GB 15998-1995 Diagnostic criteria and management principles for pertussis
Some standard content:
GB 15998-
3—1995
Pertussis is a respiratory infectious disease caused by Bordetella pertussis. It is highly contagious and has a high mortality rate in infants and young children. The clinical manifestations are characterized by paroxysmal spasmodic coughs and cock-like inspiratory roars at the end of spasmodic coughs. It is more common in children and the course of the disease can be up to 2 to 3 months. In the process of formulating this standard, reference was made to the diagnostic criteria in the "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" and the "Implementation Measures for the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" formulated by the Ministry of Health in 1989, and the epidemiology, clinical practice and local conditions of pertussis in my country were combined as much as possible to facilitate implementation and application.
Appendix A of this standard is the appendix to the standard;
Appendix B of this standard is the appendix to the suggestion.
This standard was proposed by the Ministry of Health of the People's Republic of China. The drafting units of this standard are: Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Ditan Hospital, Beijing, and Capital Institute of Pediatrics.
The main drafters of this standard are: Zhang Rongzhen, Yang Lixin, and Wang Shushan. This standard is interpreted by the Ministry of Health's technical unit, the Office of Supervision and Administration of Communicable Disease Prevention and Control. 1 Scope
National Standard of the People's Republic of China
Diagnostic criteria and principles of management of pertussis
Diagnostic criteria and principles of management of pertussis
This standard stipulates the diagnostic criteria and management principles of pertussis. GB15998-1995
This standard applies to the diagnosis, reporting and treatment of pertussis patients by medical, health and health care institutions and personnel at all levels and types. 2 Diagnostic principles
Clinical diagnosis should be made based on epidemiological data and clinical manifestations. Confirmation requires positive culture of Bordetella pertussis or detection of specific antibodies against pertussis.
3 Diagnostic criteria
3. 1 Epidemiological history
Contact with pertussis patients within three weeks, or pertussis is prevalent in the area. 3.2 Clinical manifestations
3.2.1 Patients with paroxysmal cough during the epidemic season. 3.2.2 Coughing is accompanied by vomiting, and in severe cases, there is subconjunctival hemorrhage or tongue frenulum ulcer. 3.2.3 Newborns or infants with unexplained paroxysmal cyanosis or asphyxia, usually without typical cough. 3.2.4 Continuous cough for more than two weeks, and other causes can be ruled out. 3.3 Laboratory diagnosis
3.3.1 The total white blood cell count is significantly increased, with lymphocytes accounting for more than 50%. 3.3.2 Bordetella pertussis is isolated from the patient's sputum or throat secretions, see Appendix A. 3.3.3 The convalescent serum agglutinating antibodies are more than four times higher than the acute phase antibodies, see Appendix B. 3.4 Case classification
3.4.1 Suspected cases
Those who meet any of the four items 3.2.1, 3.2.2, 3.2.3, 3.2.4, or are accompanied by item 3.1. 3.4.2 Clinically diagnosed cases
Add 3.3.1 to suspected cases.
3.4.3 Confirmed cases
Add 3.3.2 or 3.3.3 to suspected cases.
