Some standard content:
317009-1997
Rubella is an acute respiratory infectious disease caused by rubella virus. It is distributed worldwide and is classified as a statutory Class C infectious disease in my country. It can occur all year round, with the disease occurring more frequently in winter and spring. The susceptible age is mostly between 1 and 5 years old, so it is more common in preschool children. Since the advent of rubella vaccine, the incidence rate has dropped significantly. Rubella has mild clinical symptoms, but after the first infection of rubella virus in pregnant women in early pregnancy, the virus can enter the fetus through the blood-fetal barrier, often causing congenital fetal malformations, stillbirth, and premature birth. Therefore, early diagnosis and prevention of rubella are extremely important. 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 Chinese Academy of Preventive Medicine, the technical unit entrusted by the Ministry of Health. 333
1 Scope
National Standard of the People's Republic of China
Diagnostic criteria and principles of management for rubella
Diagnostic criteria and principles of management for rubella
This standard specifies the diagnostic criteria and management principles for rubella. GB 17009
This standard applies to the diagnosis, reporting and management of rubella patients by medical and health care institutions, health and epidemic prevention institutions and personnel at all levels. 2 Diagnostic principles
Typical cases can be clinically diagnosed based on clinical manifestations combined with epidemiology, and atypical cases need to be confirmed based on the detection of serum rubella antibodies or the isolation of rubella viruses. 3 Diagnostic criteria
3.1 Rubella
3.1.1 Epidemiological history
History of contact with confirmed rubella patients within 14 to 21 days. 3.1.2 Clinical symptoms
3.1.2.1 Fever.
3.1.2.2 Red maculopapular rash appears on the skin of the whole body within 1 to 2 days of onset. 3.1.2.3 Lymph node enlargement or conjunctivitis behind the ear, occipital area, and neck, or accompanied by joint pain (or arthritis). 3.1.3 Laboratory diagnosis
3.1.3.1 Rubella virus is isolated from throat swab specimens (see Appendix A), or rubella virus nucleic acid is detected. 3.1.3.2 Rubella IgM antibody is detected in serum of patients who have not received attenuated live rubella vaccine within 1 month (see Appendix B). 3.1.3.3 The serum rubella IgG antibody titer of patients in the recovery period is 4 times or more higher than that in the acute period, or the antibody in the acute period is negative but the antibody in the recovery period is positive (see Appendix B).
3.1.4 Case classification
3.1.4.1 Suspected cases: meet the criteria of 3.1.2.2 and 3.1.2.1 or 3.1.2.3. 3.1.4.2 Clinically diagnosed cases: add 3.1.1 to suspected cases. 3.1.4.3 Confirmed cases: add 3.1.3.1 or 3.1.3.2 or 3.1.3.3 to suspected cases. 3.2 Congenital rubella syndrome
3.2.1 Clinical manifestations
3.2.1.1 Neonatal cataract/congenital glaucoma, congenital heart disease, hearing loss, pigmentary retinopathy, cleft lip, microcephaly, X-ray bone abnormalities,
3.2.1.2 Purpura, splenomegaly, jaundice, mental retardation, meningoencephalitis. 3.2.2 Laboratory confirmed cases of mothers with a history of rubella virus infection in early pregnancy State Administration of Technical Supervision Approved on October 6, 1997 53.1
Implementation on October 1, 1998
3.2.3 Laboratory diagnosis
3.2.3.1 Infant serum rubella IgM antibody positive. GB17009-1997
3.2.3.2 Infant rubella IgG antibody level persists and exceeds the level of passively acquired maternal antibody (more than 4 times). 3.2.3.3 Rubella virus is isolated from infant throat swabs, blood, urine, cerebrospinal fluid or organ biopsy specimens or rubella virus RNA is detected. 3.2.4 Case classification
3.2.4.1 Suspected cases: meet any of 3.2.1.1 or may be accompanied by any of 3.2.1.2. 3.2.4.2 Clinically diagnosed cases: any of 3.2.1.1 or any of 3.2.1.2, and 3.2.2. 3.2.4.3 Confirmed cases: clinically diagnosed cases plus 3.2.3.1 or 3.2.3.2 or 3.2.3.3. 4 Treatment principles
4.1 Isolation and treatment of patients
Suspected or diagnosed cases of rubella should be isolated immediately, generally for 5 days after the rash appears. No quarantine is conducted for general contacts. Symptomatic treatment and supportive treatment are given to patients. Children with congenital rubella syndrome are treated by specialists according to their symptoms. For early pregnant women diagnosed with rubella virus infection, it is recommended to terminate pregnancy in time to prevent possible congenital malformations of the fetus. 4.2 Prevention of rubella in pregnant women
