This standard specifies the diagnostic criteria and treatment principles for epidemic Japanese encephalitis. This standard applies to the diagnosis, reporting and treatment of epidemic Japanese encephalitis by medical, health care and epidemic prevention institutions at all levels across the country. WS 214-2001 Diagnostic criteria and treatment principles for epidemic Japanese encephalitis WS214-2001 standard download decompression password: www.bzxz.net

WS214—2001
Epidemic Japanese encephalitis is an acute infectious disease that causes damage to the central nervous system caused by Japanese encephalitis virus through mosquito vectors. It is a natural zoonotic disease. Most people infected with Japanese encephalitis virus show subclinical infection, and about 1% (or less) of the infected people have typical encephalitis symptoms. my country is an endemic area for epidemic Japanese encephalitis. Except for a few areas such as Tibet, Qinghai, and Xinjiang, there are reports of cases throughout the country. Henan, Hunan, Hubei, Guizhou, Jiangxi, Guangdong, and Guangxi are all relatively serious endemic areas. At present, vaccines are available for prevention, but there is no specific and effective treatment. The "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" lists epidemic Japanese encephalitis as a Class B infectious disease. In order to better guide the prevention and control work, this standard is specially formulated. Appendix A of this standard is the appendix to the standard. Appendix B of this standard is the appendix to the suggestion.
This standard is proposed by the Department of Disease Control of the Ministry of Health. The drafting units of this standard: Institute of Virology, Chinese Academy of Preventive Medicine, and Beijing Ditan Hospital. The main drafters of this standard are Zhang Libi and Xu Daozhen. The Ministry of Health has entrusted the Office of Communicable Disease Supervision and Management to interpret this standard. 359
1 Scope
Health Industry Standard of the People's Republic of China
Diagnostic criteria and principles ofmanagement of japanese B encephalitis This standard specifies the diagnostic criteria and principles of management of japanese B encephalitis (hereinafter referred to as JE). WS 214 2001
This standard is applicable to the diagnosis, reporting and management of JE by medical, health care and epidemic prevention institutions at all levels across the country. 2 Diagnostic principles
Clinical diagnosis is made through comprehensive analysis based on epidemiological history, symptoms and signs, laboratory tests, and the diagnosis must be confirmed by serological or etiological tests.
3 Diagnostic criteria
3.1 Epidemiology
Living in areas where JE is prevalent, developing the disease during the mosquito biting season, or traveling to JE prevalent areas during the mosquito biting season within 25 days before the onset of the disease.
3.2 Symptoms and signs
3.2.1 Acute onset, fever, headache, projectile vomiting, lethargy, and may be accompanied by meningeal irritation symptoms. 3.2.2 Acute onset, with varying degrees of consciousness disturbance after 2-3 days of fever, such as coma, convulsions, convulsions, limb spastic paralysis and other central nervous system symptoms, or developing into central respiratory and circulatory failure. 3.2.3 Cerebrospinal fluid: increased pressure, non-suppurative inflammatory changes (clear appearance, slightly increased protein, normal sugar and chloride, increased cellularity, mostly (50-500)×10°/L, mainly multinuclear cells in the early stage, mainly mononuclear cells in the later stage). 3.2.4 Those who have not received JE vaccine within one month and have positive anti-JE IgM antibodies in blood or cerebrospinal fluid. 3.2.5 Those whose anti-JE IgG antibodies or neutralizing antibodies in the convalescent serum are more than 4 times higher than those in the acute phase, or those whose anti-JE IgG antibodies are negative in the acute phase but positive in the convalescent phase.
