Relationship of cytomegalovirus load assessed by real-time PCR to pp65 antigenemia in organ transplant recipients

BACKGROUND: Cytomegalovirus (CMV) infection in immunocompromised patients can lead to viremia associated with morbidity and mortality. Monitoring of viral loads in blood is critical for initiating and monitoring antiviral treatment. OBJECTIVES: Validate quantitative real-time PCR assay targeting the US17 and UL54 regions of the CMV genome for automated DNA and extraction and amplification. STUDY DESIGN: 3422 blood specimens from organ transplant recipients, including longitudinal specimens from 12 organ transplant recipients, were tested by CMV PCR and pp65 antigenemia. RESULTS: CMV PCR for both US17 and UL54, was more sensitive and detected CMV DNA earlier and for longer than the CMV pp65 antigenemia test. Using antigenemia results as a reference standard, an optimal cutoff of 500 normalized copies was calculated for both US17 and UL54 PCR targets based on high sensitivity, specificity, and positive and negative predictive values. CMV DNA levels tracked well with clinical symptoms, response to treatment, and antigenemia. CONCLUSIONS: Detection of persistent increases in CMV DNA levels above 500 normalized copies by this real-time PCR assay is indicative of symptomatic CMV disease in organ transplant recipients. Quantitative real-time PCR for CMV DNA can be used in lieu of antigenemia for monitoring CMV infection and determining when to initiate preemptive treatment.     

Real-time PCR assay compared to nested PCR and antigenemia assays for detecting cytomegalovirus reactivation in adult T-cell leukemia-lymphoma patients

We analyzed the efficiency of the quantitative real-time PCR assay for cytomegalovirus (CMV) reactivation in adult T-cell leukemia-lymphoma (ATL) patients and compared the results with those obtained with qualitative nested PCR and antigenemia assays. The viral load obtained by the real-time PCR assay closely paralleled the number of antigen-positive cells obtained with the antigenemia assay. Real-time PCR revealed that a large number of DNA copies could be present even in samples assessed as negative or low in antigen-positive cells (0 to 10 antigen-positive cells/50,000 cells) by antigenemia assay. CMV copy numbers did not differ between the negative and low-antigen-positive groups. When the input concentration for real-time PCR assay was 2,500 to 5,000 copies/ml, the positivity rate for the nested PCR assay was 47.3%, while the positivity rate was more than 90% at an input concentration of >/=50,000 copies/ml. Real-time PCR is more sensitive than the antigenemia and nested PCR assays. Moreover, real-time PCR was able to detect CMV reactivation earlier than the antigenemia and nested PCR assays through the use of longitudinal analysis in four ATL patients with CMV pneumonia. In longitudinal assessments, analysis of the results suggested that a cutoff level of 5,000 copies/ml might be used to initiate treatment. Real-time PCR is more suitable for monitoring CMV reactivation in ATL patients than the antigenemia and nested PCR assays. CMV viral loads of 5,000 copies/ml are proposed as the cutoff for initiating antiviral therapy in ATL patients.     

Algorithm based on CMV kinetics DNA viral load for preemptive therapy initiation after hematopoietic cell transplantation

Preemptive therapy in hematopoietic cell transplantation is initiated by a diagnostic technique at first detection of cytomegalovirus (CMV). The aim of this study was to use the viral dynamics of CMV DNA viral to start preemptive therapy, and to prospectively compare the CMV viral load kinetics to pp65 antigenemia. Two hundred sixty-three blood samples were collected prospectively from 93 patients. All clinical decisions regarding use of preemptive therapy were based on CMV antigenemia. Based on the positivity of the antigen assay and clinical CMV outcome in allotransplant patients, an optimal threshold of 3.05 log 10 (1,130 copies/ml) was found to discriminate patients who required preemptive therapy and those who did not (sensitivity, 71%; specificity, 65%). A DNAemia level increase of 2.24 log 10 (174 copies/ml) per day was the optimal threshold to discriminate between patients who required preemptive therapy and those who did not (sensitivity, 93%; specificity, 43%). Sensitivity of PCR assay was 92.4% compared with 39% for the antigen assay (P < 0.001). A standardized real-time PCR assay is more appropriate than the antigen assay for detecting CMV. It allowed earlier diagnosis of active CMV infection and monitoring of the response to anti-CMV treatment.     