For women of childbearing age, blood rubella IgG antibodies should be checked before marriage or before pregnancy. If they are confirmed to be susceptible, they should be vaccinated against rubella. Pregnant women should avoid contact with rubella patients in the early stage of pregnancy (within 3 months). If contact is made, serum rubella IgG antibodies can be tested during the incubation period. If positive, it indicates that there has been rubella virus infection in the past. If negative, serum IgG and IgM antibodies should be tested again about 1 month after contact. If positive, it can be judged as rubella virus infection. 4.3 Immunization and preventionwww.bzxz.net
The effective method to prevent rubella is to vaccinate with live attenuated rubella vaccine, which can be implemented for susceptible populations. 335
A1 Virus isolation
Collect specimens from the patient's respiratory tract to isolate pathogens. A1.1 Specimen collection
GB 17009--1997
Appendix A
(Standard Appendix)
Etiological diagnosis method
Rubella patients should take throat swab specimens 4 to 5 days before the rash and 1 to 2 days after the rash. In addition to throat swab specimens, children with congenital rubella syndrome can also take urine, tears, cerebrospinal fluid, bone marrow and other specimens as soon as possible after birth. Dip a sterile cotton swab in liquid (2% bovine serum Eagle solution) and smear the patient's throat several times, then dip the cotton swab in the above specimen solution (1-1.5mL) and squeeze it repeatedly. Add sufficient antibiotics (1000u/mL of penicillin and streptomycin) and 50μg/mL of nystatin (or 5μg/mL of amphotericin). A1.2 Inoculation
Take 0.1~~0.2mL of specimen solution and inoculate it on a monolayer of primary rabbit kidney cells (PRK) (or other primary cells such as primary human embryonic kidney, rabbit embryonic fibroblasts, human embryonic diploid cells, etc.) grown in a test tube, place at 37℃ for adsorption for 1-2h, then discard the solution, add 1mL of maintenance solution containing double the amount of conventional antibiotics, and culture at 33℃. Change the solution every 4 days and observe continuously for 8-14 days. If no cytopathic changes are visible, freeze at 30℃ for 3 times and blindly passage. Each passage can be inoculated on RK13, Vero, BHK21, LLC-MK cells, and blindly passage for 3 generations. A1.3 Observation of cytopathic changes
Cytopathic changes usually appear in about 10 days. If no lesions appear, 100TCID5n of ECH()1 virus or a certain amount of Sindbis, or VSV, or polio virus can be used to attack 14 days after inoculation, and a normal cell virus control can be set. If lesions appear in the control, there are no lesions in the inoculated specimen tube, indicating that the original inoculated cells are infected with the virus. A1.4 Virus strain identification
Use known rubella virus immune serum to observe whether it can work with the new strain. Traditionally, neutralization test, hemagglutination inhibition test or immunofluorescence method are used for virus identification.
A2 Virus nucleic acid detection
Rubella virus nucleic acid is detected using molecular hybridization technology or PCR technology. Appendix B
(Standard Appendix)
Serological Diagnostic Method
B1 Hemagglutination Inhibition Test
B1.1 Principle
There are rubella virus receptors on goose erythrocytes (or pigeon erythrocytes), which can produce agglutination when encountering rubella virus. If the antibody and virus (hemagglutinin) are pre-incubated and then added to erythrocytes, 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 (sera) of different dilutions is the titer of the hemagglutination inhibition antibody. B1.2 Main Materials
B1.2.1 Hemagglutination plate: a U-shaped plate with 6×12 or 8×12 circular holes (with smooth bottom of the holes). B1.2.2 Quantitative pipette: the quantitative pipette uses 25μL (i.e. 0.025ml.). B1.2.3 Conical bottom small test tube: shaped like a centrifuge tube, it is convenient for the absorption of serum. B1.3 Preparation of Rubella Hemagglutinin
Take the virus to infect BHK21 cells or Vero cells, and change the medium for culture as usual. For the last time, change to 2% bovine serum 199 comprehensive medium, and culture at 33 (536
GB17009-1997
for more than 67 days. When the cells show +++ pathological changes, harvest them, freeze them, and take the supernatant and treat it with Tween-80 ether to obtain the hemagglutinin. If the culture medium of infected cells is prepared with 2% bovine serum treated with white clay to prepare the hemagglutinin, Tween-80 ether treatment is not required. B1.4 Hemagglutination titer
All solutions used are precooled at 4℃, and the microplate method is used. The hemagglutinin is diluted with glucose-gelatin-barbital buffer in a 1:2 ratio, and then 0.25% fresh goose red blood cell (or pigeon red blood cell) suspension is added, mixed, and placed at 4℃ for at least 1h. The results are observed and the highest value is +++ (4 units). The dilution degree is the hemagglutination titer.
