Threshold levels of CMV-specific T-cell populations presumably affording protection from active CMV infection in allo-SCT recipients have been proposed, but lack extensive validation. We quantified CMV pp65 and immediate-early 1-specific IFN-c CD8 þ and CD4 þ and CD4 þ T-cell markers were 68.1 and 61.8%, respectively. Recipients of grafts from CMV seropositive, related or HLA-matched donors, or receiving non-myeloablative conditioning had nonsignificant tendencies to reach more frequently protective levels of both T-cell subsets at early and late (day þ 365) times after transplantation. The use of antithymocyte globulin and umbilical cord blood transplantation were associated with impaired CMV-specific T-cell reconstitution. CMV-specific IFN-c CD8 þ and CD4 þ T-cell recovery occurred irrespective of detectable CMV DNAemia.

Cytomegalovirus (CMV) reactivation occurs frequently in critically ill patients. The natural course of CMV infection and the interaction between CMV and the adaptive immune system in this setting remain poorly defined. Fifty‐three CMV‐seropositive patients in a surgical and trauma intensive care unit were included in this study. The CMV DNA load in tracheal aspirates (TA) and plasma (PL) was monitored by qPCR. CMV‐specific T‐cell immunity was assessed by intracellular cytokine staining. Plasma TNF‐α levels were determined by ELISA. CMV reactivation occurred in 39.7% of patients (23% had CMV DNA detected only in TA). The analysis of TA allowed an earlier diagnosis in 28% of patients. Clearance of CMV DNAemia preceded that of CMV DNA in TA in some episodes. Peak CMV DNA levels were significantly higher in TA than in PL (P = 0.02). CMV reactivation developed in the presence of CMV‐specific T cells. Termination of CMV reactivation was associated with an expansion of functional CMV‐specific T cells. Plasma levels of TNF‐α did not allow for the prediction of the occurrence of CMV reactivation. CMV‐specific T‐cell immunity is preserved in most critically ill patients experiencing CMV reactivation. Analysis of respiratory specimens is imperative for an optimal monitoring of CMV reactivation in this setting. J. Med. Virol. 82:1384–1391, 2010. © 2010 Wiley‐Liss, Inc.

Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n ‫؍‬ 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.Quantitative real-time PCR (QRT-PCR) assays are being increasingly used for the surveillance of active cytomegalovirus (CMV) infection in allogeneic stem cell transplant (Allo-SCT) recipients (23). Several CMV QRT-PCR assays targeting different CMV genes and using different chemistries are commercially available (23). The analytical performance and clinical usefulness of these assays have been assessed, mostly in comparison with the pp65 antigenemia test or quantitative endpoint PCR assays (1, 2, 6, 9, 10-13, 15, 17, 19, 21, 22, 24-26). Limited data are available on how these QRT-PCR tests compare to each other for the quantitation of CMV plasma DNAemia (5,11,12,18,24). This information may allow, at least to some extent, direct comparisons of CMV DNA loads measured at different centers.Nucleic acid extraction is a critical step in QRT-PCR testing, and it has been shown to be a major source of assay variability in viral DNA quantitation (7). Automated nucleic acid extraction systems are less time-consuming, less prone to analytical errors, and, overall, more efficient than manual methods. A few studies have directly compared the extraction efficiency of different automat...

The performance of the QuantiFERON-cytomegalovirus (CMV) assay was compared to that of a flow cytometry intracellular cytokine staining (ICS) method for the detection of CMV-specific gamma interferon (IFN-␥)-producing CD8؉ T-cell responses in allogeneic stem cell transplant (allo-SCT) recipients and for estimations of their magnitude and functionality. A total of 90 whole-blood specimens from 23 allo-SCT recipients was analyzed by both methods. Overall, the percentage of specimens that yielded concordant results by both methods was 68.8% ( -cell responses, as quantitated by ICS. In summary, the data indicated that the QuantiFERON-CMV assay is less sensitive than the ICS method for the detection of CMV-specific IFN-␥-producing CD8؉ T-cell responses in the allo-SCT setting. Nevertheless, it allowed the estimation of the total and polyfunctional CMV-specific IFN-␥-producing CD8 ؉ T-cell responses in specimens that tested positive by both methods. The assessment of the magnitude and the functionality of T-cell immunity against cytomegalovirus (CMV) is emerging as a clinically useful tool for the therapeutic management of CMV infection in the allogeneic stem cell transplantation (allo-SCT) setting (7, 13). The monitoring of CMV-specific CD8 ϩ or CD4 ϩ T-cell responses may allow for the optimization of preemptive antiviral therapy regimens on an individual basis and the identification of patients who may benefit from prophylactic antiviral or adoptive T-cell transfer therapeutic strategies (1,7,13). In recent years, several methods have been developed for the ex vivo quantitation and functional characterization of T-cell responses, among which flow cytometry for surface immunophenotyping and intracellular cytokine staining (ICS) are currently considered the "gold standard" (13). The QuantiFERON-CMV assay (Cellestis Ltd., Melbourne, Australia) is a commercially available test that allows the inference of the size of the CMV-specific T-cell response by quantitating the level of gamma interferon (IFN-␥), produced mostly by CMV-specific CD8 ϩ T cells, upon the stimulation of whole blood with a number of immunogenic peptides mapped within IE-1, IE-2, pp65, pp50, and gB and restricted by several widespread HLA-I haplotypes (17). The performance of the QuantiFERON-CMV assay has been assessed mostly in the solid-organ transplant (SOT) setting (see reference 4 for a review). Recently reported data, although preliminary, lend support to the suitability of the QuantiFERON-CMV assay for the monitoring of the CMV-specific IFN-␥-producing CD8 ϩ T-cell responses in allo-SCT recipients (2). Nevertheless, it is largely unknown how this method compares to ICS assays for such a purpose. In previous studies, we reported the development and clinical utility of an ICS assay for the quantitation of CMV-specific IFN-␥-producing CD8 ϩ and CD4 ϩ T cells (11,12,(14)(15)(16). The assay was found to reliably predict protection from the development of CMV DNAemia in allo-SCT recipients, and it was recently used in the setting of a novel strateg...

Preemptive antiviral therapy strategies for active cytomegalovirus (CMV) infection occurring in allogeneic stem cell transplant recipients should be optimized to avoid overtreatment. The current study was aimed at determining whether the analysis of the kinetics of CMV DNA load in plasma may provide useful information for the therapeutic management of active CMV infection in this setting. A total of 59 consecutive patients were included in the study, of which 40 (67.8%) developed 1 (n = 21) or more (n = 19) episodes of CMV DNAemia. The need for antiviral therapy for initial or secondary episodes of CMV DNAemia could not be predicted on the basis of the CMV DNA load value in the first plasma testing positive by polymerase chain reaction (PCR). In contrast, in the absence of antiviral therapy, an increase of ≥3-fold between the baseline CMV DNA load and that measured a median of 6 days later discriminated between initial episodes eventually requiring antiviral treatment and those resolving spontaneously (sensitivity, 76.4%; specificity, 89.4%; positive predictive value, 86.6%; negative predictive value, 80.9%). This criterion was not useful for identifying recurrent episodes of CMV DNAemia that required antiviral therapy. The CMV doubling time and CMV DNA loads at the time of the first positive PCR and at initiation of preemptive therapy did not differ significantly between episodes that responded immediately to antiviral therapy from those showing a delayed response. The analysis of the dynamics of CMV DNA load in plasma in the absence of antiviral therapy allowed early recognition of episodes of CMV DNAemia that eventually needed to be treated, but did not permit prediction of the kinetics of CMV DNA clearance in response to antiviral therapy.