This standard specifies the diagnostic criteria, treatment methods and prevention and treatment principles of kala-azar. This standard is applicable to the prevention and treatment of kala-azar by professional institutions in epidemic areas and the diagnosis and treatment of kala-azar patients by medical and health institutions at all levels and types across the country. GB 15986-1995 Diagnostic criteria and treatment principles for kala-azar GB15986-1995 standard download decompression password: www.bzxz.net

National Standard of the People's Republic of China
Diagnostic criteria and principles of management of kala-azar
Diagnostic criteria and principles of management of kala-azar GB15986—1995
This standard is formulated in accordance with the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases and the Measures for the Implementation of the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases. 1 Subject matter and scope of application
This standard specifies the diagnostic criteria, treatment methods and prevention and control principles of kala-azar. This standard is applicable to the prevention and control of kala-azar by professional institutions in epidemic areas and the diagnosis and treatment of kala-azar patients by medical and health institutions of all levels and types across the country.
2 Diagnostic principles
Diagnosis is made based on epidemiological history, clinical manifestations, parasitological examinations and serological immunological methods. 3 Diagnostic criteria
3.1 Kala-azar
Kala-azar is transmitted through white bites, and the main lesions occur in the internal organs. In a few special cases, skin lesions or simple lymphadenopathy are the main symptoms, which are called skin or lymphadenopathy respectively. Dogs can also get this disease, called canine visceral leishmaniasis, which is the main animal host of kala-azar in the hilly areas of my country. Most of the local people are infected by sick dogs. 3.1.1 Residents in the kala-azar epidemic area, or people who have lived in the epidemic area during the white season (May to September). 3.1.2 Long-term irregular fever, progressive enlargement of the spleen, mild or moderate enlargement of the liver, decreased white blood cell count, anemia, thrombocytopenia or nasal and gingival bleeding.
3.1.3 Leishmania is found in the smear of the puncture material such as bone marrow, spleen or lymph nodes, or the puncture material is injected into the NNN medium to culture the promastigotes of Leishmania (see Appendix A for details). 3.1.4 The antibody test is positive by indirect fluorescent antibody test (IFA), enzyme-linked immunosorbent assay (ELISA), PVC film rapid ELISA, indirect hemagglutination (IHA) and other methods; or the circulating antigen test is positive by monoclonal antibody spot-ELISA (McAb spot-ELISA direct method) or monoclonal antibody-antigen spot test (McAb-AST method) and monoclonal antibody-enzyme-linked immunosorbent electrophoresis transfer blot test (EITB) (see Appendix B for details). Suspected cases: meet 3.1.1 and 3.1.2. Confirmed cases: add 3.1.3 to suspected cases. Clinical diagnosis: add 3.1.4 to suspected cases. 3.2 Cutaneous kala-azar
3.2.1 Most cases have a history of kala-azar several years or more than ten years ago, and it may also occur during the course of kala-azar; a few patients have no history of kala-azar and are primary cases.
3.2.2 Skin nodules, papules and erythema are present on the face, limbs or trunk, with occasional depigmented spots. The white blood cell count may increase to 10,000/mm2, and the eosinophil count is usually above 5%.
3.2.3 Leishmania parasites are found in the tissue fluid or skin tissue scrapings from the nodules and papules. 3.2.4 The circulating antigen is positive when detected by McAbdot-ELISA. Approved by the State Administration of Technical Supervision on December 15, 1995, 300
Implemented on July 1, 1996
Suspected case: meet 3.2.1 and 3.2.2. Confirmed case: Suspected case plus 3.2.3. Clinical diagnosis: Suspected case plus 3.2.4. 3.3 Lymph node type kala-azar
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3.3.1 Patients are mostly adults who live or travel from non-endemic areas to kala-azar endemic areas (deserts, hilly areas) where infants and young children are the main victims during the white season.
3.3.2 Lymph nodes in the neck, behind the ears, in the armpits, in the groin or on the trochlea are swollen, the size is peanuts to broad beans, shallow, and movable. The liver and spleen are not enlarged, and eosinophils increase.
3.3.3 Leishmania can be found in tissue fluid smears from swollen lymph nodes or in tissue sections of lymph nodes. Suspected cases: meet 3.3.1 and 3.3.2. Confirmed cases: Suspected cases plus 3.3.3. 4 Treatment principles
4.1 Treatment (see Appendix C for details)
4.1.1 Kala-azar
4.1.1.1 Initial treatment:
Six-day treatment with antimony sulphate (pentavalent sodium antimony gluconate): 120~150mg antimony/kg for adults, 200~240mg antimony/kg for children, divided into 6 times, injected intramuscularly or intravenously once a day, 6 days as a course of treatment. Three-week treatment with antimony sulphate: 133mg antimony/kg for adults, 200mg antimony/kg for children, divided into 6 times, injected intramuscularly or intravenously twice a week, 3 weeks to complete a course of treatment. This method is suitable for patients with poor physical constitution or severe illness.
4.1.1.2 Patients who have not recovered or relapsed after one course of treatment: The dose of antimony sulphate should be increased by 1/3 on the basis of the six-day treatment, and the eight-day treatment should be used instead.
