This standard specifies the diagnostic criteria and treatment principles for hepatitis D. This standard is applicable to medical and health departments at all levels for the diagnosis and prevention of hepatitis D. GB 15999-1995 Diagnostic criteria and treatment principles for hepatitis D. GB15999-1995 Standard download decompression password: www.bzxz.net

GB15999—1995
Hepatitis D (hereinafter referred to as D-hepatitis) is caused by the D-hepatitis virus (HDV). HDV is a defective RNA virus. It must rely on the assistance of the hepatitis B virus (HBV) to infect the human body and can easily lead to severe hepatitis, chronic hepatitis and cirrhosis. my country is a high-incidence area for HBV infection, so HDV infection can affect and threaten many HBV-infected people, making their condition worse. Therefore, the prevention and treatment of D-hepatitis is also very important. For this reason, the formulation of the diagnostic criteria and treatment principles for D-hepatitis applicable to the whole country has far-reaching significance for the prevention and treatment of D-hepatitis.
Appendix A of this standard is the appendix of the standard.
This standard is proposed by the Ministry of Health of the People's Republic of China. The drafting units of this standard are: Beijing Medical University First Hospital, Beijing Ditan Hospital, Beijing You'an Hospital. The main drafters of this standard are: Wang Qinhuan, Xu Daozhen, Lin Xiuyu. This standard is interpreted by the Ministry of Health's technical management unit, the Office of Infectious Disease Supervision and Management of the Ministry of Health. 108
1 Scope
National Standard of the People's Republic of China
Diagnostic criteria and principlesof management of viral hepatitis D This standard specifies the diagnostic criteria and management principles of hepatitis D. This standard is applicable to the diagnosis and prevention of hepatitis D by medical and health departments at all levels. 2 Referenced standards
GB15999—1995
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard is published, the versions shown are valid. All standards will be revised, and parties using this standard should explore the possibility of using the latest version of the following standards. GB15990--1995 Diagnostic criteria and management principles of hepatitis B 3 Definitions
3.1 Coinfection
Infection with two pathogens at the same time.
3.2 Superinfection
Infection with another pathogen on the basis of infection with one pathogen. This refers to HDV infection on the basis of HBV infection. 4 Diagnostic principles
A comprehensive diagnosis must be made based on epidemiological data, clinical symptoms and signs, and laboratory tests. The diagnosis must rely on the detection of HDV infection markers in the patient's serum or liver tissue. If necessary, liver puncture must be performed, and pathological histological examination of liver tissue and immunohistochemistry or molecular hybridization methods must be used for etiological examination.
5 Diagnostic criteria
5.1 Epidemiological data
Same as hepatitis B [refer to GB159903.2.1.1a Epidemiological data of acute non-jaundice hepatitis], or those with a history of close contact with hepatitis D patients and HBsAg positive should pay more attention. 5.2 Symptoms and Signs
5.2.1 Co-infection of HDV/HBV
Most patients present with acute self-limited hepatitis. The symptoms and signs are the same as those of acute hepatitis B (refer to 3.2.1.1b) and 3.2.1.1c) of GB15990 in the diagnostic criteria for acute hepatitis B). If the patient has a biphasic increase in serum ALT and bilirubin, co-infection should be suspected. A small number of patients present with acute severe hepatitis [refer to 3.2.1.5a) 1) and 2) of GB15990 in the diagnostic criteria for acute severe hepatitis B].
5.2.2 HDV/HBV overlapping infection
For patients who were originally serum HBsAg positive (including HBsAg carriers and chronic hepatitis B patients), whose condition suddenly becomes active, or whose progressive disease develops into cirrhosis, chronic active liver or severe hepatitis should pay attention to the possibility of overlapping HDV infection. 5.3 Liver function test
Same as liver function test for acute, chronic or severe hepatitis B. Refer to 3.2.1.1d), 3.2.1.3b), 3.2.1.4c) and 3.2.1.5a) of GB15990 for liver function test for acute, chronic and severe hepatitis B. 5.4 Detection of HDV infection markers
5.4.1 Serum hepatitis D virus antigen (HDAg) see A3 in Appendix A. Intrahepatic HDAg can also be detected if necessary. 5.4.2 Blood and (or) intrahepatic HDV RNA.