4 Treatment principles
4.1 Isolation of patients
Isolate for 40 days from the onset of the disease, or isolate for 30 days after the onset of controlled cough. Observe contacts for 21 days. Approved by the State Administration of Technical Supervision on December 15, 1995 402
Implemented on July 1, 1996
4.2 Treatment of patients
GB15998—1995
Give antibiotic treatment and symptomatic treatment as soon as possible to prevent and treat complications. 4.3 Emergency measures in case of an outbreak
4.3.1 Emergency vaccination of susceptible children who have not been vaccinated with pertussis vaccine with a combination of diphtheria, pertussis and tetanus. 4.3.2 Susceptible children who have a history of close contact with pertussis patients can use drugs for prevention. 4.4 Immunization and prevention of pertussis
Pertussis is an infectious disease that can be prevented by vaccines. Vaccination of susceptible children with a combination of diphtheria, pertussis and tetanus is an important measure to prevent the disease. Domestic and foreign studies have shown that the prevention effect of pertussis vaccines is positive. According to the current immunization program in my country, children start primary immunization in the third month after birth, and the full course of immunization is 3 shots, with a protection rate of more than 90%, and a booster shot is given in the second year. 403
A1 Pathogen isolation
GB15998—1995
Appendix A
(Standard Appendix)
Etiological diagnosis method of pertussis
Collect specimens from the patient's nasopharynx or isolate pathogens by cough dish method. A1.1 Collection of specimens
A1.1.1 Cough plate method
When collecting samples using the cough plate method, when the patient coughs, the Bordet-Gengou culture plate should be opened and placed 10 cm in front of the patient's mouth, and the patient should cough several times towards the surface of the plate to directly collect the droplets coughed out by the patient. Then cover the plate with a flat blood cover and send it for inspection. The exposure time of flat blood should be 15 s.
A1.1.2 Nasopharyngeal swab
Since Baigan cough is common in children, when sampling, the patient's head is tilted against the mother's chest, and the mother fixes the patient's head with both hands. The doctor holds the cotton swab in his right hand and presses the top of the patient's head with his left hand, so that the cotton swab with a slightly downward bend at the top enters from the anterior nostril and slowly penetrates backward along the bottom of the inferior nasal cavity. Due to the arc shape of the nasal cavity, do not use too much force to avoid trauma and bleeding. When the top of the cotton swab reaches the back wall of the nasopharyngeal cavity, leave the cotton swab for a while to wait for a reflex cough, then gently rotate it for a circle and slowly remove the cotton swab. A1.2 Inoculation and strain identification
A1.2.1 Bacterial type
B. pertussis should be a Gram-negative, oval, short rod with a body length of 0.5~1.0um. A1.2.2 Colony
B. pertussis is inoculated in Bao-Jiang Ershi medium and cultured at 37℃ for 3~4 days. Its colonies are round, grayish white, smooth in surface, protruding in the center, neat at the edges, and pearl-like luster. There are diffuse hemolytic rings around the colonies. A1.2.3 Biochemical and antigenic characteristics
B. pertussis has extremely inactive biochemical reactions, does not ferment any carbohydrates, does not liquefy gelatin, does not produce hydrogen sulfide, does not form indole, does not reduce nitrates, does not utilize citrate, is oxidase-negative, and more than 70% of strains are catalase-positive. It does not produce urease and phenylalanine deaminase.
The newly isolated strain of Bordetella pertussis has a capsule, strong virulence, and smooth colonies, which are called phase I bacteria. Bordetella pertussis has 6 agglutination factors, of which 1, 2, and 3 are the main ones. Based on this, phase I bacteria can be divided into three serotypes: 1.2, 1.31.2.3. Serotypes are relatively stable, but can vary. Serotypes can vary not only in natural infection but also in artificial subculture. A2 Skin necrosis test
A2.1 At least two healthy rabbits or guinea pigs are used for each test, and several strains can be tested on each rabbit or guinea pig at the same time. A2.2 The bacterial lawn on the third or fourth generation 20% sheep blood bag-Jiang Ershi medium cultured for 40 to 48 hours is suspended in buffered saline or physiological saline.
A2.3 Filter the bacterial solution with sterilized absorbent cotton and determine the concentration. A2.4 Use physiological saline to dilute the bacterial solution to two concentrations of 4×108/mL and 2×10%/mL. A2.5 Depilate healthy rabbits or guinea pigs one day before the test. A2.6 Disinfect with 75% alcohol before injection, and then use a small test tube dipped in pigment to print on both sides of the spine. Four strains can be tested on each rabbit. A2.7 Inject 0.1mL intradermally in each circle. When removing the needle, twist it slightly in the skin to prevent the bacterial solution from flowing out. A2.8 Keep a record after the injection.