Validation of clinical application of cytomegalovirus plasma DNA load measurement and definition of treatment criteria by analysis of correlation to antigen detection

Successful preemptive cytomegalovirus (CMV) therapy in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infections. The pp65 antigenemia assay has been used for this purpose with considerable success. Quantification of CMV DNA is currently regarded to be an alternative diagnostic approach. The precise relationship between these two methods has still to be defined, but is essential to compare diagnostic results. This study compared the results of both assays with a large series of transplant recipients in different categories. An internally controlled quantitative real-time CMV DNA PCR was used to test 409 plasma samples from solid organ transplant (SOT) and stem cell transplant (SCT) patients. Levels of CMV DNA in plasma correlated well with classified outcomes of the pp65 antigenemia test. Despite this correlation, the quantitative CMV PCR values in a class of antigen test results were within a wide range, and the definition of an optimal cutoff value for initiating treatment required further analysis by a receiver-operating characteristic curve analysis. This is essential for reactivating infections in particular. For the SCT patients the optimal cutoff value of CMV DNA load defining relevant viral reactivation (in this assay, 10,000 copies/ml) was slightly higher than that for the SOT patients (6,300 copies/ml). Based on a comparison with the established pp65 antigenemia assay, quantification of CMV DNA in plasma appeared to be capable of guiding the clinical management of transplant recipients. This approach may have important advantages, which include a superior reproducibility and sensitivity, allowing the inclusion of kinetic criteria in clinical guidelines.     

Quantification of DNA in plasma by an automated real-time PCR assay (cytomegalovirus PCR kit) for surveillance of active cytomegalovirus infection and guidance of preemptive therapy for allogeneic hematopoietic stem cell transplant recipients

The performance of a plasma real-time PCR (cytomegalovirus [CMV] PCR kit; Abbott Diagnostics) was compared with that of the antigenemia assay for the surveillance of active CMV infection in 42 allogeneic hematopoietic stem cell transplantation (Allo-SCT) recipients. A total of 1,156 samples were analyzed by the two assays. Concordance between the two assays was 82.2%. Plasma DNA levels correlated with the number of pp65-positive cells, particularly prior to the initiation of preemptive therapy. Fifty-seven episodes of active CMV infection were detected in 37 patients: 18 were defined solely by the PCR assay and four were defined on the basis of the antigenemia assay. Either a cutoff of 288 CMV DNA copies/ml or a 2.42-log₁₀ increase of DNAemia levels between two consecutive PCR positive samples was an optimal value to discriminate between patients requiring preemptive therapy and those not requiring therapy on the basis of the antigenemia results. The real-time PCR assay allowed an earlier diagnosis of active CMV infection and was a more reliable marker of successful clearance of CMV from the blood. Analysis of the kinetics of DNAemia levels at a median of 7 days posttreatment allowed the prediction of the response to CMV therapy. Two patients developed CMV colitis. The PCR assay tested positive both before the onset of symptoms and during the disease period. The plasma real-time PCR from Abbott is more suitable than the antigenemia assay for monitoring active CMV infection in Allo-SCT recipients and may be used for guiding preemptive therapy in this clinical setting.     

