WS/T 590-2018Diagnosis for chikungunya fever (English PDF)

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009.

This standard was drafted. Guangdong Provincial Center for Disease Control and Prevention, Guangzhou Eighth People's Hospital, China Center for Disease Control and Prevention, China Center for Disease Control and Prevention, Center for Disease Control and Prevention, Dongguan People's Hospital.

1 Scope

This standard specifies the diagnostic basis, diagnostic principles, diagnosis and differential diagnosis of Chikungunya fever.

This standard is applicable to the diagnosis of Chikungunya fever by various medical and health institutions at all levels and their medical staff.

2 Abbreviations

The following abbreviations apply to this document.

CHIKV. chikungunya virus ELISA. enzyme-linked immunosorbent assay IgG. immunoglobulin G (immunoglobulin G) IgM. immunoglobulin M (immunoglobulin M) RNA. ribonucleic acid RT-PCR. reverse transcription-polymerase chain reaction

3 Diagnosis basis

4 Diagnostic principles

Comprehensive judgment based on the patient's epidemiological history, clinical manifestations and laboratory findings. Has never happened in Chikungunya fever the area should be based on laboratory inspections.

5 Diagnosis

6 Differential diagnosis

Chikungunya fever should be associated with dengue fever, alphavirus infection, infectious erythema, post-infection arthritis (including rheumatic fever), Scarlet fever, rickettsial disease (typhus, ascariasis), measles, drug eruption, etc. See Appendix C and Appendix D.

Appendix A

(normative appendix) Chikungunya fever serological test method A.1 Enzyme-linked immunosorbent assay for chikungunya virus (CHIKV) IgM antibody (IgM antibody capture enzyme linked Immunosorbent assay A.1.1 Principle First, a 96-well plate is coated with an anti-human IgM µ-chain antibody (capture antibody), and if a CHIKV anti-IgM antibody is present in the serum to be tested, the captured antibody binds and then binds to the CHIKV antigen, and finally binds to the enzyme-labeled anti-CHIKV antibody, and the enzyme interacts with the chromogenic substrate.

A color reaction will occur with the use.

A.1.2 Materials and reagents Materials and reagents are as follows.

a) coating solution. sodium carbonate/sodium bicarbonate buffer, pH 9.6, 1.59 g Na2CO3 and 2.93 g NaHCO3 dissolved in 1 L of water;

b) lotion. phosphate buffered saline (PBS), containing 0.05% Tween 20, pH 7.2;

c) blocking solution. PBS containing 5% milk powder and 0.05% Tween 20;

d) coated antibody. anti-human IgMµ chain antibody;

e) viral antigen. inactivated purified CHIKV antigen, non-infectious;

f) cell antigen. purified uninfected virus cell culture medium;

g) enzyme-labeled antibody. horseradish peroxidase-labeled CHIKV monoclonal antibody;

h) enzyme substrate. TMB;

i) Stop solution. 2N H2SO4;

j) Clinical specimens. serum and/or cerebrospinal fluid specimens from patients with acute phase; positive and negative serum controls;

k) Equipment. flat bottom 96-well plate, plate washer, microplate reader, incubator, single-channel and multi-channel pipettes, reagent reservoir, fresh-keeping Bag, absorbent paper.

A.1.3 Detection method Follow these steps.

a) Mark the experiment number on the plate with a marker to determine the location of each clinical specimen.

b) Dilute the anti-human IgMµ chain antibody in a 1.2000 ratio with a pH 9.6 coating and add to the well of a 96-well plate at 75 µL/well.

Incubate overnight at 4 °C.

c) Pour off the antibody coating and blot the remaining liquid on the plate to blotting paper. Add.200 µL of blocking solution to each well to block the reaction plate. room Incubate for 30 min at room temperature.

d) Wash the plate 5 times with the washing machine on the automatic washer. Make sure that each well is filled with lotion for each wash.

e) The patient's serum is diluted 1.100 with a dilution, and the cerebrospinal fluid is diluted 1.10, then 50 µL/well, 2 wells each.

At the same time, the positive control serum and the negative control serum were diluted 1.100 with a diluent, and 2 wells were added for each. Incubate for 1 h in a 37 °C wet box.

