| BackgroundHepatitis C is a major blood borne disease caused by virus(HCV),which is a global epidemic.The incidence of occult,atypical symptoms,if not diagnosed early,easy to miss the best time of treatment,which poses a serious threat to the health of the masses.At present,the preventive vaccine has not been successfully developed to control the spread of hepatitis C,hepatitis C improve treatment effect mainly depends on the timely and accurate diagnosis laboratory.The main methods of laboratory detection include anti-HCV detection,HCV antigen detection,HCV RNA detection and HCV genotype detectionHCV antibody was born in the early 1990s,is the earliest method for HCV laboratory diagnosis,has the advantages of simple operation,short time and other advantages,is widely used in the screening of blood products and clinical diagnosis.Usually in clinical diagnosis of advanced first The screening test for antibody detection,the samples results showed reactive,it need to further recombinant immunoblot assay(RIBA)or nucleic acid detection test;Detection of HCV antigen is a novel detection index of HCV developed in recent years,patients with hepatitis C virus infection in after 1-4 weeks can detect HCV antigen,diagnosis significantly shorten the window period,conducive to the early diagnosis of hepatitis C,antigen detection can be used for the screening of blood donors,judgment and prognostic evaluation of the efficacy of antiviral therapy and in immunocompromised patients such as HIV infection,long-term kidney dialysis patients,the diagnosis of hepatitis C patients with organ transplantation or innate immune deficiencies,and identification of HCV infection or current infection,As a new detection method,it can make up for the deficiency of antibody detection method,and does not need special equipment,simple operation,short time;HCV RNA at 1-2 weeks after infection can be detected,the HCV RNA is HCV in vivo activities of the most direct indicators,is conducive to the early diagnosis of HCV infection,is of great significance for the treatment and prognosis of the disease,usually detection of HCV using fluorescence quantitative method of viral load;there is a close relationship between HCV genotype and disease progression and response to interferon treatment,different genotype on response to antiviral treatment,different treatment taken,there is a certain effect on HCV RNA quantitative results,genotype determination helps determine the antiviral treatment and drug dose.Object1 To build HCV antibody serum panel,HCV antigen serum panel,HCV nucleic acid serum panel and HC V genotype serum panel,the assessment of antibody,antigen,nucleic acid and genotyping diagnostic kits for overall performance,The sensitivity,specificity,analytical sensitivity and analytical specificity of the testing kits will be evaluated.2 The clinical samples are detected antibody,nucleic acid and genotype,analyze the relationship between nucleic acid detection results and ELISA,RIBA,and analyze the distribution of RIBA of each band,explore the relationship between age,gender,and viral load,to discuss the relationship between the distribution of genotype and other areas,gender,age,viral load.MethodsThe first part is evaluation methodology:from the national reference laboratory sample inventory selected serum samples,to build HCV antibody serum panel,HCV antigen seru.m panel,HCV nucleic acid serum panel and HCV genotype serum panel,various indexes of serum panels constructed include reference lab basic serum panel,reference lab analytical specificity serum panel,reference lab characteristic serum panel,reference lab linear dilution serum panel,reference lab sensitivity serum panel,and then select the antibody,antigen,nucleic acid and genotyping diagnostic kits are detected serum panels,statistical analysis of test results,The sensitivity,specificity,analytical sensitivity and analytical specificity of the testing kits will be analyzed.The second part is the diagnosis of clinical samples,which is collected from 1597 clinical samples of HCV infection in high risk population,the background information of samples were collected,and then the first part evaluates the better performance of kit for detecting antibody,nucleic acid and gene type,first diagnose antibody using ELISA kit for screening test,screening test show positive samples,then using RIBA detection and quantitative detection of nucleic acid,The positive of nucleic acid were genotyped using diagnostic kit for HCV genotyping,the samples with uncertain genotype sample are by direct sequencing to detect gene types,and various indicators of detection for statistical analysis,compare the clinical significance between them.Results1 participate in the evaluation of serum antibody panel of two kinds of domestic antibody diagnose kit and an imported antibody detection kit,the detection sensitivity is 50/50,specificity is between 48/50-50/50;antibody seroconversion panel test results for the liquidation of import kit than domestic two kinds of antibody reagents prolong the average days of small tips in early discovery the ability of infection,the imported reagent has more advantages than the two domestic reagents;analysis of specific serum antibody interference panel test results for three kinds of antibody detection kit were 45/45 for the detection of abnormal samples without false positive results;the test results for linearity serum panel C manufacturer kit detection linear the sensitivity of serum dilution panel than the other two kits more sensitive.The evaluation of two kinds of domestic kit antigen specificity and sensitivity for HCV antigen basic serum panel.The results show that the sensitivity of B and A manufacturers were 9/50,16/50,specificity are 48/50,47/50,illustrate the performance of domestic antigen kit has yet to be improved,is not a substitute for clinical diagnosis.The nucleic acid for HCV nucleic acid basic serum panel showed specificity for 50/50,the sensitivity of 49/50 to a low concentration of nucleic acid sample check phenomenon;evaluation and analysis of domestic linear nucleic acid kit using commercial HCV nucleic acid serum linearity panel,the result showed that there was good linear relation.The calculated regression formula is y = 1.0189x-0.1693,the correlation coefficient R2=0.9916>0.95;nucleic acid sensitivity results showed that serum panel detection limit sensitivity can be set to 250IU/ml;RNA interference serum panel results showed that the analytical specificity of 40/40;With high concentration