4.7.1.3 For cases resistant to antimony agents, one of the following options may be selected: 4 mg/kg of pentapeptide is injected intramuscularly once daily or every other day, with a total amount of 60 mg/kg; 2-3 mg/kg of hydroxystilbene is injected intramuscularly or intravenously each time, with a total amount of 85 mg/kg. 4.1.2 Skin-type kala-azar
Six-day or eight-day treatment with stiboglossine for 2-3 consecutive courses. Stiboglossine has a good effect, 4 mg/kg each time, intramuscularly, with a total amount of 60-80 mg/kg, and can generally cure the disease. If the skin lesions have not completely disappeared, another course of treatment may be given. 4.1.3 Lymph node-type kala-azar
The dosage and course of stiboglossine are the same as those in 4.1.1.1. 4.2 Prevention
4.2.1 Elimination of sick dogs: In hilly areas where kala-azar is prevalent, parasitological examinations and serological immunological methods should be used in a timely manner to detect dogs infected with visceral leishmaniasis and kill them. In areas where there are many sick dogs, the masses should be mobilized to keep fewer or no domestic dogs. 4.2.2 Elimination of larvae: In plain areas where kala-azar is still prevalent, if the density of vector larvae is high after monitoring, insecticides should be used to spray houses and livestock houses at the beginning of the white season. In hilly and desert areas, after patients are found during the white season, insecticides can be used to spray the patient's home and houses and livestock houses within a radius of 15m around it to eliminate the larvae that invade the room from the wild. 4.2.3 Prevention
Use mosquito nets and mosquito coils, burn dry wild wormwood smoke cane to repel; do not sleep outdoors, and it is recommended to install fine-mesh screen doors and windows. In hilly areas where kala-azar is prevalent, insecticides (such as cypermethrin, etc.) can be used to spray dogs during the white season to kill or repel larvae that come to bite and suck blood. Personnel on duty in the wild in desert areas at night should apply repellents to exposed parts of the body. 301
A1 Pathogenic examination
A1.1 Iliac puncture
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Appendix A
Etiological diagnosis
(Supplement)
A1.1.1 The patient lies on his side, exposing the iliac part, and uses his fingers to identify the superior spine of the iliac bone. The skin around the area is disinfected with iodine and alcohol. This is usually done under local anesthesia.
A1.1.2 The size of the puncture needle varies depending on the age of the patient. Infants and young children use a 20-gauge puncture needle, older children and young people can use a 5-cm-long 18-gauge lumbar puncture needle, and adults use a 17-gauge puncture needle. All of them need to be sterilized by pressure steam. A1.1.3 The puncture site is about 1 cm behind the anterior superior spine of the iliac bone. First, pierce the skin, then stand the needle upright so that it is 70°~80° with the horizontal line. After passing through the subcutaneous tissue and periosteum, you can feel that the needle has touched the surface of the bone. You can use a rotating motion to drill the needle tip into the bone. A1.1.4 According to the age and weight of the patient, the puncture depth is 0.5~1.0cm, from shallow to deep. As long as the needle does not tilt after letting go, it means that the needle tip has entered the bone. The needle shaft can be pulled out and connected to a 2mL or 5mL syringe. After the bone marrow is drawn, the puncture needle should be pulled out immediately, and the bone marrow should be made into a smear for examination.
A1.1.5 After the bone marrow smear is made, let it dry naturally and number it with a marker. Fix it with methanol before staining, prepare a 3% dilution of Giemsa solution with water, and stain for 30 minutes, or add 3 drops of Giemsa solution to 2mL of water, drop it on the smear, and stain for 20 minutes. Then rinse gently with running water and let dry, then examine with an optical microscope (oil objective). A1.2 Lymph node puncture
A1.2.1 Generally, the groin area is selected. If the lymph nodes in other parts are swollen, they can also be used for puncture. A1.2.2 After local disinfection of the swollen lymph node, hold a lymph node with the clean left thumb and index finger, lift it up, and fix it between the two fingers. Be careful not to contaminate the puncture site. Take the high-pressure sterilized needle in the right hand, first pierce the skin, then pierce the lymph node, and pull out the needle after a few seconds without suction with a syringe. A1.2.3 Shoot the lymph tissue fluid in the needle onto the glass slide. Since the amount of fluid obtained is very small, it should be carefully made into a smear. A1.2.4 Smear staining microscopy: See A1.1.5. A1.3 Skin scraping examination
After disinfecting the skin around the skin, pinch the skin nodules with the clean left thumb and index finger to fix them between the two fingers, then use a sterilized and dry scalpel to gently cut the skin, scrape the skin tissue on both sides of the incision, make a smear, and stain for microscopic examination. A1.4 Preparation of tri-en base: 14.0g agar, 6.0g sodium chloride, 900mL distilled water, put them into a flask, heat and melt, and divide them into test tubes, 3mL per tube. Sterilize with pressure steam, wait for a little cooling, add 1/3 of the defibrinated rabbit blood to each test tube, mix evenly, and place it on an angle to cool. After cooling, add 0.5mL of Locke's solution to each tube and refrigerate at 4℃ for later use. A1.5 Protozoan culture: Under strict aseptic operation, inject the aspirated bone marrow and lymph into the culture medium, or cut a small piece of skin tissue from a patient with skin kala-azar and put it into Trinki, and culture it in a 22-24℃ incubator. After 15 days, use a platinum ear to take a small amount of culture fluid and examine it under a microscope. If the promastigotes of Leishmania are found, the diagnosis can be confirmed. 302
B1 Antibody detection
B1.1 Indirect fluorescent antibody test (IFA)
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Appendix B
Serological diagnosis
(Supplement)
-Generally, the patient's serum is used for the test. For infants and young children, it is inconvenient to collect blood, and the filter paper dried blood drop method can be used. B1.1.1 Antigen slice: Collect promastigotes of Leishmania after culture in NNN medium for about 10 days, centrifuge (3000r/min) for 15min, discard the supernatant, add physiological saline to rinse, centrifuge and wash 3 times, fix with phosphate buffered saline (PBS) containing 0.2% formalin 0.01mol/L pH7.2, put in the refrigerator for 1h, take out, centrifuge and precipitate, discard the supernatant, wash once with PBS, dilute to 50-100 promastigotes per field of view, drop on the glass slide, and blow dry with an electric fan. This antigen slice can be placed in a 20℃ refrigerator for later use. B1.1.2 Preparation of dried blood drops: Draw a circle with a diameter of 1.2cm on filter paper, drop 2 drops (equivalent to 20mm2) of patient's earlobe blood in the circle, put it in a plastic bag with desiccant after drying, and store it in the refrigerator for inspection. B1.1.3 Cut the dried blood drop from the filter paper, soak it in 0.2mL0.01mol/LpH7.2 PBS, which is equivalent to a 1:20 serum dilution (if the sample to be tested is serum, dilute it 1:20), and place it in the refrigerator overnight. During the test, continue to dilute it to 1:320 or 1:640, drop the serum or dried blood drop soaking solution of different dilutions on the antigen piece, incubate it in a humid box at 37℃ for 30min, slowly wash away the serum or dried blood drop soaking solution with PBS at pH7.2-8.0, soak it in PBS for 10min, continue to wash it with distilled water once, and blow it dry with an electric fan. B1.1.4 Add 1:10 diluted fluorescent-labeled goat anti-human IgG respectively, incubate it in a humid box at 37℃ for 30min, wash it as before, blow it with an electric fan for ten minutes, and wait for inspection.