B1.5 Hemagglutination inhibition test
Original serum 0.1ml. + 0.7ml. manganese chloride heparin solution, after acting at 4℃ for 20min, add 1 drop of backlogged goose red blood cells (or pigeon red blood cells) to the 1:8 diluted serum, shake 2-3 times within 1h, place at 4℃ overnight, and then centrifuge at 2000r/min for 10min, and take the supernatant for inspection. The supernatant to be inspected is diluted in multiple proportions with pH6.2 glucose-gelatin-barbital buffer, and 0.025ml. hemagglutinin is added to each well (containing 4 units: if it is a pH9 hemagglutinin, it should be adjusted to about pH6.2 with 0.1mol/L hydrochloric acid before use). After the antigen and antibody are allowed to act at 4℃ for 1h, add 1 drop of 0.25% goose red blood cells (0.025mL ), and the result is determined after 2 hours at 4°C, with complete inhibition of blood coagulation as the endpoint. The following controls are preset for each test:
Positive serum control: a certain known titer should appearNegative serum control: a negative result should appearTested serum control: there should be no nonspecific agglutinationAntigen control:
Agglutination phenomenon of ++→++ (4 units), ++ (2 units), ++~+ (1 unit), ± (1/2 unit) should appear
Hemocyte control:
Should be positive
HI antibody positive standard judgment uses 1:16 as the positive standard. Glucose-gelatin-barbital buffer (DGV) preparation: barbital
barbital sodium
anhydrous CaCl2
MgsO,.H ,O
Glucose
Barbital and gelatin can be dissolved in 250ml distilled water respectively, and then other drugs can be added in turn. After dissolution, add double distilled water to 1000ml, and divide into 10 bottles for disinfection. Before use, adjust the pH to 6.2 with 0.1mol/L hydrochloric acid, and use it to dilute the serum to be tested, prepare hemagglutinin and red blood cells.
Preparation of manganese chloride-heparin solution
160,000 units
Add double distilled water to 100ml. Dilute 4 times (1:3) with double distilled water when using. B2 Enzyme-linked immunosorbent assay to detect rubella IgG antibodies (ELISA indirect method) B2.1 Principle
Rubella antigen is coated on a solid phase carrier, and the serum to be tested is added, and then the specific antibody is indirectly detected with enzyme-labeled anti-antibody. B2.2 Materials
B2.2.7 Polystyrene ELISA plate.
B2.2.2 Rubella negative and positive sera.
B2.2.3 Rubella antigen.
GB17009-1997
B2.2.4 Anti-IgG horseradish peroxidase conjugate. B2.2.5 Enzyme substrate o-phenylenediamine-hydrogen peroxide. B2.3 Operation procedure
B2.3.1 Dilute rubella antigen and cell antigen not infected with rubella virus (control antigen) with pH 9.6 carbonate buffer. B2.3.2 Add 0.1 mL/well of rubella antigen and control antigen to the single-row well and double-row well of a 40-well polystyrene plate, respectively. No antigen is added to the last well on the lower right as a blank control, and incubate at 4°C overnight. B2.3.3 Wash the plate three times with 0.05% Tween20 saline (NS-T) and discard the washing solution. B2.3.4 Dilute the test serum into four dilutions of 1:20 to 1:1280 with 2% bovine serum NS-T in the hemagglutination plate. Add 1 well of rubella antigen and control antigen (0.1 mL, the same below) to each dilution, shake gently and place at 37°C for 1 to 2 hours. B2.3.5 Wash three times with NS-T, pour off the washing solution, add 0.1 mL 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°C for 1 to 2 hours. B2.3.6 Wash three times with NS-T, pour off the washing solution, add 0.1 mL of o-phenylenediamine-hydrogen peroxide substrate solution to each well (including blank control well), place at 37°C for 10 to 20 minutes in the dark, and observe at any time. When the control well begins to turn light yellow, stop with 2 mol/L sulfuric acid, and add 50 μL to each well. B2.3.7 Use the blank control well to adjust the zero point, and use the microplate reader (492nm) to measure the OD value of each well. OD values less than 0.05 are counted as 0.05. If the OD value of the rubella antigen well/OD value of the control antigen well is > 2.1 at the same dilution, the P/N is positive. The reciprocal of the highest positive dilution is the antibody titer of the serum. Those less than 1:20 are considered negative. B2.3.8 Each experiment should be controlled by positive and negative serum with known titers. B3 Enzyme-linked immunosorbent assay for detection of rubella IgM antibodies (antibody capture ELISA method) B3.1 Principle
Use anti-human IgMμ chain antibodies coated on a solid phase carrier to capture IgM in the serum to be tested, and then use rubella virus antigens and known enzyme-labeled rubella virus antibodies to detect whether the IgM in the serum is anti-rubella antibodies. Use the substrate to make the enzyme color and detect. B3.2 Materials
B3.2.1 Polystyrene 40-well or 96-well plates. B3.2.2 Anti-human IgMu chain antibody.