5.4.3 Serum hepatitis D virus antibodies;
5.4.3.1 Anti-HD IgM see A1 in Appendix A.
5.4.3.2 Anti-HD see A2 in Appendix A.
5.5 Detection of HBV infection markers, refer to Appendix A of GB15990. Among the above 5 items, HBsAg positive in 5.5, one or more positive in 5.4 and abnormal liver function in 5.3 can be diagnosed as hepatitis D. 5.1 and 5.2 are for reference.
In 5.4 and 5.5, if the clinical and etiological diagnosis is consistent with acute hepatitis B, with one or more positive HDV infection markers, it can be diagnosed as HDV/HBV co-infection; if the clinical and etiological diagnosis is consistent with invasive hepatitis B virus infection, with one or more positive HDV infection markers, it can be diagnosed as HDV/HBV superinfection (refer to Appendix A of GB15990). 6 Principles of prevention and treatment
6.1 Same as hepatitis B (refer to Chapters 4 and 5 of GB15990). 6.2 Hepatitis D patients among hospitalized patients should be isolated to prevent the spread of HDV among HBV positive patients. 410
GB15999--1995
Appendix A
(Appendix to the standard)
Specific detection of HDV infection
This standard requires the use of ELISA to detect HDV infection markers and requires the use of diagnostic kits approved by the Ministry of Health. A1 Detection of anti-HDIgM by enzyme-linked immunosorbent assay 1.1 Principle
According to the principle of specific binding between antigen and antibody, anti-human immunoglobulin IgM (preferably anti-& chain) is used to capture IgM in the serum to be tested, and then HDAg is added to make it bind to anti-HDIgM in the serum, and then enzyme-labeled anti-HDIgG is used, which has both the characteristics of reacting with HDAg and the catalytic amplification activity of the enzyme. When the corresponding substrate is added, the enzyme oxidizes the substrate, turning the colorless substrate into a colored one, and the presence and content of anti-HDIgM are determined by the depth of color or the high or low OD value. A1.2 Materials
A1.2.1 Polystyrene plastic plate: 40 or 96 wells. A1.2.2 Variable micropipette: 20μL and 100μL. A1.2.3 Anti-human IgM (μ chain): mouse anti-human IgM (μ chain) monoclonal antibody or rabbit (sheep) anti-human IgM (μ chain) polyclonal antibody. A1.2.4 HDAg: Use crude extract of genetically engineered antigen. A1.2.5 Anti-HD IgM negative and positive control serum. A1.2.6 HRP-anti-HD IgG.
A1.2.7 Coating solution: 0.5mol/L pH 9.5~~9.6 carbonate buffer (15.9g sodium carbonate, 29.3g sodium bicarbonate), add water to 1000mL, dilute 1:10 for use.
A1.2.8 Washing solution: 0.01mol/L, pH7.2~7.4, PBST (containing 0.85% sodium chloride, 0.05% Tween20). A1.2.9 Dilution solution: 0.01mol/L, pH7.2~7.4, PBS, used to dilute serum and labeled antibodies. A1.2.10 Substrate solution is freshly prepared before use. 4mg of o-phenylenediamine (OPD) is added with 10mL of matrix solution (18.4g of phosphate and 5.1g of sodium citrate dissolved in 1000mL of deionized water, pH5.0, stored at 4℃ for later use). When ready to use, dissolve and add 50μL of 3% hydrogen peroxide, mix well and use immediately.
