Human Cytomegalovirus (HCMV) is one of the problematic viral infections in solid organ, Hematopoietic stem cell transplant (HSCT) recipients and other immunocompromised hosts such as patients with HIV infection. Treatment of CMV infection often requires multidisciplinary specialties and a team work management. Every physician who works on CMV infection will inevitably encounter a diagnostic challenge in his routine practices.
Currently, molecular tests are very popular among physicians. The accuracy and precision (reproducibility) of the test, high sensitivity, the ability to detect the lowest concentration of virus (DNA), the lower risk of contamination and shorter turnaround time (1) are advantages of these tests over serologic and culture methods. Along with the increased usage and more access to these tests, physicians should be well familiar with the strengths and weaknesses of the test. In the absence of adequate knowledge about these tests, interpretation may mislead to inaccurate diagnosis and physicians involve in diagnostic challenges. Inaccurate timing for the right diagnosis of congenital cytomegalovirus infection is one of the well-known examples. As it is known, delay in application of the molecular DNA testing in this situation (i e, after three weeks of birthday) had a little significance and the positive results should be interpreted with caution (2).
Interpretation of CMV viral load is one of the common diagnostic challenges of practice. Common pitfalls could be categorized in pre-analytical, analytical and post- analytical errors.
Important pre-analytical errors
- Choosing different specimen type for quantitative-polymerase chain reaction (Q-PCR), which is one of the most common pitfalls. It is better to follow and monitor patients with only one sample type in blood. Although, detection of CMV DNAemia is acceptable both in whole blood and plasma but each specimen has advantages and limitations. Estimated differences between plasma and whole blood may be as great as 100 folds. Whole blood detects both cell-free and intracellular viruses, which means CMV DNA is detected more frequently and often higher in whole blood. On the other hand, detection of CMV DNA in plasma is thought to be more likely correlated with active infection. Thus, plasma samples may be more specific and whole blood samples may be more sensitive to detect CMV DNAemia (3). Regardless of these advantages, no (or low level of) HCMV-DNA copies may be detectable in plasma during the early phase of replication or in the case of latent infection reactivation. Despite all of the aforementioned notes, it is shown that HCMV replication in vivo is a highly dynamic process with a doubling time of approximately one day (4).
- Inappropriate sample transport or storage (5)
- Improper sample processing (6)
Important analytical errors (3, 7-10)
- Differences in DNA extraction methods
- Selection of primers and probes targeting with different genes
- Different ability to limit the detection
- Low sensitivity
- Poor precision and accuracy of the test
Important post-analytical errors
Wrong reporting and interpretation of results that may come from
- Biologically non-significant variability in CMV seropositive patients (less than 0.5 log10 copies/mL changes in viral load). This variability is also known as biological fluctuations (11-13).
- When detecting CMV viral load values of less than1000 copies/mL, changes of less than five-fold rarely reflect clinically important changes in viral replication. For values higher than 1000 copies/mL, at least three-fold changes in viral load may be significant (14).
- Comparison of different values (copies/mL with IU/mL) which may lead to misinterpretation of increment or decrement in viral load (3, 9).
In 2010, the world health organization (WHO) established an international standard to achieve an agreement on viral load values among laboratories (15). Additionally, WHO introduced standard sample to allow laboratories to assess the accuracy of viral load values and the estimation of converting factor. When laboratory use conversion factor, logarithmic value (copy/mL) could be report as IU/ml.
Recently, the conversion factors provided by some manufacturers were validated by some researchers, which showed significant differences with dilutions of the WHO standard samples (16, 17). This fact reemphasized that the standardization efforts must be made within each laboratory to reduce these variability.
Some useful points regarding the interpretation of CMV quantitative DNA testing include: not to compare test results from two laboratories and apply different cutoffs (from different laboratory) to read and interpret test results; use a single specimen type for monitoring, request conversion factor of each laboratory to reach identical unit of viral load counting and omit inter-assay variability; ignoring small changes in viral loads specially in the lower limit of quantification and address longitudinal changes (rapid and great increases in viral loads); interpretation of test results in the view of clinical setting (avoid treating laboratory test results instead of patients).
References
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1.
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Coll O, Benoist G, Ville Y, Weisman LE, Botet F, Anceschi MM, et al. Guidelines on CMV congenital infection. J Perinat Med. 2009;37(5):433-45. [PubMed ID: 19673682]. https://doi.org/10.1515/JPM.2009.127.
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Kraft CS, Armstrong WS, Caliendo AM. Interpreting quantitative cytomegalovirus DNA testing: understanding the laboratory perspective. Clin Infect Dis. 2012;54(12):1793-7. [PubMed ID: 22412060]. https://doi.org/10.1093/cid/cis212.