B3.2.3 Rubella virus antigen and normal control antigen. B3.2.4 Anti-Rubella virus antibody (monoclonal or polyclonal). B3.2.5 Enzyme-labeled anti-antibody: Anti-Rubella virus antibody and enzyme-labeled anti-antibody can also be replaced by enzyme-labeled anti-Rubella virus antibody. B3.2.6 Enzyme substrate - o-phenylenediamine and hydrogen peroxide. B3.2.7 Known Rubella IgM antibody positive and negative sera. B3.2.8 Coating solution: 0.05 mol/L carbonate buffer (pH 9.6), anhydrous sodium carbonate
sodium bicarbonate
distilled water
293 mg, use no more than 2 weeks
add to 100 ml
B3.2.9 Washing solution (NS-T): 0.85% saline Tween solution sodium chloride
Tween 20
distilled water
B3.2.10 The diluent is NS-T solution containing 5% bovine serum. B3.2.11 o-phenylenediamine-hydrogen peroxide substrate solution First prepare citric acid phosphate buffer (pH 5.0): disodium hydrogen phosphate
citric acid
add to 2000mL
distilled water
Prepare substrate solution before use:
o-phenylenediamine (OPD) 4mg
citric acid phosphate buffer 10mL
30% hydrogen peroxide
B3.3 Operation
GB17009—1997
add to 1000mL, divide into vials and store frozen
B3.3.1 Coat anti-human IgMμ chain antibody, 100μL per well. B3.3.24C overnight, pour off the liquid, and block with 120μL of 10% bovine serum NS-T solution. B3.3.337C After 1h, pour off the blocking solution and wash three times. B3.3.4 Add the test serum (1:100 dilution, i.e. 1μL test serum plus 100μL diluent), 100uL per well. Add each serum to 2 wells (or 4 wells), wash three times after 2h at 37℃.
B3.3.5 Add rubella antigen to 1 well (or 2 wells), 100μL per well; add normal control antigen to another well (or 2 wells), wash three times after 4C overnight.
B3.3.6 Add rubella monoclonal antibody to each well, 100μL, 37℃ for 1.5h, pour off the antibody, wash three times. B3.3.7 Add enzyme-labeled anti-mouse antibody to each well, 100μL, 37℃ for 1.5h, pour off the anti-mouse antibody, wash 4 times, and dry. Steps B3.3.6 and B3.3.7 can also be replaced by adding enzyme-labeled anti-Rubella monoclonal (or polyclonal) antibody. Add 100μL to each well, bathe at 37°C for 2h, wash three times and then dry.
B3.3.8 Add substrate o-phenylenediamine-hydrogen peroxide, 100μL to each well, 37°C for 10-20min. B3.3.9 Add the reaction solution that stops the enzyme, i.e. 2mol/L sulfuric acid, 50μL/well, and determine the result. B3.4 Result determination
B3.4.1 Visual determination method: The serum to be tested shows obvious brown reaction with the Rubella antigen well, and the normal control antigen well shows no (or slight) color. If both can be clearly detected by the naked eye, it is positive. B3.4.2 Use the 492nm light source of the microplate reader to measure the OD value. The serum is positive when P/N ≥ 2.1 (P is the OD value of the test serum and rubella antigen, and N is the OD value of the test serum and normal control antigen). When the test serum is compared with a known rubella-negative serum, P is the OD value after the test serum and rubella antigen react, and N is the OD value after the negative serum of the specimen reacts with rubella antigen.
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