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Influenza diagnostic criteria and processing principles are the criteria for the diagnosis and treatment of influenza.

Influenza diagnostic criteria and management principles

Influenza diagnostic criteria and treatment principles are diagnosis and treatment

This standard specifies the diagnostic criteria and management principles for influenza.

This standard applies to the diagnosis, reporting, and treatment of influenza at various levels of medical, health, and health care institutions and personnel.

During an influenza epidemic, a preliminary diagnosis can be made based on clinical symptoms and epidemiology. If the diagnosis is confirmed, it is necessary to isolate the virus-positive or the patient's duplicate serum antibodies, and the antibody in the recovery phase is measured to be 4 times or more higher than that in the acute phase.

2.1, emerged from chills,

4.1,

Epidemiological history

During the epidemic season, a large number of patients with upper respiratory tract infections appear in one unit or area at the same time; or there has been a significant increase in patients with upper respiratory tract infections in the area or nearby areas in the near future;

Etiological diagnosis

A1 virus isolation

Specimens were collected from the respiratory tract of a suspected flu patient to isolate the pathogen.

A2 Specimen Collection and Processing

A2.1 The time for specimen collection should be as early as possible (within 1 to 3 days).

A2.2 Specimen collection methods are usually cotton swab wiping or throat rinsing. The cotton swab wipe method is used to collect children's specimens. When collecting, sterilize the cotton swabs slightly with the specimen collection solution (pH 7.2 7.6 containing 20% 40% sterilized broth saline or Hanks solution or saline ), Repeatedly wipe the patient's pharynx several times, and then put the cotton swab into a test tube filled with the collection solution and stopper. The throat washing method is used to collect adult specimens. First, the patient is allowed to cough, and then the throat is repeatedly washed with about 10 mL of collection solution for about 1 minute, and spit into the tube. If possible, nasopharyngeal secretions can be extracted by negative pressure suction in children's specimens, and nasopharyngeal lotion in adult specimens can improve the positive rate of separation. Specimens should be sent to the laboratory as soon as possible after being collected (4 ° C).

A2.3 Specimen treatment: Use a capillary pipette to repeatedly blow the specimen solution (if it is a throat swab specimen, first squeeze the throat swab repeatedly and discard it), disperse the mucus, and let it stand at 4 ° C for 5 to 10 minutes. After the precipitation, 3 mL of the supernatant was taken, and 1,000 units of penicillin and 1000 g of streptomycin were added per milliliter, and they were mixed and treated at 4 ° C for 2 to 4 hours to inoculate. If the sample is heavily polluted, it can be treated at 4 ° C overnight. If the specimen is not expected to be inoculated within 48 hours, the specimen should be stored at a low temperature (preferably -70 ° C).

A3 Inoculation and culture method

The most commonly used method is chicken embryo culture and tissue culture. If conditions permit, virus isolation can be performed by both methods.

A3.1 Chicken embryo culture method: Take 9 to 11-day-old chicken embryos, inoculate the treated specimen solution with 0.2 mL of amniotic cavity and allantoic cavity, inoculate 3 to 4 embryos per specimen, and set at 33 to 35 ° Incubate in the incubator for 3 days, and then place the chicken embryo in a refrigerator at 4 ° C overnight (or set it at -20 ° C for 1h and then at 4 ° C for several hours). Urine and amniotic fluid can be harvested separately and used for preliminary hemagglutination. 1 to 2 drops of urine or amniotic fluid, placed in a large-well plastic plate, and then added 1 to 2 drops of 1% chicken red blood cells, shake and set the temperature at room temperature or 4 for 30 to 45 minutes to observe the results, according to the degree of blood cell aggregation ++++, +++, ++, +, ±,-), if the blood coagulation is +++ ++++, then determine the blood coagulation titer by serial dilution, if it has a certain blood The titer can be identified. If hemagglutination is negative, it can be transmitted blindly for 2 generations, and if it is still negative, discard it.