Wash 5 times.

f) Dilute viral antigens and cellular antigens. Add virus antigen to 50 µL/well on the left side of each row, and 50 µL/well on the right side of each row.

The wells are added to the cell antigen. Incubate overnight at 4 °C wet box. Wash 5 times.

g) Add horseradish peroxidase-labeled monoclonal antibody at 50 µL/well, incubate for 1 h at 37 °C in a wet box, and wash plate 5 times.

h) Add 50 µL/well of TMB substrate solution to all wells, including 1 well of unfilled blank control wells. Incubate for 10 min at room temperature in the dark.

The antibody-positive wells gradually show blue.

i) Add 50 µL/well of stop solution to all wells and the blue hole will turn yellow. Put the microplate in the microplate reader, the OD value was read at a wavelength of 450 nm.

A.1.4 Judgment of results the result judgment rules are as follows.

a) If the ratio of the optical density (OD) value of the serum antigen hole of the positive control to the OD value of the cell antigen hole is >= 2.1, the detection result is effect;

b) If the ratio of the OD value of the antigen hole of the test sample to the OD value of the cell antigen hole is >= 2.1, the CHIKV in the sample to be examined can be determined.

IgM antibody is positive.

A.1.5 Meaning

The CHIKV IgM antibody is positive in the patient's serum or cerebrospinal fluid samples, indicating that the patient is newly infected with CHIKV, which can be used as a basis for case diagnosis.

A.2 Indirect immunofluorescence assay (IFA) for detection of Chikungunya virus IgG antibody A.2.1 Principle IFA is the first reaction with the specific antibody (primary antibody) in the serum to be tested and the CHIKV antigen immobilized on the slide, if stored in serum CHIKV-specific IgM and IgG antibodies bind to fluorescently labeled anti-human IgM and IgG antibodies (second antibody) under fluorescent microscopy Specific fluorescence is visible.

A.2.2 Materials and reagents Materials and reagents are as follows.

a) Equipment. 10 µL, 100 µL,.200 µL and 1 000 µL pipettes; fluorescence microscope; CHIKV antigen tablets (available Preparation of infected cells such as BHK-21, Vero or C6/36, low temperature, dry, sealed preservation); coverslips;

b) Reagents. CHIKV antibody positive control serum, negative control serum; fluorescein labeled anti-human IgG and IgM fluorescent antibody;

Patient serum samples; sample dilutions; phosphate buffer (PBS, pH 7.2); Tween 20.

A.2.3 Detection method Follow these steps.

a) remove the antigen tablet and equilibrate to room temperature;

b) If the IgG antibody is detected, first dilute the serum to be tested with the specimen dilution, starting from 1.10, and diluting to the required titer;

c) Pipette the appropriate amount of diluted specimens into each reaction zone (the amount of specimens covers all reaction zones) to avoid spillage and production Bubble

d) The antigen-bearing piece with the specimen is placed in a wet box and incubated in a 37 ° C incubator for 30 min;

e) Rinse the antigen pellet with PBS containing 2 Torr Tween 20, then rinse for 5 min;

f) Remove the antigen sheet, wipe the lotion from the back and sides of the antigen sheet with absorbent paper (do not wipe the reaction area), add appropriate amount of fluorescence Labeling anti-human IgM or IgG fluorescent antibodies for detection of IgM or IgG antibodies (fluorescent antibodies must be used before use) the book is diluted with PBS);

g) Place the antigen in a dark box protected from light and incubate in a 37 ° C incubator for 30 min;

h) Rinse the antigen pellet with PBS containing 2 Torr Tween 20, and place the antigen pellet in PBS containing 1.8 000 Evans Blue Tween 20 Rinse for 5 min;

i) Remove the antigen sheet, wipe the wash solution from the back and sides of the antigen sheet with absorbent paper (do not wipe the reaction area), seal with glycerin buffer Tablets, cover the coverslips to avoid air bubbles;

j) Observed under a fluorescence microscope.

A.2.4 Result judgment the result judgment rules are as follows.

a) The specific fluorescent particles appear yellow-green under the microscope, and if the cytoplasm of the antigen sheet exhibits uniform granular fluorescence, it can be judged.

