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WS 269-2019: Diagnosis for brucellosis
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WS 269-2019English599 Add to Cart 5 days [Need to translate] Diagnosis for brucellosis Valid WS 269-2019
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GB 15979   GB/T 15981   WS/T 641   WS/T 268   WS 268   

Basic data

Standard ID WS 269-2019 (WS269-2019)
Description (Translated English) Diagnosis for brucellosis
Sector / Industry Health Industry Standard
Classification of Chinese Standard C59
Classification of International Standard 11.020
Word Count Estimation 26,221
Date of Issue 2019
Date of Implementation 2019-07-01
Issuing agency(ies) National Health Commission

WS 269-2019: Diagnosis for brucellosis

---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Diagnosis for brucellosis ICS 11.020 C 59 WS People's Republic of China Health Industry Standard Replacing WS 269-2007 Diagnosis of brucellosis Published on.2019 - 01 - 02 2019 - 07 - 01 implementation National Health and Wellness Committee of the People's Republic of China

Foreword

Chapter 6 of this standard is mandatory and the rest are recommended. This standard was drafted in accordance with the rules given in GB/T 1.1-2009. This standard replaces WS 269-2007 "Diagnostic Criteria for Brucellosis". Compared with WS 269-2007, the main technical changes of this standard are as follows. - Added normative references (see Chapter 2); - Added abbreviations (see Chapter 3); - Added colloidal gold immunochromatographic test (see 4.3.1.2, Appendix C.2); - Added enzyme-linked immunosorbent assay (see 4.3.1.3, Appendix C.3); - Increased Brucella culture smear Gram stain for the detection of suspected Brucella (see 4.3.1.4); -- Revised diagnostic principles and increased clinical diagnosis (see Chapters 5 and 6.2); - Increased culture of Brucella, method of culturing Brucella by blood culture apparatus (see D.1 and D.1.1); - Revised the method for culturing Brucella in a biphasic blood culture flask in the culture of Brucella (see D.1.2); -- Revised the culture method of Brucella cultured by pathological materials in the culture of Brucella (see D.1.3); - Increased identification of Brucella and related specific tests (see D.2); - Increased nucleic acid detection of Brucella and BCSP31 polymerase chain reaction (see D.2.5 and D.2.5.1); - Added AMOS polymerase chain reaction (see D.2.5.2); - Increased Brucella genomic DNA extraction method (see D.2.6); - Increased biosafety requirements for Brucella test (see D.3); - Removed the plate agglutination test (PAT) (see C.1.1 of the.2007 edition); - Removed skin allergy test (see C.2 of.2007 edition); - Removed the unfertilized chicken eggs from Brucella culture (see C.3.1.2 of the.2007 edition); -- Removed the urine culture method of Brucella culture (see C.3.2 of.2007 edition); -- Removed the biologically isolated Brucella method for blood culture (see C.3.4,.2007). This standard was drafted. China Center for Disease Control and Prevention, China Center for Disease Control and Prevention, Beijing Ditan Hospital, China Disease Prevention and Control Heart plague brucellosis prevention and control base, Shanxi Provincial Center for Disease Control and Prevention, Liaoning Provincial Center for Disease Control and Prevention, Hubei Province Disease Prevention Control Center, Inner Mongolia Autonomous Region Comprehensive Disease Prevention and Control Center, Hangzhou Municipal Center for Disease Control and Prevention, Qinghai Provincial Institute of Endemic Disease Prevention and Control. The main drafters of this standard. Cui Buyun, Li Xingwang, Wang Dali, Jiang Hai, Zhang Qiuxiang, Mao Lingling, Cheng Junfu, Mi Jingchuan, Xu Weimin, Xu Liqing, Tian Guozhong, Liu Wei. The previous versions of the standards replaced by this standard are. --WS 269-2007. Diagnosis of brucellosis

1 Scope

This standard specifies the diagnostic basis, diagnostic principles, diagnosis and differential diagnosis of human brucellosis. This standard applies to the diagnosis of brucellosis in various types of medical and health institutions and their medical staff at all levels.

