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Basic dataStandard ID: WS 295-2019 (WS295-2019)Description (Translated English): Diagnosis for meningococcal meningitis Sector / Industry: Health Industry Standard Classification of Chinese Standard: C59 Classification of International Standard: 11.020 Word Count Estimation: 19,160 Date of Issue: 2019 Date of Implementation: 2019-07-01 Issuing agency(ies): National Health Commission WS 295-2019: Diagnosis for meningococcal meningitis---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 meningococcal meningitis ICS 11.020 C 59 WS People's Republic of China Health Industry Standard Replacing WS 295-2008 Diagnosis of epidemic cerebrospinal meningitis Published on.2019 - 01 - 02 2019 - 07 - 01 implementation National Health and Wellness Committee of the People's Republic of China Foreword5.1, 5.2, and 5.3 of this standard are 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 295-2008 "Diagnostic Criteria for Epidemic Cerebrospinal Meningitis." Compared with WS 295-2008, the main technical changes of this standard are as follows. - Added abbreviations (see Chapter 2); - Revised the history of epidemiology (see 3.1, 2.1 of the.2008 edition); -- Revised clinical performance (see 3.2,.2008 version 2.2); -- Revised the "peripheral blood image" as "blood routine" (see 3.3.1, 2.3.1 of the.2008 edition); - increased the results of cerebrospinal fluid examination (see 3.3.2); - Added "defective (spot) tissue fluid" (see 3.3.3.2 and 3.3.3.3); - Revised the diagnostic principle (see 4,.2008 edition 3); - Revised "serology" to "immunology" (see 3.3.4,.2008 version 2.3.4); - Revised suspected cases (see 5.1,.2008 edition 4.2); -- Revised clinically diagnosed cases (see 5.2,.2008 edition 4.3); - Revised confirmed cases (see 5.3, 4.4 of the.2008 edition); - Added "other meningococcal disease" (see 7.2); - Added "differential diagnosis" (see Appendix B, B.4); -- Modified the laboratory test method for Neisseria meningitidis (see Appendix A); -- According to changes in the epidemiological characteristics of epidemic cerebrospinal meningitis, in Appendix B, the pathogen of epidemic cerebrospinal meningitis The relevant descriptions in the study, epidemiology and clinical manifestations are revised (see Appendix B). This standard was drafted. China Center for Disease Control and Prevention, Beijing Ditan Hospital affiliated to Capital Medical University, Shandong Provincial Disease Prevention and Control Center, Gansu Provincial Center for Disease Control and Prevention, Qilu Children's Hospital of Shandong University, Beijing Children's Hospital of Capital Medical University, Chinese Medicine Beijing Union Medical College Hospital. The main drafters of this standard. Li Yixing, Cui Fuqiang, Shao Zhujun, Li Junhong, Li Xingwang, Xu Aiqiang, Jiang Rongmeng, Zhu Bingqing, Li Hui, Gaizhong Tao, Shen Kunling, Wu Dan, Fan Hongwei. The previous versions of the standards replaced by this standard are as follows. --WS 295-2008. Diagnosis of epidemic cerebrospinal meningitis1 ScopeThis standard specifies the diagnostic basis, diagnostic principles, diagnosis and differential diagnosis of epidemic cerebrospinal meningitis. This standard is applicable to the diagnosis of epidemic cerebrospinal meningitis at various levels of medical and health institutions and their medical staff.2 AbbreviationsThe following abbreviations apply to this document. CFU/mL. colony forming unit per ml (colony-forming unit/mL) DIC. disseminated or diffuse intravascular coagulation DNA. deoxyribonucleic acid ELISA. enzyme-linked immunosorbent assay IgG. immunoglobulin G (immunoglobulin G) OD. optical density PBS. phosphate buffer saline Real-time PCR. real-time polymerase chain reaction SBA. serum bactericidal assays TTC. triphenyl tetrazolium chloride Rpm. revolutions per minute (revolutions per minute) WHO. world health organization3 diagnosis basis3.1 Epidemiological history Local occurrence or prevalence of the disease, or history of travel or travel in epidemic cerebrospinal meningitis within 10 days before onset. 