Sensitivity And Accuracy Of An Kit In Detecting Mutations Associated With Hepatitis B Antiviral Resistance Based On Pyrosequencing And Its Clinical Applications

This study consists of two parts:1. Ten common resistance mutations associated NAs standard plasmid and used to evaluate the performance of the kit. Direct polymerase chain reaction (PCR) sequencing, pyrosequencing were detected 92 HBV DNA in peripheral serum of CBH patients, clonal sequencing were performed for all resistance-positive who two results were not consistent, used to evaluate the performance of the kit.2 Sera were collected from 119 patients with AHB. Pyrosequencing was used to screen HBV reverse-transcriptase (RT) domain and clonal sequenc-ing were performed for all resistance-positive samples.Ⅰ. Sensitivity and Accuracy of a Kit in Detecting Mutations Associated with Hepatitis B Antiviral Resistance Based on PyrosequencingObjective Early detection of antiviral drug-resistant mutations enables prompt initiation of rescue therapy.The aim of this study was to determine the accuracy and sensitivity of a new line probe assay in the detection of antiviral drugresistant HBV mutations.Methods Construction consists of ten recognized NAs mutation sites related to the standard resistance plasmids and tested with kit based on mixed plasmids of different proportion. Two assays, including direct sequencing and pyrosequencing were assessed respectively.82 samples from 44 patients with virologic breakthrough or poor virologic response during entecavir, tenofovir, lamivudine or adefovir treatment and 13 samples from 13 nucleoside-naive patients were tested by direct sequencing and an pyrosequencing can detect mutations at 10 positions of the reverse transcriptase region of the HBV polymerase gene.Results 1,Plasmids were constructed successfully.2,In mixed plasmids of different proportion, Performance evaluation kit experiments and by gradient PCR mixture of different concentrations of standard plasmids tests and clinical examination to be carried out pyrosequencing serum amplified fragments showed 500-1000copes/ml above the level of the standard plasmid titer and HBV-DNA1000copes/ml titer above the level of HBV-DNA (+) serum samples were obtained effective amplification, the positive rate of 100%.Pyrosequencing of amplified products were sequenced with the Sanger method sequencing results was 100%. Different levels in the 1000copes/ml titer ratio (wild type and mutant) mixed sequencing plasmid:plasmid mutant plasmids≥10% of total time pyrosequencing method detected 100% positive rate. In 1000copes/ml degree level variant plasmid 10% of total plasmid were carried out using the kit repeated 10-20 pyrosequencing, the sequencing results was 100%.Complete concordance between Pyrosequencing and sequencing results was observed for (84/95) samples (88.4%). Testing of clonal analysis of discordant samples confirmed the presence of mutations where Pyrosequencing but not direct sequencing detected mutations. Pyrosequencing consistently detected mutations present in≥10% of the virus population when HBV DNA concentration was≥1000copes/ml copies/mL. so the research and development platform based on Pyrosequencing detected resistance mutations in HBV detection Kit is a sensitivity, accuracy, repeatability and reliable technical support Pyrosequencing detection kit.Conclusions Pyrosequencing has high concordance with direct sequencing in detecting antiviral drug-resistant mutations Developed by the Pyrosequencing-based sequencing of drug resistance mutations in HBV PCR kit Sequencing and Pyrosequencing primers for specific amplification system for gene B and C of common genotypes of HBV strains, and amplification efficiency, to ensure the detection of Pyrosequencing System specificity and sensitivity. Pyrosequencing technology platform for full and accurate test results, test sites and flexible. But its ensitivity in detecting mutations at some positions needs to be improved.Ⅱ.An Kit in Detecting Mutations Associated with Hepatitis B Antiviral Resistance Based on Pyrosequencing and Its Clinical ApplicationsObjective Pre-existing antiviral-resistanthepatitisBvirus(HBV) has been associated with primary non-response to lamivudine treatment in patients with chronic hepatitis B, but little is known of the capacity for resistant HBV to cause primary infection. The study was to investigate if patients with acute hepatitis B (AHB) are infected with drug-resistant HBV in shang hai.Methods Sera were collected from 119 patients with AHB. Direct polymerase chain reaction (PCR) sequencing and Pyrosequencing were used to screen HBV reverse-transcriptase(RT) domain and clonalsequenc- ing were performed for all resistance-positivesamples.Results Direct PCR sequencing showed that 6 samples(5.04%) were positive for drug-resistant HBV variants,comprised of 1 with rtM204I and 2 with rtM204V,3 with rtA181V;. Pyrophosphate sequencing confirmed the results of direct sequencing and 3 cases were not detected by direct sequencing of the mutant strain was detected pyrophosphate sequencing. Pyrosequencing were detected in patients infected with HBV resistant mutations in 9 cases (7.56%), including 1 with rtA181V+rtM204I,2 with rtM204I,4 with rtA181V and 2 with rtM204V. Multiple mutants coexist with wild-type strains.Conclusions:Drug-resistant HBV strains can cause acute hepatitis B in China.