Some standard content:
【GB15984—1995】
Diagnostic criteria and treatment principles for cholera
Cholera is an acute intestinal infectious disease caused by Vibrio cholerae of groups 01 and 0139. It is a Class A infectious disease with rapid onset, rapid spread, wide impact and serious harm. It is also the most serious of the three international quarantine infectious diseases today. According to the provisions of the "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" and the "Implementation Measures of the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases", the diagnostic criteria and treatment principles for this disease are formulated. 1 Subject content and scope of application
This standard stipulates the diagnostic criteria for cholera, the treatment principles for patients, epidemic sites and epidemic areas, etc., and recommends the etiological examination of cholera, serological immunological examination, identification and typing methods of Vibrio cholerae, and rehydration therapy for patients. This standard is applicable to the diagnosis and treatment of cholera caused by Vibrio cholerae of groups 01 and 0139 in health and epidemic prevention and medical care institutions at all levels across the country. 2 Diagnostic principles
During the cholera epidemic period in summer and autumn, patients with diarrhea, vomiting, detection of 01 or 0139 Vibrio cholerae in feces or vomitus and diarrhea, or serum tests showing a significant increase in antibodies to 01 or 0139 Vibrio cholerae will be diagnosed. The patient's clinical symptoms may range from mild to severe.
3 Diagnostic criteria
3.1 Diagnostic criteria for suspected cholera
a. Patients with typical clinical symptoms, such as severe diarrhea, watery stool (yellow water, clear water, rice oil or bloody), vomiting, rapid onset of severe dehydration, circulatory failure and muscle spasm (especially tibia spasm) before the etiological examination is confirmed. b. Patients with a clear history of contact during the cholera epidemic (such as those who eat, live together or care for others, etc.), and who have diarrhea and vomiting symptoms, but no other reasons can be found. Patients with any of the above items are diagnosed as suspected cholera. 3.2 Determine the diagnostic criteria
a. Anyone with diarrheal symptoms and positive stool culture for Vibrio cholerae group 01 or 0139; b. Anyone in the epidemic area during the cholera epidemic period, anyone with typical cholera symptoms (see 3.1a), negative stool culture for Vibrio cholerae group 01 and 0139, but no other cause can be found; C. Anyone with diarrheal symptoms in the epidemic area during the epidemic period, with double serum antibody titer determination, such as serum agglutination test with a 4-fold increase or Vibrio killing antibody determination with a 8-fold increase; d. Anyone with diarrheal symptoms within 5 days before and after the first stool culture detection of Vibrio cholerae group 01 or 0139 during the epidemic source inspection.
Clinical diagnosis: meet b.
Confirmed case: meet a, c, or d.
4 Clinical classification standards
4.1 Mild
Only diarrhea symptoms, rarely accompanied by vomiting, less than 10 bowel movements a day, soft stool, loose stool or yellow watery stool, some patients have mucus or blood in their stool, normal or slightly poor skin elasticity, most patients can eat and get up and move around as usual, pulse, blood pressure, and urine volume are all normal. 4.2 Moderate
Diarrhea frequency is 10 to 20 times a day, mental performance is apathetic, there is hoarseness, dry and lack of elasticity of the skin, sunken eye sockets, muscle spasms, thin and rapid pulse, blood pressure (systolic pressure) children <9.33kPa (<70mmHg), adults 12 to 9.33kPa (90 to 70mmHg), urine volume is <400mL per day, and the degree of dehydration is 5% to 10% for children of equivalent weight and 4% to 8% for adults. 4.3 Severe
Diarrhea more than 20 times a day, extreme irritability or even coma, loss of skin elasticity, deep eye sockets, obvious hair, severe muscle spasms, weak and rapid pulse, or even no pulse, blood pressure (systolic pressure) <6.67kPa (<50mmHg) for children, <9.33kPa (<70mmHg or undetectable for adults) and other manifestations of circulatory failure, urine volume <50mL or no urine per day, dehydration level equivalent to more than 10% of body weight for children and more than 8% for adults. 4.4 Toxic type (dry cholera)
is a relatively rare type. After onset, the patient quickly enters a state of shock, with no diarrhea or vomiting or mild diarrhea and vomiting, no dehydration or only mild dehydration, but severe toxic circulatory failure. 5 Treatment principles
5.1 Isolation and treatment according to Class A infectious diseases. Critically ill patients should be treated first. Provide first aid on the spot, and after the patient's condition stabilizes, send the patient to a designated isolation ward accompanied by medical staff. Confirmed and suspected cases should be isolated separately.