3.2 Clinical manifestations 3.2.1 Incubation period A few hours to 10 days, usually 2 days to 3 days. 3.2.2 Main clinical symptoms and signs 3.2.2.1 fever, headache, vomiting, and/or meningeal irritation, infants and young children can see the anterior hernia. Severe patients can have varying degrees A disturbance of consciousness and/or infection with toxic shock. 3.2.2.2 Defects (spots) appear on the skin and mucous membranes. Freckles can be quickly expanded into pieces. 3.3 Laboratory testing 3.3.1 Blood routine The total number of white blood cells and neutrophil counts were significantly increased. 3.3.2 Cerebrospinal fluid routine The typical change is pressure increase, the appearance is turbid rice soup or pus; the number of white blood cells is significantly increased, and the polymorphonuclear leukocytes are mainly increased; Sugar and chloride are significantly reduced and protein content is increased. However, in the early stage of the disease, only the pressure rises, the appearance is clear, and then a typical change occurs. Cerebrospinal fluid in patients with cerebral shock is usually clear, and there is no change in protein, cell number and sugar. 3.3.3 Pathogens 3.3.3.1 Defects (plaque) tissue fluid, cerebrospinal fluid smear test, Gram-negative kidney-type diplococcus can be seen in polymorphonuclear leukocytes or outside the cells. 3.3.3.2 Cerebrospinal fluid, blood, sputum (plaque) tissue fluid culture Neisseria meningitidis positive. 3.3.3.3 cerebrospinal fluid, blood, sputum (plaque) tissue fluid Neisseria meningitidis specific nucleic acid detection positive. 3.3.4 Immunology 3.3.4.1 Acute cerebrospinal fluid samples were positive for Neisseria meningitidis-specific polysaccharide antigens. 3.3.4.2 In the recovery period, the serum specificity of Neisseria meningitidis IgG antibody is 4 or more times higher than that in the acute phase.4 Diagnostic principlesSuspected cases and/or clinically diagnosed diseases based on epidemiological history and clinical manifestations and routine blood test results and/or cerebrospinal fluid findings The diagnosis of the case. Confirmation of Neisseria meningitidis pathogen or immunological test results, further pathogen clustering for cases with positive pathogen detection Diagnosis (see A.3 in Appendix A).5 diagnosis5.1 suspected cases It also complies with 3.1 and 3.2.2.1, and at the same time complies with any of 3.3.1 and 3.3.2. 5.2 Clinical diagnosis cases Meets 5.1 and complies with 3.2.2.2. 5.3 confirmed cases Meets 5.1 or 5.2 and complies with any of 3.3.3 and 3.3.4. 5.4 Clinical classification Clinical classification diagnosis is performed on the basis of clinical diagnosis or confirmed diagnosis (see Appendix B, B.3).6 differential diagnosisIt is mainly caused by meningitis caused by other bacteria, sepsis caused by other causes, and cyanosis caused by various causes. Infant It should also be differentiated from diseases such as febrile seizures (see Appendix B, B.4).7 other7.1 Carrier No clinical symptoms and signs, throat swab culture positive Neisseria meningitidis or N. meningitidis specific nucleic acid test positive. 7.2 Other meningococcal diseases After human infection with Neisseria meningitidis, most of them are asymptomatic carriers. In the case of symptoms, the clinical disease spectrum is complex and diverse, in addition to causing brain In addition to meningitis, it can also cause sepsis, pneumonia, and occasionally can cause diseases such as arthritis, myocarditis, pericarditis and endophthalmitis.Appendix A(normative appendix) Neisseria meningitidis laboratory test method A.1 Sample collection, processing and transportation A.1.1 Sample collection and processing A.1.1.1 Cerebrospinal fluid Aseptically collect at least 2 mL of cerebrospinal fluid and send it to