Editorial Commentary: Standardization of Viral Load Testing for Cytomegalovirus: The Long Road Just Got Longer.

Nucleic acid tests for the quantification of viremia have become essential tools in the management of viral infections. Early availability of international standards for use as calibrators and a small number of deployed tests have made for fairly (although not perfectly) standardized tests for the measurement of human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, and hepatitis B virus genomes, which have allowed for the establishment of clinically actionable quantitative cutoffs that appear to be useful in the management of patients worldwide. Unfortunately, this is not the case for measurement of cytomegalovirus (CMV), for which an international standard has arrived late (15 years into the practice of using CMV viral load testing) and in whose absence an extensive array of tests has been placed into clinical use. This situation has resulted in a quantitative odyssey that began in a wilderness of numeric disagreement described by seminal studies that delineated the dramatic disparities in measurement among quantitative CMV DNA tests [1, 2]. The greatest disagreement was observed with centerspecific, laboratory-developed real-time quantitative polymerase chain reaction (qPCR) tests compared with qPCR assays performed with commercially available primers and probes [1]. The former are used commonly for CMV quantification, making these findings all the more alarming. The consequences of this variability are profound. When up to 1000-fold differences in quantification are observed across laboratories, efforts to define actionable management thresholds are stymied and decision making for individual patients can become clouded when their CMV DNAs are measured by assays with different performance. Once the chaos of CMV quantification was appreciated and its consequences understood, the route toward quantification improvement was begun. Most of the attention to date has focused on assay calibration: the desirable properties of calibration materials, the effects of incorporation of a common calibrator across different assays, and the development of internationally recognized reference material for use in calibration. Before voyaging further, for the purpose of navigating successfully through recently published data, it is important to grasp definitions and concepts that drive this field. First is the concept of quantitative agreement. This is the quantitative equivalence between measurements obtained with different tests. The closer the difference between measurements is to zero, the better the agreement. qPCR data should be reported and interpreted as log10-transformed values. In the absence of treatment, differences ≤0.5 log10 are typically interpreted as lacking clinical significance. This practice is derived from monitoring of HIV-1 infection; off-treatment viral loads after set-point achievement could vary to this degree without indicating a change in clinical status. This range has been adopted broadly for other monitoring situations, but it reflects more a level of comfort than a span substantiated by clinical data. The second important concept is harmonization. It is the quantitative equivalence in measurement across all tests, when those tests are calibrated with a reference material whose quantity is derived by consensus. This is in contrast to standardization, which is a level of agreement that results from the use of assays calibrated with material that is a higherorder (ie, “pure”) substance quantified using gold standard measurement methods. For example, cortisol measurements are standardized because cortisol is measured directly as a physical entity and quantification tests are calibrated with gravimetrically measured cortisol preparations [3]. In contrast to cortisol quantification tests, viral load qPCRs can at best achieve harmonization. These latter tests indirectly measure nucleic acid content, based on calibrators that are complex reference materials whose concentration was determined by consensus. Furthermore, laboratories that participate in the consensus process do not use higherorder reference methods for viral nucleic acid measurement. Rather, they use the very same qPCR tests that will utilize the reference preparation as a calibrator once it is available. Received 31 May 2016; accepted 6 June 2016; published online 15 June 2016. Correspondence: A. Valsamakis, Johns Hopkins Hospital, Meyer B1-193, 600 N Wolfe St, Baltimore, MD 21287-7093 (avalsam1@jhmi.edu). Clinical Infectious Diseases 2016;63(5):590–3 © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup. com. DOI: 10.1093/cid/ciw375