GB 16394-1996 Diagnostic criteria and treatment principles for poliomyelitis
Some standard content:
GB16394-1996
Poliomyelitis is an acute intestinal infectious disease caused by poliovirus. The main clinical manifestation is acute flaccid paralysis. Some cases may have permanent limb paralysis. The World Health Assembly passed the requirement to eradicate polio in the world by 2000. The formulation of diagnostic criteria and treatment principles is not only related to the correct diagnosis of cases, statistics of epidemics, treatment of patients and disease prevention measures, but also related to whether the region has achieved the goal of eradicating polio. Judging from the number of reported cases and the distribution range of wild virus circulation in the environment, my country has entered the late stage of eradicating the disease. For this reason, there should be a unified diagnostic standard and treatment principle that is in line with international standards. This standard is formulated with reference to international standards and combined with domestic conditions. This standard is proposed by the Ministry of Health of the People's Republic of China. The responsible drafting unit of this standard is: Institute of Virology, Chinese Academy of Preventive Medicine. The main drafter of this standard is: Zhang Libi.
This standard is interpreted by the Ministry of Health's technical management unit, the Office of Communicable Disease Supervision and Management of the Ministry of Health. 476
1 Scope
National Standard of the People's Republic of China
Diagnostic criteria and principle of management for poliomyelitis This standard specifies the diagnostic criteria and management principles for poliomyelitis. GB16394—1996
This standard applies to the diagnosis, reporting and management of poliomyelitis by medical, health and epidemic prevention institutions and personnel at all levels across the country. 2 Diagnostic principles
The diagnosis of poliomyelitis must be made based on a comprehensive analysis of medical history, clinical symptoms, physical examination and laboratory tests. 2.1 Medical history
Attention should be paid to epidemiological history and contact history.
2.2 Physical examination
A comprehensive physical examination should be conducted, paying attention to systemic symptoms, limb activity and signs such as muscle strength, muscle tension and tendon reflexes. If necessary, a comprehensive examination of the nervous system and other systems should be conducted.
2.3 Laboratory examination
2.3.1 Check stool for polio virus.
2.3.2 Check cerebrospinal fluid or serum for specific IgM, IgG antibodies, or neutralizing antibodies. 2.3.3 Pathological examination is performed if necessary after the death of paralysis patients. 3 Diagnostic criteria
3.1 Suspected cases
Any acute flaccid paralysis (AFP) of unknown etiology, including cases under 15 years old with a preliminary clinical diagnosis of Guillain-Barré syndrome 3.2 Poliomyelitis clinically consistent cases and confirmed cases of wild polio virus 3.2.1 Medical history
History of contact with confirmed polio patients, after a latent period of 2 to 35 days (generally 7 to 14 days); or no obvious history of contact, with the following clinical symptoms.
3.2.2 Clinical manifestations
3.2.2.1 Early symptoms may include fever, irritability, sweating, nausea, stiff neck and back, and tenderness of intestinal muscles. Asymmetric flaccid paralysis appears after the fever subsides. Neurological examination reveals asymmetric (unilateral or bilateral) flaccid paralysis of the limbs (or/and abdominal muscles), decreased muscle tone of the body or limbs, decreased or eliminated deep tendon reflexes, but no obvious sensory impairment. 3.2.2.2 Flaccid paralysis remains 60 days after paralysis, and no other cause is found (muscle atrophy may occur in the later stage). 3.2.2.3 Suspected patients die and cannot provide evidence to deny the diagnosis of poliomyelitis. 3.2.2.4 Suspected patients are lost to follow-up after 60 days.
3.2.3 Laboratory examination
3.2.3.1 After the onset of the disease, the virus is isolated from the feces, pharynx, cerebrospinal fluid, brain or spinal cord tissue and identified as wild polio virus. Approved by the State Bureau of Technical Supervision on May 23, 1996 and implemented on December 1, 1996
GB16394-1996
3.2.3.2 The patient has not taken live polio vaccine within 6 weeks before the onset of the disease, and anti-polio virus IgM antibodies are detected in the cerebrospinal fluid or blood within 1 month after the onset of the disease.