5.2 For patients with mild dehydration, oral rehydration is the main treatment. For oral rehydration treatment methods, please refer to Chapter D1 of Appendix D.
5.3 For patients with moderate to severe dehydration, intravenous infusion should be given immediately for first aid, and oral rehydration should be used after the patient's condition stabilizes, the degree of dehydration is reduced, and vomiting stops. For infusion treatment methods, please refer to Chapter D2 of Appendix D.
5.4 At the same time as fluid therapy, antibiotics should be given to reduce the amount of diarrhea and shorten the bacterial excretion period. According to the source of the drug and the sensitivity of the Vibrio cholerae that caused the epidemic to antimicrobial drugs, a commonly used antibiotic can be selected and taken for 3 consecutive days. For commonly used antibiotics and dosages, please refer to Appendix E. ||tt ||6 Criteria for lifting isolation
6.1 After stopping the use of antimicrobial drugs, patients who have not detected Vibrio cholerae in stool culture for two consecutive days (if there is no stool, an anal swab can be used to collect stool from the rectum) can be released from isolation. 6.2 After the patient's symptoms disappear after treatment, if there is no condition for stool culture, the hospitalization and isolation period shall not be less than seven days from the date of onset.
6.3 For chronic carriers, if stool culture is negative for seven consecutive days and bile is cultured once a week, and two consecutive negative results are found, isolation can be lifted, but epidemiological observation is still required. 7 Treatment of epidemic sites and epidemic areas
The purpose of demarcating and treating epidemic sites and epidemic areas is to promptly discover and manage the source of infection, cut off the transmission route, and quickly control the epidemic. Phage-biotyping should be performed promptly on the El Tor type Vibrio cholerae detected from the first patient. If it is an epidemic strain, the epidemic site should be demarcated in a timely manner , epidemic area, and handle it according to the following provisions. If it is a non-epidemic strain, it shall be handled as a general diarrheal pathogen. 7.1 Delineation of epidemic sites and epidemic areas
a. Epidemic sites: refers to the households that enter and exit the same door as patients, suspected patients or carriers, or several households that have close living relations with them. b. Epidemic areas: It is delineated according to the geographical location, water system distribution, traffic conditions, natural villages and other characteristics of the epidemic site. Generally, in rural areas, it is one or several villages, one township or adjacent townships, and in cities, it is one or several neighborhood committees or one street. 7.2 Treatment of epidemic sites
a. Epidemic report: When the responsible epidemic reporter finds a patient, suspected patient or carrier, he shall report it to the health and epidemic prevention agency at the place of occurrence within 6 hours in urban areas and within 12 hours in rural areas by the fastest communication method, and report the infectious disease card at the same time. b. Isolation and treatment of infectious sources: Patients, suspected patients and carriers shall be isolated and treated immediately on the spot. When transferring patients, contaminated objects, ground and tools for transporting patients shall be disinfected at any time.
C. Disinfection of epidemic sites: disinfection at any time and terminal disinfection. Disinfect the vomit and diarrhea of patients, suspected patients and carriers, and the contaminated environment, objects and drinking water. d. Stool test: for all personnel in the epidemic site, stool test once a day from the start of treatment. The first stool test should be conducted before taking the medicine.
e. Preventive medication for patients and close contacts: an antimicrobial drug can be selected according to the results of drug sensitivity test and drug source, and taken for 2 consecutive days. For commonly used antimicrobial drugs and dosages, please refer to Appendix E.
