Establishment Of Real Time Allele Specific Locked Nucleic Acid Quantitative PCR For Detection Of HBV YIDD Mutation And Evaluation Of Its Clinical Application

Objectives1. To establish real time allele specific locked nucleic acid quantitative PCR(RT-AS-LNA-qPCR) for detection of HBV YIDD (ATT) mutation and to evaluate itsperformance and primary clinical application.2. To monitor the dynamic changes of quantitative hepatitis B surface antigenconcentration in HBeAg-positive treatment-naive CHB patients and to investigate itspotential role in predicting virological response to entecavir therapy.Methods1. Establishment of RT-AS-LNA-qPCR for detection of HBV YIDD (ATT)mutation1.1Establishment of RT-AS-LNA-qPCRThe present study included PCR reaction system, amplification conditions andpreparation of standard plasmids for calibration curves, etc.1.2Methodological evaluation of RT-AS-LNA-qPCR1×1010copies/μl~1×101copies/μl recombinant plasmidsincludingpMD-18-YMDD andpMD-18-YIDD were tested byRT-AS-LNA-qPCR. Themethodological evaluation of RT-AS-LNA-qPCR included linear range test, detectionlimit test, specificity test, accuracy test, reproducibility test and sensitivity test, etc.1.3Methodological comparisonThe NAs-experienced patients’ sera were parallel analyzed byRT-AS-LNA-qPCR and direct sequencing.Statistical analysis such as Cohen’s Kappatest and Pearson’s correlation analysis, etc. were performed to comparetheir performance.Clinical samples containing minor variants detected by our established assaywere analyzed by cloning sequencing to test the accuracy of RT-AS-LNA-qPCR.1.4The evaluation of RT-AS-LNA-qPCR for clinical applicationClinical samples containing different proportions of rtM204I were analyzed byRT-AS-LNA-qPCR. The minimum proportion of mutant which could be accuratelyand quantitatively measured was regarded as the sensitivity of the assay of clinicalsamples. The sensitivity of the assay in detection of clinical samples was evaluated.The serum samples from a chronic hepatitis B (CHB) patient firstly receivedLMV mono therapy and then switched to LMV+ADV combined therapy were alsodynamically analyzed for10times to evaluate the value of clinical application.2. Quantitative hepatitis B surface antigen in prediction of response totreatment-naive, hepatitis B e antigen-positive patients receiving Entecavir2.1Patients and tests26HBeAg-positive treatment-na ve CHB patients receiving ETV (0.5mg once aday) were consecutively recruited and followed up for1year from the Center of Liverdiseases of the First Affiliated Hospital of Fujian Medical University between January2011and July2013.Serums were collected at baseline and every3month (month3, month6, month9and month12, respectively.).HBsAg and HBeAg concentrations were measured using commercially availablechemiluminescence assay. HBV DNA were determined by real-time polymerase chainreaction. ALT levels were measured by velocity method.2.2Receiver operating characteristic curve (ROC) analysisROC analysis was used to explore quantitative hepatitis B surface antigen inprediction of response to treatment-naive, hepatitis B e antigen-positive patientsreceiving Entecavir and to find the best cut-off valuefor the HBsAg level for theprediction of virological response to ETV. Results1.Establishment of RT-AS-LNA-qPCR for detection of HBV YIDD (ATT)mutation.1.1Successfully establishedRT-AS-LNA-qPCRRT-AS-LNA-qPCR could significantly improvethe sensitivity and specificity ofthe assay for detection of HBV YIDD mutation.RT-AS-LNA-qPCRcould detect minorvariants which were not detectable via direct sequencing.1.2Methodological evaluation of RT-AS-LNA-qPCRThe linear range of RT-AS-LNA-qPCR was between1×109copies/μl and1×102copies/μl. The low detection limit was1×101copies/μl. The intra-run andinter-runcoefficient of variation (CV) was between0.29%~2.72%. Sensitivity of theassay were10-6,10-4and10-2in the wild-type background of1×109copies/μl,1×107copies/μl and1×105copies/μl, respectively.1.3Methodological comparisonThe complete coincidence rate between RT-AS-LNA-qPCR and directsequencing was91.2%(93/102), partial coincidence rate was8.8%(9/102), and nocomplete discordance was observed (0/102). The two assays showed a highconcordance (Kappa=0.676, P=0.000).The established assaywas consistent with the cloning sequencing finding.1.4Evaluation of RT-AS-LNA-qPCR for clinical applicationThe sensitivity of the assay in detection of clinical samples was0.03%.RT-AS-LNA-qPCR was a high sensitive assay which could applied to early detectHBV drug-resistance mutants in clinic.2. Quantitative hepatitis B surface antigen in prediction of response totreatment-naive, hepatitis B e antigen-positive patients receiving Entecavir2.1Results of quantitative detection of ALT, HBV DNA and HBsAgAfter1year of treatment,17patients achieved VR,9patients didn’t. Baselinecharacteristics of the patients were as follows: The VR+group was lower in ALTlevels than VR-group, but the difference was not statistically significant (141.82± 77.29IU/ml and134.2±49.76IU/ml, respectively, t=0.27, P=0.793). HBV DNAlevel of VR+group was significantly lower than the VR-group (6.76±1.00lg IU/mland7.65±0.87lg IU/ml, respectively, t=-2.27, P=0.033).HBsAg concentration of the VR+group was lower than the VR-group, but thedifference was not statistically significant (3.79±0.61lg IU/ml and4.19±0.43lgIU/ml, respectively, t=-1.75P=0.094). A rapid HBsAg decline during the first3months of therapy followed by a much slower decline in the subsequent period wasobserved. From baseline to month3, the reductions of HBsAg were0.32±0.29and0.14±0.10lg IU/ml in VR+and VR-groups (t=2.245, P=0.035), respectively.2.2ROC analysisThe lg HBsAg concentration at month3showed the highest area under the curve(AUC) value (AUC=0.840, P=0.005). The best cut-off value for the HBsAgconcentration at month3for the prediction of VR was3.8650lg IU/ml, with adiagnostic sensitivity and specificity of82.4%and77.8%, respectively.Conclusions1. A novel assay named RT-AS-LNA-qPCR for detection of HBV YIDD mutationwas successfully established. The assay had advantages over direct sequencing whichcould applied widely in clinical laboratories with wide linear range, high sensitive,specific, reproducibility, reliable and low detection limit, etc.2. HBsAg level less than or equal to3.8650lg IU/ml at3months of ETV treatmentmay be a useful parameter in predicting virological response to ETV inHBeAg-positive CHB patients.