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Diagnosis for dengue fever
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Diagnostic criteria and principle of management of dengue fever
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Basic data
| Standard ID | WS 216-2018 (WS216-2018) |
| Description (Translated English) | Diagnosis for dengue fever |
| Sector / Industry | Health Industry Standard |
| Classification of Chinese Standard | C59 |
| Word Count Estimation | 25,251 |
| Date of Issue | 2018-03-06 |
| Date of Implementation | 2018-08-01 |
| Older Standard (superseded by this standard) | WS 216-2008 |
| Regulation (derived from) | State-Health-Communication (2018) 4 |
| Issuing agency(ies) | National Health Commission |
WS 216-2018: Diagnosis for dengue fever
---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 dengue fever
ICS 11.020
C 59
WS
People's Republic of China Health Industry Standard
Replacing WS 216-2008
Dengue diagnosis
Published on.2018-03-06
2018-08-01 implementation
National Health and Family Planning Commission of the People's Republic of China
Content
Foreword...II
1 Scope...1
2 Terms and definitions...1
3 Diagnostic basis...1
4 Diagnostic principles... 2
5 diagnosis...2
6 differential diagnosis... 2
Appendix A (Normative Appendix) Method of dengue serological detection...3
Appendix B (Normative Appendix) Methods for detecting dengue pathogens...10
Appendix C (informative) Differential diagnosis of dengue...16
Appendix D (informative) The etiology, epidemiology and clinical manifestations of dengue...18
References...21
Foreword
Chapter 5 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 216-2008 "Diagnosis Diagnostic Criteria".
Compared with WS 216-2008, the main technical changes of this standard are as follows.
-- Revised the scope of application (see Chapter 1, Chapter 1 of the.2008 edition);
- Terms and definitions removed the beam arm test, adding severe dengue fever and NS1 antigen (see Chapter 2, Chapter 2 of the.2008 edition);
-- Revised clinical performance (see Chapter 3, 3.2 of the.2008 edition);
- Revised laboratory tests (see Chapter 3, 3.3 of the.2008 edition);
- Removed the "blood concentration; hypoalbuminemia" indicator (see 3.3.3 of the.2008 edition);
- Added "positive detection of dengue virus NS1 antigen within 5 days of onset" (see Chapter 3, 3.3 of.2008);
- Revised suspected cases (see Chapter 5, 5.1 of the.2008 edition);
-- Revised clinically diagnosed cases (see Chapter 5, 5.2 of the.2008 edition);
- Revised confirmed cases (see Chapter 5, 5.3 of the.2008 edition);
- increased severe cases (see Chapter 5, 5.4 of the.2008 edition);
- Adjusted the order of Appendix A and Appendix B;
-- Appendix A adds the method of "enzyme-linked immunosorbent assay for detection of dengue virus NS1 antigen" and deletes the original "hemagglutination inhibition (HI) test
Method for testing dengue virus hemagglutination inhibitory antibodies" (see Appendix A and Appendix B, Appendix A and Appendix B of the.2008 edition);
-- Revised the contents of Appendix D to replace the original "dengue hemorrhagic fever" and "dengue shock syndrome" with "severe dengue fever".
Indications for high-risk populations of severe dengue fever and early identification of critically ill cases are included (see Appendix D, Chapter 5, Appendix D of the.2008 edition).
This standard was drafted. Guangdong Provincial Center for Disease Control and Prevention, Guangzhou Eighth People's Hospital, China Center for Disease Control and Prevention, viral disease
Prevention and Control Institute, China Center for Disease Control and Prevention, Beijing Ditan Hospital, Capital Medical University, China Academy of Military Medical Sciences, Microbial Flow
Institute of Diseases.
The main drafters of this standard. He Jianfeng, Zhang Fuchun, Wang Shiwen, Yin Wenwu, Li Jiandong, Li Xingwang, Qin Chengfeng, Wang Jian, Wu De,
Deng Aiping.
The previous versions of the standards replaced by this standard are.
--WS 216-2001;
--WS 216-2008.
Dengue diagnosis
1 Scope
This standard specifies the diagnostic basis, diagnostic principles, diagnosis and differential diagnosis of dengue fever.