B1.1.5During the examination, add a drop of distilled water to the slide, cover it with a cover glass, and observe it under a fluorescent microscope or a 6×40 optical microscope with a high-pressure mercury lamp fluorescent light source. The cytoplasm and flagella of the positive worm body show yellow-green fluorescence, with clear outlines, while the nucleus and kinetochord generally do not show fluorescence.
B1.1.6In each test, the patient's dry blood drop (or serum) and the normal person's dry blood drop (or serum) soaking liquid and PBS are used as controls. Since the normal person's blood sample may occasionally show "ten" when diluted 1:20, "++" is used as the positive standard, and 1:20 (i.e. 1:20→+) is used as the minimum positive dilution.
B1.2PVC film rapid ELISA
B1.2.1 Antigen (soluble antigen of promastigotes): Collect promastigotes of Leishmania, wash them three times by centrifugation with saline, add 10 times the volume of 0.01% thimerosal saline according to the volume, ultrasonically treat them twice in an ice bath, 10 minutes each time, freeze and thaw them repeatedly for 5 times, centrifuge them at 4000r/min for 3 minutes, and store the supernatant at -20°C for later use. B1.2.2 Operation method:
B1.2.2.1 Number the back of the PVC sensitized film before the experiment, and then add 0.2mL of serum diluent (PBS/T) to each well. B1.2.2.2 Add the serum to be tested and the reference serum according to the number (one negative control and one positive control for each batch), 10μL to each well, mix and place at 37°C for 5 minutes (if placed at room temperature at 25°C, it will take 10 minutes). B1.2.2.3 After incubation, pour off the diluted serum, wash with washing solution (NaCI/T) for 8 times in a row, and air dry. B1.2.2.4 Add enzyme conjugate diluted according to the working concentration, 0.2mL per well, and place at 37℃ for 5min. B1.2.2.5 Pour off the enzyme conjugate, wash with washing solution 8 times, then wash with distilled water once, and air dry. B1.2.2.6 Add tetramethylbenzidine (TMBS) substrate solution with 3% hydrogen peroxide (H,O2), 0.2mL per well, react for 5~～10min, and then observe the results.
B1.2.3 Reaction standard:
Visual judgment: Judge the results according to the positive control and negative control of each batch. Positive is bright blue, and negative is basically colorless. 303
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Spectrophotometer colorimetric judgment: No hydrogen peroxide (H2O2) is used for termination, and 595nm wavelength is used for colorimetric comparison. P/N ≥ 2.1 is considered positive (P is the optical density value of the patient; N is the optical density value of a normal person). B1.3 Indirect hemagglutination method (IHA)
B1.3.1 Take the promastigotes of Leishmania cultured for 10 to 12 days, wash them with normal saline, add 4 times normal saline according to the volume, treat them with ultrasound at 150W, 18kc, 300mA power for 10s in an ice bath, and dilute them 20 times with PBS of pH 6.4 and 0.075mol/L. The protein content measured by Folin phenol test is 200ug/ml.
B1.3.2 Formalization: Use healthy human "O" type red blood cells and add 10 times normal saline to centrifuge 4 to 5 times. Add 100 ml of 1% glutaraldehyde for every 8 ml of packed red blood cells, shake in a 4C water bath for 30 min, wash the formaldehyded red blood cells 5 times each with normal saline and distilled or deionized water, finally dilute to 15%, add 0.1 part thimerosal for preservation, and store in a refrigerator. B1.3.3 Sensitization: Dilute the aldehyded erythrocytes to 1.5% with PBS (pH 7.2, 0.75 mol/L), wash three times by centrifugation, prepare a 5% cell suspension, add an equal amount of 1/10,000 acid solution, place in a 37°C water bath for 30 minutes, shake every 3 to 5 minutes, wash three times with 5 times the amount of the above PBS, and then prepare a 2.5% erythrocyte suspension with PBS (pH 6.4, 0.075 mol/L). Then, add an equal amount of PBS (pH 6.4) to the erythrocyte suspension, dilute the antigen 20 times, sensitize in a 37°C water bath for 45 minutes with shaking, centrifuge at 2000 r/min for 3 minutes, discard the supernatant, add PBS (pH 7.2), and repeat the centrifugation once. Finally, prepare a 1% suspension with PBS (pH 7.2) containing 1% normal immune serum for use. B1.3.4 Add a quantitative amount of physiological saline solution onto the dilution plate, add an equal amount of serum to be tested into the first well, mix well and dilute in multiples to concentrations such as 2-1, 2-2, or 2-12. Take 0.025mL of serum of each concentration and add it to 12 6mm×7mm V” wells of the micro-hemagglutination plate, then add an equal amount of sensitized red blood cells and shake well, place it in a wet gauze box, and judge the result after 1 hour at room temperature, and use reference positive and negative serum as a control. Patients whose serum dilution reaches 2-? agglutination are considered positive, and those without agglutination are considered negative. B2 Detection of circulating antigens
B2.1 Monoclonal antibody-antigen spot test (McAb-AST) B2.1.1 The serum to be tested is diluted in multiples, and 2μL is taken and dropped on the nitrocellulose filter membrane (0.45μm) and placed at room temperature for 30 minutes. B2.1.2 Immerse the filter membrane in standard buffer (0.1mol/L tris(hydroxymethylaminomethane) (Tris)-HCl pH7.4.0.25% gelatin, 0.5% NP-40) at room temperature for 2 hours.
