantine:
The examination of brucellosis in humans and animals by specific serology, skin test, isolation of bacteria and other methods. Elimination:
The positive livestock detected are slaughtered. 3 Diagnosis of Brucellosis
Brucellosis is a zoonotic infectious disease that seriously endangers people's health and the development of animal husbandry. Infected livestock are the main source of infection of brucellosis between humans and animals.
3.1 Epidemiology: Before the onset of the disease, the patient has a history of close contact with livestock or livestock products, Brucella culture, or lives in an epidemic area, or has a close relationship with the production, use and research of vaccine. 3.2 Clinical manifestations: fever (including low fever), sweating, muscle and joint pain, fatigue, or other suspicious symptoms and signs such as enlargement of the liver, spleen, lymph nodes and testicles that last for several days or even weeks. 3.3 Laboratory examination: positive or suspicious agglutination reaction on brucellosis slides or bengalensis plates, or skin allergy test, observed once 24 and 48 hours respectively, and the skin redness, swelling and moistening range is once 2.0cm×2.0cm and above (or more than 4.0cm2). 3.4 Bacteria isolation: Brucella was isolated from the patient's blood, bone marrow, other body fluids and excrement. 3.5 Serological examination: Standard tube agglutination test (SAT) titer is 1:100 or above; for those who have a history of Brucella vaccination within six months, even if the SAT titer is 1:100 or above, it should be re-examined after 2 to 4 weeks, and the titer should increase by 4 times or more; or the complement fixation test (CFT) should be used for examination, and the CFT titer should be 1:10 or above: the anti-human immunoglobulin test (Coomb's) titer should be 1:400 or above. Suspected cases: those who meet 3.1.3.2 and 3.3. Confirmed cases: suspected cases plus any one of 3.4 or 3.5 is positive. 4 Principles of handling epidemic areas
4.1 Verification of diagnosis: The diagnosis of confirmed patients should be verified based on epidemiological data, clinical manifestations and laboratory test results. Approved by the State Bureau of Technical Supervision on December 15, 1995 and implemented on July 1, 1996
GB15988-1995
4.2 Quarantine and elimination of epidemic livestock: All sheep, cattle and pigs in the epidemic area shall be quarantined by serological methods, and re-inspected one month after quarantine. All positive livestock detected shall be slaughtered immediately (or kept in isolation). Stop the transportation of cattle, sheep and pigs for at least one year. Livestock products shall be stored and disinfected in situ and shall not be transported out for the time being
4.3 Disinfection: Sites, utensils, pens and uneaten dairy products contaminated by abortion products of sick animals shall be disinfected. 4.4 Immunization: Livestock that have tested negative after two quarantines, as well as the threatened livestock in villages around the epidemic area, should be immunized with animal vaccines for three consecutive years. The annual immunization coverage rate should not be less than 90%. 4.5 Clinical monitoring and treatment: Serological and skin allergy tests are conducted on people who come into contact with livestock and livestock products in the epidemic area to find out the infection situation of the population. All confirmed patients should receive systemic treatment. 4.6 Publicity and education: Publicity and education on the hazards, clinical manifestations and prevention and control knowledge of brucellosis are conducted for residents and occupational groups in the epidemic area. 4.7 Verification of the treatment effect of the epidemic area: Starting from the second year after the epidemic area is treated, the epidemic area and the surrounding areas will be verified for three consecutive years. The verification method is carried out in accordance with the requirements of the "Brucellosis Epidemic Area Control Assessment Standards" jointly formulated by the Ministry of Agriculture and the Ministry of Health. 316
GB15988—1995
Appendix A
Specific laboratory examination techniques for the diagnosis of brucellosis (supplement)
A1 Isolation of Brucella from suspected brucellosis patientsA1.1 Blood culture
