Ann Clin Microbiol.  2017 Jun;20(2):21-26. 10.5145/ACM.2017.20.2.21.

Practical Aspects of Cytomegalovirus DNA Quantitative PCR

• Chang J
• Hwang SH
• Kim MN
• Sung H

• KMID: 2383227
• DOI: http://doi.org/10.5145/ACM.2017.20.2.21

Abstract

Human cytomegalovirus (CMV) is a clinically important pathogen that causes significant morbidity and mortality in immunocompromised patients and is typically monitored using real-time polymerase chain reaction (real-time PCR). International standards and certified reference materials were recently developed by the WHO, providing the opportunity to standardize viral load reporting. Clinical microbiologists who conduct quantitative CMV DNA testing should be aware of technical issues that can affect the analytical and clinical performance of the method used. These include specimen type, limits of detection and quantification, linear range, reproducibility, and wide variability in viral load values among different assays. Specimen types for testing include whole blood, plasma, serum, and peripheral blood leukocytes. The tests that use whole blood and peripheral blood leukocytes have higher sensitivities. Adsorption chromatography methods are widely used for nucleic acid extraction, and efficiencies can differ between manual and automatic processes. The most common method for quantitative CMV DNA testing is real-time PCR. All CMV testing methods require quality control at the pre-analytic, analytic, and post-analytic stages. In transplant patients, specific quantitative results and monitoring of any changes at follow-up are important. Five to seven days is an adequate follow-up interval in this regard, and drug-resistant CMV should be suspected if there is no response to therapy. One specimen type should be chosen for follow-up quantitative CMV DNA testing, optimized according to WHO standards. Further studies are needed to better standardize CMV testing approaches and to determine the appropriate clinical cut-off level.

Keyword

Cytomegalovirus; Preemptive therapy; Quantification; Real-time polymerase chain reaction; Standardization

MeSH Terms

Adsorption
Chromatography
Cytomegalovirus*
DNA*
Follow-Up Studies
Humans
Immunocompromised Host
Leukocytes
Limit of Detection
Methods
Mortality
Plasma
Polymerase Chain Reaction*
Quality Control
Real-Time Polymerase Chain Reaction
Viral Load
DNA

Figure

• 

  Fig. 1 Clinical utility of quantitative CMV DNA testing.

Reference

1. Kim T, Sung H, Park KT, Kim SC, Kim SH, Choi SH, et al. Clinical usefulness of human cytomegalovirus antigenemia assay after kidney transplantation. Infect Chemother. 2009; 41:72–77.

2. Fishman JA. Infection in solid-organ transplant recipients. N Engl J Med. 2007; 357:2601–2614.

3. Razonable RR, Hayden RT. Clinical utility of viral load in management of cytomegalovirus infection after solid organ transplantation. Clin Microbiol Rev. 2013; 26:703–727.

4. Pang XL, Fox JD, Fenton JM, Miller GG, Caliendo AM, Preiksaitis JK;. American Society of Transplantation Infectious Diseases Community of Practice. Canadian Society of Transplantation. Interlaboratory comparison of cytomegalovirus viral load assays. Am J Transplant. 2009; 9:258–268.

5. Dolan A, Cunningham C, Hector RD, Hassan-Walker AF, Lee L, Addison C, et al. Genetic content of wild-type human cytomegalovirus. J Gen Virol. 2004; 85:1301–1312.

6. Fryer JF, Heath AB, Minor PD. Collaborative Study Group. A collaborative study to establish the 1st WHO International Standard for human cytomegalovirus for nucleic acid amplification technology. Biologicals. 2016; 44:242–251.

7. Kraft CS, Armstrong WS, Caliendo AM. Interpreting quantitative cytomegalovirus DNA testing: understanding the laboratory perspective. Clin Infect Dis. 2012; 54:1793–1797.

8. Dioverti MV, Razonable RR. Clinical utility of cytomegalovirus viral load in solid organ transplant recipients. Curr Opin Infect Dis. 2015; 28:317–322.

9. Garrigue I, Boucher S, Couzi L, Caumont A, Dromer C, Neau-Cransac M, et al. Whole blood real-time quantitative PCR for cytomegalovirus infection follow-up in transplant recipients. J Clin Virol. 2006; 36:72–75.

10. Lisboa LF, Asberg A, Kumar D, Pang X, Hartmann A, Preiksaitis JK, et al. The clinical utility of whole blood versus plasma cytomegalovirus viral load assays for monitoring therapeutic response. Transplantation. 2011; 91:231–236.