B2.1.3 Take out the filter membrane and immerse it in McAb-C-2 (1:100 dilution) at 4°C overnight. B2.1.4 After washing with buffer, incubate with horseradish peroxidase (HRP) labeled rabbit anti-mouse IgG (1:1000) at room temperature for 2 hours. B2.1.5 After washing, place the filter membrane in diaminobenzidine substrate solution, react at room temperature for 30 minutes, and wash with distilled water to terminate the reaction. B2.1.6 After drying, visually inspect the filter membrane for the presence of dark brown or brown spots with clear edges and a diameter larger than that of normal human serum, which is a positive reaction. No brown spots appear, and the reaction is similar to that of normal control serum, which is a negative reaction. To read the results more accurately, wait until the filter membrane is dry and use a Shimadzu dual-wavelength TLC scanner (CS-910,S460nm) to measure the optical density area integral (Integral of surfacearea.ISA), the threshold value ≥1.0 is a positive reaction; the threshold value <1.0 is a negative reaction. B2.2 Enzyme-labeled monoclonal antibody spot-ELISA direct method (McAbdot-ELISA) B2.2.1 The monoclonal antibody purified by glutaraldehyde two-step method is prepared into HRP-McAbL12F7, which is stored at -30℃ for later use. B2.2.2 The serum to be tested is diluted in multiples to 1:8 in sequence. 2μL of sample is dripped on the nitrocellulose membrane and dried in the shade at 4℃. B2.2.3 The nitrocellulose (NC) membrane with serum is placed in a standard buffer (0.1mol/L Tris-HClpH 7.4 0.25% gelatin, 0.5% NP-40) and washed 4 times on a shaker at room temperature, each time for 10 minutes. B2.2.4 After washing, add 1:100 diluted HRP-McAb, shake at room temperature for 2 hours, and wash with standard buffer 6 times, 10 minutes each time. B2.2.5 Add substrate 4-chloroethylphenol and shake for 10 minutes, then wash with water to terminate the reaction. B2.2.6 Visual inspection indicates a positive reaction if blue-grey spots appear. Negative reaction means no blue-grey spots or only traces of serum. B2.2.7 Positive and negative controls are required for each test. B2.3 Monoclonal antibody-enzyme-linked immunosorbent electrotransfer blot technique (McAb-EITB) B2.3.1 Serum sample processing: Take 50μL of each serum to be tested, add PBS with pH 7.4 to 1mL, 3000r/min30min, take the supernatant and add 0.2mL.30% polyethylene glycol (PEG molecular weight 6000), 4C overnight, 3000r/min30min, then wash with 50% PEG, 3000r/304
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min30min, finally add 50μL PBS to dissolve the precipitate for later use. B2.3.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technology: The experiment uses 10% separation gel (concentrate 3.3μl, 3mol/L. Tris-HCl 1.25mL, 10% sodium dodecyl sulfate (SDS) 100μL, tetramethylethylenediamine (TEMED) 5μL, 10% ammonium persulfate Ap 50μl). 3% concentration gel (concentrate 0.5mL. 1mol/LTris-HCl 0.625mL, H, O, 3.8mL, 10% SDS 50μl, TEMED 5μl, 10% Ap 25μL). After the gel plate is polymerized, load 2μL of treated serum into each tank. Insert the plate into the electrophoresis tank and electrophoresed at 200V for 50min.
B2.3.3 Electrophoretic transfer: Cover the gel film on the nitrocellulose membrane and align it to remove bubbles. Turn on the power supply, the current is 200mA, the voltage is 15V, and the electrophoresis is performed for 30min.
B2.3.4 After transfer, the nitrocellulose membrane is washed three times with standard buffer (Tris-HCl pH 7.4, gelatin 0.25%, 0.5NP-40), each time for 10min.
B2.3.5 Add 1:500 diluted HRP-McAbL12H4, 37℃ for 1h, then 4C overnight, wash three times with standard buffer, DAB-H,O, system color development for 10min, and wash with water to terminate the reaction.
B2.3.6 Visually observe the specific bands, with 130kD, 100kD and 25kD as positive bands. Appendix C
(Supplement)
C1 Anti-kala-azar treatment
C1.1 Patients who have just been treated: Six-day treatment with Stiboglucon or three-week treatment for patients with severe kala-azar. Stiboglucon should be injected intravenously as much as possible, and Stiboglucon should be injected slowly from the vein. During the six-day treatment, if the patient has side effects such as high fever, pain in the nose and spleen area, the injection can be stopped for several days, and the injection can be continued after the symptoms are relieved. The total amount of the drug can be combined with the previous injection. C1.2 Uncured patients: When the patient is reexamined half a month after a course of Stiboglucon treatment, if the body temperature is still higher than normal, the white blood cell count is increased, the spleen is still swollen, and the protozoa have not disappeared, the treatment should be considered ineffective. The dose of Stiboglucon can be increased by 1/3 of the original dose, and the second course of treatment is carried out with eight days and eight injections.
C1.3 Relapse patients: After treatment, the body temperature of kala-azar has returned to normal, the general condition and blood picture have improved, the spleen has also shrunk, and the puncture examination can no longer find Leishmania. However, after a few months (generally within half a year), the body temperature rises, the spleen is enlarged again, and the protozoa are found again on the bone marrow smear, which is a relapse. Stibnitz can still be used for treatment, and the dose should be increased by 1/3 based on the original dose. C1.4 Antimony-resistant patients: Patients who have not recovered after more than three courses of antimony. Clinically, they are called antimony-resistant kala-azar patients. The following two aromatic dipeptides can be used for treatment.
Pentamidine: Before each injection, dissolve the drug in sterile distilled water to make a 4% solution, and inject it intramuscularly, 4 mg/kg each time, once a day, for 15 consecutive days as a course of treatment, and the total dose is 60 mg/kg. During the drug injection process, redness and swelling may occur at the injection site, and local heat tenderization can be used to alleviate the reaction. Occasionally, blood pressure drops and there are reactions such as pulse acceleration, dizziness, and palpitations, which can disappear after injecting epinephrine.