the microbiology laboratory immediately after collection. If conditions permit, it is recommended to vaccinate at the bedside. Cerebrospinal fluid (0.1 mL ~ 0.5 mL) was inoculated on 5% sheep blood chocolate plate (hereinafter referred to as chocolate plate) or blood plate, after three-zone scribing Send to the laboratory for cultivation. After the laboratory receives the cerebrospinal fluid sample, it should be tested within 1 h. First put the sample into a sterile test tube, 2 000 rpm ~ 3 000 Centrifuge for 20 min at rpm and aspirate the supernatant. The supernatant can be used for the detection of Neisseria meningitidis specific antigen, or it can be placed at -20 °C. Other tests are performed. The precipitate should be thoroughly shaken and mixed for culture and microscopic examination. If the cerebrospinal fluid sample is less than 1 mL, it will not be performed. Centrifugation, direct plating culture and Gram stain microscopy. A.1.1.2 Blood For nucleic acid testing (eg Real-time PCR), aseptically remove.200 μL of cerebrospinal fluid sample before centrifugation, -20 ° C Save below. Aseptically collect 5 mL to 10 mL of venous blood in the acute phase of the patient and immediately send it to the microbiology laboratory for processing and testing. build Inoculate at the bedside, immediately inject a proper amount of blood (5 mL to 10 mL) into the blood culture bottle, or inoculate 0.5 mL of blood directly into the chocolate. Tablet or blood plate. The remaining blood sample is used for Neisseria meningitidis specific nucleic acid detection or IgG antibody detection. Collecting patients with acute onset And in the recovery period, double blood samples, serum were separated, and N. meningitidis-specific IgG antibodies were detected. Should not be used during blood sample collection Anticoagulant. It is recommended to collect 1 part of each blood sample at the same time before the anti-infective treatment, and a total of 2 blood samples are cultured. Newborns take 1 blood sample Product. A.1.1.3 Defects or freckles Select fresh spots or ecchymoses on the patient's skin, first disinfect with iodophor, then wipe the iodophor with 70% alcohol until the alcohol is completely evaporated. Use a sterile needle to break through, extrude the tissue fluid, directly draw the tissue fluid smear with the disinfected slide, Gram stain microscopy; then use a sterile cotton swab The tissue fluid is inoculated on a chocolate plate or a blood plate, and the three-zone is streaked and sent to the laboratory for culture. A.1.2 Delivery of samples Neisseria meningitidis is sensitive to temperature, avoiding exposure of the sample to sunlight, high temperature or cold environment, and the temperature is too low or too high. The pathogen is dead. When transporting samples or cultures, keep samples at 25 ° C ~ 35 ° C, can not be transported at low temperature (for detection of resistance Except for samples of body and nucleic acid). A.2 Neisseria meningitidis culture and microscopy A.2.1 Cerebrospinal fluid sample culture The pellet was aspirated with a sterile capillary or micropipette tip and directly inoculated onto a chocolate plate and/or blood plate with a three-zone scribing. 5% CO2 environment, 37 ° C, culture for 24 h to 72 h, daily inspection of bacterial growth, timely transfer of suspected colonies on the plate and Identification. A.2.2 Cerebrospinal fluid sample microscopy Pipette 10 μL～20 μL of the sediment after centrifugation of the cerebrospinal fluid sample, drop it on the glass slide and spread it, and dry the slide in the biological safety cabinet. Quickly pass the flame 3 times to fix the smear, taking care to avoid burning. Crystal violet staining for 1 min, rinse the slides with running water for 1 min to remove excess Moisture; Gram iodine mordant for 1 min, rinse the slide with running water and dry; decolorize with 95% ethanol for 5 s to 10 s; counterstain with 20% dye solution s ~ 30 s, or counterstained with phenol red for 10 s ~ 15 s, rinse the slide with running water and dry or dry. Microscopic observation of the stained smear, in bright Under the lens and oil mirror, Neisseria meningitidis is located in or outside the polymorphonuclear leukocytes, which is Gram-negative and Renal-shaped. A.2.3 Separation and culture of blood samples After receiving the plate or blood culture bottle that has been inoculated with blood samples, the laboratory should immediately place it in an environment of 37 ° C and 5% CO 2 for 24 h. 72 h. If using a blood culture bottle, observe the growth of the bacteria daily, such as the growth of bacteria, aseptically take 0.5 mL to inoculate the chocolate. Plate and/or blood plate. A.2.4 Neisseria meningitidis colony morphology Neisseria meningitidis can be grown on blood plates or chocolate plates. After culturing on the blood plate for 18 h to 20 h, it is round, smooth, Wet, central bulge, clear border, gray colonies, no hemolysis, colony diameter up to 1 mm. Colonies growing on chocolate plates Large colonies, colorless or gray, opaque. A.2.5 Biochemical characteristics of Neisseria meningitidis Identification of Neisseria meningitidis was performed using an oxidase assay and a glucose metabolism assay. If the oxidase test is positive, then the glucose metabolism test Test. Neisseria meningitidis oxidizes glucose and maltose but does not oxidize lactose and sucrose. A.3 Neisseria meningitidis serological grouping A.3.1 Test method Serogroup identification of Neisseria meningitidis can be performed by serum slide agglutination. At present, Neisseria meningitidis has identified 12 different The serogroup, of which A, B, C, W, X, and Y are the six most common serogroups that cause epidemic cerebrospinal meningitis. A.3.2 Experimental operation Wipe a piece of glass with ethanol, divide the slide into multiple parts with a crayon or other marker, add at the bottom of each part 10 μL of sterile saline, pick a suitable amount of bacterial culture from a chocolate plate or blood plate with a sterile inoculating loop or needle, stick or toothpick. The culture was made into a slightly emulsion-like suspension in physiological saline for testing. Add the selected serum 10 μL or 10 to the top of each section μL saline or PBS. Mix the serum or saline with the respective bacterial suspension under light or black background, shake the slide 1 Min~2 min (time may vary depending on the serum producer). In view of biosafety, WHO recommends the use of formalin-inactivated suspension of Neisseria meningitidis instead of a physiological saline suspension of live bacteria. Formalin is a kind of Cancers should be handled with great care during storage and use, and should be performed in a biosafety cabinet. A.3.3 Reading of experimental results Serum-separable N. meningitidis should be agglutinated only with one type of serum, without agglutination with saline or PBS. If in physiology Agglutination in saline or PBS, indicating that Neisseria meningitidis has self-coagulation, and is judged as self-coagulating bacteria; if there is no self-coagulation phenomenon, but with two or more The serum is agglutinated and is judged to be a polycoa. It cannot be agglutinated with any serum or normal saline or PBS, and is judged to be non-coagulable; All cases can be reported as "serum-inseparable Neisseria meningitidis". A.4 Real-time PCR diagnosis of epidemic cerebrospinal meningitis A.4.1 species-specific Real-time PCR detection A.4.1.1 ctrA gene detection Most Neisseria meningitidis contains the ctrA gene (capsular transport gene), and a few strains will delete the ctrA gene into non-segregable bacteria. Strain. Invasive cases of Neisseria meningitidis are mostly caused by infection with capsules. When detecting samples of aseptic parts such as cerebrospinal fluid and blood, Real-time PCR was performed using the ctrA gene as a target gene. The ctrA gene