medigraphic.com
ISSN 1027-2852 (Electronic)
ISSN 0864-4551 (Print)

2016, Number 3

<< Back Next >>

Biotecnol Apl 2016; 33 (3)

Improvements in the QUALITATIVE HCV UMELOSA test to detect the RNA of the hepatitis C virus

Perea Y, Armas A, González Y, Figueredo JE, Laza CM, Acevedo BE

Language: English
References: 28
Page: 3301-3307
PDF size: 315.90 Kb.


ABSTRACT

QUALITATIVE HCV UMELOSA is a test used since 2005 to confirm HCV infection and the follow-up of the antiviral therapy in patients infected with the hepatitis C virus (HCV). The drawbacks of the assay are the need for two rounds of the polymerase chain reaction and that it does not detect amplification inhibitions, leading to false positive and negative results, respectively. Therefore, the general objective of this study was to modify the QUALITATIVE HCV UMELOSA test without affecting its analytical characteristics. RNA extraction, amplification and detection stages of HCV were standardized. An internal standard was incorporated in the extraction step and a specific primer was introduced for its detection, to detect the presence of PCR inhibitors. Appropriate primers were introduced that made it possible to eliminate the nested PCR and therefore decrease the probability of obtaining false positive results. Finally, the analytical characteristics of the new test gave good clinical and analytical results and it was 100 % consistent with the reference kit. The new standardized trial showed a sensitivity of 72.31 IU/mL, which was higher than that of QUALITATIVE HCV UMELOSA (200 IU/mL), thereby enabling its use as a confirmatory assay in therapeutic follow-up and for the screening of plasma mixtures for hemoderivative production. The modifications made on QUALITATIVE HCV UMELOSA make it socially possible to offer better care for the patient with economic benefits because of the reduction of the production cost of the kit and the cost of the Cuban health system.


REFERENCES

  1. Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology. 2013;57(4):1333-42.

  2. Colquillo B, Sanchez R, Terceros P. Nuevas estrategias para el diagnóstico y seguimiento de la Hepatitis C. Evaluación de las técnicas de laboratorio RT PCR y EIA. Biofarbo 2009:17(2):15-22.

  3. Ly KN, Xing J, Klevens RM, Jiles RB, Ward JW, Holmberg SD. The increasing burden of mortality from viral hepatitis in the United States between 1999 and 2007. Ann Intern Med. 2012;156(4):271-8.

  4. TecnoSUMA. Control de enfermedades infecciosas. 2015 [cited 2015 Dec 12]. Disponible en http://www.tecnosuma. com/programasnacionales .

  5. Ministerio de Salud Pública. Anuario Estadístico de Salud 2012. La Habana: Dirección Nacional de Registros Médicos y Estadísticas de Salud, MINSAP; 2013.

  6. Hernández J. Evolución del virus de la Hepatitis C en muestras Hospitalarias de la Comunidad Valenciana (Tesis Doctoral): Valencia: Servei de Publicaciones, Universidad de Valencia; 2005.

  7. Gonzalez-Perez I, Gonzalez Gonzalez YJ, Vina-Rodriguez A, Armas Cayarga A, Solis RL. The usefulness of Umelosa hepatitis C virus qualitative kit as supplemental test for confirmation of hepatitis C virus infection. Rev Soc Bras Med Trop. 2004;37(1):25-7.

  8. Gonzalez-Perez I, Armas Cayarga A, Garcia de la Rosa I, Josefina Gonzalez Gonzalez Y. Homemade viral RNA isolation protocol using silica columns: a comparison of four protocols. Anal Biochem. 2007;360(1):148-50.

  9. Gonzalez-Perez I, Cayarga AA, Hernandez YP, de la Rosa IG, Gonzalez YJ, Leon CS, et al. Long-term conservation of HCV RNA at 4 degrees C using a new RNA stabilizing solution. J Virol Methods. 2010;168(1-2):207-11.

  10. TecnoSUMA. Instructivo del estuche QUANTITATIVE HCV UMELOSA. Edición 2. La Habana: TecnoSUMA; 2010.

  11. Perea Y, Armas A, González YJ, Laza CM. Stability of standard curves of hepatitis C virus transcripts used for viral quantification. Biotecnol Apl. 2009;26(3):222-5.

