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National Standard of the People's Republic of China
Diagnostic criteria and principles of management of measles
Diagnostic criteria and principles of management of measlesGB15983-1995
This standard is formulated in accordance with the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases and the Measures for the Implementation of the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases. 1 Subject content and scope of application
This standard specifies the diagnostic criteria and management principles of measles. This standard is applicable to the diagnosis, reporting and management of measles patients by medical, health and health care institutions and personnel at all levels and of all types. 2 Diagnostic principles
Measles is a respiratory infectious disease caused by measles virus, which is highly contagious. After the widespread use of measles live attenuated vaccine, there are not only patients with typical symptoms of measles, but also patients with atypical symptoms. The former can be diagnosed based on clinical manifestations combined with epidemiology, while the latter needs to be diagnosed based on the detection of serum measles antibodies or the positive isolation of measles virus. 3 Diagnostic criteria
3.1 Clinical symptoms
3.1.1 Red maculopapular rash appears on the skin of the whole body. 3.1.2 Fever (38℃ or higher).
3.1.3 Cough or upper respiratory tract catarrhal symptoms, or conjunctivitis. 3.1.4 Measles mucosal plaques (Koplik's plaques) are seen on the buccal mucosa of the oral cavity in the early stage of the disease (usually on the 2nd to 3rd day of the disease). 3.1.5 Red maculopapular rash on the skin starts from behind the ear and spreads to the whole body, lasting for more than 3 days and showing a typical course. 3. 2 Epidemiological history
History of contact with patients diagnosed with measles, incubation period of 6 to 18 days. 3.3 Laboratory diagnosis
3.3.1 Measles IgM antibodies are found in serum without attenuated live measles vaccine within one month. 3.3.2 The titer of measles IgG antibodies in the serum of patients in the recovery period is 4 times or more higher than that in the acute period, or the acute period antibody is negative and the recovery period antibody turns positive.
3.3.3 Measles virus was isolated from nasopharyngeal secretions or blood (Appendix A), or measles virus nucleic acid was detected. 3.4 Case classification
3.4.1 Suspected case
Those who meet the conditions of 3.1.1 plus 3.1.2, or 3.1.3 at the same time. 3.4.2 Clinically diagnosed case
Suspected case plus 3.1.4 or 3.1.5 or 3.2. 3.4.3 Confirmed case
Suspected case plus 3.3.1 or 3.3.2 or 3.3.3. Any of the following clinical symptoms plus 3.3.1 or 3.3.2 or 3.3.3 State Administration of Technical Supervision Approved on December 15, 1995 274
Implementation on July 1, 1996
4 Treatment principles
4.1 Isolation and treatment of patients
GB15983—1995
If a suspected or diagnosed case is found, it should be isolated immediately, and the isolation period shall be extended to 5 days after the rash appears. For patients with pneumonia, the isolation period shall be extended to 10 days after the rash appears.
Symptom treatment and prevention of complications shall be given to patients. 4.2 Emergency measures for susceptible persons
4.2.1 Emergency vaccination of attenuated live measles vaccine can be implemented for susceptible people around the patient who have not developed the disease. The coverage of emergency vaccination should be wide and the implementation time should be as early as possible. Vaccination should be carried out within 3 days of contact with the patient. 4.2.2 For those who are young, weak or have contraindications to measles live attenuated vaccination, they can be injected with human immunoglobulin (plasma or placental) containing high-dose measles antibodies for passive immunization (Appendix B). 4.2.3 Susceptible children who have close contact with patients and have not been vaccinated against measles should be quarantined for 21 days. 4.3 Immunization and prevention of measles
Universal vaccination of susceptible children with attenuated measles live vaccine is the primary measure to prevent this disease. Routine immunization (primary immunization) is scheduled for 8 months of age. Based on the monitoring of measles immunity in the population, re-immunization with the vaccine should be carried out when immunity decreases. In order to improve the success rate of measles vaccination, the cold chain storage and transportation of live vaccines should be guaranteed, and sufficient doses should be administered. 275
A1 Isolation of pathogens
GB159831995
Appendix A
Etiological diagnosis method
(Supplement)
Collect specimens from the patient's respiratory tract or blood to isolate pathogens. A1.1 Specimen collection
Specimens should be collected before the rash appears and when Koplik spots appear. Dip a sterile cotton swab in liquid (2% bovine serum Eagle solution) and smear the patient's pharynx several times, then dip the cotton swab in the above specimen solution (1~～1.5mL) and squeeze it repeatedly. Add 10 times the conventional amount of penicillin and streptomycin (each 1000u/mL) and nystatin 50μg/mL to the specimen solution. Viruses can also be isolated from white blood cells in heparin-anticoagulated leukocytes. A1.2 Inoculation
Take 0.1~~0.2mL of the sample solution and inoculate it on a monolayer of primary human embryonic kidney cells (or other primary cells such as human amnion, human embryonic lung, monkey kidney, etc. or human diploid cells or passage cells such as B95a cells) grown in a test tube. After 1h at 37℃, discard the solution and add 1mL of cell maintenance solution containing double the amount of conventional antibiotics and culture at 37℃. Set up a blank control culture without inoculated samples. A1.3 Observation of cytopathic effect
Generally, fusion giant cell lesions appear in about 7 days. When the cytopathic effect accounts for more than 75% of all cells (ten ten ten), freeze and thaw three times, and take the supernatant after slow precipitation for seeding. If no lesions are seen, three generations of blind transmission are required. Each culture is observed for two weeks. If no lesions appear, it is negative.
