**4. CMV viral load threshold for treatment initiation**

The development of antiviral strategies has resulted in a large decrease in the incidence of CMV disease. Two main therapeutic strategies have been developed for the control of CMV infection and prevention of CMV disease, prophylaxis and pre-emptive therapy. Both strategies have been shown to be equally effective to protect against CMV infection. In the prophylaxis strategy antiviral treatment is administered to all patients after transplantation for a period of time between 100 and 200 days. (Boeckh M, 2003; Hebart & Einsele, 2004; Meijer et al., 2003; Zaia, 2002). In the pre-emptive strategy treatment is administered only when the CMV viral load reaches an established threshold (Gimeno et al., 2008; Machida et al., 2000). The preemptive administration of treatment consequently requires the use of highly sensitive assay for monitoring CMV viral load.

The International Consensus Guidelines on the Management of CMV after transplantation considered necessary the establishment of a universal cut-off value for initiating therapy (Razonable & Emery, 2004). Several studies have tried to establish the clinical utility of using the RT-PCR test to guide preemptive therapy in SCT recipients (Avetisyan et al., 2006; Boeckh M, 2009; Gerna et al., 2008; Gimeno et al., 2008; Harrington et al., 2007; Kalpoe et al., 2004; Lilleri et al., 2004; Limaye et al., 2001; Machida et al., 2000; Ruell et al., 2007; Verkruyse et al., 2006). However as stated earlier, there are significant differences between the different techniques used to determine the CMV viral load, thus the standardization of specific cutoff values is limited by the variations in the performance of the test, the assay design, and the diversity in the patient population studied thus results can not be extrapolated. So, it would be necessary an international reference standard between viral loads obtained with different tests. Currently, each laboratory must establish its own cutoff value and monitor clinical outcomes to verify the trigger points used.

Some authors have suggested that pre-emptive therapy should be initiated after two consecutive increased viral load values (Boeckh M, 2009; Gimeno et al., 2008). However, the inter- and intra-assay variability in some cases with variations over 30% that may represent a risk for using this treatment initiation strategy (Boeckh & Boivin, 1998; Boeckh & Nichols, 2004), with variations of the viral load less than 0.5 log may not be significant. Other authors propose considering CMV replication kinetics for the initiation of treatment. In these cases, it needs to be considered that while CMV duplicate within 1-2 days on average, the time for replication is shorter in the presence of immunosuppressant drugs, which may result in faster increase of the viral loads. In addition in these cases may be necessary taking into account the initial viral load since it is predictive of risk for developing CMV disease (Emery & Griffiths, 2000).

Some studies have established optimal cut-offs for treatment initiation, classifying patients according to the risk for developing CMV disease. A recent publication by Boeckh and Ljungman recommends initiating treatment with viral loads over 100 copies/ml in SCT recipients at high risk for CMV infection that received the transplant within the last 100 days, and 500 copies/ml for patients at low risk. For long-term SCT recipients initiation of treatment is recommended with viral loads over 1,000 copies/ml (Boeckh M, 2009).

Most of the studies to establish thresholds for the control of CMV infection have been performed using plasma for testing CMV viral load with a wide range of cut-off viral loads varying from 550 copies/ml to 10,000 copies/ml (Avetisyan et al., 2006; Gimeno et al., 2008;

The development of antiviral strategies has resulted in a large decrease in the incidence of CMV disease. Two main therapeutic strategies have been developed for the control of CMV infection and prevention of CMV disease, prophylaxis and pre-emptive therapy. Both strategies have been shown to be equally effective to protect against CMV infection. In the prophylaxis strategy antiviral treatment is administered to all patients after transplantation for a period of time between 100 and 200 days. (Boeckh M, 2003; Hebart & Einsele, 2004; Meijer et al., 2003; Zaia, 2002). In the pre-emptive strategy treatment is administered only when the CMV viral load reaches an established threshold (Gimeno et al., 2008; Machida et al., 2000). The preemptive administration of treatment consequently requires the use of

The International Consensus Guidelines on the Management of CMV after transplantation considered necessary the establishment of a universal cut-off value for initiating therapy (Razonable & Emery, 2004). Several studies have tried to establish the clinical utility of using the RT-PCR test to guide preemptive therapy in SCT recipients (Avetisyan et al., 2006; Boeckh M, 2009; Gerna et al., 2008; Gimeno et al., 2008; Harrington et al., 2007; Kalpoe et al., 2004; Lilleri et al., 2004; Limaye et al., 2001; Machida et al., 2000; Ruell et al., 2007; Verkruyse et al., 2006). However as stated earlier, there are significant differences between the different techniques used to determine the CMV viral load, thus the standardization of specific cutoff values is limited by the variations in the performance of the test, the assay design, and the diversity in the patient population studied thus results can not be extrapolated. So, it would be necessary an international reference standard between viral loads obtained with different tests. Currently, each laboratory must establish its own cutoff value and monitor clinical

Some authors have suggested that pre-emptive therapy should be initiated after two consecutive increased viral load values (Boeckh M, 2009; Gimeno et al., 2008). However, the inter- and intra-assay variability in some cases with variations over 30% that may represent a risk for using this treatment initiation strategy (Boeckh & Boivin, 1998; Boeckh & Nichols, 2004), with variations of the viral load less than 0.5 log may not be significant. Other authors propose considering CMV replication kinetics for the initiation of treatment. In these cases, it needs to be considered that while CMV duplicate within 1-2 days on average, the time for replication is shorter in the presence of immunosuppressant drugs, which may result in faster increase of the viral loads. In addition in these cases may be necessary taking into account the initial viral load since it is predictive of risk for developing CMV disease (Emery

Some studies have established optimal cut-offs for treatment initiation, classifying patients according to the risk for developing CMV disease. A recent publication by Boeckh and Ljungman recommends initiating treatment with viral loads over 100 copies/ml in SCT recipients at high risk for CMV infection that received the transplant within the last 100 days, and 500 copies/ml for patients at low risk. For long-term SCT recipients initiation of

Most of the studies to establish thresholds for the control of CMV infection have been performed using plasma for testing CMV viral load with a wide range of cut-off viral loads varying from 550 copies/ml to 10,000 copies/ml (Avetisyan et al., 2006; Gimeno et al., 2008;

treatment is recommended with viral loads over 1,000 copies/ml (Boeckh M, 2009).

**4. CMV viral load threshold for treatment initiation** 

highly sensitive assay for monitoring CMV viral load.

outcomes to verify the trigger points used.

& Griffiths, 2000).

Hong et al., 2004; Yakushiji et al., 2002). Other studies have established thresholds for the control of CMV infection of 1,000 copies/ml (Boeckh M, 2009; Harrington et al., 2007), and 10,000 (Gerna et al., 2008; Lilleri et al., 2004; Verkruyse et al., 2006) using whole blood. These cut-off values were defined to be protective independently of their CMV sero-status. Few studies have established a protective cut-off for CMV infection in leukocytes; one of these established a cut off of 1,000 CMV genomes copies per 200,000 leukocytes (Avetisyan et al., 2006).

Another important issue after transplantation is the optimal frequency of CMV monitoring which has not been defined for SCT. Most authors recommend a weekly periodicity increased twice a week once CMV replication is detected and during treatment administration, while treatment administration should be interrupted after two consecutive negative determinations (Boeckh M, 2009).

In summary, it has not been established a cutoff threshold for initiating antiviral therapy against CMV maybe due to differences in CMV serological status, immunosuppressive drug regimens and period of treatment. Further studies are necessary in large series of SCT recipients assessing safety of viral load thresholds.