A1.1.1 Biphasic culture medium: Aseptically draw 4~5mL of blood from the vein of a suspected patient, inject the blood into 5~6 medium test tubes containing biphasic culture medium near the flame of an alcohol lamp, or into 2~4 flasks containing biphasic culture medium, gently mix and tilt the blood to distribute on the agar slant, and culture it in a 37℃ incubator. If the patient is suspected of being infected with bovine Brucella, half of the specimen should be cultured in a CO2 environment, and the results should be observed after three days. If no Brucella growth is seen, the specimen can be tilted again according to the above method to smear the blood on the agar slant, and culture continued. Observe every other day. If there are suspected Brucella colonies, use a platinum ear hook to pick them out and inoculate them into the agar test tube medium to obtain pure culture, which can be further identified as Brucella. If no bacteria are found in the blood culture for thirty days, it can be determined to be negative. A1.1.2 Inoculation of unfertilized chicken eggs: Take two fresh eggs and place them on a fixed rack with the sharp end facing upward. Disinfect the eggshells with iodine and alcohol in turn. Use an ophthalmic scalpel to make a small hole in the top. Use a three-centimeter-long injection needle to slowly inject the blood to be tested into the yolk. Inoculate each egg with 0.2 ml of blood. Immediately seal the hole with sterilized paraffin and culture it in a 37°C incubator. After five days, aseptically open the eggs inoculated with blood and use a sterilized capillary to suck out 0.5~0.6 mL of the yolk and egg white of the blood-inoculated part. Inoculate it on 2~3 slant culture media and culture it at 37°C. Observe it for 2~3 days. If no suspicious colonies grow after 15 days, it is determined to be negative. A1.2 Bone marrow culture
Use a sterile catheter to drain urine into a sterile container. To concentrate bacteria and increase the detection rate, add 1% to 3% of high-priced Brucella immune serum to the urine. After mixing, place it in a 37C incubator for 2 hours, centrifuge it at high speed, take 0.5ml of the precipitate and inoculate it on a selective culture medium for culture, or inject it into guinea pigs, and use biological methods to isolate Brucella. A1.3 Culture of other pathogenic materials
Isolate Brucella from milk, cerebrospinal fluid, joint fluid and synovial fluid, and sterilely inoculate the liquid specimen onto an agar slant or culture plate. Spread it on the surface of the culture medium and observe the results with reference to the blood culture method. If there is still no growth of suspicious bacteria after 15 days, it is determined to be negative. A1.4 Biological separation of Brucella
In order to improve the detection rate of Brucella and separate Brucella from contaminated materials, the test material (solid specimens are ground into a slurry state with sterilized physiological saline) is injected subcutaneously or intraperitoneally into guinea pigs or mice. Guinea pigs are inoculated with 1ml and mice are inoculated with 0.5mL. After inoculation of guinea pigs, the serum-allergic reaction can be observed and bacteria can be isolated and cultured. Organs of mice are dissected and cultured 20 days after infection, and organs of guinea pigs are dissected and cultured 30 days after inoculation.
A2 Specific serological examination
A2.1 Plate agglutination test (PAT)
A2.1.1 Equipment and reagents
Hudson's concave glass plate or a clean, grease-free glass plate, plate agglutination antigen, test serum, known negative and positive serum, 0.1mL pipette or micropipette, toothpick or thin wire. A2.1.2 Operation method
A2.1.2.1 Prepare a clean square glass plate and divide it into 25 squares (or more), 5 horizontally and 5 vertically. Write the serum number in each square of the first column.
A2.1.2.2 Use a 0.1mL pipette to add the test serum to each square of any row (horizontally): 0.08ml in the first square, 0.04ml in the second square, 0.02mL in the third square, and 0.01mL in the fourth square. A2.1.2.3 Add 0.03mL of plate agglutination antigen to each serum square, mix with a toothpick or a thin wire, starting from the square with the smallest amount of serum. Use one toothpick to mix each serum and burn it after use. If a thin wire is used for mixing, wipe each serum with an alcohol cotton ball after mixing, and then use it as another serum.
A2.1.2.4 After mixing, place the glass plate on the flame of an alcohol lamp or an agglutination reaction box, and heat it evenly to about 30°C. Record the reaction results within 5 minutes.
A2.1.2.5 Use one negative and one positive serum as a control for each test. A2.1.2.6 Record the reaction intensity with a plus sign according to the following standards: +10+10+10: Large agglutination pieces or small particles appear, and the liquid is completely transparent, 100% agglutination. +10+10: There are obvious agglutination pieces, and the liquid is almost completely transparent, 75% agglutination. : There are visible agglutination pieces, and the liquid is not very transparent, 50% agglutination. ++
: The liquid is turbid, with only a small amount of particles, 25% agglutination. : The liquid is uniformly turbid.
A2.1.2.7 The relationship between plate agglutination reaction and test tube agglutination reaction: 0.The presence of agglutination in 0.08mL serum is equivalent to a serum dilution of 1:25 in the test tube method, 0.04mL is equivalent to 1:50, 0.02mL is equivalent to 1:100, and 0.01ml is equivalent to 1:200. A2.1.3 Judgment
Human serum 0.02mL showing agglutination of ++ or above is judged as positive, and human serum 0.04mL showing agglutination of ++ or above is judged as suspicious.