Hydroxystilbamidine: Dissolve the drug in a small amount of distilled water before each use, and then use 1% procaine solution to make a 2.5%~~5% solution, slowly inject intramuscularly, or dissolve the drug in 50% glucose solution to make a 2% solution, and inject intravenously once a day, each dose is 2~3mg/kg, and the total dosage is about 85mg/kg. Aromatic double agents are unstable in aqueous solution, easy to deteriorate, and increase toxicity. Fresh solutions must be used for each injection. C2 Symptomatic treatment
C2.1 Anemia: If the patient has moderate anemia, iron preparations should be given during treatment. For severe anemia, in addition to iron preparations, small-volume multiple blood transfusions can be given. Antimony preparations can be used for treatment after the anemia improves. 305
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C2.2 Nosebleed: First clean the nasal cavity and find the bleeding point, then use cotton to spread 1:1000 adrenaline solution and 3% ephedrine to the bleeding area, or cover the bleeding area with gelatin sponge. C3 General treatment
During the treatment, the patient should rest in bed, prevent colds, and give nutritious and high-calorie foods such as eggs, pork liver, tofu, etc., and take a sufficient amount of multivitamins orally every day to facilitate the recovery of the patient. C4
Treatment of complications
C4.1 Pneumonia: Patients with pneumonia should not be treated with antimony agents or aromatic dual-class drugs. If pneumonia occurs during the treatment of anti-kala-azar, the injection should be stopped immediately, and antibiotics should be used first. After the symptoms of pneumonia disappear, anti-kala-azar drugs should be used for treatment. C4.2 Acute granulocytopenia: Penicillin should be used immediately to prevent secondary infection. If it occurs during the treatment with antimony agents, the use of anti-kala-azar drugs should be stopped immediately, and antimony agents or aromatic diabetic drugs should be used for treatment after the symptoms disappear. Sometimes kala-azar itself can also cause this disease, and the cause is unrelated to the use of antimony agents. In this case, the use of antimony agents is not only harmless, but also can promote the recovery of granulocytes as the condition improves. C4.3 Running horse disease: Qingwusu should be used as early as possible for treatment, which has significant effects. At the same time, antimony agents should be used for treatment according to common methods. C4.4 For patients with more serious heart, liver or kidney diseases, antimony agents should be used with caution and symptomatic treatment should be given. Additional notes:
This standard was proposed by the Ministry of Health of the People's Republic of China. This standard was drafted by the Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine. The main drafters of this standard are Zhai Jingqi and Guan Liren. This standard is entrusted by the Ministry of Health to the technical coordination unit, the Office of Infectious Disease Supervision and Management of the Ministry of Health, to be responsible for the interpretation. 3066 Visual inspection indicates a positive reaction if blue-grey spots appear. Negative reaction means no blue-grey spots or only traces of serum. B2.2.7 Each test must have positive and negative controls. B2.3 Monoclonal antibody-enzyme-linked immunosorbent electrotransfer blot technique (McAb-EITB) B2.3.1 Serum sample processing: Take 50μL of each serum to be tested, add PBS with pH 7.4 to 1mL, 3000r/min for 30min, take the supernatant and add 0.2mL.30% polyethylene glycol (PEG molecular weight 6000), 4C overnight, 3000r/min for 30min, then wash with 50% PEG, 3000r/304
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min for 30min, and finally add 50μL PBS to the precipitate to dissolve for use. B2.3.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technology: The experiment uses 10% separation gel (concentrate 3.3μl, 3mol/L. Tris-HCl 1.25mL, 10% sodium dodecyl sulfate (SDS) 100μL, tetramethylethylenediamine (TEMED) 5μL, 10% ammonium persulfate Ap 50μl). 3% concentration gel (concentrate 0.5mL. 1mol/LTris-HCl 0.625mL, H, O, 3.8mL, 10% SDS 50μl, TEMED 5μl, 10% Ap 25μL). After the gel plate is polymerized, load 2μL of treated serum into each tank. Insert the plate into the electrophoresis tank and electrophoresed at 200V for 50min.
B2.3.3 Electrophoretic transfer: Cover the gel film on the nitrocellulose membrane and align it to remove bubbles. Turn on the power supply, the current is 200mA, the voltage is 15V, and the electrophoresis is performed for 30min.
B2.3.4 After transfer, the nitrocellulose membrane is washed three times with standard buffer (Tris-HCl pH 7.4, gelatin 0.25%, 0.5NP-40), each time for 10min.
B2.3.5 Add 1:500 diluted HRP-McAbL12H4, 37℃ for 1h, then 4C overnight, wash three times with standard buffer, DAB-H,O, system color development for 10min, and wash with water to terminate the reaction.
B2.3.6 Visually observe the specific bands, with 130kD, 100kD and 25kD as positive bands. Appendix C
(Supplement)
C1 Anti-kala-azar treatment
C1.1 Patients who have just been treated: Six-day treatment with Stiboglucon or three-week treatment for patients with severe kala-azar. Stiboglucon should be injected intravenously as much as possible, and Stiboglucon should be injected slowly from the vein. During the six-day treatment, if the patient has side effects such as high fever, pain in the nose and spleen area, the injection can be stopped for several days, and the injection can be continued after the symptoms are relieved. The total amount of the drug can be combined with the previous injection. C1.2 Uncured patients: When the patient is reexamined half a month after a course of Stiboglucon treatment, if the body temperature is still higher than normal, the white blood cell count is increased, the spleen is still swollen, and the protozoa have not disappeared, the treatment should be considered ineffective. The dose of Stiboglucon can be increased by 1/3 of the original dose, and the second course of treatment is carried out with eight days and eight injections.
C1.3 Relapse patients: After treatment, the body temperature of kala-azar has returned to normal, the general condition and blood picture have improved, the spleen has also shrunk, and the puncture examination can no longer find Leishmania. However, after a few months (generally within half a year), the body temperature rises, the spleen is enlarged again, and the protozoa are found again on the bone marrow smear, which is a relapse. Stibnitz can still be used for treatment, and the dose should be increased by 1/3 based on the original dose. C1.4 Antimony-resistant patients: Patients who have not recovered after more than three courses of antimony. Clinically, they are called antimony-resistant kala-azar patients. The following two aromatic dipeptides can be used for treatment.
Pentamidine: Before each injection, dissolve the drug in sterile distilled water to make a 4% solution, and inject it intramuscularly, 4 mg/kg each time, once a day, for 15 consecutive days as a course of treatment, and the total dose is 60 mg/kg. During the drug injection process, redness and swelling may occur at the injection site, and local heat tenderization can be used to alleviate the reaction. Occasionally, blood pressure drops and there are reactions such as pulse acceleration, dizziness, and palpitations, which can disappear after injecting epinephrine.
