Optimization of a real-time PCR assay for hepatitis B virus load determination in infected patients.
Optimización de un ensayo de PCR en tiempo real para la determinación de carga viral del virus de hepatitis B en pacientes infectados.
Keywords:
hepatitis B virus, viral load, optimization, qPCR, genotype
Abstract
Hepatitis B virus (HBV) infection is a significant health problem in the world, with around 294 million chronic carriers. Determination of viral load is crucial for the monitoring and treatment follow-up of HBV-infected patients. In-house methods are economical alternatives for those settings where HBV viral load determination might not be widely available commercially. Molecular diagnostic techniques need to take into account the variability of this virus. The aim of this study was the evaluation and optimization of a real-time PCR method for the determination of HBV load. After optimization, the in-house assay evaluated in this study showed acceptable sensitivity and specificity, allowing its use for monitoring patients in low-income settings.
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References
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Chen J, Li L, Yin Q, Shen T. A review of epidemiology and clinical relevance of Hepatitis B virus genotypes and subgenotypes. Clin Res Hepatol Gastroenterol. 2023; 47: 102180. https://doi.org/10.1016/j.clinre.2023.102180.
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Locarnini SA, Littlejohn M, Yuen LKW. Origins and Evolution of the Primate Hepatitis B Virus. Front Microbiol. 2021; 12: 653684. https://doi.org/10.3389/fmicb.2021.653684.
GBD 2019 Hepatitis B Collaborators. Global, regional, and national burden of hepatitis B, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet Gastroenterol Hepatol 2022; 7: 796-829. https://doi.org/10.1016/S2468-1253(22)00124-8.
MacLachlan JH, Cowie BC. Hepatitis B virus epidemiology. Cold Spring Harb Perspect Med 2015; 5: a021410. https://doi.org/10.1101/cshperspect.a021410.
Gerlich WH. Medical virology of hepatitis B: how it began and where we are now. Virol J 2013; 10: 239. https://doi.org/10.1186/1743-422X-10-239.
McNaughton AL, Revill PA, Littlejohn M, Matthews PC, Ansari MA. Analysis of genomic-length HBV sequences to determine genotype and subgenotype reference sequences. J Gen Virol 2020; 101: 271-283. https://doi.org/10.1099/jgv.0.001387.
Pujol FH, Toyé RM, Loureiro CL, Jaspe RC, Chemin I. Hepatitis B eradication: vaccine as a key player. Am J Transl Res. 2023; 15: 4971-4983.
Huzair F, Sturdy S. Biotechnology and the transformation of vaccine innovation: The case of the hepatitis B vaccines 1968-2000. Stud Hist Philos Biol Biomed Sci 2017; 64: 11-21. https://doi.org/10.1016/j.shp- sc.2017.05.004.
World Health Organization. Combating Hepatitis B and C to Reach Elimination by 2030. Available from: https://apps.who.int/iris/bitstream/handle/10665/206453/WHO_HIV_2016.04_eng.pdf. Accessed on December 22, 2024
Feld JJ, Lok AS, Zoulim F. New Perspectives on Development of Curative Strategies for Chronic Hepatitis B. Clin Gastroenterol Hepatol. 2023; 21: 2040-2050. https://doi.org/10.1016/j.cgh.2023.02.032.
WHO. Guidelines for the prevention, diagnosis, care and treatment for people with chronic hepatitis B infection. Geneva: World Health Organization; 2024. Licence: CC BY-NC-SA 3.0 IGO.
Pawlotsky JM. Virological markers for clinical trials in chronic viral hepatitis. Virological markers for clinical trials in chronic viral hepatitis. JHEP Rep. 2024; 6: 101214. https://doi.org/10.1016/j.jhepr.2024.101214.
Portilho MM, Mendonça ACDF, Bezerra CS, do Espirito-Santo MP, de Paula VS, Nabuco LC, et al. Usefulness of in-house real time PCR for HBV DNA quantification in serum and oral fluid samples. J Virol Methods. 2018; 256: 100-106. https://doi.org/10.1016/j.jviromet.2018.03.001.
Devesa M, Loureiro CL, Rivas Y, Monsalve F, Cardona N, Duarte MC, et al.. Subgenotype diversity of hepatitis B virus American genotype F in Amerindians from Venezuela and the general population of Colombia. J Med Virol. 2008; 80: 20-26. doi: https://doi.org/10.1002/jmv.21024.
Tamura K, Stecher G, and Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol Biol Evol. 2011; 38: 3022-3027. https://doi.org/10.1093/molbev/msab120.
Uzunoğlu E, Şahin AM, Avci E, Kutlu H, Güntepe G. Can HBsAg Be Used as a Viral Replication Marker in Chronic Hepatitis B Patients? Viral Hepa J. 2017; 23: 55-59.
Aynalia A, Ciftcia E, Aridogana BC, Cetina ES, Kayab S, Carsancaklia SA, Ozturka T. Evaluation of viral load distribution of HBV DNA positive patients at Suleyman Demirel University Hospital. J Exp Clin Med. 2015; 32: 147-150. https://doi.org/10.5835/jecm.omu.32.04.002.
Guney M, Bakir A, Erdal H, Gunal A, Yildiz F, Sig AK, Kurkcu MF, Yavuz MT, Gulsen M. The Correlation between Histopathological Stages and Viral Markers of Chronic Hepatitis B infection in Ankara, Turkey. Int J Innov Sci Res Technol. 2021; 6: 1066-1070.
Akoglu H. User’s guide to correlation coefficients. Turk J Emerg Med. 2018; 18: 91-93. https://doi.org/10.1016/j.tjem.2018.08.001.
HBVdb. The Hepatitis B Virus database. https://hbvdb.lyon.inserm.fr/HBVdb/HBVdbIndex. Accessed on December 27, 2024.
Pujol F, Jaspe RC, Loureiro CL, Chemin Hepatitis B virus American genotypes: Pathogenic variants? Clin Res Hepatol Gastroenterol. 2020; 44: 825-835. https://doi.org/10.1016/j.clinre.2020.04.018.
Chen J, Li L, Yin Q, Shen T. A review of epidemiology and clinical relevance of Hepatitis B virus genotypes and subgenotypes. Clin Res Hepatol Gastroenterol. 2023; 47: 102180. https://doi.org/10.1016/j.clinre.2023.102180.
Araujo NM. Hepatitis B virus intergenotypic recombinants worldwide: An overview. Infect Genet Evol. 2015; 36:500-510. https://doi.org/10.1016/j.meegid.2015.08.024.
Locarnini SA, Littlejohn M, Yuen LKW. Origins and Evolution of the Primate Hepatitis B Virus. Front Microbiol. 2021; 12: 653684. https://doi.org/10.3389/fmicb.2021.653684.
Published
2025-06-06
How to Cite
Sulbarán, M. Z., Sulbarán, Y. F., Loureiro, C. L., Rangel, H. R., Jaspe, R. C., & Pujol, F. H. (2025). Optimization of a real-time PCR assay for hepatitis B virus load determination in infected patients.: Optimización de un ensayo de PCR en tiempo real para la determinación de carga viral del virus de hepatitis B en pacientes infectados. Investigación Clínica, 66(2), 166-174. https://doi.org/10.54817/IC.v66n2a04
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Original Works
