medigraphic.com
ISSN 1561-3054 (Electronic)

2020, Number 2

<< Back Next >>

Rev Cubana Med Trop 2020; 72 (2)

Evaluation of a simple and fast real time polymerase chain reaction assay for quantification of hepatitis B virus DNA

Rodriguez LLÁ, Montalvo MC, Bello CM, López HD, Martinez MY, Marrero SB, González GYJ, Armas CA

Language: Spanish
References: 21
Page: 1-14
PDF size: 424.94 Kb.


ABSTRACT

Introduction: Assays to quantify hepatitis B virus (HBV) DNA or viral load are indispensable for the diagnosis and follow-up of patients with chronic hepatitis B, hence the availability of diagnostic kits for this purpose. The present study deals with the validation of HBV SUMASIGNAL (one step), a real time polymerase chain reaction (RT-PCR) system for quantification of the HBV genome proposed by the Immunoassay Center.
Objective: Evaluate the analytical performance of HBV SUMASIGNAL (one step).
Methods: Use was made of a panel of 80 well characterized serum samples and the Third WHO International Standard for hepatitis B virus nucleic acid amplification techniques. Determination was performed of assay characteristics such as clinical specificity, analytical specificity (cross-reactivity), linear range or linearity and accuracy, intra-assay precision and comparison with a reference assay.
Results: Analytical and clinical specificity was 100%. Evaluation of linearity and accuracy with a WHO reference standard revealed that all the differences between the log10 of the value obtained and the reference value were lower than 0.5 log10 (r= 0.9977 and r2= 0.9954). The intra-assay variation coefficients obtained were low. Comparative evaluation with the commercial Artus HBV RG PCR kit showed a strong correlation (r= 0.8882).
Conclusions: The assay HBV SUMASIGNAL (one step) is easy to conduct manually, fast and includes reagents for nucleic acid extraction. Based on the validity of the method for the use in mind, it may be recommended for incorporation into the diagnosis, surveillance and treatment of patients with chronic hepatitis B.


REFERENCES

  1. Organización Mundial de la Salud. Hepatitis B. 19 de julio de 2019. [acceso 11/09/2019]. Disponible en: https://www.who.int/es/news-room/fact-sheets/detail/hepatitis-b

  2. Organización Panamericana de la Salud. Nuevas generaciones sin la infección por el VIH, la sífilis, la hepatitis B y la enfermedad de Chagas en las Américas 2018. ETMI Plus. Washington, DC; 2019 [acceso 11/09/2019]. Disponible en: http://iris.paho.org/xmlui/handle/123456789/50993

  3. Organización Panamericana de la Salud. La hepatitis B y C bajo la lupa. La respuesta de salud pública en la Región de las Américas 2016. Washington, DC: OPS; 2016 [acceso 11/09/2019]. Disponible en: http://iris.paho.org/xmlui/handle/123456789/31447

  4. European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017 [acceso 11/09/2019];67:j370-98. Disponible en: http://doi: 10.1016/j.jhep.2017.03.021. Epub 2017 Apr 18.

  5. Armas Cayarga A, Perea Hernández Y, González González YJ, Figueredo Lago JE, Valdivia Álvarez IY, Gómez Cordero I, et al. Performance characteristics of a fast-real-time PCR assay for hepatitis B virus DNA quantification. Biologicals. 2019 [acceso 11/09/2019];58:22-7. Disponible en: https://doi.org/10.1016/j.biologicals.2019.01.003

  6. Rodríguez L, Montalvo MC, Sariego S, Bello M, Mora E, Kourí V, et al. Reacción en cadena de la polimerasa en tiempo real para la cuantificación del ADN del virus de la hepatitis B. Rev Cubana Med Trop. 2012 [acceso 11/09/2019];64(3). Disponible en: https://www.medigraphic.com/pdfs/revcubmedtro/cmt-2012/cmt123i.pdf