3.2.6 Positive JE virus isolated from cerebrospinal fluid, brain tissue, or serum (see Appendix A for details). 3.3 Case classification
3.3.1 Suspected cases 3.1 plus 3.2.1 or 3.2.2. 3.3.2 Clinically diagnosed cases Suspected cases plus 3.2.3. 3.3.3 Confirmed cases Clinically diagnosed cases plus 3.2.4 or 3.2.5 or 3.2.6. 4 Treatment principles
4.1 Treatment
Currently there is no specific antiviral drug, and the main treatment is symptomatic, supportive and comprehensive treatment. 4.1.1 For patients with Japanese encephalitis, mosquitoes should be prevented and killed indoors, and patients should be carefully cared for and monitored, and changes in their condition should be closely observed. Approved by the Ministry of Health of the People's Republic of China on November 23, 2001360
Implemented on May 1, 2002
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4.1.2 Symptomatic treatment, use physical and drug methods to control body temperature at around 38°C, anticonvulsant, anti-twitch, anti-cerebral edema, keep the airway open, perform tracheotomy as soon as possible in case of respiratory failure, and use artificial respirator when necessary. 4.1.3 For comatose patients, use nasogastric feeding of high-calorie, multi-vitamin nutritious liquid food to maintain water and electrolyte balance. 4.1.4 Prevent secondary infection, early detection and early treatment of infection. 4.1.5 For those with residual neuromuscular symptoms in the recovery period, strengthen active and passive exercise or acupuncture or physical rehabilitation treatment. 4.1.6 Hyperbaric oxygen chamber treatment can be used if necessary if conditions permit. 4.2 Prevention
4.2.1 Anti-mosquito and mosquito control.
Preventive injection of Japanese encephalitis vaccine.
A1 Etiological diagnosis
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Appendix A
(Standard Appendix)
Experimental diagnostic methods
Etiological diagnosis can be made by finding the JE capsule or antigen or specific nucleic acid in the patient's brain tissue, cerebrospinal fluid or blood.
A1.1 Virus isolation
A1.1.1 Specimen collection
Blood in the symptomatic period or cerebrospinal fluid in the early stage of the disease is collected for virus isolation, but the positive rate is not high. If brain tissue biopsy materials or household inspection materials are taken, the success rate of virus isolation is higher, but the former is not easily accepted by patients and can only be used for postmortem diagnosis in the later period. Other tissues such as liver, spleen, kidney, lymph nodes, etc. are rarely collected for virus isolation. A1.1.2 Specimen processing
Blood specimens can be sterilely defibrinated with glass beads or anticoagulated with heparin, and then directly inoculated into sensitive animals or chicken embryos, or cultured for cells. Cerebrospinal fluid can be directly inoculated. Brain tissue can be added with sterile 10% skim milk or saline, ground into a homogenate, centrifuged at 1500r/min for 30min, and the supernatant can be inoculated into animals or cells. To prevent bacterial contamination, 1000U/mL penicillin and 1000ug/mL streptavidin can be added during the grinding and homogenization process. A1.1.3 Commonly used sensitive animals and chicken embryos
Commonly used sensitive animals for isolation of epidemic encephalitis B are three-week-old mice or suckling mice (1-3 days old). Each mouse is inoculated with 0.03mL/three-week-old mouse (0.01mL/suckling mouse) in the brain. Those who develop hair count, back, limb paralysis, etc. during 3d~21d are likely to be positive, and further identification of the isolated virus is conducted. If the mice inoculated with the specimen do not develop the disease within 21 days, the mouse brain tissue should be ground into a 10% suspension, centrifuged at 15001/min for 30 minutes, and the supernatant should be taken for blind inoculation, and then inoculated into new mice or cell cultures. The three generations of blind inoculation with negative results are regarded as negative for isolated virus. Chicken embryos are sensitive to Japanese encephalitis virus. The specimen is injected into the brain or (and) chorioallantoic membrane of 7-9 day old chicken embryos, incubated at 37℃, and if the chicken embryo dies after 2-5 days, the chicken embryo or chorioallantoic membrane can be used for identification of Japanese encephalitis virus (chicken embryo virus isolation is now less used).
A1.1.4 Commonly used sensitive cells
Epidemic encephalitis virus is sensitive to many types of cells cultured in vitro, which can be used to isolate the virus. Commonly used cells include chicken embryo fibroblasts, human embryo kidney cells, human embryo lung cells, pig kidneys, hamster kidneys, and monkey kidney cells. Infected cells can show obvious cytopathic effects. Mosquito-derived cells Ca/36 strain, hamster kidney BHK2 strain or monkey kidney Vero strain are sensitive to Japanese encephalitis virus. A1.2 Virus identification
Use neutralization test for identification. If the infectivity of the virus can be neutralized by anti-Japanese encephalitis immune serum, it can be identified as Japanese encephalitis virus. The method is shown in A2.1.
