Comparative Study On The Efficacy Of HBV DNA Detection With Domestic Fluorescence Quantitative PCR Reagentd And COBAS Amplicor Assay

BACKGROUND AND AIMS Hepatitis B virus(HBV) is a well-known pathogen of acute and chronic hepatitis.By the report of World Health Organization (WHO),HBV has infected about 2 billion individuals worldwide and caused chronic liver diseases about 300～400 million.Of the disease of world's top 10 causes of death,hepatitis B accounted for the seventh place.Every year,about 1 million people of the world died of HBV-related hepatic failure,liver cirrhosis(LC-B) or hepatocellular carcinoma(HCC).China is a hyperepidemic area of HBV infection. The standardized positive rate of hepatitis B surface antigen(HBsAg) carriers is as high as 9.09%and the standardized prevalence rate of HBV is 50.04%among the general population in China in 2002.A report of nationwide hepatitis B seroepidemiological survey published by the Ministry of Health of China in 2006 showed the following results.The national positive rate of HBsAg carriers was 7.18%in 1～59-year-old crowd.The national positive rate of people with hepatitis B antibody was 50.09%.The annual incidence of decompensated liver cirrhosis in people with chronic Hepatitis B was about 3%.The cumulative incidence of 5-year was about 16%,and 6%to 15%in those people who might develop into HCC.Therefore,HBV is a major public health problem in China and has a substantial impact on the health of people.HBV DNA is a direct index about the activity of HBV replication and the infectivity of HBV.It is also one of the most important index of the antiviral efficacy and prognosis,It can direct the application of antiviral drugs.It is found that baseline level of HBV DNA is significantly related with liver cirrhosis.A "dose - effect" relationship between the baseline levels of HBV DNA and HCC has been observed. The persistent high HBV DNA load is one of the most important factor for the development of chronic hepatitis B(CHB).The quantitative test for HBV DNA overcomes the limitations of direct detection means,such as immunological methods. It reflects the level of virus by testing the level of viral nucleic acid,in vivo.There are two kinds of HBV DNA quantitative detection methods.One is based on polymerase chain reaction(PCR),such as real-time fluorescence quantitative PCR(FQ-PCR),and the other based on nucleic acid hybridization,such as dot-blot hybridization and branched DNA technology(bDNA).Roche COBAS Amplicor,which is approvaled by the US Food and Drug Administration(FDA),is expensive and must be dedicated by Roche PCR instrument.It is difficult to be widely used in China.Currently,the State Food and Drug Administration(SFDA)of China has approved a number of real-time FQ-PCR reagents for HBV DNA detection,but the difference between the results detected with them and COBAS Amplicor which is recognized in international scope is still unknown.The study was conducted to investigate the situation of domestic real-time FQ-PCR diagnostic kits in Shandong region and compare the efficacy of domestic FQ-PCR diagnostic kits for HBV DNA detection with COBAS Amplicor HBV DNA monitor.METHODS The domestic FQ-PCR reagents used in 32 medical institutions' laboratories in 17 cities of Shandong province from December 2007 to April 2008 were statisticsed.Reagent A was used in 34%of the medical institutions' laboratories,and reagent B 50%.Serum samples from 151 patients with chronic hepatitis B(CHB) were included according to their HBV DNA levels determined by COBAS Amplicor HBV DNA monitor(Reagent A),and were retested by FQ-PCR with domestic reagent A and B,respectively.The concordance and correlation of the results were assessed and the sensitivity and specificity of Reagent A and Reagent B were evaluated.11 serum samples were chosed from the 151 serum samples,and they were detected with reagent A and B again.The statstics were made by SPSS for windows.Diference were considered as statistically significant at a P value of＜0.05(two sides).RESULTS In 151 serum samples,103 samples had the Ct reports of all three reagents.The serum HBV DNA levels detected with COBAS Amplicor,reagent A and B were 5.87±1.64,5.11±1.34 and 4.93±1.35 log₁₀copies/mL,respectively(P＜0.05). And the HBV DNA level detected with COBAS Amplicor was higher than the other two(P＜0.05),while those with reagent A and B were comparable(P＞0.05).The correlation coefficient between results detected with COBAS Amplicor and reagent A, COBAS Amplicor and B were 0.947 and 0.937,respectively.The correlation between results detected with reagent A,B and COBAS Amplicor for low virus load samples was not as good as those for high load samples.The overall sensitivity of reagent A and B were 90.4%and 77.0%,and the overall specificity were 56.3%and 100%, respectively.There was no difference between the two results of the 11 serum specimens。CONCLUSIONS Both of the domestic FQ-PCR reagents A and B are analysised according to the results detected with COBAS Amplicor.Although there are significant differences between the results detected with the two domestic real time FQ-PCR diagnostic kits and COBAS Amplicor,there is a good correlation between the results detected with reagent A,B and COBAS Amplicor(r＞0.85).And both of them have good stability.Reagent A,B are fairly good for the detection of serum HBV DNA,although with less reliability than COBAS Amplicor.The two reagents test results can help clinicians to make clear the level of virus replication in vivo and the effect of antiviral therapy in patients with CHB.There is a better correlation between the results detected with reagent A,B and COBAS Amplicor for the high virus load groups than for the low virus load groups.It suggestes that reagent A,B have a narrower linear range.The reliability is not good for lower virus load samples. Therefore we should consider biochemistry and immunology indicators when we monitored the effect of anti-viral therapy.When virus load is low,we should consider other test results and clinical performance in order to exclude the false positive and false negative.HBV DNA should been tested again when necessary.COBAS Amplicor is more sensitive than regent A and B.No significant difference is observed in the results detected with regent A and B.Regent A is more sensitive while regent B is more specific.A higher false positive rate is observed in the results detected with reagent A and a higher false negative rate with reagent B for a low virus load. Reagent A,B had their own characteristics.Both of the 2 domestic reagents are fairly good for the detection of serum HBV DNA,although with less reliability for lower virus load samples.Same reagent should be chosed in the course of treatment in order to compare the effect and monitor the occurrence of early drug resistance.COBAS Amplicor should be used when necessary.