2 Normative references

The following documents are indispensable for the application of this document. For dated references, only the dated version applies to this document. For undated references, the latest edition (including all amendments) applies to this document. Regulations for the Administration of Highly Pathogenic Microorganisms (Poisonous) Species or Samples for Infection of Humans, Ministry of Health Order No. 45,.2005 List of Pathogenic Microorganisms Infected by Humans, Ministry of Health (Wei Shi Jiao Fa [2006] No. 15) Technical Regulations for the Safe Transport of Dangerous Goods, Airline (Doc 9284)

3 Abbreviations

The following abbreviations apply to this document. CFT. complement fixation test DNA. deoxyribonucleic acid dNTPs. deoxy-ribonucleoside triphosphate ELISA. enzyme linked immunosorbent assay GICA. gold immunochromatography assay PBS. phosphate buffer saline PCR. polymerase chain reaction RBT. rose bengal plate agglutination test RTD. routine test dilution SAT. serum agglutination test

4 diagnosis basis

4.1 Epidemiological history Before the onset, the patient has close contact with livestock and livestock products suspected of Brucella infection, or raw food for cattle, goat milk and meat products, or raw Live in brucellosis; or engage in Brucella culture, testing or Brucella vaccine production and use. Other epidemiological parameters See Appendix A. 4.2 Clinical manifestations 4.2.1 There is fever (including low fever), sweating, fatigue, muscle and joint pain for several days or even weeks. 4.2.2 Some patients have lymph nodes, liver, spleen and testicular swelling, a small number of patients may have a variety of rashes and jaundice; patients in acute and chronic phase Can manifest as bone and joint system damage. See Appendix B for specific clinical manifestations. 4.3 Laboratory inspection (see Appendix C, Appendix D for experimental methods) 4.3.1 Laboratory screening 4.3.1.1 The results of the tiger red plate agglutination test (RBT) were positive. 4.3.1.2 Colloidal gold immunochromatographic test (GICA) results were positive. 4.3.1.3 The results of the enzyme-linked immunosorbent assay (ELISA) were positive. 4.3.1.4 Brucella culture smear Gram staining detected suspected Brucella. 4.3.2 Laboratory diagnosis 4.3.2.1 Brucella is isolated from cultures of any pathological material such as blood, bone marrow, other body fluids, and excretions. 4.3.2.2 Test tube agglutination test (SAT) titer of 1.100 and above, or patients with a disease duration of more than one year and still have clinical symptoms The titer is 1.50 and above. 4.3.2.3 Complement binding test (CFT) titer is 1.10 and above. 4.3.2.4 Anti-human immunoglobulin test (Coomb's) titer of 1.400 and above.

5 Diagnostic principles

The occurrence, development and outcome of brucellosis are complicated, and its clinical manifestations are diverse. It is difficult to determine the diagnosis with a certain symptom. The diagnosis of brucellosis should be combined with the patient's epidemiological exposure history, clinical manifestations and laboratory tests.

6 diagnosis

6.1 Suspected cases Meets 4.1 and meets 4.2 at the same time. 6.2 Clinical diagnosis cases Meet the suspected case and comply with any of 4.3.1. 6.3 confirmed cases Meet suspected or clinically diagnosed cases and meet any of 4.3.2. 6.4 latent infection Meets 4.1 and complies with any of 4.3.2 and does not comply with 4.2.