A2.9 Observe the necrosis 48 to 72 hours after injection and measure the size of the necrosis. If one of the two rabbits is injected with 4×108 bacteria, necrosis appears at the injection site, which is positive.
GB15998—1995
A3 Immunofluorescence detection of Bordetella pertussis (direct method) A3.1 Principle
The known pertussis antiserum is chemically combined with fluorescent pigments to make fluorescent antibodies, which are used to stain unknown bacteria fixed on the glass slide. If it is the corresponding bacteria, the binding surface of the two will remain on the glass slide and will not be washed away by the buffer. Fluorescence appears under the fluorescence microscope. A3.2 Materials
A3.2.1 Fluorescent antibody
Before use, dilute with pH7.3 phosphate buffered saline according to the instructions and store in a refrigerator at 4℃ for later use. A3.2.2 pH 7.3 Phosphate buffered saline
Sodium chloride
Disodium hydrogen phosphate
Sodium dihydrogen phosphate
Distilled water
A3.2.3 Fluorescence microscope
A3.2.4 Other materials
1 000 mL
0.8~1.2 mm thick slide glass. 0.17 mm thick cover glass. Glycerol buffer (90 mL analytical grade glycerol plus 10 mL pH 8.2 phosphate buffer), immersion oil can be replaced by liquid paraffin. A3.3 Method
A3.3.1 Use an inoculation loop to spread the specimen to be tested on a glass slide, about 1 cm2 in size, fix it with a flame, and circle the specimen with a crayon. A3.3.2 Add the diluted fluorescent antibody to the specimen, place it in a humidified box, and place it in a 37°C incubator for 30 minutes. A3.3.3 Take out the glass slide, use a dropper to absorb 3~~5mL of phosphate buffered saline to rinse the fluorescent antibody on the specimen, and then immerse it in three cylinders containing phosphate buffer in turn, soaking each cylinder for 3~~5 minutes, and shaking from time to time. A3.3.4 Dry, seal, and examine under a microscope.
A3.3.5 In order to make the results correct, the following controls are also required: a) Negative control of the specimen: No fluorescent antibody is added to the specimen, only phosphate buffered saline is added. b) Positive control: Fluorescent antibody is added to the specimen of known positive strains. c) Blocking test: first add the same unlabeled antiserum to the specimen for 30 minutes, wash thoroughly and then add the labeled antibody for staining. If it is a positive specimen, there should be no specific fluorescence.
d) Add the normal unimmunized labeled antibody to the specimen, and the result should be no specific fluorescence. A3.4 Result judgment
A3.4.1 Fluorescence intensity and morphological characteristics Ten Ten Ten Ten: The bacteria are significantly swollen, the black core is clear, and the fluorescence is bright. Ten Ten Ten: The bacteria are swollen, the black core is clear, and the fluorescence is bright. Leaf Ten: The bacteria are slightly swollen, the shape is clear, there is a black core, and the fluorescence is weak. Ten: The bacteria are not swollen, the shape is unclear, and the fluorescence is weak. One: The bacteria have no fluorescence.
A3.4.2 Judgment criteria
Positive: The bacterial fluorescence is above "ten ten", and the bacteria with typical morphology can be seen scattered or piled on the slide. The positive control is "ten ten to ten ten", and the negative control is negative.
B1 Pertussis tube agglutination test (half-volume method) B1.1 Principle
GB15998-1995
Appendix B
(Suggested Appendix)
Pertussis serological diagnosis method
Bacteria, red blood cells and other particulate antigens, added to serum containing specific antibodies, in the presence of appropriate electrolytes, after a certain period of time, visible agglutination groups may appear. The method of using this phenomenon to quantitatively determine the corresponding antibody of a known antigen or to determine the antigen with a known antibody is a quantitative agglutination test. B1.2 Materials
B1.2.1 Pertussis 1st phase standard serum
The titer is 1:12800.