7.3 Release of epidemic sites: after the isolation of the source of infection, the epidemic site can be released if the stool test of all personnel in the epidemic site is negative for two consecutive times and no secondary patients or carriers appear. If there is no condition for fecal examination, it can also be released if no new cases appear within 5 days after the epidemic site is treated. 7.4 Treatment of epidemic areas: strengthen drinking water disinfection and water source management, food hygiene and market trade management, do a good job in fecal management, and improve environmental hygiene. Discover the source of infection promptly and handle it according to the provisions of 7.2 to prevent transmission
Appendix A
A1 Collection of specimens
Pathological examination
(Supplement)
The specimen is mainly the patient's feces. 1-3mL of watery stool is collected, and the amount of feces the size of a fingernail is collected for formed stool. It can also be collected by rectal cotton swab or stool collection tube inserted from the anus into the rectum 3-5cm.
A2 Submission of specimens for examination
The collected specimens should be inoculated into the culture medium immediately. If it cannot be examined immediately, it should be placed in alkaline protein water or Cary-Blair preservation solution or inserted into Cary-Blair semi-solid preservation medium. The ratio of specimen to preservation solution is about 1:5. A3 Isolation and culture
A3.1 Direct isolation and culture: When the watery stool specimens of patients in the acute phase are cultured for bacterial growth, mucus flocs can be taken or cotton swabs can be directly inoculated on selective culture medium. At present, the commonly used strong selective culture media include gentamicin agar, No. 4 agar and TCBS agar. A3.2 Isolation and culture after bacterial enrichment: All fecal specimens should be inoculated with alkaline protein aged water culture medium, and after 6-8 hours of bacterial enrichment at 37℃, take an inoculation loop of culture from the surface below the bacterial film and inoculate it on strong selective agar.
A3.3 Identification: Pick suspicious colonies from the isolation culture medium and perform slide agglutination test with multivalent diagnostic serum for Vibrio cholerae group 01 and diagnostic serum for Vibrio cholerae group 0139. The titer of the serum for slide agglutination should generally be 1:40-1:50. If the suspicious colonies quickly show obvious agglutination visible to the naked eye in the serum (generally within 10 seconds), and do not agglutinate in physiological saline, it is judged as positive. When there are many suspicious colonies, more than 5 colonies should be selected for slide agglutination examination one by one. If necessary, take the transparent part of the edge of the original streaked colony for slide agglutination again. If all are negative, it can be reported that Vibrio cholerae group 01 and group 0139 were not detected. The strain of the first case needs to be sent to the first-level laboratory for further identification or re-examination. Appendix B
Serological examination
(reference)
B1 Serological examination is helpful for epidemiological traceability diagnosis. The composition of the test antibody is anti-bacterial antibody. For serological diagnosis, two serum samples are needed from the patient. The first serum sample is collected on the 1st to 3rd day of onset, and the second serum sample is collected on the 15th to 20th day. Agglutination test and Vibrio killing test are commonly used to check antibacterial antibodies in serum. The Vibrio killing test is more sensitive than the agglutination test. B2 test tube agglutination test: dilute the serum to be tested with saline solution, and each tube contains 0.5mL of diluted serum. The 16-18h culture of standard bacteria on nutrient agar is made into a suspension containing about 1.8 billion bacteria/mL (equivalent to the concentration of bacterial standard turbidimetric tube) with 0.2% formalin saline solution, and 0.5mL is added to each diluted serum tube; the bacterial suspension is added to 0.5mL of saline solution as a control. Shake well, place at 37C for 3h to observe the preliminary results, and then put in the refrigerator (4℃) or at room temperature overnight to observe the final results. The physiological saline control does not show natural agglutination, and can make the bacteria agglutinate at the bottom of the tube into an umbrella shape. The supernatant is translucent and is judged as 100. The highest dilution of serum that can make the bacteria agglutinate is judged as the serum agglutination titer. The second serum titer is 4 times or more higher than the first serum titer, which has diagnostic significance.