  7. Fryer JF, Heath AB, Wilkinson DE, Minor PD. A collaborative study to establish the 3rd WHO international standard for hepatitis B virus for nucleic acid amplification techniques. Biologicals 2016 [acceso 11/09/2019];46:57-63. Disponible en: https://doi.org/10.1016/j.biologicals.2016.12.003

  8. Burd E. Validation of laboratory developed molecular assays for infectious diseases. Clin Microbiol Rev. 2010;23:570-6. doi:10.1128/CMR.00074-09

  9. Valsamakis A. Molecular Testing in the Diagnosis and Management of Chronic Hepatitis B. Clin Microbiol Rev. 2007;20(3):426-39. doi:10.1128/CMR.00009-07

  10. Datta S, Chatterjee S, Veer V. Recent advances in molecular diagnostics of hepatitis B virus. World J Gastroenterol. 2014 [acceso 11/09/2019];20(40):4615-25. Disponible en: http://www.wjgnet.com/1007-9327/full/v20/i40/14615.htm DOI: http://dx.doi.org/10.3748/wjg.v20.i40.14615

  11. Torres Santos AP, Levi JE, Figueiredo Lemos M, Calux SJ, Takano Oba I, Moreira RC. An in-house real-time polymerase chain reaction: standardization and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA. Mem Inst Oswaldo Cruz. 2016;111(2):134-40. doi: 10.1590/0074-02760150415.

  12. Liu C, Chang L, Jia T, Guo F, Zhang L, Ji H, Zhao J, Wang L. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets. Virology J. 2017;14:94. DOI 10.1186/s12985-017-0759-8

  13. Baylis SA, Wallace P, McCulloch E, Niesters HGM, Nübling CM. Standardization of nucleic acid tests: the approach of the World Health Organization. J Clin Microbiol. 2019;57:e01056-18. Disponible en: https://doi.org/10 .1128/JCM.01056-18

  14. Barriga Angulo G, Solís Trejo M Aceves Rosas A. Estudio comparativo de dos pruebas para determinar carga viral para VIH-1 y VHC basadas en RT-PCR de tiempo real. Rev Mex Patol Clin. 2009 [acceso 11/09/2019];56(4):247-56. Disponible en: https://www.medigraphic.com/pdfs/patol/pt-2009/pt094c.pdf

  15. Ding C, Cantor CR. Quantitative analysis of nucleic acids-the last few years of progress. J Biochem Mol Biol. 2004;37(1):1-10. DOI: 10.5483/bmbrep.2004.37.1.001

  16. de Oliveira dos Santos, Botelho Souza LF, Borzacov LM, Villalobos-Salcedo JM, Souza Vieira D. Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil. Virology J. 2014;11:16. doi:10.1186/1743-422X-11-16

  17. Aguiar J, Rodríguez L, León Y, Freyre F, Silva JA, Montalvo MC, Gell O, Estrada R, Canales E, Muzio V, Guillén G, Pentón E, Aguilar JC. Hepatitis B virus DNA quantification by a cost effective and simple real time PCR in Cuban carriers. J Infect Dis Therap. 2014 [acceso 11/09/2019];2:49-58. DOI: Disponible en: http://dx.doi.org/10.14205/2310-9386.2014.02.02.4

  18. Ballagi-Pordany A, Belak S. The use of mimics as internal standards to avoid false negatives in diagnostic PCR. Mol. Cell Probes. 1996;10:159-64. DOI: 10.1006/mcpr.1996.0022

  19. Terrault NA, Lok ASF, McMahon BJ, Chang KM, Hwang JP, Jonas MM, Brown RS Jr., Bzowej NH, Wong JB. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology. 2018;67:1560-99. DOI 10.1002/hep.29800

  20. Ministerio de Salud Pública. Plan Estratégico Nacional para la prevención y control de las ITS, el VIH y las hepatitis 2019-2023. La Habana: Ministerio de Salud Pública; 2019. p. 159-60.

  21. Chu CJ, Hussain M, Lok AS. Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection. Hepatology. 2002;36:1408-15.




2020     |     www.medigraphic.com