CHIKV-specific antibodies were positive.

b) According to the brightness of fluorescent light and the proportion of positive cells in the total number of cells, the fluorescence reaction can be roughly divided into "~", those without specific fluorescence were negative. When the antibody titer is detected, the antibody titer is the highest fluorescence dilution when the specific fluorescence reaches " ".

number.

c) If the positive control shows non-specific fluorescence or the negative control shows clear fluorescence, the test is not established and repeated testing is required.

A.2.5 Meaning

During the recovery period, serum IgG antibody positive conversion, or IgG antibody titer increased 4 times or more compared with the acute phase, indicating that the patient recently infected with CHIKV, can be used as a basis for case diagnosis.

A.3 plaque reduction neutralization test for chikungunya virus neutralizing antibody A.3.1 Principle the CHIKV neutralizing antibody in serum can block the virus from adsorbing cells, while the unneutralized virus still has the ability to infect cells, which can lead to single Layer cell lesions, shedding, etc., form a localized degenerative cell area called plaque. Covering monolayer cells with reactive staining the nutrient agar of the material can indicate the amount of plaque. "Erosion" is a virus-infected cell that cannot be dyed with color after the lesion is dead.

The plaque is formed, and the cells that are not infected with the virus can be colored by the reactive dye, and the amount of plaque can be determined by color contrast. Detection of serum neutralizing antibodies when the reference strain with known plaque titer is used, it should react with the serum to be tested. The decrease of the number of plaques indicates that the serum has neutralizing activity, and after the neutralization reaction, the reciprocal of the serum dilution that causes a 50% reduction in plaque is the titer of the plaque-reducing neutralizing antibody. Plaque reduction and neutralization test for Chikungunya Virus neutralization antibody assays should be performed in a BSL-3 laboratory.

A.3.2 Materials and reagents Materials and reagents are as follows.

a) Equipment. 4 ° C refrigerator, 37 ° C water bath, carbon dioxide incubator, sterile flat bottom 6-well tissue culture plate.

b) Susceptible cells. healthy monolayer Vero cells.

c) Cell growth solution. 100 mL of growth solution containing 88 mL of Eagle's MEM solution and 1 mL of 10 000 IU/mL streptomycin solution.

1% glutamine 1 mL, fetal bovine serum 10 mL, adjusted to pH 7.2 with 7.5% sodium bicarbonate solution, and mixed before use.

d) Cell maintenance solution. 100 mL of maintenance solution containing 96 mL of Eagle's MEM solution and 1 mL of 10 000 U/mL streptomycin solution.

1% glutamine 1 mL, fetal bovine serum 2 mL, adjusted to pH 7.2 with 7.5% sodium bicarbonate solution, and mixed before use.

e) Cell Digestion. 2.5 g of trypsin is dissolved in 1 000 mL of calcium-free magnesium phosphate buffer and sterilized by filtration.

f) Virus stock solution and its titration. CHIKV is inoculated with Vero cells, and the cells have a cytopathic effect (CPE) the virus was obtained, and the supernatant was dispensed into a sterile 2 mL screw serum tube after centrifugation, and stored in a -70 ° C refrigerator as a virus stock solution.

Take a virus solution for virus plaque forming unit (PFU) titration before use. The specific steps are as follows.

1) Prepare 3 VERO cell 6-well cell culture plates that have grown into a single layer and number them;

2) Dilute the virus solution to 10-8 serially with the maintenance solution, discard the cell culture medium, and then each from the highest dilution.

Dilute the virus solution into a 6-well cell plate, add 2 wells per dilution, 0.1 mL per well, and the last two wells Control, adsorption at 37 ° C for 1 h;

3) Prepare 1% agar medium, keep it at 43 ° C, add agar medium to each cell well after virus adsorption.

3 mL per well, gently shake and mix to ensure that the agar completely covers the virus solution. After about 20 minutes, the agar is completely solidified.

The plate was placed upside down in a CO2 incubator and cultured at 37 ° C;

4) Observe the amount of plaque on a daily basis and circle the plaque with a marker on the back of the cell plate; if a virus of 10-4 dilution is inoculated the plaque of the hole cannot be counted, and there are an average of 100 plaques in the two cell wells of the 10-5 dilution virus.

The titer was 107 PFU/mL.

g) Other reagents. positive control serum, negative control serum, serum sample to be tested, bovine serum albumin (BSA), Tris pH 7.4 Buffer, agarose, neutral red.