Hydroxystilbamidine: Dissolve the drug in a small amount of distilled water before each use, and then use 1% procaine solution to make a 2.5%~~5% solution, slowly inject intramuscularly, or dissolve the drug in 50% glucose solution to make a 2% solution, and inject intravenously once a day, each dose is 2~3mg/kg, and the total dosage is about 85mg/kg. Aromatic double agents are unstable in aqueous solution, easy to deteriorate, and increase toxicity. Fresh solutions must be used for each injection. C2 Symptomatic treatment
C2.1 Anemia: If the patient has moderate anemia, iron preparations should be given during treatment. For severe anemia, in addition to iron preparations, small-volume multiple blood transfusions can be given. Antimony preparations can be used for treatment after the anemia improves. 305
GB15986—1995
C2.2 Nosebleed: First clean the nasal cavity and find the bleeding point, then use cotton to spread 1:1000 adrenaline solution and 3% ephedrine to the bleeding area, or cover the bleeding area with gelatin sponge. C3 General treatment
During the treatment, the patient should rest in bed, prevent colds, and give nutritious and high-calorie foods such as eggs, pork liver, tofu, etc., and take a sufficient amount of multivitamins orally every day to facilitate the recovery of the patient. C4
Treatment of complications
C4.1 Pneumonia: Patients with pneumonia should not be treated with antimony agents or aromatic dual-class drugs. If pneumonia occurs during the treatment of anti-kala-azar, the injection should be stopped immediately, and antibiotics should be used first. After the symptoms of pneumonia disappear, anti-kala-azar drugs should be used for treatment. C4.2 Acute granulocytopenia: Penicillin should be used immediately to prevent secondary infection. If it occurs during the treatment with antimony agents, the use of anti-kala-azar drugs should be stopped immediately, and antimony agents or aromatic diabetic drugs should be used for treatment after the symptoms disappear. Sometimes kala-azar itself can also cause this disease, and the cause is unrelated to the use of antimony agents. In this case, the use of antimony agents is not only harmless, but also can promote the recovery of granulocytes as the condition improves. C4.3 Running horse disease: Qingwusu should be used as early as possible for treatment, which has significant effects. At the same time, antimony agents should be used for treatment according to common methods. C4.4 For patients with more serious heart, liver or kidney diseases, antimony agents should be used with caution and symptomatic treatment should be given. Additional notes:
This standard was proposed by the Ministry of Health of the People's Republic of China. This standard was drafted by the Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine. The main drafters of this standard are Zhai Jingqi and Guan Liren. This standard is entrusted by the Ministry of Health to the technical coordination unit, the Office of Infectious Disease Supervision and Management of the Ministry of Health, to be responsible for the interpretation. 3066 Visual inspection indicates a positive reaction if blue-grey spots appear. Negative reaction means no blue-grey spots or only traces of serum. B2.2.7 Each test must have positive and negative controls. B2.3 Monoclonal antibody-enzyme-linked immunosorbent electrotransfer blot technique (McAb-EITB) B2.3.1 Serum sample processing: Take 50μL of each serum to be tested, add PBS with pH 7.4 to 1mL, 3000r/min for 30min, take the supernatant and add 0.2mL.30% polyethylene glycol (PEG molecular weight 6000), 4C overnight, 3000r/min for 30min, then wash with 50% PEG, 3000r/304
GB15986—1995
min for 30min, and finally add 50μL PBS to the precipitate to dissolve for use. B2.3.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technology: The experiment uses 10% separation gel (concentrate 3.3μl, 3mol/L. Tris-HCl 1.25mL, 10% sodium dodecyl sulfate (SDS) 100μL, tetramethylethylenediamine (TEMED) 5μL, 10% ammonium persulfate Ap 50μl). 3% concentration gel (concentrate 0.5mL. 1mol/LTris-HCl 0.625mL, H, O, 3.8mL, 10% SDS 50μl, TEMED 5μl, 10% Ap 25μL). After the gel plate is polymerized, load 2μL of treated serum into each tank. Insert the plate into the electrophoresis tank and electrophoresed at 200V for 50min.
B2.3.3 Electrophoretic transfer: Cover the gel film on the nitrocellulose membrane and align it to remove bubbles. Turn on the power supply, the current is 200mA, the voltage is 15V, and the electrophoresis is performed for 30min.
B2.3.4 After transfer, the nitrocellulose membrane is washed three times with standard buffer (Tris-HCl pH 7.4, gelatin 0.25%, 0.5NP-40), each time for 10min.
B2.3.5 Add 1:500 diluted HRP-McAbL12H4, 37℃ for 1h, then 4C overnight, wash three times with standard buffer, DAB-H,O, system color development for 10min, and wash with water to terminate the reaction.
B2.3.6 Visually observe the specific bands, with 130kD, 100kD and 25kD as positive bands. Appendix C
(Supplement)
C1 Anti-kala-azar treatment
C1.1 Patients who have just been treated: Six-day treatment with Stiboglucon or three-week treatment for patients with severe kala-azar. Stiboglucon should be injected intravenously as much as possible, and Stiboglucon should be injected slowly from the vein. During the six-day treatment, if the patient has side effects such as high fever, pain in the nose and spleen area, the injection can be stopped for several days, and the injection can be continued after the symptoms are relieved. The total amount of the drug can be combined with the previous injection. C1.2 Uncured patients: When the patient is reexamined half a month after a course of Stiboglucon treatment, if the body temperature is still higher than normal, the white blood cell count is increased, the spleen is still swollen, and the protozoa have not disappeared, the treatment should be considered ineffective. The dose of Stiboglucon can be increased by 1/3 of the original dose, and the second course of treatment is carried out with eight days and eight injections.
C1.3 Relapse patients: After treatment, the body temperature of kala-azar has returned to normal, the general condition and blood picture have improved, the spleen has also shrunk, and the puncture examination can no longer find Leishmania. However, after a few months (generally within half a year), the body temperature rises, the spleen is enlarged again, and the protozoa are found again on the bone marrow smear, which is a relapse. Stibnitz can still be used for treatment, and the dose should be increased by 1/3 based on the original dose. C1.4 Antimony-resistant patients: Patients who have not recovered after more than three courses of antimony. Clinically, they are called antimony-resistant kala-azar patients. The following two aromatic dipeptides can be used for treatment.
