| BackgroundHCV nucleic acid test is the gold standard for the diagnosis of hepatitis C,among which HCV nucleic acid qualitative test is mainly used for clinical diagnosis and blood donor screening,while HCV nucleic acid quantitative test can be used for the confirmation of present infection,treatment monitoring and prognosis judgment.In 2018,WHO recommended the use of quantitative HCV nucleic acid testing for clinical diagnosis in Guidelines for the care and treatment of persons diagnosed with chronic hepatitis C virus infection.In the same year,a quantitative HCV RNA assay with a lower limit of detection≤1000 IU/ml(3.0 log10 IU/ml)was recommended by the European Society of Hepatology for diagnosis and management of HCV in low and middle income countries,and in specific settings in high income countries,which was a weak recommendation of medium quality.In addition,the combination of different detection methods to form a testing strategy can make up for the limitations of a single detection method.At the same time,the purpose of the test,the performance of the test method and the infection rate of the tested population should be considered in the development of the test strategy.Therefore,if HCV nucleic acid quantitative detection was used for clinical diagnosis,the performance of HCV RNA quantitative detection reagent and the HCV infection rate of the population to be tested should be considered.In addition,the application of threshold value in HCV nucleic acid quantitative detection should also be considered.Objective1.To understand the analytical performance of some domestic registered reagents and put forward the clinical diagnostic testing strategy suitable for the clinical diagnosis of hepatitis C in China.2.To explore the diagnostic threshold of HCV nucleic acid quantitative detection,so as to provide data support for the revision of the Technical Specifications for Laboratory Detection of Hepatitis C Virus in China,and promote the application of HCV nucleic acid quantitative detection for clinical diagnosis in China.MethodsFour HCV nucleic acid quantitative detection reagents were used to blind test the established commercial serum panel and laboratory serum panel.The positive coincidence rate,negative coincidence rate,analytical specificity,analytical sensitivity,limit of detection,the detection rate of different genotypes and the linear correlation coefficient of the reagents were obtained.Different tesing strategies were combined with different detection methods and 2093 samples from different populations were uesd to validate different testing strategy.The positive detection rate and positive missed detection rate of different testing strategies were calculated,and the cost-benefit analysis was carried out.In addition,the application of diagnostic thresholds was determined according to the distribution of HCV viral load.ResultsThe positive coincidence rate and negative coincidence rate of four HCV RNA quantitative detection reagents were 20/20 and the analysis specificity of common clinical interference samples were 40/40 and HCV 1-6 genotype can be detected by them.In addition,they had the same analytical sensitivity,with positive detection rates of 92.9%and average prolongation days of 15.5 for samples of commercial seroconversion panel,respectively.The results of commercial and laboratory linearity panel showed that the linear correlation coefficients(r)of reagent A,B,C and D were 0.998,0.9989;1.000,0.9403;0.996,0.991 and 0.996,0.9932(P<0.01),respectively.Total CV%,intra-assay CV%and inter-assay CV%of four reagents were less than 5%at different concentration levels,in which intra-assay CV%of reagent A was the smallest.The minmum total CV%and intra-assay CV%at P1(105IU/ml)and P2(104IU/ml)levels were found in reagent D(1.74%,1.78%)and reagent A(2.67%,2.22%).The limit of detection of reagent A,B,C and D were 25IU/ml,50IU/ml,50IU/ml,50IU/ml,respectively.The positive rate of anti-HCV in the total population was 17.73%,and the viral load was mainly distributed between 105IU/ml-107IU/ml(48.09%).When the sample Ct value was 38,the area under the ROC curve reached the maximum,and the corresponding load value was 2.685 log10 IU/ml.The two-step strategy A cost 33,299 yuan in total,the positive detection rate of HCV in the total population was 94.94%(225/237),and the missed detection rate was 5.06%(12/237).The two-step strategy B cost a total of 147,221 yuan,and the positive detection rate of HCV in the total population was 100%,but it could not distinguish the previous infected persons.The three-step method cost 43,059 yuan in total,and separated 137 cases of HCV previous infection.The positive detection rate of HCV in the total population was 94.09%(223/237),and the missed diagnosis rate of HCV was 5.91%(14/237).ConclusionsFour HCV RNA quantitative detection reagents had good positive predictive value,negative predictive value,analytical specificity,analytical sensitivity,linearity,precision,limit of detection and the ability to detect HCV 1-6 genotype,and all of them met the quality requirements of the National Medical Products Administration for registered reagents.By contrast,reagent A has better linearity,precision and lower the limit of detection.This study preliminarily explored that the diagnostic threshold of viral load of HCV infected people is 2.685 logio IU/ml,which means that if the value of viral load≥2,685 logio IU/ml,it is considered positive,otherwise negative.The two-step strategy can shorten the turnaround time,and strategy A has moderate detection cost and high positive detection rate.Two-step strategy B has the higher positive detection rate than that of strategy A,but the cost is expensive and does not distinguish between previous infections.The three-step method has a relatively long turnaround time and a certain amount of missed detection,but the cost of detection is the lowest.In addition,it can exclude the false positives in the initial screening of anti-HCV and distinguish the previous infected persons. |
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