A1.2.11 Terminator: 2 mol/IH,SO4. A1.3 Test steps
A1.3.1 Dilute the anti-human IgM (μ chain) monoclonal antibody with 0.05mol/L pH9.5~~9.6 phosphate buffer, coat the polystyrene plate, 100uL per well at 4℃ overnight.
A7.3.2 Wash three times with PBST and pat dry.
A1.3.3 Add 100μL of 1:1000 diluted serum to be tested and positive and negative control serum (3 negative control wells, 2 positive control wells) to each well, 37℃, 1h.
A1.3.4 Wash the plate three times with PBST under negative pressure and drain. A1.3.5 Add 50~100μL of appropriately diluted HDAg2~4 ELISA units to each well and place at 45℃ for 1h. A1.3.6 After washing three times, add 50μL HRP-anti-HDIgG antibody (in PBS containing 10% calf serum) to each well, 42-45°C, 1h.
A1.3.7 After washing three times, add 50μL freshly prepared substrate solution, place in dark place at room temperature for color development for 15-30min. When the positive control turns yellow, add 1 drop (25μl.) 2mol/L sulfuric acid to each well to terminate the reaction. A1.4 Result judgment
A1.4.1 Self-test method: brown-yellow, yellow is positive; light yellow is suspicious; colorless is negative. A1.4.2 Colorimetric method: Determine the OD value at 492nm and calculate its S/N value according to formula (A1). Any S/N greater than or equal to 2.1 is positive: 11
Otherwise, it is negative.
A1.5 Significance
GB15999—1995
OD49 of the sample to be tested
Average of OD492 of the negative control
Anti-HDIgM positive indicates acute or recent HDV infection; anti-HDIgM is often transiently positive in cases of HDV/HBV co-infection; if the two are superimposed, they are often persistently positive. If anti-HBcIgM and anti-HBcIgG are detected simultaneously, it can also be used to distinguish between HDV/HBV co-infection or superimposed infection. Therefore, anti-HDIgM detection has important diagnostic value. A2 Enzyme-linked immunosorbent assay to detect anti-HD total antibodies, mainly anti-HDIgG. A2.1 PrinciplebzxZ.net
Use the specific reaction between antigen and antibody to determine the ability of antibodies in the serum to be tested to block the specific binding between enzyme-labeled anti-HD and HDAg.
A2.2 Materials
A2.2.1 Anti-HDIgG polyclonal or monoclonal antibodies are used for coating. A2.2.2 Anti-HDV positive and negative control serum. A2.2.3 HDAg, HRP-anti-HDIgG, coating solution, diluent and washing solution are the same as above. A2.3 Test steps
A2.3.1 Dilute the anti-HD antibody to an appropriate concentration with the coating solution and coat the polystyrene plate, 100μL per well, incubate at 37℃ for 1h, and incubate at 4℃ overnight. A2.3.2 Wash the plate three times under negative pressure and drain, add 50μL of HDAg to each well, and incubate at 42℃ for 2h. A2.3.3 After washing three times, add 50μL of the serum to be tested and the negative and positive control serum (set 3 wells for negative control and 2 wells for positive control) to each well, and add anti-HD-HRP at 37℃ for 1h. A2.3.4 After washing three times, add 50μL of freshly prepared substrate solution to each well and place it in the dark at room temperature for color development for 15 to 30 minutes. When the negative control develops color, add 1 drop of 2mol/L sulfuric acid to each well to terminate the reaction. A2.4 Result judgment
Visual method: colorless or light yellow is anti-HD positive; brown or yellow is anti-HD negative. Colorimetric method: Determine the OD492 of the sample to be tested and the negative control well, and calculate the blocking rate according to formula (A2). Blocking rate (%) Negative control double amount = sample ODaz× 100 Negative control OD492
Any sample with a blocking rate greater than 50% is positive, otherwise it is negative. A2.5 Significance
Anti-HD positive indicates a past HDV infection. A high titer may also indicate current infectionA3 Detection of HDAg by double antibody sandwich ELISAA3.1 Principle
HDAg is coated with HBsAg and is released only after being lysed by a detergent (Tween20 or NP40). Using the immunological principle of antigen-antibody binding, double antibody sandwich ELISA is used to detect HDAg. A3.2 Materials
A3.2.1 Anti-HD IgG purified antibody for coating. A3.2.2 Anti-HBc monoclonal antibody, ELISA titer 1:100,000, used to dilute HRP-anti-HD. A3.2.3 10% Tween 20.