3.2.3.3 After the onset of the disease, the patient has not taken polio vaccine or been exposed to vaccine virus, but the serum neutralizing antibody or specific IgG antibody titer of the patient in the recovery period is 4 times higher than that in the acute period, or the specific IgG antibody in the cerebrospinal fluid is significantly elevated, and the ratio of blood to cerebrospinal fluid IgG antibody titer is abnormal or inverted (the normal value is 200-400:1 when the blood-brain barrier is intact). Poliomyelitis clinically consistent cases: suspected cases plus 3.2.1 plus 3.2.2.1; or suspected cases plus 3.2.2.2; or suspected cases plus 3.2.2.3; or suspected cases plus 3.2.2.4; or suspected cases plus 3.2.3.2; or suspected cases plus 3.2.3.3. Confirmed cases of wild poliomyelitis virus: add 3.2.3.1 to suspected cases. 3.3 Excluded cases
3.3.1 Suspected cases Cases confirmed by laboratory and clinical examinations to be non-poliomyelitis a) Guillain-Gallery syndrome (diagnosed clinically and/or with cerebrospinal fluid protein and cell detection); b) Other enterovirus infections confirmed by pathogen isolation or serological evidence; transverse myelitis;
traumatic neuritis;
other diseases (the name and basis of the diagnosis should be indicated). e)
3.3.2 Suspected cases Follow-up 60 days after paralysis: no residual paralysis, no wild poliomyelitis virus isolated from stool specimens, or negative serum or cerebrospinal fluid IgM antibodies within two weeks after paralysis.
3.3.3 Suspected cases with residual paralysis 60 days after paralysis, but within two weeks of onset of the case, two stool specimens were collected at intervals of 24~~48h and blindly propagated twice in RD and Hep-2 cells, and no wild polio virus was isolated. 3.3.4 Suspected cases with residual paralysis 60 days after paralysis, but within four weeks of onset of the disease, cerebrospinal fluid or blood specific IgM antibodies were negative, and the neutralizing antibody or specific IgG antibody titer in the recovery serum was not more than 4 times higher than that in the acute phase. Judgment: Poliomyelitis can be ruled out for those who meet 3.3.1 or 3.3.2 or 3.3.3 or 3.3.4. 3.4 Vaccine-related cases
The incidence is extremely low and often seen in children with low immune function. 3.4.1 Vaccine-related cases in vaccine recipients
Fever occurs within 4 to 35 days after taking live vaccine (mostly seen in the first dose of vaccine), and acute flaccid paralysis occurs 6 to 40 days later, without obvious sensory loss, and the clinical diagnosis is consistent with poliomyelitis. After paralysis, no live polio vaccine is taken again, and only polio vaccine strains are isolated from stool specimens. If there is a positive serological test for polio IgM antibody, or a 4-fold increase in neutralizing antibodies or IgG antibodies and the virus type is identical to the isolated vaccine strain, the diagnosis is more sufficient. 3.4.2 Vaccine-related cases in vaccine contacts
Close contact history with live vaccine recipients within 35 days after taking the vaccine, acute flaccid paralysis occurs 6 to 60 days after contact, which is consistent with the clinical diagnosis of poliomyelitis. For patients who have not taken polio vaccine again after paralysis and only polio vaccine virus is isolated in the feces, if there is a positive serological specific IgM antibody or IgG antibody (or neutralizing antibody) that is 4 times higher and consistent with the isolated vaccine virus type, the diagnosis is more sufficient.
4 Treatment principles
4.1 Reporting and investigation of infectious diseases
4.1.1 Immediately report to the local epidemic prevention unit within 12 hours in cities and 24 hours in rural areas after the discovery of suspected cases of poliomyelitis. If confirmed or excluded later, further exclusion or confirmation report will be made. 4.1.2 Individual investigation must be conducted on reported cases within 48 hours. Within 14 days of onset and before taking the vaccine again, collect two feces (24-48 hours apart, each specimen is 5-8g) and send them to the provincial laboratory for virus isolation under refrigerated conditions (below 4°C). If conditions permit, blood or cerebrospinal fluid should be collected at the same time to make a rapid diagnosis.
4.2 Immunization and prevention
GB16394-1996
4.2.1 The trivalent mixed attenuated live poliomyelitis vaccine can effectively prevent the onset of poliomyelitis. The basic immunization is to take a dose of live vaccine at the age of two, three and four months after the birth of the baby, with an interval of one month. A booster immunization is given at the age of four. 4.2.2 According to the requirements of poliomyelitis eradication, the health and epidemic prevention departments can implement supplementary immunization in addition to basic immunization, such as emergency immunization and enhanced immunization activities of different scopes as needed. 4.2.3 Enhanced immunization should generally be carried out in winter and spring. For children of a certain age group (such as under 4 years old), regardless of whether they have taken vaccines in the past, two rounds of immunization should be carried out, with an interval of one month. 4.3 Isolation and treatment of patients
4.3.1 The patient should be isolated from the digestive tract for 40 days, and the excrement should be disinfected before discharge. 4.3.2 Early patients should rest in bed and receive symptomatic treatment. 4.3.3 For patients with limb paralysis, the paralyzed limbs should be properly protected from trauma and compression and placed in a functional position. 4.3.4. For those with respiratory muscle paralysis and breathing difficulties, artificial respirators can be used. If there is a bacterial infection, antibacterial drugs can be given. 4.3.5 For those with flaccid paralysis, attention should be paid to clearing throat secretions to prevent suffocation. Those with swallowing difficulties can be given nasogastric feeding. 4.3.6 For those with advanced paralysis, massage, physical therapy, acupuncture, body therapy, surgery and other rehabilitation treatments can be given. 4.4 Detection of wild viruses in the natural environment
4.4.1 For each suspected polio patient, the stool of five close contacts (under 5 years old) shall be collected within one month of the onset of the disease and sent to the designated laboratory for virus isolation, typing and identification of the strain. 4.4.2 In areas without polio cases, the stool of normal and healthy children in nurseries can be collected for polio virus isolation, typing and identification of the strain.