A2.2 Red tiger plate agglutination test (RBPT) A2.2.1 Equipment and reagents
Clean defatted glass slides or glass slides with concave holes, 0.1mL pipette or micropipette, toothpick or thin wire, red tiger plate agglutination antigen, and serum to be tested.
A2.2.2 Operation method
Add 0.03mL of the serum to be tested on the slide, then add 0.03mL of bengalen red plate antigen, shake well or mix with a toothpick, and determine the result within 5 minutes.
A2.2.3 Determination
Determination of agglutination degree (1 to 10 10 10) is the same as plate agglutination reaction; it can also be divided into only two categories: (10) positive and (1) negative. A2.3 Test tube agglutination test (SAT)
A2.3.1 Equipment and reagents
Test tube agglutination antigen, test serum, 0.5% carbolic saline, pipette, agglutination test tube, incubator and test tube rack, etc. A2.3.2 Operation method
A2.3.2.1 Dilution of the serum to be tested: In general, use 5 small test tubes (caliber 8-10mm) for each serum. Add 2.3mL of carbolic saline to the first tube, do not add to the second test tube, add 0.5mL to the third, fourth and fifth tubes respectively, use a 1mL pipette to draw 0.2mL of the serum to be tested, add it to the first tube, and mix it. After mixing, use the pipette to draw 0.5mL of serum from the first tube and add it to the second and third tubes respectively. Use the pipette to mix the third tube, and draw 0.5mL and add it to the fourth tube, and mix it. Draw 0.5mL from the fourth tube and discard it. After dilution in this way, the serum dilutions from the second tube to the fifth tube are 1:12.5, 1:25, 1:50 and 1:100 respectively. A2.3.2.2 Add antigen: First dilute the antigen stock solution appropriately with 0.5% carbolic saline (usually 1:10 dilution). Add the diluted antigen to each diluted serum tube (do not add the first tube, as the serum control), add 0.5mL to each tube and mix well. After adding the antigen, the total volume of each tube is 1mL, and the serum dilutions from the second to the fifth tubes are 1:25, 1:50, 1:100 and 1:200 respectively. Aspirate 0.5mL from the first tube again, leaving 1mL.
A2.3.2.3 Control: For negative serum control, add antigen after serum dilution (similar to the tested serum control). For positive serum control, dilute the serum to the original titer and add antigen. For antigen control, add carbonate water to the appropriately diluted antigen. A2.3.3 Judgment
A2.3.3.1 Judgment Preparation of turbidimetric tubes: Turbidimetric tubes must be prepared for each test as the basis for judgment. The preparation method is: take 5-10mL of the original dilution of anti-318
GB15988-1995
used in this test, add an equal amount of 0.5% carbonate saline to make a multiple dilution, and prepare a turbidimetric tube according to Table A1. Table A1 Turbidimetric tube preparation
Antigen dilution, mL
Carbonate saline, mL
Clearness
A2.3.3.2 After all test tubes, control tubes and turbidimetric tubes are fully shaken, place them in a 37℃ incubator for 20-22h, take them out and place them at room temperature for 2h, and then use the turbidimetric tube as the standard to determine the results.
A2.3.3.3 Record the results: Record the results according to the clarity of the upper liquid in each tube. In particular, the 50% clarity (++) has a greater impact on the determination results, and must be compared with the turbidimetric tube for determination. +++ Ten: Complete agglutination, the upper liquid is 100% clear. Ten + Ten: Almost complete agglutination, 75% of the upper liquid is clear. + Ten: Significant agglutination, 50% of the liquid is clear. Ten: Slight agglutination, 25% of the liquid is clear. One: No agglutination, the liquid is not clear. The titer of each serum is determined by the highest serum dilution that shows agglutination of ten or more. A2.4 Anti-human immunoglobulin test (COOMBS) A2.4.1 Equipment and reagents
In addition to the general equipment and reagents required for the test tube agglutination test, anti-human immunoglobulin serum and a common centrifuge are also required. A2.4.2 Operation method
A2.4.2.1 Test tube agglutination test stage: Perform the test tube agglutination test according to A2.3. A2.4.2.2 Anti-immunoglobulin reaction stage: Select the suspected reaction tubes and all negative reaction tubes of the test tube agglutination test, record the tube number, centrifuge at 4000r/min for 15min, wash three times with physiological saline, then add 0.5mL of physiological saline and anti-human immunoglobulin serum of a certain dilution (generally 1:20 times dilution) to each tube, mix well, place the reaction tube in a 37C incubator for 20-22h, take it out and place it at room temperature for 2h before judging the result.