A3.2 Tissue culture method: 0.2mL treated sample solution is used to inoculate monolayer primary human embryonic kidney (or monkey kidney or hamster kidney), or dog kidney passage cells (Madin Darby Canine Kidney, MDCK) Inoculate 4 tubes of each sample, set the sample solution at 37 ° C for 1 to 2 hours, discard the sample solution, add 1 mL of 199 maintenance solution containing 2 g of trypsin per milliliter, incubate at 33 ° C for 7 to 10 days, and observe the lesions daily. From the third day, every other day, use 0.4% chicken or guinea pig red blood cells to perform a red blood cell adsorption test on the culture tube (aseptically harvest the cell culture supernatant before the test). If the result is negative, pass the harvested supernatant blindly, such as If positive, the hemagglutination titer of the supernatant was measured. If the first-generation red blood cell adsorption test of the specimen is negative, the entire supernatant collected can be used for blind passage for 2 passages. If it is still negative, discard it.

Identification of A4 new isolates

Newly isolated influenza viruses can be identified using test methods such as hemagglutination inhibition, complement binding, neutralization, and blood cell adsorption inhibition. The most common and simplest method is the hemagglutination inhibition test, and complement complement testing is used if necessary.

A4.1 Hemagglutination inhibition test: Hemagglutination inhibition test is performed with the immune sera of the current popular influenza A, influenza A and B virus representative strains and newly isolated influenza viruses to observe whether the new isolates can be suppressed by the immune sera. The method of hemagglutination inhibition test is shown in Appendix B.

A4.2 Erythrocyte adsorption inhibition test: Take the tissue culture tube and normal cell control tube that are positive for the erythrocyte adsorption test, wash the cells twice with Hanks solution, and add 0.2 mL of immune serum treated with cholera filtrate (RDE) (1:10). Then add 0.6mL Hanks, and after 30 minutes at room temperature, add 0.2mL of 0.4% chicken (or guinea pig) red blood cells, and set it at room temperature for 30 minutes to observe the presence or absence of blood cell adsorption under a common light microscope. If blood cell adsorption is inhibited by the immune sera used, the isolated virus is consistent with or close to the type of immune sera used.

A4.3 Complement binding test: If the immune sera of known influenza A (A3, A1) or influenza B or C viruses cannot inhibit hemagglutination of a newly isolated virus, it should be considered as possible influenza A New subtypes of the virus can be determined by using complement sera against the nuclear proteins of influenza A and B viruses as complement complement tests.

Serological diagnosis

B1 Hemagglutination inhibition test method

B1.1 Principle

Some animal red blood cells (such as chickens, guinea pigs, etc.) and human "O" type red blood cells have influenza virus receptors. In the event of influenza virus, it can cause agglutination of red blood cells, referred to as hemagglutination. If specific antibodies and influenza virus (hemagglutinin) are added in advance to red blood cells, no agglutination occurs, which is called hemagglutination inhibition. After quantifying hemagglutinin with serum antibodies of different dilutions, the highest dilution that can completely inhibit hemagglutination is the titer of hemagglutination inhibiting antibodies.

B1.2 Main materials

Large-well plastic plate, 1 mL pipette or 1 mL pipette.

B1.3 Collection and processing of duplicate serum

The serum in the acute phase was taken within 3 days of the onset, and the serum in the recovery phase was taken 2 to 4 weeks after the onset. Take 0.1mL of separated serum and add 0.9 (or 0.4) mL of cholera filtrate (RDE) (diluted 1:10 or 1: 5), shake well, and remove the non-specific inhibin in the serum overnight at 37 ° C. The next day was placed in a 56 ° C water bath for 50 min to inactivate excess RDE.

B1.4 Preparation of influenza virus hemagglutinin

Allantoic fluid harvested 48 hours after influenza virus inoculation to chicken embryos was added with 1/10000 thimerosal to preserve. In order to delay the precipitation of urine salt, first make an equal dilution with sterile normal saline and set it at 4 for later use.

B1.5 Preparation of chicken red blood cell suspension

Normal healthy chicken blood was drawn from the vein or heart, stored in Alsevr's solution, and stored at 4 ° C. Wash 3 times with physiological saline before use, and centrifuge at 2000 r / min for 10 min at the last time, and red blood cells were made into 1% concentration with physiological saline.

B1.6 Hemagglutinin titration

Dilute influenza virus hemagglutinin in a large-well plastic plate with physiological saline starting from 1: 5. Make 0.25mL of each well and add the same amount of 1% chicken red blood cells. After the observation, the highest dilution of hemagglutinin with ten hemagglutination was taken as its hemagglutination titer, that is, 1 hemagglutination unit, and 4 hemagglutinin units were used in the experiment, for example, the hemagglutinin titer was 1 : 320, is 1 unit, 4 units is 1:80 dilution. Before the formal experiment, 4 hemagglutination units were taken for proofreading.