A.3.3 Detection method Follow these steps.

a) The serum to be tested is inactivated in a water bath at 56 ° C for 30 min to remove complement and other interfering factors in the serum.

b) Dilute the virus with a maintenance solution containing 1% BSA to contain.200 plaque units (200 PFU/0.1 mL) per 0.1 mL.

c) Dilute the test serum with a maintenance solution containing 1% BSA first, then dilute it to 2.160 continuously or as needed the titer is guaranteed to be 0.1 mL per dilution.

d) Take 0.1 mL of diluted.200 PFU/0.1 mL of virus solution, add 0.1 mL of serum of different dilutions, and mix well.

After that, incubate overnight at 4 ° C or incubate for 1 h at 37 ° C.

e) Take 0.5 mL of diluted.200 PFU/0.1 mL of virus solution, add an equal amount of maintenance solution containing 1% BSA, and then mix the virus solution is diluted 10 times in a row, including. 10-1 and 10-2. The final virus content of each of these mixed dilutions is 100.

PFU/0.1 mL, 10 PFU/0.1 mL, and 1 PFU/0.1 mL, and finally back to the virus solution, and the virus back drops and virus the serum mixture was incubated under the same conditions.

f) After the incubation, the monolayer of cell culture medium in the 6-well plate is aspirated, and then the virus-serum mixture is added, 0.1 mL per well.

A virus dilution for virus back-drip was added to another cell culture plate, two wells were added for each dilution, and adsorption was carried out for 1 h at 37 °C.

g) Prepare a 1% agar or agarose medium containing neutral red, heat to completely dissolve it, and place it in a 43 ° C water bath to keep it in a liquid state.

The nutrient agar formulation containing neutral red dye is. 1% agar and 0.4% neutral red in the maintenance solution.

h) Add the above 1% agar medium to the virus serum reaction well and the virus back to the well, add 3 mL per well, gently shake and mix Evenly, make sure the agar completely covers the virus-serum mixture.

i) After about 20 minutes, the agar is completely solidified, and the plate is placed upside down in a CO2 incubator and cultured at 37 °C. Observation ecg Spot size and circle the plaque with a marker on the inverted plate.

A.3.4 Judgment of results the result judgment rules are as follows.

a) The virus back drop test is to determine whether the amount of virus added is appropriate, calculate the number of plaques in the virus back drop test, and calculate parallel holes.

The average number of plaques in the test, the plaque amount in the test hole is calculated only when the plaque amount is between 30 and 100, and finally the neutralization resistance is calculated.

Body titer.

b) positive serum control is established, the antibody titer is determined within the range of the next dilution of the known antibody titer;

According to the wells, the results of the control cells were normal and the results were reliable. Only 90% of the test holes have a reduction in the amount of plaque.

It can be determined that an immune neutralization reaction has occurred. For example, reverse titration indicates that the known attack virus titer is 100 PFU/0.1 mL, then A serum dilution of <= 10 plaques is the end point.

c) The reciprocal of the highest serum dilution that neutralizes the virus is the plaque-reducing neutralizing antibody titer.

A.3.5 Meaning

The serum antibody titer in the recovery period is 4 times or more than the acute phase or the antibody is positive, indicating that the patient has recently been infected with CHIKV.

The basis for the diagnosis of the case.

Appendix B

(normative appendix) Chikungunya fever pathogen detection method B.1 Chikungunya virus isolation and identification (cell culture) B.1.1 Principle Chikungunya virus (CHIKV) can infect mosquito-derived C6/36 cells and African green monkey kidney cells (Vero) and mammalian cells such as golden hamster kidney cells (BHK21) and cause cytopathic effect (CPE). Cell culture isolate the presence or absence of CHIKV can be identified by a specific immunofluorescence assay or a specific nucleic acid amplification assay.

B.1.2 Materials and reagents Materials and reagents are as follows.

a) Equipment. carbon dioxide incubator, biological safety cabinet, inverted microscope;

b) susceptible cells. C6/36 or Vero or BHK21;

c) Cell digestive juice. 2.5 g of trypsin is dissolved in 1 000 mL of calcium-free magnesium phosphate buffer, and filtered and sterilized;

d) Cell growth solution. 100 mL of growth solution containing 88 mL of Eagle's MEM solution and 1 mL of 10 000 IU/mL streptomycin solution.