  12. TecnoSUMA. Instructivo del estuche QUALITATIVE HCV UMELOSA. Edición 2. La Habana: TecnoSUMA; 2012.

  13. Máster Diagnóstica. Instructivo del estuche HPV Direct Flow Chip Kit, v 1.0. Granada: Máster Diagnóstica; 2014.

  14. Esteban J, Alonso-Rodriguez N, del- Prado G, Ortiz-Perez A, Molina-Manso D, Cordero-Ampuero J, et al. PCR-hybridization after sonication improves diagnosis of implant-related infection. Acta Orthop. 2012;83(3):299-304.

  15. Finney DJ. PROBIT analysis; parallel line analysis. Tercera edición. Cambridge: Cambridge University Press; 1971.

  16. Jose M, Gajardo R, Jorquera JI. Stability of HCV, HIV-1 and HBV nucleic acids in plasma samples under long-term storage. Biologicals. 2005;33(1):9-16.

  17. Gonzalez-Perez I, Gonzalez Gonzalez YJ, Armas Cayarga A, Vina-Rodriguez A, Medina Concepcion A, Trujillo Pelegrin N, et al. Validation of a nested PCR assay UMELOSA HCV CUALITATIVO for the detection of Hepatitis C virus. Biologicals. 2003;31(1):55-61.

  18. Tabor E. Workshop on implementation of Nucleic Acid Testing. December 14, 1999. Bethesda: National Institute of Health, Bethesda; 1999.

  19. Saldanha J, Heath A, Lelie N, Pisani G, Nubling M, Yu M. Calibration of HCV working reagents for NAT assays against the HCV international standard. The Collaborative Study Group. Vox Sang. 2000;78(4):217-24.

  20. Fryer JF, Minor PD. Standardisation of nucleic acid amplification assays used in clinical diagnostics: a report of the first meeting of the SoGAT Clinical Diagnostics Working Group. J Clin Virol. 2009;44(2):103-5.

  21. Martins PP, Lampe E, Lewis-Ximenez LL, de Souza PS, Fernandes CA, Villar LM. Performance of molecular methods for hepatitis C virus diagnosis: usefulness among chronic cases and during the course of infection. Clin Lab. 2013;59(9-10):1031-9.

  22. Cabezas-Fernandez M, Cabeza-Barrera M. Introduction of an automated system for the diagnosis and quantification of hepatitis B and hepatitis C viruses. Open Virol J. 2012;6:122-34.

  23. Vermehren J, Yu ML, Monto A, Yao JD, Anderson C, Bertuzis R, et al. Multi-center evaluation of the Abbott RealTime HCV assay for monitoring patients undergoing antiviral therapy for chronic hepatitis C. J Clin Virol. 2011;52(2):133-7.

  24. Gonzalez-Horta EE, Marante J, Amador- Canizares Y, Alvarez-Lajonchere L, Guerra I, Martinez-Donato G, et al. Analysis of hepatitis C virus core encoding sequences in chronically infected patients reveals mutability, predominance, genetic history and potential impact on therapy of Cuban genotype 1b isolates. Eur Rev Med Pharmacol Sci. 2011;15(11):1320-7.

  25. Roca J, Ahmad M, Panda SK, Padrón G, Jameel S. Hepatitis C virus genotyping in developing countries: Results from Cuba, India and Turkey. Biotecnol Apl. 1999;16(2):88-92.

  26. Rodriguez Lay Lde L, Villalba MC, Corredor MB, Frometa SS, Hernandez JM, Carrera SD, et al. HCV genotype determination in monoinfected and HIV co-infected patients in Cuba. Trans R Soc Trop Med Hyg. 2012;106(12):711-7.

  27. Somma M. Sesión 4. Extracción y purificación de ADN. In: Querci M, Jermini M, Van den Eede G (eds.). Análisis de la presencia de microorganismos genéticamente modificados en muestras de alimentos. Ispra: Institute for Health and Consumer Protection; 2002; 1-18.

  28. Hedman J, Radstrom P. Overcoming inhibition in real-time diagnostic PCR. Methods Mol Biol. 2013;943:17-48.




2020     |     www.medigraphic.com