B1.7 Hemagglutination inhibition test procedure

B1.7.1 Dilute the above-mentioned processed test serum (1:10 or 1: 5) with physiological saline to make a serial multiple dilution, and add 0.25mL to each well.

B1.7.2 Add 4 units of hemagglutinin in the same amount.

B1.7.3 Add 0.25mL of 1% chicken erythrocytes to each well, and shake to room temperature or 4 for 30 45min.

B1.8 Observation of results

When observing the results, tilt the hemagglutination plate for tens of seconds, and read the results when the red blood cells in the negative wells slide freely into a teardrop shape. The reciprocal of the highest dilution of the serum in which complete inhibition occurred was used as the antibody titer. It is necessary to perform the same test in the detection of the patient's double serum, and the serum antibody titer in the recovery phase is 4 times or more than that in the acute phase to be positive.

B2 specific complement binding assay

B2.1 Principle

The three influenza A, B, and C viruses each have specific soluble antigens (nucleoprotein antigens), and each subtype of influenza A virus has the same soluble antigen, which is completely different from the soluble antigens of type B or C, so it can be Use complement binding tests to distinguish them. When a pandemic is caused by a large mutation or a new subtype of the influenza virus, because it is too late to fully grasp the properties of the newly isolated strain, it is often necessary to use a complement binding test and a hemagglutination inhibition test to serologically diagnose the new strain. .

Preparation of type B2.2 specific immune serum

B2.2.1 Antigens for immunization

Type A PR8 strain and Type B Lee strain were mouse adaptive strains. 12 to 16 g of mice were infected with nasal cavity under ether anesthesia, 0.05 mL per mouse, and 3 to 5 days after infection, when most of the mice became sick and died, the mice were dissected. Lungs were ground and made into a 10% suspension with normal saline, and coarse blocks were removed by low-speed centrifugation. The supernatant of the mouse lung suspension was taken as the antigen for immunization.

B2.2.2 Preparation of immune serum

Select healthy guinea pigs weighing 300-500g, collect 3 5mL of blood before exemption, and separate and sera the frozen serum for comparison. Guinea pigs are gently anesthetized with ether, and 0.5 mL of the above-mentioned mouse lung suspension antigen is dripped into the nasal cavity, and a total of 3 to 4 times of immunizations are made, with an interval of one week at the same time as the last immunization. If the serum titer of the isotype urine membrane antigen exceeds 1:80 and there is no cross-reaction with the isotype antigen, immediately collect blood to separate the serum and store it at low temperature (below -20 ° C). Before the test, the serum was inactivated in a water bath at 56 ° C for 30 minutes.

B2.3 Preparation of soluble antigen

Allantoic fluid is routinely inoculated and harvested with influenza A or B virus or newly isolated virus. If urine hemagglutination is above 1:40, the allantoic membrane is harvested, the allantoic membrane is washed once with saline, and sterile Scissors cut the membrane into small pieces and put them into test tubes. Add 1 mL of sterile normal saline to the allantoic membrane of each chicken embryo, and thaw three times. After the last thawing, centrifuge at 3000 r / min for 15 min. The supernatant is aspirated as soluble antigen. Add 1: 10000 thimerosal for antisepsis and put it in a refrigerator at 4 for at least one month.

B2.4 Duplicate serum collection

Sera were collected from patients during the acute phase (within 3 days of onset) and during the recovery phase (after 10-30 days of onset). The serum was inactivated at 56 ° C for 30 min before the test.

B2.5 The preparation of 1% sheep red blood cell suspension, the preparation and titration of hemolysin, complement and conventional methods.

B2.6 Complement binding test procedure

Proceed as usual.

B2.7 Judgment of results

B2.7.1 A positive reaction between the urinary membrane antigen of the tested virus and the immune sera of influenza A (or B) viruses can be diagnosed as influenza A (or B) viruses. If both A and B are negative, C should be considered. Influenza virus or other virus.

B2.7.2 If the double sera have a 4-fold or more increase in A (or B) antigen, it can be diagnosed as an A (or B) influenza virus infection.