A2 Serological diagnosis method
A2.1 Neutralization test
A2.1.1 Principle
Neutralizing antibodies bind to viruses and can change the conformation of the virus surface, so that the virus cannot bind to the receptors on the surface of sensitive cells and cannot cause virus infection in sensitive cells to produce cytopathic effects. The neutralization test is based on the determination of the infectivity of the virus and must be carried out in sensitive animals or cells. There is a qualitative and quantitative correlation between the virus and the neutralizing antibodies. Japanese encephalitis virus can only be neutralized by anti-Japanese encephalitis neutralizing antibodies. The amount of antibodies determines the completeness of the virus neutralization. Therefore, the neutralization test can be used to identify viruses and can also be used to measure antibodies. The level of neutralizing antibody titer is positively correlated with the body's antiviral protection. 362
A2.1.2 Fixed virus dosage
A2.1.2.1 Virus
Diluted serum method
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First, determine the 50% cytopathic dose (TCIDs.) of the virus in sensitive cells or animals or the median lethal dose (LD5.) of animals. The virus dosage used for the fixed virus-diluted serum neutralization test is 100TCIDso/0.1mL, and the allowable range is 32~320TCIDso. A2.1.2.2 Inactivation of serum
Add an equal amount of Hank's solution to the serum sample and inactivate it at 56C for 30 minutes. A2.1.2.3 Prepare several tubes of cells that have grown into sheets and have good morphology. Operation steps:
a) Dilute the inactivated serum with Hanks solution in two times, i.e. 14, 18, 1:16, 132, 1:64, 1:128. The amount of each dilution is determined according to the number of test tubes to be inoculated. For example, if 0.1mL is inoculated for each dilution, 0.5mL is required, and 0.4mL is actually required.c
b) Take a virus with a concentration of 100TCIDso/0.1mL and add an equal amount of a series of diluted serum, shake well, and place in a 37C water tank for 1 hour, shaking 2 to 3 times during the process.
c) Take out the above virus and serum suspension, inoculate 4 tubes for each dilution, 0.2 mL per tube, and add maintenance solution to 1 mL. d) Set up another virulence titer control for the same batch of viruses, select 10-410-8 virus, inoculate 4 tubes for each dilution, 0.1 mL per tube in the same batch of cell tubes and add maintenance solution to 1 mL.
e) Take another 4 cell tubes from the same batch, add 1 mL of maintenance solution as normal cell control. f) After all the above cell tubes are clearly labeled, plug them with rubber stoppers and culture at 37℃, observe and record the results every day, and generally observe for 7 days to 14 days.
Calculate the titer of neutralizing antibodies according to formula (1) and (2) based on the cytopathic effect, see formula (A1) and (A2). Table A1 Calculation of 50% serum neutralization endpoint
Serum dilution
1 + 4(10-06)
1:8(10-0.9)
1 + 16(10-1.2)
132(10-1.5)
1 : 64(10-1.B)
1 + 128(10-2.1)
Number of cytopathic tubes/total tubes
Wherein: A——distance ratio,
X——pathological change rate below 50%;
X,——pathological change rate above 50%,
Y,—neutralizing titer of serum;
Cytopathic change distribution
（) tubes
（—) tubes
50%一X
IgYi = IgK, + A·IgK2
K,—dilution of serum with pathological change rate below 50%, K --dilution coefficient.