This standard applies to all types of medical and health institutions and their medical staff for the diagnosis of dengue cases.
2 Terms and definitions
The following terms and definitions apply to this document.
2.1
Severe dengue fever severe
A serious type of dengue, clinical manifestations of severe bleeding, shock, severe organ damage.
2.2
NS1 antigen NS1 antigen
A glycoprotein in a non-structural protein of dengue virus, which is abundantly present on the surface of infected cells and can be used as a specific reference for early diagnosis.
Standard.
3 diagnosis basis
3.1 Epidemiological history
Within 14 days before the onset of illness, dengue fever cases have occurred in dengue fever epidemic areas, or within 1 month of living places or workplaces.
3.2 Clinical manifestations
3.2.1 acute onset, sudden high fever, obvious fatigue, anorexia, nausea, etc., often accompanied by more severe headache, eyelid pain, body muscle pain,
Symptoms such as bone and joint pain, may be accompanied by facial, neck, chest flushing, conjunctival congestion and so on.
3.2.2 Rash. On the 3rd to 6th day of the disease, a congestive rash or a spotted rash occurs in the limbs of the face. Typical rash is seen in the limbs
Needle-like bleeding points and "Pice Island"-like performance. The rash is distributed on the torso or head and face of the limbs. It is often itchy and does not desquamate. Lasting 3 days~
5 d.
3.2.3 bleeding tendency. some patients may have different degrees of bleeding, such as subcutaneous hemorrhage, injection site defect, gum bleeding, nose
衄 and beam arm test positive and so on.
3.2.4 severe bleeding. subcutaneous hematoma, gross hematuria, digestive tract, chest and abdomen, vagina, intracranial and other parts of the bleeding.
3.2.5 Severe organ damage. acute myocarditis, acute respiratory distress syndrome, acute liver injury, acute renal insufficiency, central nervous system
System damage and other performance.
3.2.6 shock. tachycardia, wet end of the limbs, prolonged capillary filling time > 3s, weak pulse or undetectable, reduced pulse pressure difference or
Blood pressure can not be measured.
3.3 Laboratory inspection
3.3.1 Reduced white blood cell count and/or thrombocytopenia.
3.3.2 Dengue virus IgM antibody positive (see A.1, A.2 in Appendix A).
3.3.3 Positive detection of dengue virus NS1 antigen within 5 days of onset (see A.3).
3.3.4 The titer of serum-specific IgG antibody in the recovery period of dengue virus is 4 times or more higher than that in the acute phase or positive (see A.4, A.5).
3.3.5 Dengue virus is isolated from blood, cerebrospinal fluid or tissue of patients with acute phase (see B.1, B.2 in Appendix B).
3.3.6 Detection of dengue virus nucleic acid by RT-PCR or real-time fluorescent quantitative RT-PCR (see B.3, B.4).
4 Diagnostic principles
Comprehensive judgment based on the patient's epidemiological evidence, clinical manifestations and laboratory findings.
5 diagnosis
5.1 suspected cases
A suspected case can be diagnosed if one of the following is true.
a) Compliance with 3.1 and 3.2.1 at the same time.
b) Comply with 3.2.1 and 3.3.1 at the same time.
5.2 Clinical diagnosis cases
One of the following can be diagnosed as a clinically diagnosed case.
a) Comply with 5.1a) and comply with any of 3.2.2, 3.2.3 and 3.3.1.
b) Comply with 5.1 and comply with any of 3.3.2 and 3.3.3.
5.3 confirmed cases
It can be diagnosed as a confirmed case if it meets 5.1 or 5.2 and meets any of 3.3.4, 3.3.5 and 3.3.6.
5.4 severe dengue fever
Conforms to 5.2 or 5.3, and at the same time meets any of 3.2.4, 3.2.5, 3.2.6 to diagnose severe dengue fever.
6 differential diagnosis
Dengue fever should be differentiated from measles, rubella, scarlet fever, influenza, Chikungunya fever, Zika virus disease; severe dengue fever should be
Identification of leptospirosis, hemorrhagic fever with renal syndrome, and tsutsugamushi. See Appendix C and Appendix D.