A1.4 Identification of strains
Use known measles virus immune serum to observe whether it can work with new strains. The traditional method is neutralization test. A2 Virus nucleic acid detection
The virus nucleic acid is determined by molecular hybridization technology or polymerase chain reaction (PCR) technology. Appendix B
Reference dose of human immunoglobulin preparation for passive immunization (supplement)
B1 Types
Immunoglobulin is referred to as "gamma globulin" and is an artificial passive immunization preparation. The "gamma globulin" currently available includes human plasma gamma globulin (HGG) and placental gamma globulin (PGG). The former is extracted from the plasma of healthy people, and the latter is extracted from the placental blood of healthy pregnant women. The gamma globulin containing high-valent measles antibodies can be used for measles passive immunization. B2 Vaccination targets
Susceptible populations among those who are young, weak or have contraindications to measles live attenuated vaccination among those who have close contact with measles patients. B3 Usage and dosage
For passive immunization of measles contacts, it is emphasized that injection should be given immediately after contact. The earlier the injection, the better the preventive effect. Using sufficient gamma globulin within 5 days after contact can achieve the goal of preventing the disease; injection after 5 days can only alleviate the symptoms. Commonly used is 10% human blood immunoglobulin, the dose is 0.2mL/kg body weight, or human placental blood immunoglobulin, the dose is 0.5mL/kg body weight, and is injected intramuscularly. Its immune effect can generally only last for 2 to 4 weeks. Appendix C
Serological diagnostic methods
(reference)
C1 Hemagglutination inhibition test
C1.1 Principle
There are measles virus receptors on monkey red blood cells, which can produce agglutination when encountering measles virus. If the antibody and virus (hemagglutinin) are pre-incubated and then added to red blood cells, no agglutination will occur, which is called hemagglutination inhibition. The highest serum dilution that can completely inhibit hemagglutination after the quantitative hemagglutinin reacts with antibodies (surface serum) of different dilutions is the antibody titer. C1.2 Main materials
C1.2. 1 Blood coagulation plate
6×12 or 8X12 round holes, U-shaped plate with smooth bottom. C1.2.2 Dilution stick or quantitative pipette
The dilution stick is made of high-quality stainless steel, with a tip with fractional petals. The volume between petals should be accurately 0.025mL. The dilution stick should be standardized before use. The quantitative pipette should be 25μl. www.bzxz.net
C1.2.3 Quantitative dropper or quantitative pipette
The dropper is made of glass or a ground glass tube with a syringe needle installed to make a quantitative dropper. The dropper is vertically controlled to drip 0.025ml per drop.