Pentamidine: Before each injection, dissolve the drug in sterile distilled water to make a 4% solution, and inject it intramuscularly, 4 mg/kg each time, once a day, for 15 consecutive days as a course of treatment, and the total dose is 60 mg/kg. During the drug injection process, redness and swelling may occur at the injection site, and local heat tenderization can be used to alleviate the reaction. Occasionally, blood pressure drops and there are reactions such as pulse acceleration, dizziness, and palpitations, which can disappear after injecting epinephrine.
Hydroxystilbamidine: Dissolve the drug in a small amount of distilled water before each use, and then use 1% procaine solution to make a 2.5%~~5% solution, slowly inject intramuscularly, or dissolve the drug in 50% glucose solution to make a 2% solution, and inject intravenously once a day, each dose is 2~3mg/kg, and the total dosage is about 85mg/kg. Aromatic double agents are unstable in aqueous solution, easy to deteriorate, and increase toxicity. Fresh solutions must be used for each injection. C2 Symptomatic treatment
C2.1 Anemia: If the patient has moderate anemia, iron preparations should be given during treatment. For severe anemia, in addition to iron preparations, small-volume multiple blood transfusions can be given. Antimony preparations can be used for treatment after the anemia improves. 305
GB15986—1995
C2.2 Nosebleed: First clean the nasal cavity and find the bleeding point, then use cotton to spread 1:1000 adrenaline solution and 3% ephedrine to the bleeding area, or cover the bleeding area with gelatin sponge. C3 General treatment
During the treatment, the patient should rest in bed, prevent colds, and give nutritious and high-calorie foods such as eggs, pork liver, tofu, etc., and take a sufficient amount of multivitamins orally every day to facilitate the recovery of the patient. C4
Treatment of complications
C4.1 Pneumonia: Patients with pneumonia should not be treated with antimony agents or aromatic dual-class drugs. If pneumonia occurs during the treatment of anti-kala-azar, the injection should be stopped immediately, and antibiotics should be used first. After the symptoms of pneumonia disappear, anti-kala-azar drugs should be used for treatment. C4.2 Acute granulocytopenia: Penicillin should be used immediately to prevent secondary infection. If it occurs during the treatment with antimony agents, the use of anti-kala-azar drugs should be stopped immediately, and antimony agents or aromatic diabetic drugs should be used for treatment after the symptoms disappear. Sometimes kala-azar itself can also cause this disease, and the cause is unrelated to the use of antimony agents. In this case, the use of antimony agents is not only harmless, but also can promote the recovery of granulocytes as the condition improves. C4.3 Running horse disease: Qingwusu should be used as early as possible for treatment, which has significant effects. At the same time, antimony agents should be used for treatment according to common methods. C4.4 For patients with more serious heart, liver or kidney diseases, antimony agents should be used with caution and symptomatic treatment should be given. Additional notes:
This standard was proposed by the Ministry of Health of the People's Republic of China. This standard was drafted by the Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine. The main drafters of this standard are Zhai Jingqi and Guan Liren. This standard is entrusted by the Ministry of Health to the technical coordination unit, the Office of Infectious Disease Supervision and Management of the Ministry of Health, to be responsible for the interpretation. 3066 Visually inspect the specific bands, with 130kD, 100kD and 25kD as positive bands. Appendix C
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C1 Anti-kala-azar treatment
C1.1 Patients who have just been treated: Six-day treatment with Stiboglucon or three-week treatment for patients with severe kala-azar, intravenous injection should be used as much as possible, and Stiboglucon should be slowly injected into the vein. During the six-day treatment, if the patient has side effects such as high fever, pain in the nose and spleen area, the injection can be stopped for several days, and the injection can be continued after the symptoms are relieved. The total amount of the drug can be combined with the previous injection. C1.2 Uncured patients: When the patient is reexamined half a month after a course of Stiboglucon treatment, if the body temperature is still higher than normal, the white blood cell count is increased, the spleen is still swollen, and the protozoa do not disappear, the treatment should be considered ineffective. The dose of Stiboglucon can be increased by 1/3 of the original dose, and the second course of treatment is carried out with eight days and eight injections.
C1.3 Relapse patients: After treatment, the body temperature of kala-azar has returned to normal, the general condition and blood picture have improved, the spleen has also shrunk, and the puncture examination can no longer find Leishmania. However, after a few months (generally within half a year), the body temperature rises, the spleen is enlarged again, and the protozoa are found again on the bone marrow smear, which is a relapse. Stibnitz can still be used for treatment, and the dose should be increased by 1/3 based on the original dose. C1.4 Antimony-resistant patients: Patients who have not recovered after more than three courses of antimony. Clinically, they are called antimony-resistant kala-azar patients. The following two aromatic dipeptides can be used for treatment.
Pentamidine: Before each injection, dissolve the drug in sterile distilled water to make a 4% solution, and inject it intramuscularly, 4 mg/kg each time, once a day, for 15 consecutive days as a course of treatment, and the total dose is 60 mg/kg. During the drug injection process, redness and swelling may occur at the injection site, and local heat tenderization can be used to alleviate the reaction. Occasionally, blood pressure drops and there are reactions such as pulse acceleration, dizziness, and palpitations, which can disappear after injecting epinephrine.