1% glutamine 1 mL, fetal bovine serum 10 mL, adjusted to pH 7.2 with 7.5% sodium bicarbonate solution, mixed before use;

e) Cell maintenance solution. 96 mL of maintenance solution containing Eagle's MEM solution 96 mL, 10 000 U/mL streptomycin solution 1 mL, 1% glutamine 1 mL, fetal bovine serum 2 mL, adjusted to pH 7.2 with 7.5% sodium bicarbonate solution, and mixed before use.

B.1.3 Detection method (taking Vero cells as an example) Follow these steps.

a) grow to a single layer of cell tube, discard the culture solution, add.200 µL of diluted serum sample solution, and use serum for each serum sample.

The maintenance solution was diluted 1.10, 1.100, two tubes were inoculated for each dilution, and adsorbed in a 5% CO2 incubator at 37 ° C for 1 h (C6/36) the cells were adsorbed at 28 ° C for 1 h);

b) After adsorption for 1 h, aspirate the sample solution in the cell tube, add 1 mL of cell maintenance solution, and set a normal cell control, set 5% CO2 Incubate at 37 °C (C6/36 cells at 28 °C) for 7 days in the incubator, observe whether CPE appears every day; if it does not appear after 7 days CPE, then need to pass blindly for 3 generations.

B.1.4 Result judgment CHIKV-infected Vero cells can exhibit typical CPE, which is characterized by cell shedding, necrosis, and fragmentation. But identify the virus Separation success requires identification of isolates or diseased cells by virus-specific RT-PCR or indirect immunofluorescence assays.

B.1.5 Meaning

The separation of CHIKV from the patient's blood sample can be used as a basis for case diagnosis.

B.2 Chikungunya virus nucleic acid detection B.2.1 TaqMan probe real-time fluorescence reverse transcription polymerase chain reaction (Real-time RT-PCR) B.2.1.1 Principle Viral RNA produces cDNA under the action of reverse transcriptase and serves as a template for polymerase chain (PCR) amplification reactions. TaqMan probe real time in the case of real-time PCR, a pair of virus-specific primers and a fluorophore-labeled virus-specific probe are added to the reaction system. TaqMan the two ends of the probe are labeled with a fluorescent reporter group and a quenching group, respectively. When the probe is intact, the fluorescent signal emitted by the reporter group is quenched.

The group absorbs and thus the fluorescent signal is not detected. During PCR amplification, probes and primers bind to the target sequence as the nascent strand follows when the cDNA template is extended to the fluorescently labeled probe binding site, the Taq enzyme functions as a 5'->3' exonuclease, and the fluorescent reporter group the quenching group is separated and the fluorophore releases the fluorescent signal. Each time the template is amplified, a free fluorescent molecule is generated, using fluorescence.

Signal accumulation monitors the entire PCR process in real time, and finally quantifies the unknown template through a standard curve. The RNA reverse transcription process can be Simultaneously with the PCR amplification reaction, reverse transcription reaction may be performed first and then PCR amplification may be performed. Other similar mechanisms of fluorescent probes can also be used for the nucleus Acid detection.

B.2.1.2 Materials and reagents Materials and reagents are as follows.

a) Equipment. pipettes (1 mL,.200 µL, 100 µL, 20 µL, 10 µL, and 2 µL) and matching tips; centrifuge tubes (1.5 mL, 0.2 mL and 0.1 mL); test tube racks (5 mL, 1.5 mL, and 20 µL); high speed refrigerated centrifuge; vortex mixer;

Photometric PCR instrument, etc.;

b) Reagents. viral nucleic acid extraction kit; fluorescent quantitative RT-PCR kit; primers and probes (reference sequence is as follows).

CHIKV-FP. 5'-TTTAGCCGTAATGAGCRTCGG-3' CHIKV-RP. 5'-CCGTGTTCGGGATCACTGTTA-3' CHIKV-P. 5'-FAM-TGCCCACACTGTGA-BHQ1-3' (underlined bases require LNA modification).

B.2.1.3 Detection method Follow these steps.

a) Viral RNA extraction. the specimen to be tested is extracted ...