7 differential diagnosis

Mainly should be differentiated from rheumatic fever, typhoid fever, paratyphoid fever, tuberculosis, rheumatoid arthritis, spondylitis, meningitis, orchitis Broken, see Appendix E for details. AA

Appendix A

(informative appendix) Brucellosis epidemiology A.1 Brucellosis Brucellosis is an infectious-allergic disease caused by bacteria in the genus Brucella that invade the body. A.2 Storage host and source of infection There are many storage hosts for Brucella, and more than 60 kinds of animals (livestock, poultry, wild animals, domesticated animals) are known as blue. The bacterium stores the host. Brucellosis is often transmitted first in livestock or wild animals, and then spreads to humans. It is a zoonotic infectious disease. Plague Livestock is the main source of infection of brucellosis. In most parts of China, sheep is the main source of infection. In some places, cattle are the source of infection. Pigs in the province can be used as a source of infection. Economic animals such as deer and dogs can also be sources of infection. A.3 Transmission routes and transmission factors Pathogens can invade the body through the skin mucosa, digestive tract, and respiratory tract. Human infections and occupations, diet, lifestyle related. Various contaminants and foods containing Brucella can be used as a medium of transmission, mainly for diseased animal products, milk, meat and internal organs of sick animals. Fur, water, soil, dust, etc. contaminated by Brucella. A.4 susceptible population Humans are generally susceptible to Brucella. The prevalence of brucellosis in the population is related to the chances and extent of close contact with the source of infection and the vector. Brucella can be repeatedly infected in patients with brucellosis. A.5 distribution A.5.1 Occupation There is obvious occupationality, and the incidence of contact with sick animals and infected animals is high. Farmers, herders, veterinarians, fur and milk, meat plus The infection rate of workers and related experimental personnel is higher than that of the average person. A.5.2 Gender People are susceptible to Brucella and have no gender differences, depending on how many exposures they have. A.5.3 Age All age groups can be infected with the disease. Since young adults are the main labor force and are exposed to sick animals, the infection rate is higher than other age groups. A.5.4 Season It can occur every month of the year. There is a clear seasonal peak in the endemic area of Brucella. The peak incidence of population in the farming and pastoral areas in northern China From April to May, summer wool and dairy foods increase, and a small peak incidence can occur. Bovine species, Brucella of Brucella The seasonality of the bacterial disease is not obvious. A.5.5 Region The infection rate of brucellosis is higher in towns and pastoral areas than in towns. People in agriculture and pastoral areas have frequent contact with livestock, and there are many opportunities for infection. In some fur processing companies. A.6 Epidemic characteristics of different epidemic areas A.6.1 Breeding area of Brucella The main source of infection in the Brucella epidemic area of sheep is diseased sheep. Brucella 1, 2, and 3 biotypes have strong invasiveness against humans and animals. And pathogenicity, easy to cause brutality epidemics in humans and animals, and the epidemic is heavy. Most typical clinical signs and symptoms appear. A.6.2 Bovine Brucella infection area The main source of infection in the Bovine Brucella epidemic area is sick cattle. Bovine species of Brucella are more biological and have different virulence. In general, cattle breeds Brucella is weakly toxic, but it has strong invasiveness. Even attenuated strains can cause fulminant abortion or infertility in cattle, which seriously affects livestock. Livestock development. However, it is mild to people, has a high infection rate and a low incidence rate, and is sporadic. Clinical symptoms and signs are more atypical, with a short course, after Less sequelae. A.6.3 Brucella infection in pig breeds The main source of infection in the Brucella epidemic area of pig breeds is sick pigs. Usually caused by pig type 1 and pig type 3 Brucella, the virulence is between the sheep Between the bacteria and the bovine species Brucella. The same biotype strain has both virulent strains and attenuated strains. The pig breed Brucella is highly pathogenic to pigs. It is weaker to sheep and cattle. It is more virulence to human than Brucella bovine, except for a few cases, most of which have no acute phase clinical table. Now. A.6.4 Dog breed Brucella infection area The main source of infection in the dog breed Brucella is the sick dog. Dog breed Brucella can cause cat abortion in addition to invading dogs. Animals such as pigs, rabbits, sika deer, and mice are infected to produce antibodies against Brucella. People can also be infected and rarely develop symptoms. A.6.5 Mixed Brucella infection area Two or more types of Brucella are present in an affected area at the same time, which is related to the grazing or circumcision of a sheep or cattle in a pasture. due to Close contact with each other, different strains can be transferred, from the transmission of Brucella to the cattle, and the transmission of Brucella to the pig; Pig breeds, Bovine Brucella can also be transferred to sheep. The prevalence of mixed-type epidemic areas depends on the main species present in the area. BB

