Dr Kavita Ramanathan

Running Head: ROLE OF QUANTITATIVE HBV DNA PCR TESTS IN HEPATITIS B INFECTION 1 REVIEW OF LITERATURE TO UNDERSTAND ROLE OF QUANTITATIVE HBV DNA PCR TEST IN PATIENTS WITH HEPATITIS B INFECTION ROLE OF QUANTITATIVE HBV DNA PCR TESTS IN HEPATITIS B INFECTION 2 Hepatitis B, despite having an effective vaccine, is a major cause of chronic liver disease world-wide. Diagnosis involves screening for Hepatitis B surface antigen (HBsAg) by serology and subsequent Nucleic acid testing (NAT) for quantification of viral load and genotyping. Viral load estimation is proven to be predictive of disease stagei, response to therapyii and infectivity. Amplicor HBV Monitor on Roche has a sensitivity of 102 copies/mL, and range of 102 to 107 copies/mLiii. However, 1. End-point PCR based assays are shown to have limited dynamic range usually 4-log, 2. Comparability between values and units of reporting generated by different methods made follow-up difficult, 3. Missed mutant variants resulted in under-reporting. Real-time PCR was shown to be more accurate as it quantified the DNA during the exponential phase of the reaction and has wider dynamic rangeiv. To ensure comparability over platforms and methodology, WHO came up with the first standard for HBV DNA quantitation test. Taqman assay has a sensitivity of 10 copies/mL, range of 10 to 109 copies/mL and specificity of 100%v. In 2003, Roche launched the Cobas TaqMan Analyzer, and in 2007, the Cobas AmpliPrep / Cobas TaqMan (fully automated with sample preparation and RT-PCR), which reduced assay time and chances of contamination. The Cobas AmpliPrep / Cobas TaqMan fulfils the need for a sensitive assay, calibrated to WHO standard for genotypes A-G, with a broad measuring range. It has almost 7-log dynamic range upto 1.1E+08IU/mL with a sensitivity of 4-12 IU/mL and good and comparable detection of genotypes A-G along with prevalent pre-core mutantvi. Abbott RealTime HBV assay is a RT-PCR platform with a sensitivity of 98.2% and specificity of 100%. Studies have found excellent correlation between RealTime and Taqman with R2=0.961vii, that is independent of HBeAg, genotype, core promoter and pre-core region mutation. Laboratories across the globe have tried to develop economic in-house methods and seminested PCRs have been shown to correlate wellviii. Also, some countries have tried developing their own automated systems and kits, e.g.: Sansure Magb in Chinaix. Concerns over underestimation of viral load due to mismatch in the primer or probe sequences and the mutant HBV DNA found justification in studies showing discordance in the Chinesex. Digital droplet PCR to detect RNA is found to be superior to RT-PCR in detection of covalently closed circular DNA that is responsible for drug resistance and relapsesxi. i Harald H. Kessler, Sabine Preininger, Evelyn Stelzl et al. Identification of Different States of Hepatitis B Virus Infection with a Quantitative PCR Assay. Clinical and Diagnostic Laboratory Immunology Mar 2000, 7 -. ii Nagata I, Colucci G, et al. The role of HBV DNA quantitative PCR in monitoring the response to interferon treatment in chronic hepatitis B virus infection. J of Hepatology 30 (6), 965-969 (June 1999). iii Gerken G, Gomes J, Lampertico P, et al. Clinical evaluation and applications of the Amplicor HBV Monitor test, a quantitative HBV DNA PCR assay. Journal of Virological Methods. 1998 Oct;74(2):155-165. ROLE OF QUANTITATIVE HBV DNA PCR TESTS IN HEPATITIS B INFECTION 3 iv Tiziano Allice, Francesco Cerutti, Fabrizia Pittaluga, Silvia Varetto, Silvia Gabella, Alfredo Marzano, Alessandro Franchello, Giuseppe Colucci, Valeria Ghisetti. COBAS AmpliPrep-COBAS TaqMan Hepatitis B Virus (HBV) Test: a Novel Automated Real-Time PCR Assay for Quantification of HBV DNA in Plasma. Journal of Clinical Microbiology Mar 2007, 45 -. v Keith Loeb, Keith R et al. High-throughput quantitative analysis of Hepatitis B virus DNA in serum using TaqMan Fluorogenic Detection system. Hepatology J , 32 (3), 626629 (Sept 2000) vi Hochberger S, Althof D et al. Fully automated quantitation of Hepatitis B virus (HBV) DNA in human plasma by the COBAS AmpliPrep / COBAS TaqMan system. J of Clin Virology, 35 (4), 373-380 (April 2006). vii Yeh ML, Huang CF et al. Comparison of the Abbott RealTime HBV assay with the Roche Cobas AmpliPrep / Cobas TaqMan HBV assay for HBV DNA detection and quantification. J of clin Virology, 60 (3), 206-214 (2014). viii Moyra Machado, Portilho Marciaet al. Usefulness of in-house PCR methods for hepatitis B virus DNA detection. J of Virological Methods, 223, 40-44 (2015). ix Fu X, Tan D et al. A multi-center clinical study comparing Sansure Magb and CAP/CTM HBV tests in the quantitative detection of HBV DNA. J of Infection in developing countries, 10(7),-). x Liu, C., Chang, L., Jia, T. et al. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets. Virol J 14, 94 (2017). xi Limothai U, Chuaypen N, Poovorawan K, et al. Reverse transcriptase droplet digital PCR vs reverse transcriptase quantitative real-time PCR for serum HBV RNA quantification. Journal of Medical Virology. 2020 Mar .