Quantification of cytomegalovirus DNA by a fully automated real-time PCR for early diagnosis and monitoring of active viral infection in solid organ transplant recipients

BackgroundQuantification of cytomegalovirus (CMV) DNA by real-time PCR is currently considered an alternative diagnostic approach for the evaluation of active infection in transplant patients. The pp65 antigenemia assay has been used as reference test for monitoring active CMV infection and guiding preemptive therapy in transplant recipients. However, this assay suffers from some limitations: need for immediate processing of the samples, labour-intensive process, lack of standardization and subjective result interpretation.ObjectivesThe aim of this study was to evaluate the performance of a new commercially available real-time PCR assay coupled with a fully automated DNA extraction system (COBAS Ampliprep/COBAS Taqman CMV Test, Roche Diagnostics) for the detection of CMV-DNA in plasma comparing it with pp65 antigenemia assay for monitoring active CMV infection in solid organ transplant recipients (SOTRs).Study designA total of 266 consecutive samples from 45 SOTRs were monitored with pp65 antigenemia and in parallel with CMV-DNA quantitation by real-time PCR assay.ResultsFifty-eight samples resulted PCR-positive, 163 negative and for 45 samples the CMV-DNA values obtained were below the lower limit of quantification (<150 copies/ml); pp65 antigen was detected in 47 samples and resulted negative in 219 specimens. Concordance between the two evaluations was 76.7%; also a good correlation was observed (r = 0.718). Considering the existing treatment criteria based on pp65 antigenemia evaluation corresponding to pp65 levels ≥ 20 positive cells/200,000, preemptive therapy was administered to four asymptomatically infected patients. The corresponding cut-off value of CMV-DNA load calculated for discrimination between self-clearing infections and those requiring therapy was 2500 copies/ml (or 2275 IU/ml).ConclusionThe fully automated real-time PCR from Roche provided specific and sensitive results and represented a rapid and simple assay for the evaluation and monitoring of CMV infection in SOTRs. Further studies are required to validate the threshold level for the initiation of preemptive therapy.     

Analysis of a quantitative PCR assay for CMV infection in liver transplant recipients: an intent to find the optimal cut-off value.

BACKGROUND: Preemptive therapy required highly predictive tests for CMV disease. CMV antigenemia assay (pp65 Ag) has been commonly used for rapid diagnosis of CMV infection. Amplification methods for early detection of CMV DNA are under analysis. OBJECTIVES: To compare two diagnostic methods for CMV infection and disease in this population: quantitative PCR (qPCR) performed in two different samples, plasma and leukocytes (PMNs) and using a commercial diagnostic test (COBAS Amplicor Monitor Test) versus pp65 Ag. STUDY DESIGN: Prospective study conducted in liver transplant recipients from February 2000 to February 2001. RESULTS: Analyses were performed on 164 samples collected weekly during early post-transplant period from 33 patients. Agreements higher than 78% were observed between the three assays. Optimal qPCR cut-off values were calculated using ROC curves for two specific antigenemia values. For antigenemia >or=10 positive cells, the optimal cut-off value for qPCR in plasma was 1330 copies/ml, with a sensitivity (S) of 58% and a specificity (E) of 98% and the optimal cut-off value for qPCR-cells was 713 copies/5x10(6) cells (S:91.7% and E:86%). Using a threshold of antigenemia >or=20 positive cells, the optimal cut-off values were 1330 copies/ml for qPCR-plasma (S 87%; E 98%) and 4755 copies/5x10(6) cells for qPCR-cells (S 87.5%; E 98%). Prediction values for the three assays were calculated in patients with CMV disease (9 pts; 27%). Considering the assays in a qualitative way, the most sensitive was CMV PCR in cells (S: 100%, E: 54%, PPV: 40%; NPV: 100%). Using specific cut-off values for disease detection the sensitivity, specificity, PPV and NPV for antigenemia >or=10 positive cells were: 89%; 83%; 67%; 95%, respectively. For qPCR-cells >or=713 copies/5x10(6) cells: 100%; 54%; 33% and 100% and for plasma-qPCR>or=1330 copies/ml: 78%, 77%, 47%, 89% respectively. CONCLUSIONS: Optimal cut-off for viral load performed in plasma and cells can be obtained for the breakpoint antigenemia value recommended for initiating preemptive therapy with high specificities and sensitivities. Diagnostic assays like CMV pp65 Ag and quantitative PCR for CMV have similar efficiency and could be recommended as methods of choice for diagnosis and monitoring of active CMV infection after transplantation.     