B3 Vibrio killing antibody detection: The modified micro-volume method can be used, and the detection procedure is as follows: a. Preparation of 2,3,5-triphenyltetrazolium chloride solution (TTC): Prepare 0.5% (W/V) solution with sterile distilled water, heat to dissolve, place in a brown bottle with pyrolysis bacteria, and store at 4℃ for later use. b. Preparation of complement indicator bacteria suspension: Select standard 01 group and 0139 group Vibrio cholerae as indicator bacteria, separate them on nutrient agar plates twice, select 2-3 smooth colonies, pick a small amount and inoculate them in 5mL nutrient broth, culture at 37℃ for 6h, take 0.1mL and transfer it to 5mL nutrient broth, culture at 37℃ for 2h, measure the absorbance (OD) value at 492nm with a spectrophotometer, and adjust the OD value to 0.1 as the stock solution. Dilute the stock solution 1:50 and mix it with an equal amount of 1:10 complement to obtain the complement indicator bacteria suspension. c. Dilution of serum to be tested: Add one drop (0.025 mL) of sterilized phosphate buffered saline (PBS) or physiological saline to each well of a microplate or 4×10-well polystyrene plastic plate that has been washed, soaked in anhydrous ethanol for 5 minutes, and sterilized by ultraviolet light for 1 hour using a micropipette, and then add one drop (0.025 mL) of inactivated serum to be tested to the first well of each row, and make consecutive double dilutions from the first well to the tenth well.
d. Add complement indicator bacteria suspension: Add one drop of complement indicator bacteria suspension to each well of diluted serum, cover, oscillate on a micro-oscillator for 1 to 2 minutes, and incubate in a 37°C water bath for 30 minutes. e: Add TTC nutrient broth: Add 0.15 mL (2 large drops) of nutrient broth containing 0.01% TTC to each well, mix well, and continue to incubate in a 37°C water bath for 4 hours. f. Observation results: The highest serum dilution without color change in the well is the titer of the Vibrio-killing antibody of the tested material. The second serum titer is 8 times or more higher than the first serum titer, which is of diagnostic significance.
C1 Serotyping
Appendix C
01 Identification and typing of group cholerae Vibrio
(reference)
Use bacterial lawn on ordinary agar or alkaline agar medium for serotyping. Typing uses monovalent sera of Ogawa type and Inaba type for slide agglutination reaction. C1.1 Those that agglutinate in Ogawa type monovalent serum but not in Inaba type monovalent serum are Ogawa type.
C1.2 Those that do not agglutinate in Ogawa type monovalent serum but agglutinate in Inaba type monovalent serum are Inaba type.
C1.3 Those that show obvious agglutination in both Ogawa type and Inaba type monovalent sera are Hikoshima type. C2 tube agglutination test
Use physiological saline to dilute the 01 group cholera polyvalent serum in multiple times from 1:20, and each tube contains 0.5mL of diluted serum. Use 0.2% formalin physiological saline to make a suspension containing about 1.8 billion bacteria per milliliter (equivalent to the concentration of bacterial standard turbidity tube) from the 16-18h culture of the test bacteria on nutrient agar, and add 0.5mL to each diluted serum tube; add 0.5mL of bacterial suspension to 0.5mL of physiological saline as a control. Shake well, place at 37℃ for 3h to observe the preliminary results. Place at 4℃ or room temperature overnight and observe the final results. The physiological saline control does not show natural agglutination. The highest dilution of serum that can make the test bacteria agglutinate ten times is the agglutination titer. The agglutination titer reaches or exceeds half of the original serum titer and can be determined as 01 group cholerae Vibrio. This test should be performed on the first batch of case strains of an epidemic and strains with atypical slide agglutination reactions.