4.4.3 Collect domestic sewage and, after treatment, isolate, type and identify the strain of polio virus according to international regulations. 479
A1 Collection and transportation of specimens
GB16394—1996
Appendix A
(Standard Appendix)
Isolation and typing of viruses
Feces are the main specimens for isolating poliovirus. Since the feces of polio patients mainly excrete the virus in the early stage of paralysis and within two weeks after paralysis, and the excretion is intermittent, specimens should be collected within 14 days after paralysis. The interval between the two collections is 24~48h, and the amount of each specimen is 5~8g.
Due to the instability of the virus to heat and ultraviolet rays, the collected feces should be placed in a sterile clean container, refrigerated and transported with ice below 4, and should be labeled according to regulations after collection and sent to the designated laboratory for virus isolation within one week. Isolation of A2 virus
A2.1 Place half of the stool specimen (4 g) into a 50 mL trifluoromethane-resistant plastic (with lid) centrifuge tube containing small glass beads, add pH 7.0 phosphate buffer containing 1/10 trifluoromethane (or 1/10 chloroform Hank's solution), tighten the lid and shake vigorously for 10 minutes to make a 20% stool suspension.
A2.23000r/min, centrifuge for 30 minutes, aseptically aspirate the suspension layer without chloroform, inoculate cells with a portion, and store the rest at -30C for later use.
A2.3 Inoculate the specimen suspension into the well-growing and newly formed Hep-2 cells and RD cell tubes. Inoculate at least 2 tubes of each cell type, add 0.1ml of suspension to each tube, and add 0.9mL of Eagle's solution containing 2% bovine serum (pH7.2, containing 100 units/mL of penicillin and 100μg/mL of streptomycin). Reserve two tubes of normal cells without virus and add only 1mL of Eagle's solution containing 2% bovine serum as normal controls. A2.4 Place the test tubes at 36℃～37C and tilt them at 5° for static culture. A2.5 Observe the cell lesions in the test tubes under a microscope every day for at least 10 to 14 days. When "10+10~+10++10+" appears (i.e. 75% to 100% lesions), freeze the test tubes below 20C for virus typing and intratypic identification. A2.6 If no cytopathic effect is observed in the first generation of culture, freeze the cells three times after 10 days of culture, take 0.1 mL of the suspension and inoculate it into the same cell tube for blind subculture (the inoculation and culture methods are the same as those of the first generation), observe the cytopathic effect every day, and freeze and store for later use if positive. If still negative after 14 days, treat it as negative for virus isolation.
A3 Identification and typing of viruses
A3.1 Prepare 4 sets of standard antisera for poliovirus. The combinations of each set are as follows: 1) Combined anti-type 1, 2, and 3 sera, each type of sera contains 20 units of neutralizing antibodies in 0.05 mL; 2) Combined anti-type 1 and 2 sera, each type of sera contains 20 units of neutralizing antibodies in 0.05 mL; 3) Combined anti-type 1 and 3 sera, each type of sera contains 20 units of neutralizing antibodies in 0.05 mL; 4) Combined anti-type 2 and 3 sera, each type of sera contains 20 units of neutralizing antibodies in 0.05 mL. A3. Use a pipette to pipette 0.05 mL of each of the 24 combined sera into the designated wells of a 96-well microplate, as shown in Figure A1, 4 wells in each group, and only 0.05 mL of maintenance solution is added to the other 4 wells. A3.3 Use the maintenance solution to dilute the virus of the isolated strain to 10- and 10°4 respectively. A3.4 Add 0.05mL of 10-3 virus solution to 4 groups of wells with mixed antiserum, add only 2 wells in each group and add 10-* virus suspension to the other 2 wells.
Virus isolate ×—3
Virus isolate Y-3
Titration isolate X
Isolate Y
GB163941996
P1+P2+P3
O=no CPE
Virus control
①=Unused well
Figure A1 Identification of poliovirus isolates using 96-well microplate cell control
A3.5 The virus control wells contain 0.05mL10-3 (or 10-4) virus plus 0.05mL maintenance solution, and the cell control wells contain 0.1mL maintenance solution plus 0.1ml of cell suspension each.