AA
Appendix A
(normative appendix)
Dengue fever serological test method
A.1 Detection of dengue virus IgM antibody by IgM capture enzyme-linked immunosorbent assay (Mac-ELISA)
A.1.1 Principle
According to the principle of antigen-antibody specific binding, the anti-human μ chain monoclonal antibody is used to capture IgM in the serum to be detected, and then add specific
The antigen and the corresponding enzyme-labeled monoclonal antibody are added to the substrate for color development. The degree of color development was positively correlated with the specific IgM antibody content.
A.1.2 Materials and reagents
The required materials and reagents are as follows.
a) Washing machine, microplate reader, thermostat or water bath (37 °C ± 2 °C);
b) 10μL ~ 100μL adjustable pipette, 10mL straw;
c) test tubes and absorbent paper for dilution of serum;
d) distilled or deionized water;
e) Dengue virus IgM antibody capture ELISA diagnostic kit.
A.1.3 Detection method
Specific steps are as follows.
a) in a series of test tubes, the negative, positive control serum, the critical value is compared with the quasi serum and the test serum to 1.100;
b) aspirate the required amount of purified dengue virus antigen and an equal volume of horseradish peroxidase-labeled monoclonal antibody to another clean glass
In a bottle or test tube, mix and set at room temperature for 1 h;
c) After mixing the labeled monoclonal antibody and antigen, draw 100 μL of diluted patient specimen and control serum into each 10 min.
The corresponding micropores on the test plate were applied at 37 ° C for 1 h;
d) Discard the serum, wash it repeatedly 6 times with the washing solution, buckle the blotting paper, add 100 μL of the above-mentioned dengue virus antigen
And the enzyme-labeled monoclonal antibody complex, at 37 ° C for 1 h;
e) Discard the dengue virus antigen and the enzyme-labeled monoclonal antibody complex, wash it repeatedly 6 times with the washing solution, buckle the absorbent paper, and add in each well.
100 μL of TMB (tetramethylbenzidine), after standing at room temperature for 10 min to fully visualize blue, add 100 μL of termination to each well.
Liquid, mix;
f) Read the absorbance of each well at a wavelength of 450 nm in 30 min.
A.1.4 Judgment of results
A.1.4.1 Determination of the results of the microplate reader
The judgment criterion is. in the case of NC/CO< 1 and PC/CO>1, if S/CO< 0.9, the result is negative, and if S/CO>1.1, the knot is
If the result is positive, if S/CO = 0.9 to 1.1, the specimen should be redone.
Note. S is the absorbance of the serum to be tested; NC is the absorbance of the serum of the negative control; PC is the absorbance of the serum of the positive control; CO is the absorbance of the critical serum
Average value.
A.1.4.2 Visual inspection
Before the stop solution is added, if the positive control serum is dark blue and the negative control serum is colorless, if the color ratio of the sample well is critical
The positive serum depth was positive, and the color of the sample well was negative compared with the critical serum.
A.1.5 Meaning
IgM antibody positive, indicating that the patient is newly infected with dengue virus, suitable for early diagnosis of dengue fever.
A.2 Detection of dengue virus IgM antibody by indirect enzyme-linked immunosorbent assay (ELISA)
A.2.1 Principle
According to the principle of specific binding of antigen-antibody, the antigen-coated plastic plate is expressed by the purified dengue virus gene engineering, and the diluted sample is to be inspected.
Specific antibody binding in serum, wherein the serum IgM moiety binds to the enzyme-labeled anti-human IgM added later, through the action of the enzyme and the substrate
A visible color response was produced and the degree of color development was positively correlated with the specific IgM antibody content.
A.2.2 Materials and reagents
The required materials and reagents are as follows.
a) Washing machine, microplate reader, thermostat or water bath (37 °C ± 2 °C);
b) 10μL ~ 100μL adjustable pipette, 10mL straw;
c) test tubes and absorbent paper for dilution of serum;
d) distilled or deionized water;
e) Dengue virus IgM antibody enzyme-linked immunoassay kit.