Comparison of PCR, antigenemia assay, and rapid blood culture for detection and prevention of cytomegalovirus disease after lung transplantation

The goal of this study was to evaluate serial cytomegalovirus (CMV) blood culture, antigenemia testing, and qualitative and quantitative plasma CMV PCR for their ability to predict CMV disease and thus to direct preemptive therapy after lung transplantation. Forty-one patients provided 414 samples for blood culture, 290 samples for antigenemia testing, and 432 samples for PCR. Seven patients developed 11 episodes of CMV disease. CMV PCR had sensitivity, specificity, and positive predictive and negative predictive values of 79, 99, 84, and 99%, respectively, compared with 48, 99, 85, and 98%, respectively, for antigenemia testing, and 8, 100, 100, and 97%, respectively, for culture. Only quantitative CMV PCR correlated with disease stage: asymptomatic patients had a mean of 1,500 CMV DNA copies/ml, whereas patients who developed CMV disease had 5,087 copies/ml 12 to 4 weeks before symptoms and 32,000 copies/ml at diagnosis. Furthermore, CMV PCR-measured DNA increased 5- to 10-fold immediately preceding symptoms. PCR and antigenemia test values decreased with anti-CMV therapy. CMV DNA (as detected by PCR), but not antigenemia, persisted in patients who later developed recurrent CMV disease. The data indicate that lung transplant recipients will benefit from monitoring of CMV disease by plasma CMV PCR.     

Monitoring cytomegalovirus infection in adult and pediatric bone marrow transplant recipients by a real-time PCR assay performed with blood plasma

The objective of this study was to evaluate the advantages of cytomegalovirus (CMV) real-time PCR in blood plasma to monitor CMV infection in a population of adult and pediatric bone marrow recipients in comparison with the pp65 antigenemia method. Fifty allogeneic bone marrow transplant recipients from our center, including 23 adults and 27 children, were enrolled. A CMV real-time PCR designed to amplify a well-conserved region of the UL123 gene was evaluated for its results with whole blood and blood plasma. The CMV real-time PCR assay and the CMV antigenemia method were performed in parallel with 558 blood samples. The results obtained by the two techniques were significantly correlated (r = 0.732; P < 0.0001). Twenty patients developed at least one episode of CMV replication, with a total of 24 episodes detected by CMV PCR; antigenemia assays were positive in 17 of these 24 episodes. The first positive PCR test preceded the first positive antigenemia by a median of 8 days. The median time interval necessary to obtain a negative CMV PCR test after implementation of preemptive treatment was 28 days. CMV PCR of plasma was positive in two children with CMV disease (one with early CMV pneumonia and one with CMV gastroenteritis), while CMV antigenemia remained negative. The use of CMV PCR with plasma to guide both implementation and discontinuation of CMV preemptive therapy might reduce the risk of occurrence of CMV disease since patients would be treated earlier, and it might also help to reduce the duration of treatment, which could attenuate the side effects of antiviral drugs.     

Monitoring of cytomegalovirus infection in solid-organ transplant recipients by an ultrasensitive plasma PCR assay

Early and accurate monitoring of cytomegalovirus (CMV) infection in solid-organ transplant recipients is of major importance. We have assessed the potential benefit of an ultrasensitive plasma-based PCR assay for renal transplant recipients. The pp65 CMV antigen (pp65 Ag) assay using leukocytes was employed as a routine test for the monitoring of CMV in 23 transplant recipients. We compared the pp65 antigenemia with the CMV load quantified by an ultrasensitive PCR (US-PCR) with a limit of detection of 20 CMV DNA copies/ml of plasma. CMV infection was detected in 215 (67%) of 321 plasma samples by the US-PCR compared with 124 (39%) of 321 samples by the pp65 Ag assay. The US-PCR assay permitted the detection of CMV infection episodes following transplantation a median of 12 days earlier than the pp65 Ag assay. Moreover, during CMV infection episodes, DNA detection by the US-PCR was consistently positive, whereas false negative results were frequently observed with the pp65 Ag assay. We found a good correlation between the two assays, and the peak viral loads were significantly higher in patients with CMV-related complications (median, 5000 DNA copies/ml) than in those without symptoms (1160 DNA copies/ml) (P = 0.048). In addition, patients that did not require preemptive therapy based on the results of the pp65 assay had CMV loads significantly lower (median, 36 DNA copies/ml) than those that needed treatment (median, 4703 DNA copies/ml) (P < 0.001). These observations provided cutoff levels that could be applied in clinical practice. The ultrasensitive plasma-based PCR detected CMV infection episodes earlier and provided more consistent results than the pp65 Ag assay. This test could improve the monitoring of CMV infection or reactivation in renal transplant recipients.     