A2.4.2.3 Judgment: The standard and degree of judgment are the same as those of the test tube agglutination test. A2.5 Complement fixation test (CFT)
A2.5.1 Equipment and reagents
37C water bath, ordinary centrifuge, ordinary refrigerator, pipettes of various capacities, flasks, agglutination tubes and test tube racks, normal saline, complement (fresh guinea pig serum or freeze-dried complement), 2% sheep red blood cell suspension, hemolysin, complement fixing antigen, negative and positive serum, and test serum. A2.5.2 Operation Room Method
A2.5.2.1 Complement Titer: When performing CFT, complement must be titrated on the same day. The complement should be diluted to 1:20 with normal saline. Usually, different amounts of 1:20 diluted complement (0.02~0.2mL) are added to ten agglutination tubes in turn. Then, 2 units of antigen solution (0.2ml) are added to each tube. Then, each tube is filled to 0.6mL with normal saline. After mixing, place in a 37℃ water bath for 30 minutes. Then, 0.2mL of hemolysin (2 units) and 0.2mL of 2% red blood cells are added, mixed, and placed in a 37℃ water bath for 30 minutes. The results are determined (see Table A2). Table A2
1: 20 complement volume
2 units of antigen
Normal saline volume
2 units of hemolysin volume
2% SRI3C volume
Result example
Complement titer procedure and results
37℃ water bath for 30min
37℃ water bath for 30min
++++++
++++ ++++
++++ +++
Hemolysin
GB15988-1995
In the above example, the tube that produces complete hemolysis and has the least complement volume is the eighth tube, which is defined as one exact unit, and the previous tube (i.e., the seventh tube) is a complete unit. In the formal test, two complete units of complement are used, and the complement dilution multiple X is calculated according to formula (A1). 20 : 2YX : 0.2
Wu Zhong: Y
1 complete unit of complement.
X=20x0.2_2
.Y=0.08,
=25. That is, complement is diluted 1:25 times.
A2.5.2.2 Titration of hemolysin and antigen: When performing CFT, hemolysin and antigen also need to be titrated, but it is not necessary to do it on the day of the test; moreover, when these two reagents are purchased, the units sold (or provided) have been titrated, and the units need to be diluted according to the instructions. A2.5.2.3 Inactivation of the serum to be tested: The temperature for inactivating complement in human serum is 56℃, and the time is 30min. A2.5.2.4 This test: The inactivated test serum is diluted starting from 1:5, and then diluted in multiples. The amount of diluted serum in each tube is 0.2 mL. Then add 0.2 mL of 2 units of antigen and 0.2 ml of 2 units of complement to each tube. Mix well and place in a 37°C water bath for 30 minutes. After taking out, add 0.4 mL of hemolysin to each tube, and place in a 37°C water bath for 30 minutes. Determine the results (see Table A3). Table A3CFTTest procedure
Test serum
2 units of antigen
2 units of complement
Normal saline
Hemolysin
(Hemolysin + sheep red
blood cells SRBC)
Result example
A2.5.3Assessment
Serum dilution
1:40
1:10
37℃ water bath for 30 min
37℃ water bath for 30 min
++++ ++++ +++
Serum control
Complement control
0.5 unit1.0 unit 2 unit
++++: No hemolysis, SRBC sinks to the bottom of the tube or is suspended. +++: 25% hemolysis. ++: 50% hemolysis. +: 75% hemolysis. 100% hemolysis. The titer of CFT is determined by 50% (++) and above without hemolysis. In order to prevent errors in judgment, a standard blood vessel can be prepared (see Table A4). Table A4 Preparation of standard blood vessel
2%SRBC
Skin allergy test
A3.1 Equipment and reagents
2%SRBC Hemolysin
Brucellarin, 75% alcohol cotton ball, tuberculin syringe, intradermal injection needle, measuring ruler. A3.2 Operation method
Normal saline
Standard (hemolysis degree)
After disinfection with alcohol cotton balls, dry, and inject 0.1mL of brucellin intradermally at the inner 1/3 of the forearm of the subject, and observe twice at 24 and 48 hours after injection.
A3.3 Judgment
—1995
GB15988
Two observations, the result with the strongest reaction shall prevail. If the injection site is congested and the infiltration is 2.0cm×2..0cm or more (or the reaction area is ≥4.0cm2), it is judged to be positive.
Additional instructions:
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 Shang Deqiu and Shu Guangya. This standard is interpreted by the Ministry of Health's technical coordination unit, the Office of Supervision and Administration of Communicable Disease Prevention and Control. 321
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