A.2.3 Detection steps
Specific steps are as follows.
a) Dilute the test serum with a sample dilution of 1.50. (First add 10 μL of serum to 90 μL of sample dilution)
Evenly, mix 1.10 diluted serum with the sample dilution and mix well 1.5. Specimens should be used within 2 hours after dilution. )
b) Add the concentrated washing solution to 720 mL (96T) distilled water and mix for use.
c) Remove the coated plate, add 100 μL/well of diluted serum, and set 2 holes for each of the negative, positive control and blank control (yin and yang)
The control control wells were directly added to the corresponding control serum 100 μL/well, and the blank control was added to the washing solution 100 μL/well).
After homogenization, incubate in a 37 ° C water bath for 40 min.
d) After incubation, remove the liquid from the plate, fill each well with washing solution, let stand for 1 min, dry, repeat washing 5 times, and buckle.
e) Add 50 μL/well of enzyme label (no blank), shake well, and incubate in a 37 ° C water bath for 30 min.
f) After incubation, remove the liquid from the plate, fill each well with washing solution, let stand for 1 min, dry, repeat washing 5 times, and buckle.
g) Add 50 μL of substrate A and B to each well, and incubate at 37 ° C for 10 min in the dark, then add stop solution 50 μL/well and measure at 450 nm.
OD value.
A.2.4 Result judgment
Cut-off = 0.2 mean value of negative control (calculated as 0.05 at N < 0.05).
The OD value of the sample is greater than the critical value, and the OD value of the sample is less than the critical value. The OD value of the sample is within the range of plus or minus 10% of the critical value.
Doubt, it is recommended to try again with other methods.
A.2.5 Meaning
IgM antibody positive, indicating that the patient is newly infected with dengue virus, suitable for early diagnosis of dengue fever.
A.3 Method for detecting dengue virus NS1 antigen by enzyme-linked immunosorbent assay
A.3.1 Principle
A monoclonal antibody against dengue virus is pre-coated on the microporous strip, which is compatible with the NS1 antigen in dengue virus in the serum of dengue cases
Specific binding, followed by a second incubation with horseradish peroxidase-labeled anti-NS1 antibody reagent, when dengue virus NS1 antigen is present in the sample
A "coated antibody-NS1-enzyme-labeled antibody" complex will be formed. Adding a color developer, a horseradish peroxidase-catalyzed display agent attached to the complex
The reaction produces a blue product which, after termination of the reaction, turns yellow and does not show if there is no dengue virus NS1 antigen in the sample.
A.3.2 Materials
10μL, 100μL,.200μL, 1mL pipette each; one thermostat, one plate washer, one microplate reader. Dilution serum
Test tube, absorbent paper; distilled water or deionized water; dengue virus NS1 antigen enzyme-linked immunoassay kit.
A.3.3 Detection steps
Specific steps are as follows.
a) Take the kit out of the refrigerator, let it stand at room temperature for 30 minutes, mix gently by shaking the reagent before use;
b) dosing. dilute the concentrated washing solution in the kit with distilled water 20 times;
c) No.. The sample corresponds to the microplate number, each plate is provided with 3 holes for the negative control, 2 holes for the positive control and 1 hole for the blank control;
d) Add diluent. add 50 μL of diluent to each well, except for blank wells;
e) Adding. Add 50 μL each of the sample to be tested or the positive control of the negative in the corresponding well, except for the blank hole;
f) Incubation. after sealing with a sealing film, incubate at 37 ° C for 60 min;
g) Add 50 μL of enzyme labeling reagent to each well, except for blank wells, gently shake and mix;
h) Incubation. after sealing with a sealing film, incubate at 37 ° C for 30 min;
i) Washing the board. Carefully remove the sealing film, wash it 5 times with a washing machine, and try to buckle as much as possible;
j) Color development. Add 50 μL of each of the developer A and B solutions to each well, gently shake and mix, and develop at 37 ° C for 15 min in the dark;
k) Measurement. 50 μL of stop solution was added to each well, and the results were measured within 10 min. Set the microplate reader wavelength to 450nm [recommended to use dual wave
The detection was performed at a length of 450 nm/(600 to 650 nm), and the value of each well was measured by zeroing with a blank hole.
A.3.4 Result determination
Threshold calculation. Threshold = 0.10 Negative control well A value mean (negative control well A value below 0.05 is calculated as 0.05).