A2.1.3.1 Preparation of virus, serum and mice 16
Percentage
·(A1)
.(A2)
A2.1.3.1.1 Aseptically obtain brain tissue of dying mice, weigh it, add diluent (10% skim milk or buffer) to make a 2×10-1 suspension (i.e. add 4 mL of liquid per gram of brain tissue), grind it and centrifuge it at 3000r/min for 20min, and take the upper suspension for later use. 363
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A2.1.3.1.2 Aseptically obtain blood, centrifuge it at 1500r/min for 15min after coagulation, separate the serum, and inactivate it at 56C for 30min. A2.1.3.1.3 Several healthy mice aged three weeks (7-9g). A2.1.3.2 Operation steps
A2.1.3.2.1 Aseptically dilute the 2×10- virus suspension 10 times continuously, i.e. 2×10~22, 2×10-3**, the method is basically the same as above. A2.1.3.2.2 Take several sterile test tubes and arrange them in a square array, each row consists of 5-6 tubes. Add one serum to be tested to each row, add another serum to be tested to the second row, the amount of serum added to each tube is 0.1mL, and the last row is added with 0.1mL of diluent as the virus control group. A2.1.3.2.3 Add the same amount of virus suspension of different dilutions to each tube, 0.1mL/tube, shake well and place at 37℃ for 1h. The selection of dilution should be appropriate with the highest dilution requiring all animals alive and the lowest dilution requiring all animals dead. A2.1.3.2.4 Use a sterile syringe to absorb the virus-serum mixture and inject it into the mouse brain, 0.03mL/mouse, 5 mice for each dilution, and keep them in the same mouse tank.
A2.1.3.2.5 Observe the death of animals every day. The observation time depends on the virus and the inoculation route. Intracerebral inoculation of Japanese encephalitis virus is generally observed for 14 days.
A2.1.3.3 Calculation of neutralization index
The Reed and Muench methods are used to calculate the median lethal dose (LDs.) of the control group and the IDs (LDs, calculated as shown in formulas (A3) and (A4)) of the neutralization group with immune serum.
Table A2Calculation of LDso
Dilution
Where: A——distance ratio;
X3—mortality percentage higher than 50%;
X,-—mortality percentage lower than 50%;
Y2——LD50 of the virus;
K,~dilution percentage higher than 50% mortality;
K dilution factor.
Cumulative total
Y2 = lgK, + AlgK,
Dead rat card
Mortality rate
·(A3)
·(A4)
A2.1.3.4 Result judgment: For those with fixed serum diluted with virus, when the neutralization index is greater than 50, it means that the unknown serum has neutralizing antibodies, 10~～49 is suspicious, and less than 10 means no neutralization ability. A2.1.4 Micro-neutralization test
(1) The micro-neutralization test is the same as the fixed virus-dilution antibody method (see A2.1.2), except that it is carried out in cells cultured in 40-well or 96-well plastic plates, with one well replacing one test tube. (2) The amount of virus and serum used is changed to 0.025 mL each. (3) The cells on the plastic plate are covered and placed in an incubator at 37°C with 5% carbon dioxide. The simplified process is as follows: 0.025 ml of virus suspension (containing 100 TCIDsc virus) plus 0.025 ml of serum to be tested is allowed to combine at 35-37°C for 1 hour, during which time it is shaken 2-3 times. After sufficient binding, 0.05 ml of cell suspension (pH 7.2-7.4, cell volume of about 1×101/0.1 mL) is added to each well, covered and placed in an incubator at 37°C with 5% CO2. The cell pathological changes are observed daily and the results are recorded. The observation can be carried out for 7 days or longer. The method for calculating the titer of 364
antibodies is the same as A2.1.2. A2.2 Capture ELISA to detect IgM antibodies to Japanese encephalitis A2.2.1 Principle
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After infection with Japanese encephalitis virus, the earliest immune response of the body is to produce IgM. Since the incubation period of Japanese encephalitis is 4 to 21 days, with an average of 10 to 14 days. Therefore, if neurological symptoms occur, specific IgM antibodies may be detected in the blood or cerebrospinal fluid. Generally, IgM antibodies persist for about a month. If IgM antibodies are detected, especially in cerebrospinal fluid, combined with symptoms, Japanese encephalitis can be diagnosed. The capture ELISA test uses anti-human IgM antibodies coated on polystyrene plastic plates to capture IgM antibodies in the infected specimens, and uses the specific binding of Japanese encephalitis antigens and IgM antibodies, and then uses enzyme-labeled anti-Japanese encephalitis antibodies to detect Japanese encephalitis antigens that have been bound to IgM antibodies. The method is specific and sensitive, and the results are fast, which is suitable for early rapid diagnosis. A2.2.2 Steps
