Hematopoietic stem cell transplantation (HSCT) is an important therapeutic tool for treating malignant and non-malignant disorders, and the human cytomegalovirus (CMV) reactivation is common in these cases due to the immunocompromised state of patients [1].

Monitoring of its reactivation and preemptive or prophylactic treatment using ganciclovir are critical for HSCT recipients. However, because of the myelotoxicity of ganciclovir and its prolongation of periods of neutropenia, the prognosis in patients at low risk of developing CMV disease is not necessarily improved. Identification of patients at high risk of developing CMV is therefore believed to be important in management of HSCT recipients [2].

Despite major advances in prevention and preemptive treatment, CMV infection still causes significant morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT) [3] and occurs in 50-90% of allogeneic transplant recipients [4].

While detection of CMV pp65 antigenemia is still widely used for monitoring CMV infection and guide preemptive therapy in patients at risk of developing CMV disease [5, 6], the quantification of CMV DNA in blood by PCR is emerging as an alternative to the pp65 antigenemia assay and may soon become the standard for the surveillance of CMV infection in allogeneic HSCT recipients [7], because it presents several advantages over the pp65 antigenemia assay, including an increased sensitivity for the detection of CMV reactivation, the reliable detection of CMV reactivation during severe neutropenia in the early post-transplant period, the shorter time required for the procedure, and the convenient processing of large numbers of specimens [8].

The aim of this study was to diagnosis and monitor active CMV infection in allogeneic HSCT patients. In addition, we have determined the proper cutoff level of CMV DNA load by real-time PCR for preemptive therapy, in order to switch the monitoring method from the pp65 antigenemia assay to real-time PCR method.

We have established a new method of diagnosis of CMV infection using real-time PCR based on TaqMan^® technology. Several studies, such as Machida et al. [2], reported that PCR is a very useful method for early diagnosis of CMV disease, but it may be frequently too sensitive for clinical use and do not necessarily indicate an imminent risk of developing CMV disease. However, we overcame this problem using receiver operating characteristic (ROC) plot analysis to determine a threshold value of the CMV DNA load for initiating treatment. The sensitivities and specificities for US17 were calculated to determine optimal cutoff value for CMV DNA copies.

In this study, we used three different detection assays (pp65 antigenemia, nested-PCR and real-time PCR) for monitoring active CMV infection in allogeneic stem cell transplant recipients. We could observe a high incidence of active CMV infection occuring during the second post-transplant month (31 - 60 days), with a maximum value of probability density 44.4 days after HSCT, corroborating data in the literature, which reports that the highest incidence of CMV infections occur during the first three post-transplant months [14], secondary to the greatest degree of immunosuppression [15, 16].

The incidence of active CMV infection was high (27 of 30 patients, 90%) after a median of 33 days (range 0-119 days), and only two (6.7%) patients had CMV disease, both in the gastrointestinal tract. Only one (3.3%) of 30 patients monitored in this study died of CMV disease, in agreement with the low rates of CMV-related mortality cited in published reports [17, 18].

The incidence of active CMV infection was comparable to that found in previous reports on HSCT. Bonon et al. [11] noted active CMV infection in 55 (79.7%) of 69 analyzed patients, considering that six (8.7%) developed CMV disease. Gentile et al. [19] evaluated 38 HSCT recipients. Among them, 25 (70%) had active CMV infection and three (7.9%) were pathologically diagnosed with CMV colitis.

The high incidence of active CMV infection detected in our patients is associated to no prophylactic treatment and pretransplant CMV sorostatus of donors and recipients (D+/R+:100%). This group is identified as high-risk patients to develop active CMV infection and CMV disease [20].

In this study, the absence of statistical significance among active CMV infection and recurrence of CMV infection, CMV disease, GVHD, opportunist infection, chronic rejection and death may be influenced by the small number of patients, the preexisting heterogeneity of conditioning regimens, GVHD prophylaxis and hematological diseases, according to Gentile et al. [19].