Real-time PCR primers and probe sequences are as follows. ctrA-F. 5'-TGTGTTCCGCTATACGCCATT-3' ctrA-R. 5'-GCCATATTCACACGATATACC-3' ctrA-Pb. 5'-(FAM)AACCTTGAGCAA"T"CCATTTATCCTGACGTTCT (SpC6) -3' The "T" marks the quenching group BHQ1. A.4.1.2 sodC gene detection Neisseria meningitidis contains sodC gene (copper-zinc superoxide dismutase gene), and other species of bacteria such as Haemophilus also include There is a sodC gene. If you consider the non-segregable Neisseria meningitidis can also cause disease by accident, detect the sodC gene, its primers and probe sequences. The columns are as follows. sodC-F. 5'-GCACACTTAGGTGATTTACCTGCAT-3' sodC-R. 5'-CCACCCGTGTGGATCATAATAGA-3' sodC-Pb. 5'-(FAM)CATGATGGCACAGCAACAAATCCTGTTT(BHQ1)-3' A.4.2 serogroup-specific Real-time PCR detection A sample positive for ctrA or sodC gene can be judged to be Neisseria meningitidis infection, and serogroup-specific gene detection is required to determine Neisseria meningitidis serogroup. The corresponding primers and probe sequences are shown in Table A.1. Table A.1 Neisseria meningitidis serogroup identification Real-time PCR primers and probe sequences Serogroup detection gene primer/probe sequence (5'-3') A sacB F. AAAATTCAATGGGTATATCACGAAGA R.ATATGGTGCAAGCTGGTTTCAATAG Pb.FAM-CTAAAAG"T"AGGAAGGGCACTTTGTGGCATAAT-SpC6 B synD F.GCTACCCCATTTCAGATGATTTGT R. ACCAGCCGAGGGTTTATTTCTAC Pb.FAM-AAGAGATGGGYAACAAC"T"ATGTAATGTCTTTATTT-SpC6 C synE F. CCCTGAGTATGCGAAAAAAATT R.TGCTAATCCCGCCTGAATG Pb.FAM-TTTCAATGC"T"AATGAATACCACCGTTTTTTTGC-SpC6 W synG F. TATTTATGGAAGGCATGGTGTATG R. TTGCCATTCCAGAAATATCACC Pb.FAM-AAATA"T"GGAGCGAATGATTACAGTAACTATAATGAA-SpC6 X xcbB F.TGTCCCCAACCGTTTATTGG R. TGCTGCTATCATAGCCGCC Pb. FAM-TGTTTGCCCACATGAATGGCGG-BHQ1 Y synF F.TCCGAGCAGGAAATTTATGAGAATAC R.TTGCTAAAATCATTCGCTCCATAT Pb.FAM-TATGGTG"T"ACGATATCCCTATCCTTGCCTATAAT-SpC6 Note. The "T" marks the quenching group BHQ1. A.4.3 DNA processing of clinical samples Extraction of Neisseria meningitidis from clinical samples such as cerebrospinal fluid, blood, and sputum (plaque) tissue fluid using a commercially available DNA extraction kit DNA, direct boiling is not recommended. Depending on the type and amount of clinical samples, and the likelihood of other Gram-positive infections, When extracting the pre-treatment of clinical sample DNA, it is recommended to add lysozyme and lysozyme. A.4.4 Real-time PCR reaction system and reaction conditions The PCR reaction system is 20 μL, 10 μL of 2×PCR reaction mixture, 2 μL of upstream and downstream primers and probes (upstream primers of ctrA) The final concentrations of the probes were 0.9 μmol/L, 0.9 μmol/L, and 0.1 μmol/L, respectively. The final concentrations of the sodC primers and probes were 0.3. Μmol/L, 0.6 μmol/L, 0.1 μmol/L, the final concentration of the primers and probes were 0.6 μmol/L, 0.6 μmol/L, 0.1 Molmol/L), according to the requirements of the fluorescence PCR instrument to determine whether to add reference fluorescence Rox and the amount of addition, DNA template 2 μL, ultrapure water is added to 20 μL. Reaction conditions. annealing temperature 60 ° C, a total of 50 cycles; other reaction conditions according to the description of the 2 × PCR reaction mixture used The book is adjusted. A.4.5 Judging the results of Real-time PCR detection The amplification plot should be a smooth S-shaped curve. If the shape of the curve changes, it should be judged as negative or retested. Manually dragging the threshold line The Ct value was read after going up to the baseline, and the test results were judged according to the following criteria. "Positive". Ct value ≤ 36; "negative". Ct value ≥ 40; "suspicious". Ct value between 36 and 40 The Ct value is between 36 and 40. The result is suspicious and should be retested. There are two ways to retest. a) Repeat the test by diluting the template 4 to 10 times and replacing the 2 μL template with 4 μL. 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