Measurement of human cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients

Quantitative monitoring of human cytomegalovirus (HCMV) infection is helpful in determining appropriate antiviral management of transplant recipients. Quantitative PCR technologies have demonstrated accuracy in measuring systemic HCMV loads. A total of 298 consecutive whole-blood specimens submitted to the Clinical Virology Laboratory at Vanderbilt University Medical Center from 15 February to 31 October 1999 were included in the study. In addition to a qualitative colorimetric microtiter plate PCR assay (MTP-PCR) and a semiquantitative pp65 antigenemia assay, each specimen was measured for HCMV loads by a quantitative PCR assay performed on an ABI PRISM 7700 Sequence Detection System (TaqMan). Compared to results of the MTP-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value were 70.5, 97.5, 87.8, and 92.8% for the antigenemia assay and were 96.7, 92.0, 75.6, and 99.1% for the TaqMan assay, respectively. There was a high correlation between antigenemia values and HCMV loads as determined by the TaqMan (r = 0.989; P < 0.001). Antigenemia values of 0, 1 to 10, 11 to 100, 101 to 1,000, and over 1,000 positive cells per 2 x 10(5) leukocytes corresponded to median HCMV loads measured by TaqMan of 125, 1,593, 5,713, 16,825, and 5,425,000 copies/ml, respectively. Corresponding to antigenemia values of 1 to 2, 10, and 50 positive cells per 2 x 10(5) leukocytes, HCMV viral loads of 1,000, 4,000, and 10,000 copies/ml are proposed as cutoff points for initiating antiviral therapy in patient groups with high, intermediate, and low risk of CMV diseases.     

Clinically-based determination of safe DNAemia cutoff levels for preemptive therapy or human cytomegalovirus infections in solid organ and hematopoietic stem cell transplant recipients

Transplantation Centers using human cytomegalovirus (HCMV) antigenemia-based preemptive therapy will need to replace in the near future the antigenemia assay with a more standardized and automatable assay, such as a molecular assay quantifying HCMV DNA in blood (DNAemia). Thus, in view of replacing antigenemia with clinically safe cutoff values, DNAemia levels corresponding to antigenemia cutoffs guiding HCMV preemptive therapy were determined retrospectively in solid organ and hematopoietic stem cell transplant recipients (HSCTR) using an "in-house" quantitative PCR (QPCR) method. Since preemptive therapy had prevented appearance of HCMV disease in all patients tested, DNA cutoffs determined retrospectively had to be considered as safe clinically as antigenemia cutoffs used prospectively. However, in solid organ transplant recipients (SOTR), initiating preemptive therapy upon an antigenemia cutoff of 100 pp65-positive leukocytes, a DNAemia cutoff of 300,000 copies/ml blood had positive and negative predictive values of >90%, indicating that a DNAemia cutoff could achieve, in terms of prevention of HCMV disease, the same clinical results as the antigenemia cutoff. In HSCTR, initiating preemptive therapy upon first antigenemia positivity, a DNAemia cutoff of 10,000 copies/ml blood had a positive predictive value of >90%, indicating that the great majority of patients treated under the antigenemia guidance would have been treated also using this DNA cutoff. On the other hand, the negative predictive value of 28.6% indicated that two out of three HSCTR had been treated under the antigenemia guidance having the same levels of viral DNA as the untreated patients. The data suggest that a quantitative cutoff could be adopted as a guiding criterion for preemptive therapy also in HSCTR. Regression analysis allowed to determine the DNAemia (corresponding to QPCR) cutoff values for two commercial assays tested both in solid organ and HSCTR. Retrospective DNAemia cutoff values will be verified for safety in prospective trials.     

Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR

Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.     

Differences between the quantitative antigenemia assay and the cobas amplicor monitor quantitative PCR assay for detecting CMV viraemia in bone marrow and solid organ transplant patients.

The relationship between quantitative PCR (COBAS Amplicor CMV Monitor, Roche Diagnostics) and quantitative antigenemia (Monofluor pp65, Sanofi Diagnostics) was examined for monitoring CMV viraemia. A total of 469 specimens from immunocompromised haematology and solid organ transplant patients were tested by quantitative antigenemia and qualitative PCR. Quantitative PCR (QPCR) was performed on the 245 specimens in which CMV DNA was detected by qualitative PCR. To exclude any effect due to specific anti-CMV treatment, analysis of antigenemia and QPCR results was only performed on the 164 of 245 specimens collected from patients not on ganciclovir or foscarnet treatment. Forty seven specimens had <400 CMV copies/mL and a negative antigen result, four specimens were antigen positive (all between 1 to 10 positive CMV cells/2 x 10(5) leucocytes) and had <400 CMV copies/mL. Fifty-one specimens had a CMV viral load > or = 400 copies/mL and a negative antigen result and 62 specimens had a CMV viral load > or = 400 copies/mL and a positive antigen. The viral load was shown to be as high as 43,000 copies/mL in some patients with a negative antigen and occurred in non-neutropenic patients. The correlation coefficient for antigen and QPCR results for specimens from bone marrow transplant patients, was 0.69 with an average CMV viral load of 3,200 copies/mL (SEM = 800) and an average antigen of nine positive CMV cells/2 x 10(5) leucocytes (SEM = 3). In the corresponding solid organ transplant group, the correlation coefficient for antigen and QPCR results was 0.71 with an average CMV viral load of 9,900 copies/mL (SEM = 2,100) and an average antigen of 26 positive CMV cells/2 x 10(5) leucocytes (SEM = 6). Both the average viral load and the average antigen result in specimens from solid organ transplant patients, were significantly higher than the average viral load and antigen result in the corresponding group of bone marrow transplant patients (Two-Sample-for-Means z-Test, P = 0.001 and P = 0.003, respectively). The differences in the kinetics of the two assays in monitoring CMV and their ability to predict CMV disease was also assessed in a sub-group of patients. In conclusion, the two assays used in this study do not always show parallel changes in CMV viral load, but may be complementary for the diagnosis and management of CMV disease. The observation that non-neutropenic patients can have a high viral load in plasma and a negative antigenemia has implications for laboratories using antigenemia alone to monitor patients for CMV disease.     

Determination,validation and standardization of a CMV DNA cut-off value in plasma for preemptive treatment of CMV infection in solid organ transplant recipients at lower risk for CMV infection

BackgroundValganciclovir preemptive therapy guided by the viral load is the current strategy recommended for preventing CMV disease in CMV-seropositive Solid Organ Transplant Recipients (SOTR) at lower risk for developing CMV infection. However, universal viral load cut-off has not been established for initiating therapy.ObjectivesOur goal was to define and validate a standardized cut-off determined in plasma by real-time PCR assay for initiating preemptive therapy in this population.Study designA prospective cohort study of consecutive cases of CMV-seropositive SOTR was carried out. The cut-off value was determined in a derivation cohort and was validated in the validation cohort. Viral loads were determined using the Quant CMV LightCycler 2.0 real-time PCR System (Roche Applied Science) and results were standardized using the WHO International Standard for human CMV.ResultsA viral load of 3983 IU/ml (2600 copies/ml) was established as the optimal cut-off for initiating preemptive therapy in a cohort of 141 patients with 982 tests and validated in a cohort of 252 recipients with a total of 2022 test. This cut-off had a 99.6% NPV indicating that the great majority of patients at lower risk will not develop CMV disease without specific antiviral therapy. The high sensitivity and specificity (89.9% and 88.9%, respectively) and the relatively small numbers of patients with CMV disease confirm that real-time PCR was optimal.ConclusionsWe have established a cut-off viral load for starting preemptive therapy for CMV-seropositive SOT recipients. Our results emphasized the importance of a mandatory follow-up protocol for CMV-seropositive patients receiving preemptive treatment.     