B2.7.3 The complement-binding antibody titer of the normal population is generally below 1:16. If the serum antibody in a single recovery period is above 1:32, it can be used as a reference for diagnosis in combination with clinical symptoms.

B2.7.4 In the survey of serum antibody levels in the population, those with complement-binding antibodies above 1:32 can be used as evidence of recent influenza infection.

B3 neuraminidase inhibition test

B3.1 Principle

After Fetuin substrate is treated with neuraminidase of influenza virus, N-acetylneuraminic acid is released, and N-acetylneuraminic acid is converted to -formaldehyde lactone acid by oxidation of iodate. The latter is formed by thiobarbituric acid to form a chromophore. The chromophore is extracted with an organic solvent and colorimetrically determined on a spectrophotometer. By using the above reaction, the neuraminidase activity of the influenza virus can be measured, and the darker the reaction color, the stronger the enzyme activity. And choose the standard dose for neuraminidase inhibition test. The neuraminidase inhibition test is to incubate a standardized dose of neuraminidase (that is, a certain dilution of influenza virus allantoic fluid) and a series of diluted test serum and normal serum to determine that the serum acts on neuramin The inhibitory titer of the enzyme activity, and the neuraminidase inhibitory titer of the serum was calculated.

B3.2 Equipment and reagents

B3.2.1 Small test tube, 10mL pipette and spectrophotometer.

B3.2.2 Phosphate buffer (PBS pH5.9) and physiological saline.

B3.2.3 Fetuin substrate solution: 450-500mg of freeze-dried fetuin and 10mL of distilled water, dissolve and store at 4 ° C, and prepare it every week.

B3.2.4 Sodium periodate solution: Weigh 4.8g of sodium periodate (NaIO (subscript start) 4 (subscript end)) in 38mL of distilled water, add 62mL of orthophosphoric acid (concentrated), mix thoroughly and place in brown Store in a ground-mouth bottle at room temperature.

B3.2.5 Arsenic reagent: Weigh 10g of sodium arsenate (NaAsO (beginning of the subscript) 2 (end of the subscript)) and 7.1g of anhydrous sodium sulfate in 100mL of distilled water, add 0.3mL of concentrated sulfuric acid, and place in a glass stopper. Store in a bottle at 4 ° C.

B3.2.6 Thiobarbituric acid reagent: Weigh 1.2g of thiobarbituric acid and 14.2g of anhydrous sodium sulfate into 200mL of distilled water, heat and dissolve it in a boiling water bath, and prepare it every week.

B3.2.7 Acidified butanol reagent: Add 5 mL of concentrated hydrochloric acid to 100 mL of n-butanol, place in a brown bottle with a glass stopper, and store at room temperature.

B3.3 Neuraminidase activity determination

B3.3.1 The virus is serially diluted (1: 2 to 1:32) with physiological saline in aliquots into small test tubes, each containing 0.1mL. Neuraminidases of standard and unknown viruses should be determined in the same test.

B3.3.2 Add 0.1 mL of normal saline to each tube, and then add 0.1 mL of fetuin substrate prepared with pH5.9 PBS. The control tube should be replaced with saline, and then mixed in a 37 ° C water bath for 18 hours.

B3.3.3 Remove the test tubes from the water bath and cool them at room temperature. Add 0.1 mL of periodate reagent to each tube, mix thoroughly, and let stand at room temperature for 20 minutes (time should be accurate).

B3.3.4 Add 1 mL of arsenic reagent to each tube. As the release of iodine appears brown, shake the test tube until the brown disappears.

B3.3.5 Add 2.5mL of thiobarbitur reagent to each tube and mix thoroughly (this reagent needs to be added after boiling and dissolving), and plug the tube with a cotton plug.

B3.3.6 Immediately put the test tube in a boiling water bath and boil for 10 minutes. The red color appears, which is the manifestation of neuraminidase activity. The darker the color, the higher the enzyme activity.

B3.3.7 After cooling the test tube in ice water, remove the tampon, add 4 mL of butanol reagent, replace with a clean rubber stopper, shake the test tube vigorously by hand, and extract the color to turn it into the organic (butanol) phase.

B3.3.8 Centrifuge at 1500 r / min for 5 to 10 minutes to make the butanol layer clear and transparent. Carefully aspirate the upper layer of butanol and put it into a clean test tube for inspection.