B1.2.2 Pertussis bacterial solution
The agglutination titer of the bacterial solution should reach more than half of the titer of the 1st phase standard serum. The concentration of the bacterial solution is set at 2×10°/mL. B1.2.3 0.85% saline solution
B1.2.4 Serum to be tested
Patient's ear blood or venous blood, separate the serum and test it. B1.2.5 Agglutination tube and test tube rack
B1.2.6 Graduated pipette (1mL, 5mL, 10mL)B1.3 Test method
Take 0.1mL of serum to be tested + 0.4mL of saline solution, dilute it 5 times, and then dilute it in multiples to 1:10, 1:20, 1:40, 1:80, 1:160, etc., 0.25mL per tube. And add 2×10°/mL concentration of pertussis bacteria solution, 0.25mL per tube. Shake the hook thoroughly and place it at 35C overnight. Take it out the next day and place it at room temperature for 1h to observe the results. Each time a test is performed, a negative control tube should be set up, i.e., pertussis liquid plus physiological saline, and a positive control tube should be set up. A row of test tubes should be used, and pertussis phase 1 standard serum should be used for dilution according to known titer. Then pertussis liquid should be added to each tube and the test group should be observed under the same conditions. B1.4 Result determination
Ten: The liquid in the tube is turbid, and a small amount of agglutination blocks sink to the bottom of the tube. Ten Ten: The liquid in the tube is semi-clear, and some agglutination blocks sink to the bottom of the tube. Ten Ten Ten Ten: The liquid in the tube is not completely clear, and all agglutination blocks sink to the bottom of the tube. Ten Ten Ten Ten: The liquid in the tube is completely clear, and all agglutination blocks sink to the bottom of the tube. If the antibody titer in the recovery period is more than 4 times higher than that in the acute period, it is very meaningful for confirmed cases. B2 Microagglutination test (hemagglutination plate method)
B2.1 Materials
B2.1.1 Pertussis phase 1 standard serum
Titer 1: 12800.
B2.1.2 Tested bacterial solution
The titer of the bacterial solution should reach half of the titer of the first phase standard serum, and the concentration of the bacterial solution should be 3×108～4×10%/ml. B2.1.3 Diluent
0.85% physiological saline.
B2.1.4 U-shaped well hemagglutination plate
8 rows×12 wells.
B2.1.5 Adjustable micropipette
B2.1.6 Multi-channel pipette
B2.1.7 35℃～37C constant temperature box
B2.1.8 Refrigerator
B2.1.9 Micro-oscillator
B2.1.10 Sugar porcelain plate with cover
B2.2 Test method
GB 15998
—1995
B2.2.1 Take 0.1mL of standard serum and add 9.9ml. physiological saline to dilute it 100 times. B2.2.2 On the U-shaped well hemagglutination plate, no saline is added to the first well, 0.025mL of saline is added to the second to tenth wells respectively, and 0.025ml. of saline is also added to the 12th well as a negative control well. B2.2.3 Use a micropipette to add 0.025 mL of 100-fold diluted standard serum to each of the first and second wells. B2.2.4 Use a multi-channel pipette to dilute from the second well to the tenth well. B2.2.5 Add 0.025 mL of bacterial solution containing 3×10°~4×10° bacteria per mL to each well of the diluted serum. B2.2.6 Oscillate with a microvibrator for 3~5 minutes. Place the blood coagulation plate in a covered porcelain plate with gauze underneath, and then place it in a 35℃ incubator overnight. B2.2.7
B2.2.8 The next day, take out the blood coagulation plate and place it at room temperature for a while, then place the blood coagulation plate on a fluorescent lamp to observe the results. B2.3 Result judgmentwww.bzxz.net
B2.3.7 The bottom of the well is in the shape of dots, and the bacterial sediment is the same as the control well (I). B2.3.2 There are no dots at the bottom of the well, and the bacterial sediment is finely agglutinated and scattered (+). B2.3.3 Agglutination is obvious, and the supernatant is slightly turbid (++). B2.3.4 Agglutination is obvious, and the supernatant is clear (+++). B2.3.5