Appendix B

(informative appendix) Clinical manifestations of brucellosis B.1 Main symptoms B.1.1 fever It is a common clinical manifestation of brucellosis. Typical cases are characterized by wavy fever, often accompanied by symptoms such as chills, which can be seen in all stages of patients. unit Sub-cases can be characterized by low heat and irregular heat, and occur mostly in the afternoon or at night. Patients with brucellosis are conscious and have less pain when they are hot, but their symptoms are aggravated when their body temperature drops. This kind of high fever and disease The phenomenon of shield is unique to brucellosis. B.1.2 sweaty It is a common clinical manifestation of brucellosis. In the acute phase, sweating is particularly heavy, and when the body temperature drops, it can be aggravated, and it can be wet and wet. Feeling nervous and annoyed. B.1.3 Muscle and joint pain It is a common clinical manifestation of brucellosis, which is systemic muscle and multiple, migratory joint pain. In some cases, there may also be a spine (waist Vertebral main) bone and joint involvement, manifested as pain, deformity and dysfunction. B.1.4 Weakness Almost all cases have fatigue fatigue. B.1.5 Other In a few cases, there may be headache, heart, kidney and nervous system involvement. B.2 main signs B.2.1 Liver, spleen and lymphadenopathy More common in acute cases, patients with liver and splenomegaly recovered slowly. B.2.2 Other Male cases can be associated with orchitis, and ovarian inflammation can be seen in female cases. Patients with acute phase can have a variety of rashes, some patients can In the presence of jaundice, patients with chronic phase manifest as damage to the bone and joint system. B.3 Clinical staging B.3.1 Acute phase With the above clinical manifestations, the course of disease within 3 months, a confirmed serological positive reaction. B.3.2 Subacute phase With the above clinical manifestations, the course of disease ranged from 3 months to 6 months, and a confirmed serological positive reaction occurred. B.3.3 Chronic phase The disease has not healed for more than 6 months, has symptoms and signs of brucellosis, and has a confirmed serological positive reaction. B.4 Incubation period The incubation period for brucellosis is generally from 1 week to 3 weeks. CC