Normal range of negative control. negative control well A ≤ 0.1 (if 1 well A > 0.1 should be discarded, if two or more negative control >
0.1, the experiment should be repeated).
Normal range of positive control. A≥0.8.
Positive judgment. The sample A value ≥ critical value is positive for dengue virus antigen.
Negative judgment. The sample A value < critical value is negative for dengue virus antigen.
A.3.5 Meaning
A positive result indicates that the patient has newly developed dengue virus infection and is suitable for early diagnosis of dengue fever.
A.4 Detection of dengue virus IgG antibodies by immunofluorescence (FA/IFA)
A.4.1 Principle
Certain fluorescein (usually fluorescein isothiocyanate) binds to antibody protein molecules without loss of antibody activity, and still resists
A specific immune response occurs to produce an immune complex. This complex exhibits fluorescence under a fluorescence microscope due to the involvement of fluorescent pigments.
Indicates the presence of a specific antigen. Direct immunofluorescence can be used to detect specific antigens in infected cells, and indirect immunofluorescence can be used.
Check for specific antibodies in serum.
A.4.2 Instruments and reagents
The required instruments and reagents are as follows.
a) 10μL～100μL, one set of 100μL～1000μL adjustable pipettes;
b) Dengue virus type 1~4 antigen tablets (denture standard strain is infected with BHK, Vero or C6/36 cells, dried at low temperature)
And corresponding monoclonal antibodies (positive control), negative serum;
c) goat anti-human (rabbit anti-human) IgG fluorescent antibody;
d) the serum of the patient to be tested (acute phase and recovery period);
e) Common dilutions. pH 7.2 ~ 7.4 PBS, Evans, sealant, etc.;
f) Fluorescence microscopy.
A.4.3 Detection steps
Specific steps are as follows.
a) take out the antigen slice and blow it dry with cold air;
b) Dilute the serum to be tested with pH 7.2 ~ 7.4 PBS, starting from 1.20, and diluting to the required dilution;
c) Pipette the diluted test sera from the high dilution to the low dilution one by one in four types of dengue virus antigen slices.
In each case, add 2 wells of each sample to be tested for serial dilution of serum. The serum amount should be completely covered by the antigen surface without overflowing the pores.
Incubate for 30 min in a 37 ° C water bath (simultaneous control for each test);
d) Rinse 3 times with pH 7.2 ~ 7.4 PBS, about 30s ~ 1min each time, then wash once with distilled water, blow dry with cold air;
e) Dilute the fluorescent antibody with pH 7.2 ~ 7.4 PBS to contain 2 working units and 1.8000 Evans blue fluorescent antibody, plus
Into each well, so that the antigen surface is completely covered, placed in a wet box, 37 ° C water bath for 30 min, taken out, rinsed and blown as above;
f) Mount with a sealant (or glycerol buffer) and observe the results by fluorescence microscope.
A.4.4 Judgment of results
The specific fluorescence is yellow-green particles distributed in the infected cell cytoplasm. According to the ratio of fluorescence brightness and positive cells in the total number of cells
For example, the fluorescence reaction can be roughly classified as "~", and those without fluorescence are "-". When detecting antibody titers, specific fluorescence reaches " "
The reciprocal representation of the highest serum dilution.
A.4.5 Meaning
A positive result can only indicate that the subject may have had a dengue virus infection, but a serum antibody titer of 1.80 or above has a diagnostic reference.
Significance, if the serum antibody titer in the recovery period is 4 times or more higher than the serum antibody titer in the acute phase, the dengue virus infection can be confirmed recently.
A.5 Neutralization test (NT)
A.5.1 Principle
When the human body is infected with dengue virus, a protective neutralizing antibody can be produced in the serum. After the dengue virus interacts with this specific antibody, it can
It is specifically "neutralized" to inhibit the replication and reproduction of dengue virus, making it incapable of infecting. Neutralization tests include animal neutralization tests,
Tissue culture neutralization test and plaque reduction neutralization test, each neutralization test is divided into fixed virus dilution serum method and fixed serum dilution
There are two kinds of virus methods.