C1.2.4 Small test tube with pointed bottom
Shaped like a centrifuge tube, it is convenient for the absorption of serum. C1.3 Preparation of measles hemagglutinin
C1.3.1 Preparation of crude hemagglutinin
Select measles strains with good hemagglutination properties and inoculate them into primary cells (human amnion, monkey kidney, etc.) with vigorous growth and good morphology, human diploid cells or passage cells (KB.MERN.FL.BHK, etc.). The inoculated virus dose is determined according to the virus titer. After the primary human amniotic cells are inoculated with the virus, they are cultured at 37°C. After the hemagglutinin appears in about 2 to 3 weeks, the supernatant is harvested every 3 to 5 days and replaced with fresh maintenance medium for culture. After repeated cultivation for several times, the supernatant is harvested. The cells and the culture medium are frozen below 30°C for the last time, and the cell residues are precipitated. The supernatant harvested several times is the hemagglutinin. After the primary monkey kidney cells, diploid cells and passaged cells are inoculated with measles virus, the lesions develop rapidly. When the lesions reach "ten ten ten +" and 50% of the lesion cells fall off, the cells and the culture medium are placed below 30°C and repeatedly frozen and thawed three times. After low-speed precipitation, the supernatant is the hemagglutinin. The titer of hemagglutinin should be above 1:32 and stored at 30°C for later use. C1.3.2 Treatment of hemagglutinin
Put a certain amount of crude hemagglutinin into a conical flask or test tube, add 5% Tween-80 (Tween-80) to make the final concentration 0.125%, shake at room temperature for 5 minutes, then add ether (analytical grade) equal to the crude hemagglutinin, shake vigorously for 12 minutes, then transfer to a sedimentation tube and centrifuge at 2000r/min for 10 minutes, carefully aspirate the lower water layer, and drain the ether until there is no ether smell. This is the hemagglutinin used in this experiment. Add 100u of penicillin and 100μg of streptomycin and store at 4℃. C1.4 Preparation of monkey erythrocyte suspension
Select monkeys with high agglutination titer with measles hemagglutinin, collect blood from the vein and place it in Alsever solution (the volume of Alsever solution is 4 times that of monkey erythrocytes), store at 4C, and it can generally be used for two weeks. Wash three times with saline before use, centrifuge for 10 minutes at 2000r/min for the last time, discard the supernatant, and prepare the packed monkey red blood cells with saline to a concentration of 1% for the test. C1.5 Serum treatment
GB15983-1995
After serum separation, take 0.1mL, add an equal amount of saline and inactivate in a 56℃ water bath for 30min, add 0.1mL of packed monkey red blood cells centrifuged at 2000r/min for 10min to each tube, shake well, keep at 4℃ overnight, and take the supernatant for determination the next day (the serum dilution is 1:2 at this time).
C1. 6 Hemagglutinin titration
C1.6.1 Titer titration
Add 0.025mL of physiological saline to each of the 1st to 10th wells of the hemagglutinin plate, add 0.025mL of hemagglutinin to the 1st well, mix well, and transfer 1 drop (0.025mL) from the 1st well to the 2nd well. Dilute in this way to the 9th well (1:2 to 512). No hemagglutinin is added to the 10th well as a red blood cell control. Add 1 drop (0.025mL) of 1% monkey red blood cells to each well, shake well, and let stand at 37℃ for 1h to read the results. The highest dilution of hemagglutinin when the hemagglutination reaches 10+ is its titer, that is, 1 hemagglutination unit. Two units of hemagglutinin are used in the formal test. C1.6.2 Unit titration
According to the above titer, dilute the hemagglutinin into 2u (e.g., if the hemagglutinin titer is 1:128, dilute it into 1:64), 1u, 1/2u, 1/4u, dilute each well with physiological saline in multiple ratios, add 1 drop of 1% monkey red blood cells, shake well, place at 37℃ for 1h to determine the result, and the agglutination of "+++ ...
Add 1 drop of 2U hemagglutinin to each of the 1st to 8th wells, and 1 drop of physiological saline to the 9th well. Shake and place at 37℃ for 30min. Then add 1 drop of 1% monkey red blood cells to each well, shake and place at 37℃ for 1h to determine the result. When determining the result, the hemagglutination plate should be tilted first and the red blood cells in the serum control well slide freely in a teardrop shape before reading the result. The red blood cells that slide down the same as the control are completely inhibited. The highest dilution of serum that completely inhibits is the titer of the antibody. The serum control should not agglutinate, otherwise the judgment should be cautious or reabsorbed.
C2 Measles passive hemagglutination test
C2.1 Principle
A serological method that uses red blood cells as carriers, hangs antigens, and uses known antigens to detect unknown antibodies based on the principle of antigen-antibody reaction.