A3.2.4 HRP-anti-HD and other materials are the same as above. A3.3 Test steps
GB15999—1995
A3.3.1 Dilute anti-HD IgG with coating solution, coat polystyrene plate, 100uL per well, 37C, 1h, 4℃ overnight. A3.3.2 After washing three times, add 50uL of the plate supernatant and negative and positive control serum to each well (3 wells of negative control serum, 2 wells of positive control) and 50μL of 10% Tween20, mix well and place at room temperature overnight to lyse HBsAg coated on the surface of HDV and release HDAg. After washing three times, add 50μL of HRP-anti-HD (the diluent contains 10% calf serum and 10 ELISA units of anti-HBc monoclonal antibody) to each well and place at 45℃ for 1h (or 37℃ for 2h). A3.3.3 After washing three times, add 50uL of freshly prepared substrate solution to each well and place in the dark at room temperature for 15-30 minutes for color development. Then add 25μL of 2mol/L sulfuric acid to each well to terminate the reaction.
A3.4 Results
Visual inspection: Brown or yellow indicates HDAg positive; colorless indicates negative. Colorimetric method: Determine the OD492 of the sample and negative control, calculate the S/N value according to formula (A3), and the S/N value greater than or equal to 2.1 indicates positive. Sample OD492
S/N=negative control OD value
A3.5 Significance
HDAg can exist in infected hepatocytes and blood. HDAg positivity indicates HDV replication in vivo. A3.6 Precautions
....(A3 )
A3.6.1 HDV and HBV infection coexist. After the blood sample is treated with a detergent, HDAg and HBcAg may still coexist. Therefore, attention should be paid to prevent the interference of HBcAg. Therefore, a monoclonal antibody that specifically neutralizes HBcAg must be added to the enzyme marker to exclude it. A3.6.2 Because the amount of HDAg in peripheral blood is small, at least 30~50μL of serum should be used for testing. A3.6.3 If the negative control OD value is 0, 0.05 is generally taken as the negative control value for calculation. 4135 Significance
Anti-HD positive indicates a past HDV infection. A high titer may also indicate current infectionA3 Detection of HDAg by double antibody sandwich ELISAA3.1 Principle
HDAg is coated with HBsAg and is released only after being lysed by a detergent (Tween20 or NP40). Using the immunological principle of antigen-antibody binding, double antibody sandwich ELISA is used to detect HDAg. A3.2 Materials
A3.2.1 Anti-HD IgG purified antibody for coating. A3.2.2 Anti-HBc monoclonal antibody, ELISA titer 1:100,000, used to dilute HRP-anti-HD. A3.2.3 10% Tween 20.
A3.2.4 HRP-anti-HD and other materials are the same as above. A3.3 Test steps
GB15999—1995
A3.3.1 Dilute anti-HD IgG with coating solution, coat polystyrene plate, 100uL per well, 37C, 1h, 4℃ overnight. A3.3.2 After washing three times, add 50uL of the plate supernatant and negative and positive control serum to each well (3 wells of negative control serum, 2 wells of positive control) and 50μL of 10% Tween20, mix well and place at room temperature overnight to lyse HBsAg coated on the surface of HDV and release HDAg. After washing three times, add 50μL of HRP-anti-HD (the diluent contains 10% calf serum and 10 ELISA units of anti-HBc monoclonal antibody) to each well and place at 45℃ for 1h (or 37℃ for 2h). A3.3.3 After washing three times, add 50uL of freshly prepared substrate solution to each well and place in the dark at room temperature for 15-30 minutes for color development. Then add 25μL of 2mol/L sulfuric acid to each well to terminate the reaction.