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Lübeck PR, Berger A, Doerr HW, Rabenau HF. Can the HCMV-pp65-antigenemia assay completely be replaced by HCMV-PCR in monitoring patients after bone marrow transplantation? Rev Infect. 2010;1(3):166-71.
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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(9):2224-8. [PubMed ID: 9276392].
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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(6):968-73. [PubMed ID: 11923702].
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Hayden RT, Yan X, Wick MT, Rodriguez AB, Xiong X, Ginocchio CC, et al. Factors contributing to variability of quantitative viral PCR results in proficiency testing samples: a multivariate analysis. J Clin Microbiol. 2012;50(2):337-45. [PubMed ID: 22116152]. https://doi.org/10.1128/JCM.01287-11.
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Lee AV, Atkinson C, Manuel RJ, Clark DA. Comparative evaluation of the QIAGEN QIAsymphony(R) SP system and bioMerieux NucliSens easyMAG automated extraction platforms in a clinical virology laboratory. J Clin Virol. 2011;52(4):339-43. [PubMed ID: 22014618]. https://doi.org/10.1016/j.jcv.2011.08.016.
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Pang XL, Fox JD, Fenton JM, Miller GG, Caliendo AM, Preiksaitis JK, et al. Interlaboratory comparison of cytomegalovirus viral load assays. Am J Transplant. 2009;9(2):258-68. [PubMed ID: 19178413]. https://doi.org/10.1111/j.1600-6143.2008.02513.x.
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Mengelle C, Mansuy JM, Da Silva I, Davrinche C, Izopet J. Comparison of 2 highly automated nucleic acid extraction systems for quantitation of human cytomegalovirus in whole blood. Diagn Microbiol Infect Dis. 2011;69(2):161-6. [PubMed ID: 21251559]. https://doi.org/10.1016/j.diagmicrobio.2010.08.011.
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Atabani SF, Smith C, Atkinson C, Aldridge RW, Rodriguez-Peralvarez M, Rolando N, et al. Cytomegalovirus replication kinetics in solid organ transplant recipients managed by preemptive therapy. Am J Transplant. 2012;12(9):2457-64. [PubMed ID: 22594993]. https://doi.org/10.1111/j.1600-6143.2012.04087.x.
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Munoz-Cobo B, Solano C, Costa E, Bravo D, Clari MA, Benet I, et al. Dynamics of cytomegalovirus (CMV) plasma DNAemia in initial and recurrent episodes of active CMV infection in the allogeneic stem cell transplantation setting: implications for designing preemptive antiviral therapy strategies. Biol Blood Marrow Transplant. 2011;17(11):1602-11. [PubMed ID: 21871245]. https://doi.org/10.1016/j.bbmt.2011.08.014.
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Cardenoso L, Pinsky BA, Lautenschlager I, Aslam S, Cobb B, Vilchez RA, et al. CMV antigenemia and quantitative viral load assessments in hematopoietic stem cell transplant recipients. J Clin Virol. 2013;56(2):108-12. [PubMed ID: 23146665]. https://doi.org/10.1016/j.jcv.2012.10.001.
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Chemaly RF, Yen-Lieberman B, Chapman J, Reilly A, Bekele BN, Gordon SM, et al. Clinical utility of cytomegalovirus viral load in bronchoalveolar lavage in lung transplant recipients. Am J Transplant. 2005;5(3):544-8. [PubMed ID: 15707409]. https://doi.org/10.1111/j.1600-6143.2005.00747.x.
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Fryer JF, Heath AB, Anderson R, Minor PD, Unit B, et al. Collaborative study to evaluate the proposed 1st [first] WHO international standard for human cytomegalovirus (HCMV) for nucleic acid amplification (NAT)-based assays. 2010. Available from: http://apps.who.int/iris/handle/10665/70521.
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Furione M, Rognoni V, Cabano E, Baldanti F. Kinetics of human cytomegalovirus (HCMV) DNAemia in transplanted patients expressed in international units as determined with the Abbott RealTime CMV assay and an in-house assay. J Clin Virol. 2012;55(4):317-22. [PubMed ID: 22989927]. https://doi.org/10.1016/j.jcv.2012.08.017.
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Schnepf N, Scieux C, Resche-Riggon M, Feghoul L, Xhaard A, Gallien S, et al. Fully automated quantification of cytomegalovirus (CMV) in whole blood with the new sensitive Abbott RealTime CMV assay in the era of the CMV international standard. J Clin Microbiol. 2013;51(7):2096-102. [PubMed ID: 23616450]. https://doi.org/10.1128/JCM.00067-13.