Real-time automated PCR for early diagnosis and monitoring of cytomegalovirus infection after bone marrow transplantation

The purpose of this study was to assess the usefulness of real-time automated PCR as a quantitative, highly reproducible, and sensitive method to detect cytomegalovirus (CMV) DNA in blood specimens. Intra- and interassay precision rates were 0.89% (small number of copies [L]), 1.43% (middle number of copies [M]), and 1.12% (high number of copies [H]), and 4.46% (L), 1.51% (M), and 2.28% (H), respectively. The linearity of this assay was obtained between 10 and 10(7) copies/well, with a minimum detection limit of 20 copies/well. Specimens from 55 of 70 healthy subjects were found to be positive for CMV antibody, but CMV DNA was not detected in any of them. In the qualitative assessment of each specimen, the results of the CMV antigenemia assay and those of the real-time PCR assay agreed in 80% (plasma specimens), 79% (all nucleated cells), and 86% (blood) of the cases examined. For eight patients diagnosed as having CMV infection or disease, no sample was positive in the antigenemia assay earlier than in the real-time PCR assay. Furthermore, the results of this assay could be obtained within 8 h. We concluded that the real-time PCR assay is useful for rapid diagnosis of CMV infection and monitoring of clinical courses.     

CMV antigenemia and quantitative viral load assessments in hematopoietic stem cell transplant recipients

BackgroundSensitive and reliable diagnostic tests are essential for the prevention of cytomegalovirus (CMV) disease after hematopoietic stem cell transplantation (HSCT). pp65 antigenemia and polymerase chain reaction (PCR) assays are commonly used to monitor CMV in HSCT recipients. However, there is considerable intra- and inter-laboratory variability in the results, which impact comparability and clinical practice.Objectives/study designUsing 380 samples from 135 HSCT recipients, we compared the new FDA approved quantitative PCR assay, COBAS^® AmpliPrep/COBAS^® TaqMan^® CMV test (CAP/CTM CMV test) developed and standardized using the 1st WHO International Standard for CMV with pp65 antigenemia and COBAS^® AMPLICOR MONITOR CMV tests.ResultsThe median time between transplantation and testing samples was 57 days (range, 0–207 days). The median CMV load (log₁₀) was 3.17 IU/mL (3.21 copies/mL). Among samples with detectable CMV load, 52% were negative by pp65 antigenemia. CMV loads were higher in pp65 antigenemia-positive than in negative samples. One pp65-antigenemia-positive cell per 100,000 leukocytes corresponded to a median CMV load of 1200 IU/mL. CMV loads determined by the CAP/CTM CMV test were slightly lower than the ones by the AMPLICOR MONITOR CMV test (?0.15 [95% CI, ?0.18 to ?0.13] copies/mL), but slope differences indicated only limited co-linearity.ConclusionsThe CAP/CTM CMV test is more sensitive than pp65 antigenemia and the AMPLICOR MONITOR CMV test in HSCT recipients. The lower limit of quantification and co-linearity with the international WHO standard renders the CAP/CTM CMV test suitable for future clinical trials defining viral load thresholds of CMV therapy.     