Animal neutralization experiments are difficult to achieve in general laboratories because of the need for special infections in animal rooms, so more laboratories use groups.
Woven culture neutralization test and plaque reduction neutralization test, in which the plaque reduction neutralization test is more sensitive and specific than the tissue culture neutralization test, but by
In the former, the requirements for the operation technology are higher, and the virus plaque is small, the spotting time is long, and the requirement for the cell covering medium is also high, so generally
The most common use in the laboratory is the tissue culture neutralization test.
A.5.2 Instruments and reagents
The required instruments and reagents are as follows.
a) 50μL ~.200μL, 1mL adjustable pipette,.200μL, 1mL sterile tip;
b) sterile cells blowpipes and straws;
c) sterile flat-bottom micro-well 96-well tissue culture plates;
d) sterile 1.5mL plastic centrifuge tube, ice box;
e) dengue virus type 1 to 4 standard strain, C6/36 cells, positive and negative control serum;
f) CO2 incubator, biological safety cabinet, inverted microscope;
g) Growth solution (200 mL). 174 mL of Eagle's MEM solution, 3% glutamine 2 mL, 10,000 units of streptomycin 2 mL, 7.5%
2 mL of sodium bicarbonate, 20 mL of newborn calf serum, and mix and mix before use;
h) Preparation of maintenance solution (200mL). 190mL of Eagle's MEM solution, 2mL of 3% glutamine, 2mL of 10,000 units of streptomycin,
7.5% sodium bicarbonate 2mL, 4mL of newborn calf serum, mix and mix before use.
A.5.3 Detection steps
A.5.3.1 Determination of virus titer
Specific steps are as follows.
a) Preparation and preservation of virus suspension. tissue culture virus suspension or milk of dengue virus type 1 to 4 standard strain harvested freshly.
The virus suspension prepared from the brain tissue of mice was centrifuged and precipitated, and the supernatant was aspirated. After adding 20% antibody-free calf serum, the mixture was dispensed.
In the cell cryotube, quickly put it in the refrigerator below -70 °C.
b) Virus inoculation. 1 small tube of frozen virus is taken from each type of dengue virus, rapidly dissolved in cold water, and each type of virus is treated with maintenance solution.
Dilute 10 times separately, from 10-2 to 10-7, using a sterile 1.5 mL plastic centrifuge tube on an ice box. Each dilution virus
Add 4 wells, add 50 μL per well, then add 50 μL of C6/36 cells (concentration 1×106/mL) to each well, and place CO2 incubator.
Internal culture, temperature 33 ° C, humidity 80%. The cytopathic lesions were observed every day for 7 days and the observations were recorded.
c) Calculation of TCID50. Calculate TCID50 based on cytopathic effect, see Table A.1.
Table A.1 Calculation of TCID50
Virus dilution
Number of cytopathic holes /
Number of vaccinations
Cumulative distribution
Ratio
Cytopathic
Fraction ratio
(+) hole (-) hole (+) hole (-) hole
4/4 4 0 9 0 9/9 100
3/4 3 1 5 1 5/6 83
2/4 2 2 2 3 2/5 40
0/4 0 4 0 7 0/7 0
In this example, a virus dilution that causes 50% of tissue culture plates to develop cytopathic effects is between 10-5 and 10-6, calculated as follows.
Distance ratio = (more than 50% of cytopathic percentage -50) ÷ (more than 50% of cytopathic percentage - less than 50% of cytopathic
Score) = (83-50) ÷ (83-40) = 0.7
The distance ratio plus the logarithm of the 50% cytopathic dilution is TCID50 (10
-4.7). This gives 100TCID50 of 10
-2.7.
A.5.3.2 Tissue culture neutralization test (fixed virus dilution serum method)
Specific steps are as follows.
a) Dilute the serum to be tested in series, 1.2, 1.4, 1.8, 1.16, 1.32 according to the estimated serum titer
The dilution factor was performed on a ice box using a sterile 1.5 mL plastic centrifuge tube.
b) The above-prepared virus was diluted to 100 TCID50/0.2 mL and placed on an ice box using a sterile 1.5 mL plastic centrifuge tube.
c) Mix the diluted serum and the diluted vi...
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