A3.6 At the same time, perform titration of virus isolates in rows E to H, add 4 wells for each titer, starting from 10-8 to 10-3A3.7 Shake thoroughly, mix and place at 37℃ for 30min, add 0.1mL of cell suspension to each well of the 96-well plate, containing 10/0.1mL of cells. A3.8 Cover and shake well again and place in a 37℃ carbon dioxide incubator for culture, observe the cell pathology of each well daily, and observe for at least 7 to 10 days. A3.9 The results are determined as follows:
P1→P2+P3
+=lesions (CPE)
A4 Identification of wild mold strains and vaccine strains
The methods used internationally before the figure are:
1) Monoclonal combined antibody neutralization test;
POLIO virus type 1
POLIO virus type 2
POLI0 virus type 3
Virus identification
POLIO virus type 1 and 2 mixed
POLIO virus type 1 and 3 mixed
POLIO virus type 2 and 3 mixed
POLIO Mixture of 3 virus types
Not POLIO virus or POLIO
Virus mixed with other enterovirus
0-No lesion (no CPE)
2) Vaccine strain nucleic acid probe hybridization test;
3) Polymerase chain reaction amplification test;
GB 16394-1996
4) Polymerase chain reaction amplification followed by endonuclease electrophoresis analysis; 5) Nucleic acid sequence analysis;
6) ELISA intratype difference detection.
According to international regulations, only the results of designated national laboratories or regional reference laboratories and the WHO headquarters reference laboratory are recognized, so the method is omitted
Appendix B
(Standard Appendix)
Specific IgM antibody determinationWww.bzxZ.net
The immune response of IgM antibodies to poliovirus infection can be detected by capture ELISA as early as 10 to 15 days after infection. It usually lasts for one month and then disappears. The detection of IgM antibodies in the blood of suspected poliomyelitis patients, especially in the cerebrospinal fluid, is helpful for the diagnosis of the disease. Because the method is simple and can be diagnosed by typing, the results can generally be obtained within 2 days, so it is an early rapid and specific diagnosis method. Early diagnosis is conducive to buying time to take emergency prevention and control measures. However, the IgM antibodies currently measured cannot distinguish between vaccine strains and wild strains, so the significance of IgM positivity can only be applied when it is clear that there is no vaccination in the near future. However, if specific IgM antibodies are found in the cerebrospinal fluid, if the blood-brain barrier is normal, it can be diagnosed as a vaccine-related case or a wild virus case in combination with the medical history. If the AFP patient fails to collect qualified stool specimens for some reason, if the blood IgM antibody is negative within one month of paralysis, it will help to exclude the diagnosis. B1 Capture ELISA method for detecting IgM antibodies
B1.1 Add 0.1mL of anti-human IgMμu chain antibody to a plastic microplate and incubate at 37℃ overnight. B1.2 Pour out the liquid, without washing, and seal each well with 0.2mL of 10% bovine serum and 0.05% Tween (Tween-20) saline, and place at 37.
B1.3 After 1 hour, pour out the bovine serum blocking solution and add 1:100 diluted patient serum (or 1:2 diluted patient cerebrospinal fluid) to be tested, and place at 37 (for 1.5-2 hours.
B1.4 Pour out the serum (or cerebrospinal fluid) to be tested, wash three times with 0.05% Tween saline, and then add known 1., 2 and 3 type polio antigens to the above wells, add 2 wells for each type, 0.1mL per well, and place at 4℃ overnight. B1.5 Aspirate the antigen, wash 3 times, and add 0.1mL of known corresponding antiserum to each corresponding well (i.e., add type 1 antiserum to the well with type 1 virus antigen, and add type 2 serum to the well with type 2 antigen).
B1.637C After 1~1.5h, pour off the antibody, wash three times, and add 0.1mL of enzyme-labeled anti-antibody to each well. B1.737C After binding for 1~1.5h, pour off the enzyme-labeled antibody, wash three times with washing solution, and add o-phenylenediamine substrate 1, 0.1mL per well. B1.8 Protect from light for 5~15min.
B1.9 When the wells with normal cells as control antigen are about to develop color, add 0.05mL of 2mol/L sulfuric acid to each well to terminate the reaction. B1.10 Determine the OD value under a 490mm light source. B1.11 Judgment of the results
The OD value of the virus antigen well is positive when the OD value of the blank control well is ≥2.1.
OD value of normal antigen wells = OD value of blank control wells"1) The preparation of o-phenylenediamine substrate is 10mL pH5.0 citric acid phosphate buffer plus 4mg o-phenylenediamine and 5ul 30% hydrogen peroxide. 2) Blank control is the control of anti-μ chain antibody coated wells plus o-phenylenediamine substrate plus sulfuric acid. 482
A positive well of type 1 antigen is positive for type 1 IgM; a positive well of type 2 antigen is positive for type 2 IgM, and so on. 183
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