C3 Differentiation of Classical Type and El Tor Type
C3.1 Differentiate the two biotypes of group 01 Vibrio cholerae by the tests listed in Table C1. Table C1www.bzxz.net
Differentiation of the two biotypes of group 01 Vibrio cholerae Tests
1. Lysis of group IV cholera cells (10/mL) 2. Polymyxin B count
3. Agglutination of chicken balls
High blood flow
Note: The results in brackets are for a few strains.
C3.2 Test method:
a. Group IV cholera phage lysis test: group 01 stenotype
El Tor
This test is carried out on the same plate as the phage typing. The method is shown in C4.1.2. b. Polymyxin B sensitivity test: Heat and melt 1.5% ordinary agar, wait until it cools to about 50℃, add 50 units of polymyxin B per milliliter of culture medium, shake well and pour into flat blood, and set aside after coagulation. Use a glass pen to draw several squares on the back of the flat blood. Place an inoculation loop of the broth culture of the tested bacteria for 2 to 3 hours on the surface of the culture medium, wait until it is dry, and culture it at 37℃ overnight to observe the results. If the tested bacteria do not grow or grow less than 10 colonies, it is sensitive and recorded as number 10. c. Chicken blood agglutination test: Draw squares in clean flat blood, use an inoculation loop with a diameter of 4mm to take a drop of physiological saline and drop it in each square, take a small amount of the 18h agar culture of the tested bacteria and make a concentrated bacterial suspension in physiological saline. Then use an inoculation loop to add a drop of 2.5% chicken blood saline suspension that has been washed three times, shake well, and observe the results with the naked eye. Those with blood cell agglutination within 1 minute are positive, and those with blood cells in a uniformly dispersed state are negative. El Tor type is positive, while classical type is generally negative.
dV-P test: Inoculate the tested bacteria into glucose phosphate protein aged water and culture at 37℃ for 2-3 days. Then take out 1mL of culture and add 0.6mL of 5% α-naphthol ethanol solution, shake for 5s, add 0.2mL of 40% potassium hydroxide solution, shake for another 5s, remove the cotton plug and place at room temperature. Those with red reaction within 1h are positive. Negative ones gradually turn light brown after the reagent is added. Classical type is negative, while El Tor type is mostly positive. e: Hemolysis test: Take 1mL of 24h ordinary broth (8-10mL in medium test tube) culture of the tested bacteria, 1mL of 1% sheep red blood cells (washed 3 times with saline, the last centrifugation speed is 2000r/min, centrifugation for 10min), mix well and place at 37℃, observe the preliminary results for 2h, and then place in a 4℃ refrigerator overnight to observe the final results. The test should have known hemolytic strains, non-hemolytic strains and broth tubes as controls. Hemolysis is positive when half of the blood cells are lysed. To prove that it is a heat-labile hemolysin, the culture of the tested bacteria can be heated to 56℃ for 30 minutes before the hemolysis test. The classical type does not produce soluble hemolysin, while some of the El Tor type produce and some do not produce heat-labile soluble hemolysin.
C4 Phage-biotyping
C4.1 Phage typing
C4.1.1 Five strains of Vibrio phages isolated in China (VP1～VP5) were used to divide the El Tor type of Vibrio cholerae into 32 phage types (Table C2). Table C2 Phage typing of El Tor type Vibrio cholerae Sensitivity
Strong bacteria type
Continued Table C2
Bacteria-killing type
C4.1.2 Phage typing method: On the back of a 1.5% ordinary agar plate, mark 9 squares with a glass pen, then add 0.2 mL of the 2-3 h broth culture of the test bacteria to the 0.7% 4mL semi-solid agar, mix well and pour on the agar plate. After that, add the stock solution of VP1~VP5 typing phage (10~10/mL) to the 1st to 5th grids respectively, and add the stock solution of Group IV cholera phage (10/mL) and conventional diluted
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