C2.2 Reagents (stored at 4°C, used within the validity period) C2.2.1 Measles-sensitized blood cells: freeze-dried product, add 5mL 1% rabbit serum saline and gently shake to make a uniform suspension before use. C2.2.2 Control blood cells: freeze-dried product, add 2.5mL 1% rabbit serum saline. C2.2.3 Measles-positive reference serum: freeze-dried product, add 0.2mL 1% rabbit serum saline and dissolve to obtain the original serum. It has been inactivated at 56°C. Before the test, it is adsorbed by aldehyde-treated blood cells in the same way as the serum to be tested. C2.2.4 10% aldehyde-treated blood cells (5mL/tube): After washing 3 times with saline, it is reduced to 5mL with saline. C2.2.5 Rabbit serum: freeze-dried product, dissolve with 1mL saline to obtain the original serum. C2.3 Test procedure
C2.3.1 Serum to be tested is treated with 0.05 mL of serum inactivated at 56℃ for 30 minutes, such as an equal amount of 10% aldehyde-treated blood cells, and placed at 37℃ for 1 hour or 4℃ overnight. The supernatant is a 1:2 diluted serum.
C2.3.2 Titer determination: Serum is diluted on a V-type micro-hemagglutination plate in multiple ratios, with dilutions ranging from 1:2 to 1:1024. 0.025 mL of diluted serum is added to each well, and 0.025 mL of 1% sensitized blood cells is added. After sufficient shaking, the plate is placed at 37℃ for 1 hour. The result is determined, and the reciprocal of the highest dilution that produces "++" agglutination is the titer of the serum.
C2.3.3 Set up the following controls:
Sensitized blood cell control: 1% rabbit serum saline + sensitized blood cells, the result is no agglutination. 278
GB15983—1995
Positive serum control: The method is the same as the test serum. The titer of the test should be within the known concentration ± 1 titer. Test serum control: 1:2 diluted test serum + control blood cells. The result is no agglutination. C3 Enzyme-linked immunosorbent assay for detection of measles IgG antibodies (ELISA indirect method) C3.1 Principle
Measles antigen is coated on a solid phase carrier, and the test serum is added. The specific antibody is then indirectly detected with enzyme-labeled anti-antibody. C3.2 Materials
C3.2.1 Polystyrene ELISA plate.
C3.2.2 Measles negative and positive sera.
C3.2.3 Measles antigen.
C3.2.4 Anti-human IgG horseradish peroxidase conjugate. C3.2.5 Enzyme substrate: o-phenylenediamine and hydrogen peroxide (H,O,). C3.3 Operation procedure
C3.3.1 Dilute measles antigen and cell antigen not infected with measles virus (control antigen) with pH 9.6 carbonate buffer. C3.3.2 Add 0.1mL/well of measles antigen and control antigen to the single-row wells and double-row wells of a 40-well polystyrene plate. No antigen is added to the last well on the lower right as a blank control and incubate at 4°C overnight. C3.3.3 Wash the plate three times with 0.05% Tween-20 saline (NS-T) and discard the washing solution. C3.3.4 Dilute the serum to be tested with 2% bovine serum NS-T in a hemagglutination plate to four dilutions of 1:200, 1:800, 1:3200, and 1:12800. Add 1 well of measles antigen and control antigen (0.1mL, the same below) to each dilution, shake gently and place at 37°C for 1 to 2 hours. C3.3.5 Wash three times with NS-T, discard the washing solution, add 0.1mL of anti-human IgG horseradish peroxidase conjugate diluted with 2% bovine serum NS-T to each well (no addition for blank control), and place at 37℃ for 1-2h. C3.3.6 Wash three times with NS-T, discard the washing solution, add o-phenylenediamine and hydrogen peroxide (H,O2) substrate solution, 0.1mL to each well (including blank control well), place at 37℃ for 10-20min away from light, observe at any time, and stop with 2mol/L sulfuric acid when the control well begins to turn light yellow, adding 50μL to each well.
C3.3.7 Use the blank control well to adjust the zero point, and measure the optical density (OD) value of each well with an enzyme marker (492nm). OD values less than 0.05 are counted as 0.05. Any OD value of the measles antigen well/OD value of the control antigen well>2.1 (i.e. P/N) at the same dilution is positive. The reciprocal of the highest positive dilution is the antibody titer of the serum. Those <1:200 are considered negative. C3.3.8 Each experiment should be controlled with positive and negative sera of known titer. C4 Enzyme-linked immunosorbent assay for measles IgM antibody (antibody capture ELISA) C4.1 Principle
Use anti-human IgMμ chain antibodies coated on a solid phase carrier to capture IgM in the serum to be tested.Then use measles virus antibody and known enzyme-labeled measles virus antibody to detect whether the IgM in the serum is anti-measles antibody. Use the substrate to make the enzyme color and detect. C4.2 Materials
C4.2.1 Polystyrene 40-well or 96-well plate. C4.2.2 Anti-human IgMμ chain antibody.