A2.2.2.1 Coat each well of a 40-well or 96-well plastic plate with goat anti-human IgMμ chain antibody, 100uL per well, in a 37°C water bath for 2h, then overnight at 4°C, and spin dry the next day. www.bzxz.net
A2.2.2.2 Block each well with 10% normal bovine serum or 1% bovine serum albumin saline, 150μL at 37°C for 1h, then discard the liquid. A2.2.2.3 Add 1:100 diluted serum to be tested (or 1:1 cerebrospinal fluid) to each well, 100μL at 37°C for 1.5h, discard the serum, and wash three times with saline containing Tween-20, then spin dry. A2.2.2.4 Add 100μL of Japanese encephalitis antigen to two wells in the wells where serum (or cerebrospinal fluid) is added, and add normal control antigen to the other two wells. Incubate at 37℃ for 3h (or overnight at 4℃), wash three times with washing solution, and spin dry. A2.2.2.5 Add 100μL of rabbit anti-Japanese encephalitis serum to each well. Incubate at 37℃ for 1h, wash three times, and spin dry. A2.2.2.6 Add 100μI./well of goat anti-rabbit IgG serum-horseradish peroxidase conjugate. Incubate at 37℃ for 1h, wash three times with washing solution, and spin dry. A2.2.2.7 Add 100uL/well of o-phenylenediamine-hydrogen peroxide substrate to each well. Incubate at 37℃. When the control antigen well begins to show color (usually 2 to 10 minutes), immediately add 50μL of 2MHzSO solution to terminate the reaction. A2.2.2.8 Measure the OD value of each well.
A2.2.2.9 Judge the result according to formula (A5)
M, = N, = N. ≥ 2.1
Wherein: M--MI greater than 2.1 is judged as positive; N,--OD value of the specimen + Japanese encephalitis antigen well;
N.--OD value of the blank well;
N2--OD value of the specimen + normal antigen control well. Appendix B
(Suggested Appendix)
Indirect ELISA to check Japanese encephalitis IgG antibody
B1 Principle
Use Japanese encephalitis antigen coated in plastic wells to adsorb human anti-Japanese encephalitis antibodies, and then use rabbit anti-human IgG enzyme marker and enzyme colorimetric agent to detect the presence of human I IgG is adsorbed by the antigen, thus detecting the presence of anti-Japanese encephalitis IgG antibodies. Different coating antigen components are used to detect the corresponding antibodies. Generally, the whole virus antigen of Japanese encephalitis is used for detection, so it detects not only neutralizing antibodies, but also non-neutralizing antibodies of Japanese encephalitis.
.B1.1 Operation steps
B1.1.1 Quantify the Japanese encephalitis antigen and normal control antigen and coat them in the odd and even wells of a 40-well (or 96-well) plastic plate, respectively. Use 100μL per well and coat at 37℃ for 2h or 4℃ overnight. 365
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B1.1.2 Pour off the antigen (or normal antigen) and wash three times with washing solution. After drying, add 10% bovine serum or 1% bovine serum albumin saline solution 150L to each well, block at 37℃ for 1-2h, dry and add the diluted plate clearing (1:100, 1:200, 1:400, 1:800, 1:1600, 1:6400, 1:12800**, 4-fold serial dilution can also be used to save reagents) to different wells, 100L of plate clearing in each well, add one coated antigen well and one coated normal control antigen well respectively, and incubate at 37℃ Let it act for 2h. B1.1.3 After discarding the serum sample, wash three times with washing solution and spin dry. Then add anti-human IgG-horseradish peroxidase labeled antibody, 100μL per well, react at 37℃ for 1 minute. B1.1.4 Pour out the anti-human IgG labeled with enzyme, wash three times, spin dry, add 100μL per well of phosphophenylene diamine, react at 37℃ for 2~~10min, when the control antigen begins to show color (usually 10min or less), immediately add 50uL2mol/lH,SO to all wells, and end the positive reaction. B1.1.5 Measure the OD value of each well, see formula (B1). Na No
Where: MM2 greater than 2.1 is 2.1
N. OD value of virus antigen well;
N. OD value of blank well,
N. OD value of normal control antigen well.
When the ratio of the highest dilution well is ≥2.1, the reciprocal of the serum dilution is the antibody titer of the serum. For example, 1:400 dilution is ≥2.1, 11800<2.0, then the antibody titer of the serum is 400.366
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