Qualitative Plasma PCR Assay (AMPLICOR CMV Test) versus pp65 Antigenemia Assay for Monitoring Cytomegalovirus Viremia and Guiding Preemptive Ganciclovir Therapy in Allogeneic Stem Cell Transplantation

The performances of a commercially available qualitative plasma PCR assay (AMPLICOR CMV test; Roche Diagnostics) and the pp65 antigenemia assay (AG) were evaluated for the monitoring of cytomegalovirus (CMV) viremia in 43 allogeneic stem cell transplant recipients. In addition, the suitabilities of both assays for triggering the initiation of preemptive ganciclovir therapy were assessed. A total of 37 CMV viremic episodes were detected in 28 patients. Positivity of plasma PCR testing in one or more consecutive specimens was the only marker of CMV viremia in 18 of the 37 episodes (PCR positive and AG negative, n = 50 specimens). Five episodes were diagnosed on the basis of a single positive AG result (AG positive and PCR negative, n = 5 specimens); both assays were eventually positive (PCR positive and AG positive, n = 27 specimens) for 14 viremic episodes; for these episodes, conversion of the PCR assay result to a positive result occurred an average of 1 week before conversion of the AG result. Overall, the concordance between the two methods was 90%, and the sensitivities of the plasma PCR assay and AG for the detection of CMV viremic episodes were 86.5 and 51.3%, respectively. Two patients who tested positive by both assays simultaneously progressed to CMV end-stage organ disease, despite the initiation of preemptive ganciclovir therapy. Conversion of the AG result to a negative result upon administration of preemptive ganciclovir therapy occurred a median of 7.5 days earlier than conversion of the plasma PCR assay result. Nineteen of the 28 patients with CMV viremia received AG-guided preemptive ganciclovir therapy; had the positivity of the plasma PCR assay triggered the initiation of preemptive therapy, 9 additional patients would have been unnecessarily treated since none of them developed CMV end-stage organ disease. Although the AMPLICOR CMV assay is more sensitive than AG, the latter appears to be more suitable both for guiding the initiation of preemptive therapy and for monitoring a patient's response to antiviral therapy.     

Paradoxical rising cytomegalovirus antigenemia during preemptive ganciclovir therapy in hematopoietic stem cell transplant recipients: incidence, risk factors, and clinical outcomes

Preemptive ganciclovir (GCV) therapy is adopted increasingly in hematopoietic stem cell transplant (HCT) recipients, but occasional cases of increasing cytomegalovirus (CMV) antigenemia levels occur during preemptive GCV therapy. This prospective study investigated the incidence, risk factors, and clinical outcomes of paradoxical responses during GCV therapy. Adult patients receiving allogeneic HCTs during a 24-month period were enrolled. Patients were prospectively monitored for CMV antigenemia once a week until 3 months after engraftment. Paradoxical responders were defined as patients exhibiting CMV antigenemia levels elevated from the baseline after the first week of preemptive GCV therapy. Of 252 HCT recipients, 97 (38%) received preemptive GCV therapy due to CMV infection. Of these 97 patients, 23 (24%) were classified as paradoxical responders. Risk factors for paradoxical response were a low white blood cell (WBC) count (P = 0.02) and a prolonged duration of CMV antigenemia (P = 0.04) before preemptive therapy. There were no significant differences in rates of successful viral clearance and secondary episodes of CMV infection between paradoxical responders (87% [20/23] and 26% [6/23]) and nonparadoxical responders (95% [70/74] and 23% [17/74], respectively). However, breakthrough CMV disease during preemptive GCV therapy was significantly more frequent in paradoxical responders (17% [4/23]) than in nonparadoxical responders (3% [2/74], P = 0.03). Paradoxical responses occurred in one-quarter of the HCT recipients receiving preemptive GCV therapy. A low WBC count and a long duration of CMV antigenemia before GCV therapy were associated with paradoxical responses, and breakthrough CMV disease during preemptive GCV therapy occurred more frequently in paradoxical responders.     

Cytomegalovirus Infection Management in Allogeneic Stem Cell Transplant Recipients: a National Survey in Spain

This study gathered information about current practices of cytomegalovirus (CMV) infection management in allogeneic stem cell transplant recipients at Spanish centers. A wide variety of preemptive antiviral therapy strategies for CMV infection guided by real-time PCR assays was found, yet the incidence of CMV disease was low (<3%).