C4.2.3 Measles virus antigen and normal control antigen. C4.2.4 Anti-measles virus antibody (monoclonal or polyclonal). C4.2.5 Enzyme-labeled anti-measles virus antibody
Anti-measles virus antibody and enzyme-labeled anti-measles virus antibody can also be replaced by enzyme-labeled anti-measles virus antibody. C4.2.6 Enzyme substrate: o-phenylenediamine and hydrogen peroxide. C4.2.7 Known measles IgM antibody positive and negative serum. C4.2.8 Coating solution: 0.05mol/L carbonate buffer (pH9.6). 279
Anhydrous sodium carbonate
Sodium bicarbonate
Distilled water
GB15983-1995
293mg, use no more than 2 weeks
Add to 100mL
C4.2.9 Washing solution (NS-T): 0.85% saline Tween solution. Sodium chloride
Tween 20
Distilled water
Add to 2000mL
0 Dilution is 5% bovine serum NS-T solution.
O-phenylenediamine and hydrogen peroxide substrate solution.
First prepare citric acid phosphate buffer (pH 5.0): disodium hydrogen phosphate
citric acid
distilled water
add to 1000mL, divide into small vials and store in ice
Prepare substrate solution before use:
o-phenylenediamine (OPD) 4mg
citric acid phosphate buffer 10mL
30%H02
C4.3 Operation
C4.3.1 Coat anti-human IgMμ chain antibody, 100μuL per well. C4.3.2Incubate at 37℃ overnight, discard the liquid, and block with 120μL of 10% bovine serum NS-T solution. C4.3.3After 1h at 37℃, discard the blocking solution and wash three times. C4.3.4 Add 100μL of the test serum (1:100 dilution, i.e. 1μL of the test serum plus 100μL of the diluent) to each well. Add each serum to 2 wells (or 4 wells), incubate at 37℃ for 2h, and wash three times.
C4.3.5 Add measles antigen to 1 well (or 2 wells), 100μL to each well; add normal control antigen to another well (or 2 wells), incubate at 4℃ overnight, and wash three times.
C4.3.6 Add 100μL of measles monoclonal antibody to each well, incubate at 37℃ for 1.5h, and then discard the antibody and wash three times. C4.3.7 Add 100μL of enzyme-labeled anti-mouse antibody to each well, incubate at 37℃ for 1.5h, and then discard the anti-mouse antibody, wash four times, and dry. Steps C4.3.6 and C4.3.7 can also be replaced by adding enzyme-labeled anti-measles monoclonal (or polyclonal) antibodies in one step. Add 100μL to each well, bathe at 37°C for 2h, wash three times and then dry.
C4.3.8 Add substrate, generally o-phenylenediamine-hydrogen peroxide (H2O2), 100μL to each well, 37°C for 10-20min. C4.3.9 Add the reaction solution that stops the enzyme, i.e. 2mol/L sulfuric acid, 50μL/well, and determine the result. C4.4 Result determination
C4.4.1 Visual determination method: The serum to be tested and the measles antigen well show obvious brown reaction, and the normal control antigen well does not (or slightly) show color. If both can be clearly detected by the naked eye, it is positive. C4.4.2 Use the 492nm light source of the microplate reader to measure the optical density (OD) value. The serum is positive when P/N ≥ 2.1 (P is the OD value of the test serum and measles antigen, and N is the OD value of the test serum and normal control antigen). When the test serum is compared with a known measles-negative serum, P is the OD value of the test serum after the measles antigen, and N is the OD value of the specimen negative serum after the measles antigen. 280
Additional Notes:
GB15983-1995
This standard was proposed by the Ministry of Health of the People's Republic of China. This standard was drafted by the Institute of Infectious Diseases of Zhejiang Medical University and the Institute of Virology of the Chinese Academy of Preventive Medicine. The main drafters of this standard are Liu Kezhou and Zhang Libi. This standard is interpreted by the Ministry of Health and entrusted by the Ministry of Health to the Office of Infectious Disease Supervision and Management.
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