Agglutination is blocky, and the supernatant is clear (++++). 10785% saline solution
B1.2.4 Serum to be tested
Patient's ear blood or venous blood, separate the serum and test it. B1.2.5 Agglutination tube and test tube rack
B1.2.6 Graduated pipette (1mL, 5mL, 10mL)B1.3 Test method
Take 0.1mL of serum to be tested + 0.4mL of saline solution, dilute it 5 times, and then dilute it in multiples to 1:10, 1:20, 1:40, 1:80, 1:160, etc., 0.25mL per tube. Add 2×10°/mL pertussis solution, 0.25mL per tube. Shake well and place at 35C overnight. Take it out the next day and place it at room temperature for 1h to observe the results. Each time a test is performed, a negative control tube should be set up, i.e., pertussis liquid plus physiological saline, and a positive control tube should be set up. A row of test tubes should be used, and pertussis phase 1 standard serum should be used for dilution according to known titer. Then pertussis liquid should be added to each tube and the test group should be observed under the same conditions. B1.4 Result determination
Ten: The liquid in the tube is turbid, and a small amount of agglutination blocks sink to the bottom of the tube. Ten Ten: The liquid in the tube is semi-clear, and some agglutination blocks sink to the bottom of the tube. Ten Ten Ten Ten: The liquid in the tube is not completely clear, and all agglutination blocks sink to the bottom of the tube. Ten Ten Ten Ten: The liquid in the tube is completely clear, and all agglutination blocks sink to the bottom of the tube. If the antibody titer in the recovery period is more than 4 times higher than that in the acute period, it is very meaningful for confirmed cases. B2 Microagglutination test (hemagglutination plate method)
B2.1 Materials
B2.1.1 Pertussis phase 1 standard serum
Titer 1: 12800.
B2.1.2 Tested bacterial solution
The titer of the bacterial solution should reach half of the titer of the first phase standard serum, and the concentration of the bacterial solution should be 3×108～4×10%/ml. B2.1.3 Diluent
0.85% physiological saline.
B2.1.4 U-shaped well hemagglutination plate
8 rows×12 wells.
B2.1.5 Adjustable micropipette
B2.1.6 Multi-channel pipette
B2.1.7 35℃～37C constant temperature box
B2.1.8 Refrigerator
B2.1.9 Micro-oscillator
B2.1.10 Sugar porcelain plate with cover
B2.2 Test method
GB 15998
—1995
B2.2.1 Take 0.1mL of standard serum and add 9.9ml. physiological saline to dilute it 100 times. B2.2.2 On the U-shaped well hemagglutination plate, no saline is added to the first well, 0.025mL of saline is added to the second to tenth wells respectively, and 0.025ml. of saline is also added to the 12th well as a negative control well. B2.2.3 Use a micropipette to add 0.025 mL of 100-fold diluted standard serum to each of the first and second wells. B2.2.4 Use a multi-channel pipette to dilute from the second well to the tenth well. B2.2.5 Add 0.025 mL of bacterial solution containing 3×10°~4×10° bacteria per mL to each well of the diluted serum. B2.2.6 Oscillate with a microvibrator for 3~5 minutes. Place the blood coagulation plate in a covered porcelain plate with gauze underneath, and then place it in a 35℃ incubator overnight. B2.2.7
B2.2.8 The next day, take out the blood coagulation plate and place it at room temperature for a while, then place the blood coagulation plate on a fluorescent lamp to observe the results. B2.3 Result judgment
B2.3.7 The bottom of the well is in the shape of dots, and the bacterial sediment is the same as the control well (I). B2.3.2 There are no dots at the bottom of the well, and the bacterial sediment is finely agglutinated and scattered (+). B2.3.3 Agglutination is obvious, and the supernatant is slightly turbid (++). B2.3.4 Agglutination is obvious, and the supernatant is clear (+++). B2.3.5
