**5. Standardization of CMV viral load quantification**

Since CMV viral loads (in copies per milliliter of body fluid) correlate with the development of CMV disease (Emery & Griffiths, 2000; Humar et al., 1999), the use of molecular diagnostics based on the measurement of the viral load has contributed to patients' management after transplantation for more than a decade. During preemptive administration of treatment, antiviral therapy is initiated when CMV replication reaches an established threshold in the peripheral blood (Lilleri et al., 2009), prior to develop clinical symptoms. Therefore, the use methods such as RT-PCR can be useful to determine when to initiate the preemptive therapy and its duration (Emery & Griffiths, 2000; Humar et al., 1999; Humar et al., 2002; Sia & Patel, 2000) as well as to monitor the response to the administered therapy. However, as previously stated the most important handicap of the available RT-PCR techniques is the high variability of CMV viral load results among different laboratories. Pang et al designed a comparative study among thirty laboratories to evaluate the reproducibility of in-house and commercial assays to detect CMV infection. They prepared a panel of samples with different CMV DNA concentrations that were evaluated in different laboratories, with several commercial available assays. While the intra-laboratory coefficient of variation was considered acceptable (around 17%), they inter-laboratory variability resulted higher than 140%. These authors considered that differences in viral load lower at <0.5 log10 are not considered clinically relevant. This difference limits the comparisons inter-laboratories and prevents the establishment of a determinate cut-off broadly applicable for making clinical decisions and monitoring the initiation of pre-emptive therapy (Caliendo et al., 2009). These discrepancies result into clinical therapeutic consequences, as a number of patients may receive treatment in one hospital while not in other hospital using a different assay (Gracia-Ahufinger et al., 2010).

The differences among assays are based on the method for nucleic acid extraction, the specimen type, the target genomic region, primers and probes used for amplification and

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detection (Caliendo et al., 2009; Ikewaki et al., 2005). Although, the variability in the results obtained with commercial assays (kits or ASRs) are proven to be lower than when using inhouse developed methods (Pang et al., 2009).

The most critical factor for standardization is the lack of using an universally accepted standard for CMV quantification will make possible the comparison among results thus establishing a common management of patients in different centers (Atkinson & Emery, 2011). The reference material used as a calibrator have to be traceable and commutable to achieve accurate clinical results ensuring consistency with clinical samples (Caliendo et al., 2009). While many laboratories produce their own calibrator as an attempt of standardizing some CMV reference material is under development and in fact there are standards commercially available for quantification. For instance, the National Institute of Standards and Technology (NIST) started the development of a reference standard for CMV based on pure CMV DNA from a Towne strain from which after some modification, the final construct will be used to produce viral DNA (Wang et al., 2004). Moreover, the OptiQuant® CMV DNA Quantification Panel from AcroMetrix has been carefully formulated to mimic naturally occurring human specimens containing CMV viral DNA. It consists of cultured CMV that has been diluted in defibrinated, delipidized normal human plasma at different concentrations. The panel can be used with any test procedure designed for measuring CMV DNA in human serum or plasma and it has been widely used as a standard in several studies (Bravo et al.; Forman et al., 2011; Hanson et al., 2007; Raggam et al., 2010) to compare techniques with the same laboratory and inter-laboratory.

In the absence of standardization the current clinical guidelines recommend to each individual laboratory to establish their own viral thresholds for CMV management, (Kotton et al., 2010; Razonable & Emery, 2004), threshold that cover a wide range of viral loads varying from 200-500 copies/mL in some laboratories (Ikewaki et al., 2005; Mori et al., 2002) to 2000-5000 copies/mL in others (Humar et al., 1999).