B3.3.9 Pour the test sample from the high dilution to the low dilution into the translucent cell in sequence, and add the virus-free control tube solution to the first translucent cell, adjust the spectrophotometer wavelength to 549nm, and control The tube is adjusted to zero and the optical density of each dilution sample is read in turn.

B3.3.10 Calculation of Enzyme Unit: The dilution of the virus with an optical density of 0.45 to 0.85 is used as an enzyme unit when the enzyme activity is measured.

B3.4 Neuraminidase inhibition test

B3.4.1 To measure the enzyme activity according to the above method, select a virus dilution of 1 enzyme unit (that is, a virus dilution with an optical density of 0.45 to 0.85 when measuring the enzyme activity), and add 0.1 mL to each tube.

B3.4.2 The serum to be tested is serially diluted. Each tube of virus is added with 0.1 mL of serum of different dilutions. The serum control tube is the lowest dilution of serum plus physiological saline instead of virus. Normal serum control group was made in the same way.

B3.4.3 Add 0.1mL of fetuin prepared in pH5.9 PBS to each tube, shake well, and incubate in a 37 ° C water bath for 18h.

B3.4.4 Take out the test tube from the water bath, and add the aforementioned reagents gradually according to B3.3.3. B3.3.7.

B3.4.5 When measuring the optical density, it should be from the low dilution serum tube to the high dilution serum tube. The detection method is the same as B3.3.9.

B3.5 Calculation method for neuraminidase inhibitor antibody titer

B3.5.1 According to the experimental results of the two tests of antiserum and normal serum, find the quotient of the optical density of the same dilution of each serum, which is the percentage of the remaining enzyme activity after the action of serum. The higher the serum dilution, the higher the residual enzyme activity. Find out the two serum dilutions before and after 50% enzyme activity and their corresponding enzyme activity percentages, and calculate the serum neuraminidase inhibitor antibody titer according to the formula.

B3.5.2 Calculation formula

In the formula: A-the reciprocal of the titer of the serum neuraminidase inhibitory antibody;

B (beginning of subscript) 1 (end of subscript)-reciprocal of serum dilution with enzyme activity above 50%;

B (beginning of subscript) 2 (end of subscript)-reciprocal of serum dilution with enzyme activity below 50%;

C (beginning of subscript) 1 (end of subscript)-the percentage of enzyme activity higher than 50%;

C (beginning of subscript) 2 (end of subscript)-percentage of enzyme activity below 50%;

50-constant.

B3.5.3 Calculation example

First calculate the enzyme activity after serum action:

Then convert logarithmic dilution to geometric dilution: for example, 10 (beginning of superscript)-3 (end of superscript) is converted to 1/1000, 10 (start of superscript)-3.5 (end of superscript) is converted to 1/3200 . Finally plug into the formula:

The neuraminidase inhibitory titer of this antiserum is 1: 1440.

Rapid flu diagnosis

C1 direct examination of airway epithelial intracellular antigen

C1.1 Principle

Because the main infection site of influenza virus is in the respiratory epithelial cells, the exfoliated cells of the patient's respiratory specimens contain the influenza virus antigen, which can be diagnosed by directly examining the antigen in the exfoliated epithelial cells. The inspection method can adopt immunofluorescence method, which can be completed within 3 to 4 hours, and can be directly shaped.

C1.2 Collection and production of specimens

Children's specimens are best collected by negative pressure suction, and adult specimens are best with nasopharyngeal lotion. Each specimen can be divided into two, one cryopreserved for virus isolation, one with pH7.2 phosphate buffered saline (PBS), the mucus is blown apart, and the supernatant is removed by centrifugation at 1500r / min for 15min. Wash with PBS 1 or 2 times, discard the supernatant after the last centrifugation, add an appropriate amount of pH 7.2 PBS to resuspend the cells, add dropwise to the circled glass slide, dry naturally, and fix with cold acetone for 10 min.

C1.3 Indirect immunofluorescence

C1.3.1 The specimens are sealed with 10% fresh sheep or bovine serum and placed in a wet box at 37 ° C for 30 min.

C1.3.2 Add appropriate dilutions of anti-A and B specific monoclonal antibodies, and place them in a wet box at 37 ° C for 1 h. Wash twice in PBS for 10 min.