Several studies have attempted to determine a CMV DNA copy number equivalent to the levels of pp65 antigenemia and to implement CMV PCR as pp65 antigenemia in clinical practice [21]. Identification of a cutoff level for real-time PCR assay would be an important indicator of time to initiate an anti-CMV treatment [20], would reduce the number of patients treated with preemptive therapy who are not destined to develop CMV disease [6] and the duration of preemptive therapy might be shortened by real-time PCR guided manner, because more rapid negative conversion of viral reactivation was detected using this assay [8].

Choi et al. [8] suggested cutoff values of approximately 3 × 10⁴ copies/mL in low-risk patients and of approximately 2 × 10⁴ copies/mL in high-risk patients. Cariani et al. [22] defined a positive cutoff value equivalent to 9960 copies/mL in HSCT. Garrigue et al. [23] reported that thresholds of 10 and 50 positive cells/2 × 10⁵ cells were equivalent to 3.3 log10 copies/mL (2000 copies/mL) and 3.8 log10 copies/mL (6300 copies/mL), respectively. Kalpoe et al. [24] suggested that preemptive therapy could be initiated at CMV DNA load of 1000 copies/mL and then increased by 10-fold per week at the first episode of CMV reactivation. Lilleri et al. [25] suggested a cutoff value of 10000 copies/mL for initiating preemptive therapy in HSCT recipients, and Graffari et al. [21] previously defined a positive cutoff value of greater than 1000 copies/2 × 10⁵ PBL in HSCT recipients.

In our study, we have calculated the optimal cutoff viral load using ROC curve for a pp65 antigenemia value ≥1 positive cell. The analysis of data indicated that a CMV DNA level of 418.4 copies/10⁴ PBL was a convenient value for discriminating between latent infections and those requiring preemptive therapy. This specific pp65 antigenemia value has been used in our Centre as the threshold for initiating preemptive therapy in HSCT recipients with an incidence of CMV disease lower than 7%.

It is difficult to compare data from different centers because real-time PCR methods are not well standardized and use different target sequences, primer sets, and extraction and detection methods, which result in different analytical performances [7].

Given the lack of standardization of CMV monitoring by real-time PCR, the cutoff definition for predicting CMV disease and initiating preemptive antiviral therapy remain a question [26]. The results shows that studies evaluating this assay are highly heterogeneous, there is no available international standard for CMV PCR, and each study determined its own assays characteristics for their own setup [23].

Most reports on detecting CMV DNA by real-time PCR have used 'in-house' assays with variable primers for the same gene or different genes (e.g. the immediate early CMV gene or the CMV DNA polymerase gene), making it difficult to extrapolate results from one institution to another [21, 27]. Assay sensitivity varies greatly depending on the methodology used. Commercial diagnostic tests could avoid variability between home-brewed assays, improving reproducibility and standardization of the results [28].

Further studies to correct current cutoff value and to validate the optimal cutoff value for the initiation of preemptive therapy are currently underway at our HSCT center.

Conversely, we thought that high negative predictive value (85.7%) could allow us to avoid unnecessary preemptive therapy with a fair aliquot of patients. The optimal cutoff value by real-time PCR for therapeutic intervention needs to be clearly defined to determine the maximal specificity for CMV disease, and to define its value for prognosis and use in therapeutic clinical trials.

The low incidence of CMV disease in HSCT recipients in our study attests to the efficacy of CMV surveillance, based on the pp65 antigenemia assay and nested-PCR (assay used in clinical routine). The quantification of CMV DNA load using real-time PCR appears to be applicable to the clinical practice and an optimal cutoff value for guiding timely preemptive therapy should be clinically validated in future studies. For this reason, we think that real-time PCR can be used complementarily to pp65 antigenemia screening to monitor preemptive therapy for presenting high sensitivity, in addition to be an alternative for CMV diagnoses in cases of samples collected before engraftment due to the lack of leukocytes during the period of aplasia, in neutropenic patients and in occurrence of CMV gastrointestinal disease.