11. Razonable RR, Brown RA, Wilson J, Groettum C, Kremers W, Espy M, et al. The clinical use of various blood compartments for cytomegalovirus (CMV) DNA quantitation in transplant recipients with CMV disease. Transplantation. 2002; 73:968–973.

12. Evans PC, Soin A, Wreghitt TG, Alexander GJ. Qualitative and semiquantitative polymerase chain reaction testing for cytomegalovirus DNA in serum allows prediction of CMV related disease in liver transplant recipients. J Clin Pathol. 1998; 51:914–921.

13. Sung H, Hwang SH, Koh YJ, Kim MN. Nucleic acid extraction for the quantification of cytomegalovirus and Epstein-Barr virus. Lab Med Online. 2016; 6:165–170.

14. Costa C, Mantovani S, Balloco C, Sidoti F, Fop F, Cavallo R. Comparison of two nucleic acid extraction and testing systems for HCMV-DNA detection and quantitation on whole blood specimens from transplant patients. J Virol Methods. 2013; 193:579–582.

15. Pillet S, Bourlet T, Pozzetto B. Comparative evaluation of the QIAsymphony RGQ system with the easyMAG/R-gene combination for the quantitation of cytomegalovirus DNA load in whole blood. Virol J. 2012; 9:231.

16. Bravo D, Clari MÁ, Costa E, Muñoz-Cobo B, Solano C, José Remigia M, et al. Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients. J Clin Microbiol. 2011; 49:2899–2904.

17. Hodinka RL. Human Cytomegalovirus. In : Carroll KC, Jorgensen JH, Pfaller MA, editors. Manual of Clinical Microbiology. 11th ed. Washington, DC: ASM Press;2015. p. 1718–1737.

18. U.S. Food and Drug Administration. Nucleic acid based tests. last visited on 4 August 2016. http://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm330711/ [Online].

19. Roberts TC, Buller RS, Gaudreault-Keener M, Sternhell KE, Garlock K, Singer GG, et al. Effects of storage temperature and time on qualitative and quantitative detection of cytomegalovirus in blood specimens by shell vial culture and PCR. J Clin Microbiol. 1997; 35:2224–2228.

20. Abdul-Ali D, Kraft CS, Ingersoll J, Frempong M, Caliendo AM. Cytomegalovirus DNA stability in EDTA anti-coagulated whole blood and plasma samples. J Clin Virol. 2011; 52:222–224.

21. Oh HB, editor. Performance Evaluation and Approval for In Vitro Diagnostic Device. 2nd ed. Seoul: Korea Medical Book Publishing Company;2015. p. 223–318.

22. Grys TE. Developing a quality system for quantitative laboratory-developed tests. Clin Microbiol Newsl. 2011; 33:179–185.

23. CLSI. Quantitative molecular methods for infectious diseases; approved guideline. CLSI document MM06-A2. Wayne, PA: Clinical and Laboratory Standards Institute;2010.

24. Emery VC, Sabin CA, Cope AV, Gor D, Hassan-Walker AF, Griffiths PD. Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation. Lancet. 2000; 355:2032–2036.

25. Humar A, Kumar D, Boivin G, Caliendo AM. Cytomegalovirus (CMV) virus load kinetics to predict recurrent disease in solid-organ transplant patients with CMV disease. J Infect Dis. 2002; 186:829–833.

26. Kotton CN, Kumar D, Caliendo AM, Asberg A, Chou S, Danziger-Isakov L, et al. Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation. 2013; 96:333–360.

27. Humar A, Snydman D. AST Infectious Diseases Community of Practice. Cytomegalovirus in solid organ transplant recipients. Am J Transplant. 2009; 9:Suppl 4. S78–S86.

28. Caliendo AM, St George K, Kao SY, Allega J, Tan BH, LaFontaine R, et al. Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients. J Clin Microbiol. 2000; 38:2122–2127.

29. Sung H, An D, Lee SO, Choi SH, Kim SH, Chi HS, et al. Detection of a UL97 gene mutation conferring ganciclovir resistance in human cytomegalovirus: prevalence of the D605E polymorphism in Korean immunocompromised patients. Ann Clin Lab Sci. 2012; 42:429–434.

30. Jeong TD, Sung H, Choi SH, Lee SO, Yoon HK, Kim MN, et al. Cytomegalovirus ventriculoencephalitis with compartmentalization of antiviral-resistant cytomegalovirus in a T cell-depleted haploidentical peripheral blood stem cell transplant recipient. Diagn Microbiol Infect Dis. 2012; 74:307–310.

31. Lurain NS, Chou S. Antiviral drug resistance of human cytomegalovirus. Clin Microbiol Rev. 2010; 23:689–712.