Appendix C

(normative appendix) Brucellosis specific laboratory testing technique C.1 Tiger Red Plate Agglutination Test (RBT) C.1.1 Equipment and reagents C.1.1.1 Equipment. clean slides, micro sampler, wooden sign, timer. C.1.1.2 Reagents. tiger red plate agglutination antigen, known negative and positive serum, serum to be tested. C.1.2 Method of operation Add 30 μL of the test serum to the slide, then add 30 μL of the agglutination antigen to the tiger red plate, shake well or mix well with a wooden stick for 5 min. Internal observation results. Negative and positive sera were used as controls for each batch of experiments. C.1.3 Result determination The macroscopic agglutination reaction was judged to be positive; the liquid was uniformly turbid, and no agglutination reaction was found to be negative. C.2 Colloidal gold immunochromatographic assay (GICA) C.2.1 Equipment and reagents C.2.1.1 Equipment. The test card package is labeled with a soluble Brucella antigen, a nitrocellulose membrane of human IgG, and a colloidal gold label. 0.1 mL pipette or micropipette. C.2.1.2 Reagents. normal saline, serum to be tested. C.2.2 Method of operation Add 10 μL of test serum to the test card well and infiltrate. 100 μL of physiological saline was added to the well to be infiltrated. The results were observed within 3 min to 20 min. C.2.3 Result determination The test card quality control area (C) showed a red line, and the test results were credible; the red line was not displayed and the test failed. Test area (T) shows a red line, the test result is positive, only a red line in the quality control area is negative. Note. Colloidal gold immunochromatographic assays may have different test cards, and the experimental methods may differ. The specific experiments refer to the instructions for testing and experimental diagnosis. C.3 enzyme-linked immunosorbent assay (ELISA) C.3.1 Kits and equipment C.3.1.1 Kit composition The composition of the kit is detailed in Table C.1. Table C.1 ELISA kit composition Name quantity/single package capacity code description Standard AD 4×2 mL CAL AD Standard AD (1 U/mL, 10 U/mL, 40 U/mL, 150 U/mL); ready to use standard A = negative control; standard B = critical Control; standard C = weak positive control; standard D = positive pair Enzyme cross-linker IgG 1×14 mL ENZCNJ IgG Use anti-human IgG, bind peroxidase, protein slow Flushing, stabilizer TMB substrate solution 1 × 14 mL TMB SUBS ready to use. contains TMB TMB Stop Solution 1×14 mL TMB STOP Ready to use. 0.5 M H2SO4 Diluent 1 × 60 mL DILBUF Ready to use. Contains PBS BSA < 0.1% NaN3 Washing solution 1 × 60 mL WASHBUF conc 10 times concentrated, containing. PBS, Tween20 Enzyme plate 1 × 12 well × 8 well MTP coated specific antigen Viscous membrane 2 sheets of FOIL cover the microtiter plate during incubation Plastic bag 1 BAG preserves adhesive film that is not used C.3.1.2 Equipment Microplate reader (absorption wavelength 450 nm, reference wavelength 600 nm to 650 nm), plate washer, pipette, 8-channel pipette, 1 mL pipetting Tube, timer, absorbent paper towel, tip, dilution plate, sample tank, marker, double distilled water or deionized water. C.3.1.3 Preparation before testing Prepare the washing solution and diluent according to the instructions. Dilute the test serum according to the instructions. C.3.2 Method of operation Proceed as follows. a) Pipette 100 μL of each negative positive control solution and diluted sample into the wells of the corresponding ELISA plate. b) Cover the plate with a viscous membrane and incubate at 18 °C ~ 25 °C for 60 min. c) Remove the film and discard the enzyme plate liquid. Add 300 μL of diluted dilution to each well, wash the plate 3 times, and place the plate on a paper towel. Remove the remaining liquid. d) Add 100 μL of enzyme cross-linker using a pipette; cover the microplate with a new membrane and incubate at 18 °C ~ 25 °C for 30 min; e) Repeat C.3.2.c). f) Use a pipette to add substrate solution and stop solution. The interval between substrate solution and stop solution should be the same. Avoid air bubbles when loading. g) Add 100 μL of TMB substrate solution to each well, cover the plate with a new adhesive film, and incubate at 18 °C ~ 25 °C for 20 min. 100 μL of TMB stop solution was added to each well to stop the enzymatic reaction, and the plate was shaken to make it mix, and the color changed from blue to yellow. h) Measure the absorbance at 450 nm within 60 min of the addition of the stop solution. C.3.3 Quality control C.3.3.1 The OD value of negative and positive standards is within the scope of quality control requirements. C.3.3.2 Test instruments and samplers must undergo rigorous calibration or calibration. C.3.3.3 Reagents should be stored under specified storage conditions and used within the validity period. C.3.4 Result determination The OD value of the sample was read using a microplate reader with reference to the instructions. The standard OD value is