and low concentration of virus load construction precision blood winding,coefficient of variation calculated respectively between 2.09%and 6.46%.The relationship between clinical samples were evaluated by HCV genotyping kit with direct sequencing,results showed that the number of samples is 33 copies of typed by HCV genotyping kit(33/45),which the same as 29 samples with direct sequencing results,4 genotype la samples misclassification genotype 1b,did not detect the 3a,3b,6a,6n;and Using commercial HCV genotype serum panel,to analysis the relationship between HCV genotyping kit,the direct sequencing and genotyping results expected,The results showed that the genotyping accuracy of HCV genotyping kit was 9/9,and the results were consistent with the results of the reference geotype type2 The clinical samples of 1597 high-risk groups were collected,and the number of positive samples was detected by enzyme-linked immunosorbent assay(ELISA)was 1278,and the number of nucleic acid positive samples was 1050.Among them,416 samples of screening test antibody positive samples were tested by Western blot,the results showed that 393 antibodies were positive,and the other 7 were indeterminate,and the 16 antibodies were negative.Then,400 samples of RIBA positive or indeterminate antibody were detected by the nucleic acid,the results showed that there were positive nucleic acid in the 315 samples,and there were 85 samples with negative nucleic acid.And 400 samples of RIBA positive and indeterminate,analysis of their band and the intensity distribution with RlIBA,the results show that five bands of color intensity greater than 1+,the probability of core area was 98.5%(394/400),NS3-1,79.8%(319/400),and the number of NS4 appeared at least and the probability of 28.3(113/400).Nucleic acid detection was performed on 319 samples with negative antibody test results,6 positive samples of nucleic acid were detected.The results of nucleic acid detection were analyzed by sex and age.Analysis the relationship between nucleic acid detection results and sexes,the result was no statistically significant(?2 =4.91,P=0.556),and the nucleic acids from different age groups were statistically significant(?2 =2412,P<0.01),indicating the viral nucleic acid of different ages in different levels.Genotyping was performed on 804 samples with high risk of positive nucleic acid,and the number of samples of HCV genotype was determined to be 765;the HCV genotyping diagnostic kit to determine the genotype of samples are 622,the number of samples amplified CORE region of genotypes were 104,and 39 samples have not successfully amplified sequence.The main subtypes of high-risk groups were 3b,lb,3a,6n and 6a,respectively 32.42%(248/765),24.97%(191/765),24.58%(188/765),7.06%(54/765),and 5.62%(43/765).And 4 domestic rare genotypes samples(rare genotypes including la,6u)and 15 cases of HCV mixed subtype infection,mixed subtype infection rate was 1.96%(15/765)(mixed subtype infection including lb/3a,lb/3b,lb/6a,2a/3b,2a/6a,3a/3b,3b/6a),The genotypes were mainly type 3 and type 6,with a low proportion of type 1 and type 2,and t:ype 4 and type 5 were no found.Conclusion1 The evaluation of one imported ELISA kit and two kinds of domestic ELISA kits had high sensitivity,specificity and sensitivity for detection of early infection capacity,they can meet the clinical antibody screening test required;Two kinds of HCV core antigen kit have a large degree of undetected phenomenon,the quality of the kit still needs to be improved,at present,the HCV core antigen can not be used as a substitute for nucleic acid for clinical diagnosis,and the detection of negative samples for the core antigen kit can not exclude HCV infection.The sensitivity,specificity,precision,specificity and other indexes of domestic HCV nucleic acid quantitative kit are high,the linearity is good,and it can meet the requirements of clinical diagnosis,however,the nucleic acid quantitative kit may be miss about detected in the low viral load samples,so the manufacturers need to further improve the sensitivity of low viral load detection.Detection of HCV genotyping kit for domestic rare genotypes may have misjudged phenomenon,however,the results of the detection of the common gene subtypes in China are consistent with the results of direct sequencing.Only a small number of samples have not been detected,and the direct sequencing method is needed to determine the genotype.2 The sensitivity and specificity of ELISA antibody detection kit were improved,and the coincidence rate of RIBA was higher,almost consistent with RIBA results.It is possible to consider the use of enzyme-linked immunosorbent assay for high risk population screening test,the test results of positive samples were nucleic acid testing,no need to use RIBA as a supplemental test Therefore,it is suggested that a high performance ELISA reagent and nucleic acid reagent should be combined for clinical diagnosis.The nucleic acid detection kit should choose a lower detection limit of HCV nucleic acid detection kit,for patients with nucleic acid diagnosis to provide more accurate results,especially for patients with antiviral therapy,efficacy prediction,detection are determined by the detection of the sensitive HCV RNA.RIBA bands showed that the probability of CORE region was higher,and the probability of NS4 region was the lowest.Therefore high variation zone bands with corresponding antigen by the reagent.The efficiency is low,also need to continue to strengthen research on purification and antigenicity on the expression of HCV NS4 protein.For high-risk groups of negative samples of antibody testing suggested that the negative set of nucleic acid detection,in particular,the early infection of patients with early diagnosis,early treatment,reduce the burden caused by hepatitis C disease.The genotype of HCV infection in high risk population were type3,type 6 and type lb,and the distribution of genotype was regionally different,and there was a correlation between sex and viral load and genotype distribution.This study is only a preliminary analysis of the distribution characteristics of hepatitis C genotype in high-risk population,but also need to collect more samples and further in-depth study of epidemiological knowledge.To provide a stronger basis for the prevention and treatment of HCV infection in high-risk population,and has an important guiding significance for the future development of HCV diagnostic kit and vaccine. |
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