C1.3.3 Add rabbit anti-mouse conjugate with appropriate dilution to 37 ° C for 1h. Wash three times in PBS for a total of 10 minutes, and pass the last time with distilled water.

C1.3.4 Observe the results under a fluorescence microscope. If more than three fluorescence-positive cells in the cytoplasm or nucleus are observed, they can be judged as positive.

C2 specimens were checked for antigen after 1 passage of sensitive cells

C2.1 Principle

Patients' respiratory tract specimens were inoculated with sensitive cells (Dog Kidney Passage Cells MDCK) to proliferate for 1 to 3 days, and then the cells were digested and dispersed to form antigen slices, and then the intracellular antigen was examined by immunofluorescence or immunoenzyme staining. Because the virus in the specimen can detect the antigen after the proliferation of sensitive cells, it can significantly improve the sensitivity of diagnosis. The antigen can be found before the appearance of cytopathies, which can be directly typed, and it is much faster than the conventional chicken embryo virus isolation method, generally 24 ~ 72h can be diagnosed. The test method can be indirect immunoenzymatic staining (C2.5) or indirect immunofluorescence (C2.4)

C2.2 Collection and processing of specimens

Pharyngeal swabs can be used for children's specimens, and negative pressure suction is preferred; throat rinses can be used for adult specimens, and nasopharyngeal lotion is preferred. Specimens are processed in accordance with A2.3 in Appendix A (Appendix to the Standard).

C2.3 Inoculation and preparation of specimens

Take 0.2 mL of the treated sample solution and inoculate it into a single layer of MDCK cells, inoculate 3 tubes per sample, set it at 37 ° C for 1 to 2 hours, discard the sample solution, and add 2 g of trypsin-containing 199 maintenance solution per ml to 1mL, incubate at 35 ° C for 24, 48, and 72h, take out one tube, collect the supernatant for later use, digest the cells with digestive juice (1 part 0.25% trypsin + 4 parts Versene), wash once with PBS, and then dropwise add Ring glass slides, dried naturally and fixed with cold acetone. Normal control cells were obtained by preparing uninfected normal MDCK cell sheets in the same way.

C2.4 Indirect immunofluorescence

C2.4.1 plus monoclonal antibody is the same as C1.3.2.

C2.4.2 Add rabbit anti-mouse fluorescein conjugate as C1.3.3.

C2.4.3 Observe the results under a fluorescence microscope and observe that the fluorescence in the cytoplasm or nucleus is observed, but the normal cell sheet does not see fluorescence, which can be judged as positive.

C2.5 Indirect immunoenzyme staining

C2.5.1 plus monoclonal antibody is the same as C1.3.2.

C2.5.2 Add sheep anti-mouse enzyme conjugate with appropriate dilution, place in a wet box at 37 ° C for 1 h, and wash twice with PBS for 10 min.

C2.5.3 Add o-phenylenediamine substrate solution, and its preparation method is: 4 mg of o-phenylenediamine + 10 mL of pH 5.0 phosphoric acid-citrate buffer solution + 15 L of hydrogen peroxide. The preparation method of pH5.0 phosphate buffer solution is: first prepare 0.1mol / L citric acid (C (subscript start) 6 (subscript end) H (subscript start) 8 (subscript end) O (subscript start) ) 7 (End of subscript) · H (Begin of subscript) 2 (End of subscript) O) (21g / 1000mL) and 0.1mol / L disodium hydrogen phosphate (Na (Begin of subscript) 2 (End of subscript) HPO (Beginning of the subscript) 4 (End of the subscript)) (28.4g / 1000mL), after mixing according to 24.3mL and 25.7mL, and then adding an equal amount (50mL) of double-distilled water and mixing to obtain pH5.0 phosphate-citric acid Buffer. Add the substrate solution for 3-5 minutes to observe the results.

C2.5.4 Observe the results under an inverted microscope (when observing the results, the cells must be wet, if the cells are dry, you can add a drop of tap water), brown-red deep-stained cells are observed, and the number of deep-stained cells is recorded as +++ +, +++, ++, +, ±,-. It is considered positive if the deep-stained cells above + are observed; sometimes it is observed that a single deep-stained cell is surrounded by colorless or light red cells, while the normal cell control is colorless or light red, which can also be determined as positive.