the y axis, the standard concentration is the x axis, and the logarithmic coordinate paper Make a standard curve on it. The concentration value of the corresponding sample is then read on the curve. The result is as follows. a) >12 U/mL is positive for the experiment; b) 8 U/mL~12 U/mL is suspicious for the experiment; c) < 8 U/mL is negative for the experiment. Enzyme-linked immunosorbent assay can detect immunoglobulins such as IgM (IgM-ELISA) and IgG (IgG-ELISA). The experimental principle and method may be There are differences, specific experiments refer to the kit instructions for testing and making experimental diagnosis. C.4 Test tube agglutination test (SAT) C.4.1 Equipment and reagents Tube agglutination antigen, known negative and positive serum, test serum, saline, 1 mL pipette, serum agglutination tube, test tube rack, 37 ° C incubator and so on. C.4.2 Method of operation Proceed as follows. a) Dilution of the serum to be tested. In general, 5 tubes per serum, 2.3 mL saline in the first tube, second The tube is not added, and the third, fourth and fifth tubes are each added with 0.5 mL. Pipette 0.2 mL of the serum to be tested into the first tube and mix. After mixing, pipette the serum from the first tube into the second and third tubes by 0.5 mL, mix the third tube with a pipette, and suck. Add 0.5 mL to the fourth tube and mix. Pipette 0.5 mL from the fourth tube into the fifth tube and mix. Then draw 0.5 mL from the fifth tube Discard. After so diluted, the serum dilutions from the second tube to the fifth tube were 1.12.5, 1.25, 1.50, and 1.100, respectively. b) Add antigen. First, properly dilute the antigen solution into the antigen application solution with normal saline (according to the instructions, generally 1.10) Dilute), the diluted antigen application solution is added to each diluted serum tube (the first tube is not added, as a serum control), each tube is added Mix 0.5 mL and mix. After adding the antigen application solution, the second tube to the fifth tube, the total amount of each tube is 1 mL, and the serum dilution is from the second tube to The fifth tube is 1.25, 1.50, 1.100, and 1.200. Aspirate 0.5 mL from the first tube and discard it, leaving 1 mL. c) Control. Negative serum control, serum diluted with antigen application solution (similar to the serum to be tested); positive serum control, The serum is diluted to the original titer, and the antigen application solution is added; the antigen control, the appropriately diluted antigen plus physiological saline. C.4.3 Result determination C.4.3.1 Preparation of reference turbidity tube. Each test shall be configured with reference to the turbidity tube as the basis for the determination. The configuration method is. taking the antigen solution for the test, Dilute into an antigen application solution by adding physiological saline, and configure a turbidity tube according to Table C.2. Table C.2 Test tube agglutination test to determine the preparation of the turbidity tube Tube number antigen application solution (/mL) saline (/mL) brightness indicator 1 0.00 1.00 100% 2 0.25 0.75 75% 3 0.50 0.50 50% 4 0.75 0.25 25% 5 1.00 0.00 0% - C.4.3.2 Judgment result. All test tubes, control tubes and reference turbidity tubes are fully oscillated and placed in a 37 °C incubator for 20 h to 22 h. After leaving the room for 2 h, then refer to the turbidiary tube as the standard judgment result. C.4.3.3 Recording results. The results are recorded according to the brightness of the upper liquid in each tube. Especially 50% clear brightness ( ) on the judgment result relationship Larger, must be compared with the comparison of the turbidity tube. . Complete agglutination, the supernatant is 100% clear. . Almost completely agglutinated, the supernatant is 75% clear. . Significant agglutination, liquid 50% clear. . There is a slight amount of agglutination, and the liquid is 25% clear. -. No agglutination, liquid is not clear. Each serum titer was determined to be the highest serum dilution with agglomeration of "" and above. Note. In order to obtain the final positive titer titer of the specimen to be inspected, you can refer to other test results to add more dilution. C.5 Complement binding test (CFT) C.5.1 Reagents and equipment C.5.1.1 Reagents. normal saline, complement (guinea pig serum or lyophilized complement), 2% sheep red blood cell suspension, hemolysin, brucella complement Binding antigen, negative and positive serum, test serum. C.5.1.2 Equipment. 37 °C water bath, ordinary centrifuge, ordinary refrigerator, 0.1 mL, 1 mL and 10 mL pipette, serum agglutination tube and test tube frame. C.5.2 Treatment and titration of five components C.5.2.1 Complement Vacuum drying of the complement maintains long-term activity. You can also choose several healthy guinea pigs, take blood to separate the serum, and mix the serum. Need to complement. Complement titration is req...

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