Hydroxystilbamidine: Dissolve the drug in a small amount of distilled water before each use, and then use 1% procaine solution to make a 2.5%~~5% solution, slowly inject intramuscularly, or dissolve the drug in 50% glucose solution to make a 2% solution, and inject intravenously once a day, each dose is 2~3mg/kg, and the total dosage is about 85mg/kg. Aromatic double agents are unstable in aqueous solution, easy to deteriorate, and increase toxicity. Fresh solutions must be used for each injection. C2 Symptomatic treatment
C2.1 Anemia: If the patient has moderate anemia, iron preparations should be given during treatment. For severe anemia, in addition to iron preparations, small-volume multiple blood transfusions can be given. Antimony preparations can be used for treatment after the anemia improves. 305
GB15986—1995
C2.2 Nosebleed: First clean the nasal cavity and find the bleeding point, then use cotton to spread 1:1000 adrenaline solution and 3% ephedrine to the bleeding area, or cover the bleeding area with gelatin sponge. C3 General treatment
During the treatment, the patient should rest in bed, prevent colds, and give nutritious and high-calorie foods such as eggs, pork liver, tofu, etc., and take a sufficient amount of multivitamins orally every day to facilitate the recovery of the patient. C4
Treatment of complications
C4.1 Pneumonia: Patients with pneumonia should not be treated with antimony agents or aromatic dual-class drugs. If pneumonia occurs during the treatment of anti-kala-azar, the injection should be stopped immediately, and antibiotics should be used first. After the symptoms of pneumonia disappear, anti-kala-azar drugs should be used for treatment. C4.2 Acute granulocytopenia: Penicillin should be used immediately to prevent secondary infection. If it occurs during the treatment with antimony agents, the use of anti-kala-azar drugs should be stopped immediately, and antimony agents or aromatic diabetic drugs should be used for treatment after the symptoms disappear. Sometimes kala-azar itself can also cause this disease, and the cause is unrelated to the use of antimony agents. In this case, the use of antimony agents is not only harmless, but also can promote the recovery of granulocytes as the condition improves. C4.3 Running horse disease: Qingwusu should be used for treatment as early as possible, which has a significant effect. At the same time, antimony agents should be used for treatment according to the usual methods. C4.4 For patients with more serious heart, liver or kidney diseases, antimony agents should be used with caution and symptomatic treatment should be given. Additional notes:
This standard was proposed by the Ministry of Health of the People's Republic of China. This standard was drafted by the Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine. The main drafters of this standard are Zhai Jingqi and Guan Liren. This standard is entrusted by the Ministry of Health to the technical coordination unit, the Office of Infectious Disease Supervision and Management of the Ministry of Health, to be responsible for the interpretation. 3066 Visually inspect the specific bands, with 130kD, 100kD and 25kD as positive bands. Appendix C
(Supplement)
C1 Anti-kala-azar treatment
C1.1 Patients who have just been treated: Six-day treatment with Stiboglucon or three-week treatment for patients with severe kala-azar, intravenous injection should be used as much as possible, and Stiboglucon should be slowly injected into the vein. During the six-day treatment, if the patient has side effects such as high fever, pain in the nose and spleen area, the injection can be stopped for several days, and the injection can be continued after the symptoms are relieved. The total amount of the drug can be combined with the previous injection. C1.2 Uncured patients: When the patient is reexamined half a month after a course of Stiboglucon treatment, if the body temperature is still higher than normal, the white blood cell count is increased, the spleen is still swollen, and the protozoa do not disappear, the treatment should be considered ineffective. The dose of Stiboglucon can be increased by 1/3 of the original dose, and the second course of treatment is carried out with eight days and eight injections.
C1.3 Relapse patients: After treatment, the body temperature of kala-azar has returned to normal, the general condition and blood picture have improved, the spleen has also shrunk, and the puncture examination can no longer find Leishmania. However, after a few months (generally within half a year), the body temperature rises, the spleen is enlarged again, and the protozoa are found again on the bone marrow smear, which is a relapse. Stibnitz can still be used for treatment, and the dose should be increased by 1/3 based on the original dose. C1.4 Antimony-resistant patients: Patients who have not recovered after more than three courses of antimony. Clinically, they are called antimony-resistant kala-azar patients. The following two aromatic dipeptides can be used for treatment.
Pentamidine: Before each injection, dissolve the drug in sterile distilled water to make a 4% solution, and inject it intramuscularly, 4 mg/kg each time, once a day, for 15 consecutive days as a course of treatment, and the total dose is 60 mg/kg. During the drug injection process, redness and swelling may occur at the injection site, and local heat tenderization can be used to alleviate the reaction. Occasionally, blood pressure drops and there are reactions such as pulse acceleration, dizziness, and palpitations, which can disappear after injecting epinephrine.
Hydroxystilbamidine: Dissolve the drug in a small amount of distilled water before each use, and then use 1% procaine solution to make a 2.5%~~5% solution, slowly inject intramuscularly, or dissolve the drug in 50% glucose solution to make a 2% solution, and inject intravenously once a day, each dose is 2~3mg/kg, and the total dosage is about 85mg/kg. Aromatic double agents are unstable in aqueous solution, easy to deteriorate, and increase toxicity. Fresh solutions must be used for each injection. C2 Symptomatic treatment
C2.1 Anemia: If the patient has moderate anemia, iron preparations should be given during treatment. For severe anemia, in addition to iron preparations, small-volume multiple blood transfusions can be given. Antimony preparations can be used for treatment after the anemia improves. 305
GB15986—1995
C2.2 Nosebleed: First clean the nasal cavity and find the bleeding point, then use cotton to spread 1:1000 adrenaline solution and 3% ephedrine to the bleeding area, or cover the bleeding area with gelatin sponge. C3 General treatment
During the treatment, the patient should rest in bed, prevent colds, and give nutritious and high-calorie foods such as eggs, pork liver, tofu, etc., and take a sufficient amount of multivitamins orally every day to facilitate the recovery of the patient. C4
Treatment of complications
C4.1 Pneumonia: Patients with pneumonia should not be treated with antimony agents or aromatic dual-class drugs. If pneumonia occurs during the treatment of anti-kala-azar, the injection should be stopped immediately, and antibiotics should be used first. After the symptoms of pneumonia disappear, anti-kala-azar drugs should be used for treatment. C4.2 Acute granulocytopenia: Penicillin should be used immediately to prevent secondary infection. If it occurs during the treatment with antimony agents, the use of anti-kala-azar drugs should be stopped immediately, and antimony agents or aromatic diabetic drugs should be used for treatment after the symptoms disappear. Sometimes kala-azar itself can also cause this disease, and the cause is unrelated to the use of antimony agents. In this case, the use of antimony agents is not only harmless, but also can promote the recovery of granulocytes as the condition improves. C4.3 Running horse disease: Qingwusu should be used as early as possible for treatment, which has significant effects. At the same time, antimony agents should be used for treatment according to common methods. C4.4 For patients with more serious heart, liver or kidney diseases, antimony agents should be used with caution and symptomatic treatment should be given. Additional notes:
This standard was proposed by the Ministry of Health of the People's Republic of China. This standard was drafted by the Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine. The main drafters of this standard are Zhai Jingqi and Guan Liren. This standard is entrusted by the Ministry of Health to the technical coordination unit, the Office of Infectious Disease Supervision and Management of the Ministry of Health, to be responsible for the interpretation. 306
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