A3.4 Results
Visual inspection: Brown or yellow indicates HDAg positive; colorless indicates negative. Colorimetric method: Determine the OD492 of the sample and negative control, calculate the S/N value according to formula (A3), and the S/N value greater than or equal to 2.1 indicates positive. Sample OD492
S/N=negative control OD value
A3.5 Significance
HDAg can exist in infected hepatocytes and blood. HDAg positivity indicates HDV replication in vivo. A3.6 Precautions
....(A3 )
A3.6.1 HDV and HBV infection coexist. After the blood sample is treated with a detergent, HDAg and HBcAg may still coexist. Therefore, attention should be paid to prevent the interference of HBcAg. Therefore, a monoclonal antibody that specifically neutralizes HBcAg must be added to the enzyme marker to exclude it. A3.6.2 Because the amount of HDAg in peripheral blood is small, at least 30~50μL of serum should be used for testing. A3.6.3 If the negative control OD value is 0, 0.05 is generally taken as the negative control value for calculation. 4135 Significance
Anti-HD positive indicates a past HDV infection. A high titer may also indicate current infectionA3 Detection of HDAg by double antibody sandwich ELISAA3.1 Principle
HDAg is coated with HBsAg and is released only after being lysed by a detergent (Tween20 or NP40). Using the immunological principle of antigen-antibody binding, double antibody sandwich ELISA is used to detect HDAg. A3.2 Materials
A3.2.1 Anti-HD IgG purified antibody for coating. A3.2.2 Anti-HBc monoclonal antibody, ELISA titer 1:100,000, used to dilute HRP-anti-HD. A3.2.3 10% Tween 20.
A3.2.4 HRP-anti-HD and other materials are the same as above. A3.3 Test steps
GB15999—1995
A3.3.1 Dilute anti-HD IgG with coating solution, coat polystyrene plate, 100uL per well, 37C, 1h, 4℃ overnight. A3.3.2 After washing three times, add 50uL of the plate supernatant and negative and positive control serum to each well (3 wells of negative control serum, 2 wells of positive control) and 50μL of 10% Tween20, mix well and place at room temperature overnight to lyse HBsAg coated on the surface of HDV and release HDAg. After washing three times, add 50μL of HRP-anti-HD (the diluent contains 10% calf serum and 10 ELISA units of anti-HBc monoclonal antibody) to each well and place at 45℃ for 1h (or 37℃ for 2h). A3.3.3 After washing three times, add 50uL of freshly prepared substrate solution to each well and place in the dark at room temperature for 15-30 minutes for color development. Then add 25μL of 2mol/L sulfuric acid to each well to terminate the reaction.
A3.4 Results
Visual inspection: Brown or yellow indicates HDAg positive; colorless indicates negative. Colorimetric method: Determine the OD492 of the sample and negative control, calculate the S/N value according to formula (A3), and the S/N value greater than or equal to 2.1 indicates positive. Sample OD492
S/N=negative control OD value
A3.5 Significance
HDAg can exist in infected hepatocytes and blood. HDAg positivity indicates HDV replication in vivo. A3.6 Precautions
....(A3 )
A3.6.1 HDV and HBV infection coexist. After the blood sample is treated with a detergent, HDAg and HBcAg may still coexist. Therefore, attention should be paid to prevent the interference of HBcAg. Therefore, a monoclonal antibody that specifically neutralizes HBcAg must be added to the enzyme marker to exclude it. A3.6.2 Because the amount of HDAg in peripheral blood is small, at least 30~50μL of serum should be used for testing. A3.6.3 If the negative control OD value is 0, 0.05 is generally taken as the negative control value for calculation. 413
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