Agglutination is blocky, and the supernatant is clear (++++). 10785% saline solution
B1.2.4 Serum to be tested
Patient's ear blood or venous blood, separate the serum and test it. B1.2.5 Agglutination tube and test tube rack
B1.2.6 Graduated pipette (1mL, 5mL, 10mL)B1.3 Test method
Take 0.1mL of serum to be tested + 0.4mL of saline solution, dilute it 5 times, and then dilute it in multiples to 1:10, 1:20, 1:40, 1:80, 1:160, etc., 0.25mL per tube. Add 2×10°/mL pertussis solution, 0.25mL per tube. Shake well and place at 35C overnight. Take it out the next day and place it at room temperature for 1h to observe the results. Each time a test is performed, a negative control tube should be set up, i.e., pertussis liquid plus physiological saline, and a positive control tube should be set up. A row of test tubes should be used, and pertussis phase 1 standard serum should be used for dilution according to known titer. Then pertussis liquid should be added to each tube and the test group should be observed under the same conditions. B1.4 Result determination
Ten: The liquid in the tube is turbid, and a small amount of agglutination blocks sink to the bottom of the tube. Ten Ten: The liquid in the tube is semi-clear, and some agglutination blocks sink to the bottom of the tube. Ten Ten Ten Ten: The liquid in the tube is not completely clear, and all agglutination blocks sink to the bottom of the tube. Ten Ten Ten Ten: The liquid in the tube is completely clear, and all agglutination blocks sink to the bottom of the tube. If the antibody titer in the recovery period is more than 4 times higher than that in the acute period, it is very meaningful for confirmed cases. B2 Microagglutination test (hemagglutination plate method)
B2.1 Materials
B2.1.1 Pertussis phase 1 standard serum
Titer 1: 12800.
B2.1.2 Tested bacterial solution
The titer of the bacterial solution should reach half of the titer of the first phase standard serum, and the concentration of the bacterial solution should be 3×108～4×10%/ml. B2.1.3 Diluent
0.85% physiological saline.
B2.1.4 U-shaped well hemagglutination plate
8 rows×12 wells.
B2.1.5 Adjustable micropipette
B2.1.6 Multi-channel pipette
B2.1.7 35℃～37C constant temperature box
B2.1.8 Refrigerator
B2.1.9 Micro-oscillator
B2.1.10 Sugar porcelain plate with cover
B2.2 Test method
GB 15998
—1995
B2.2.1 Take 0.1mL of standard serum and add 9.9ml. physiological saline to dilute it 100 times. B2.2.2 On the U-shaped well hemagglutination plate, no saline is added to the first well, 0.025mL of saline is added to the second to tenth wells respectively, and 0.025ml. of saline is also added to the 12th well as a negative control well. B2.2.3 Use a micropipette to add 0.025 mL of 100-fold diluted standard serum to each of the first and second wells. B2.2.4 Use a multi-channel pipette to dilute from the second well to the tenth well. B2.2.5 Add 0.025 mL of bacterial solution containing 3×10°~4×10° bacteria per mL to each well of the diluted serum. B2.2.6 Oscillate with a microvibrator for 3~5 minutes. Place the blood coagulation plate in a covered porcelain plate with gauze underneath, and then place it in a 35℃ incubator overnight. B2.2.7
B2.2.8 The next day, take out the blood coagulation plate and place it at room temperature for a while, then place the blood coagulation plate on a fluorescent lamp to observe the results. B2.3 Result judgment
B2.3.7 The bottom of the well is in the shape of dots, and the bacterial sediment is the same as the control well (I). B2.3.2 There are no dots at the bottom of the well, and the bacterial sediment is finely agglutinated and scattered (+). B2.3.3 Agglutination is obvious, and the supernatant is slightly turbid (++). B2.3.4 Agglutination is obvious, and the supernatant is clear (+++). B2.3.5
Agglutination is blocky, and the supernatant is clear (++++). 107
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