Improved monitoring of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation by an ultrasensitive plasma DNA PCR assay

Cytomegalovirus (CMV) DNA amplification assays in plasma have shown limited sensitivity compared to the detection of pp65 antigen in leukocytes. Our goal was to increase the sensitivity of a commercial CMV DNA PCR quantitative assay. After modification, the new assay was able to reproducibly detect 20 CMV DNA copies/ml of plasma. We compared this new ultrasensitive PCR assay with the standard PCR and the pp65 test for CMV detection and quantification in 22 consecutive allogeneic hematopoietic stem cell recipients. CMV infection or reactivation was detected in 84 of 319 (26%) samples by the ultrasensitive PCR assay compared to 38 of 319 (12%) samples by the pp65 assay (P < 0.01). All samples positive by the pp65 assay were positive by the ultrasensitive PCR, and CMV episodes were detected on average 4 days earlier and 7 days later than the first and the last pp65-positive test, respectively. In addition, during CMV episodes, the ultrasensitive assay identified positive samples that were inconsistently detected by the pp65 assay. The ultrasensitive assay was also much more sensitive than the standard PCR, with 26 versus 12% of CMV DNA-positive samples (P < 0.01). This assay improved the monitoring of CMV infection or reactivation in hematopoietic allogeneic stem cell recipients.     

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.     

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.     

Clinical and economic burden of pre-emptive therapy of cytomegalovirus infection in hospitalized allogeneic hematopoietic cell transplant recipients

Cytomegalovirus (CMV) infection remains a major complication after allogeneic hematopoietic cell transplantation (allo-HCT). We conducted a retrospective study to determine the clinical and economic burden of pre-emptive therapy (PET) for CMV infection in 100 consecutive hospitalized adult CMV positive serostatus allo-HCT recipients and compared their hospitalization cost with allo-HCT recipients hospitalized with graft vs host disease without CMV infection (control group) and across 19 US cancer centers for hospitalized patients with CMV infection between 2012 and 2015 (Vizient database). A total of 192 CMV episodes of PET for CMV infection occurred within 1 year post-HCT. PET consisted of ganciclovir (41% of episodes), foscarnet (40%), and valganciclovir (38%) with the longest average length of stay in foscarnet-treated patients (41 days). The average direct cost per patient admitted for PET was $116 976 (range: $7866-$641 841) compared with $12 496 (range: $2004-$43 069) in the control group (P < .0001). The total direct cost per encounter was significantly higher in patients treated with foscarnet and had nephrotoxicity ($284 006) compared with those who did not ($112 195). The average cost amongst the 19 US cancer centers, including our institution, was $42 327 with major disparities in cost and clinical outcomes. PET for CMV infection is associated with high economic burden in allo-HCT recipients.     

Quantification of adenovirus DNA in unrelated donor hematopoietic stem cell transplant recipients

BACKGROUND: Adenovirus (AdV) infection is a life threatening condition in immunosuppressed patients. Quantitative AdV assays can improve the clinical management of these patients. OBJECTIVES: To evaluate quantitative measurement of AdV DNA with PCR in blood from hematopoietic stem cell transplant (HSCT) recipients. STUDY DESIGN: Quantitative PCR was used to measure viral DNA levels of AdV in consecutive blood samples from 40 HSCT recipients (27 adults and 13 children) during a 1-year post-engraftment period. All patients received grafts from unrelated donors and were given anti-T-cell antibodies in the conditioning regimen. RESULTS: In the group of 40 patients, six (15%) had detectable AdV DNA in blood for different lengths of time. None of these six patients suffered from severe graft-versus-host disease. In three of the patients a high AdV viral load (>10,000copies/mL) was detected, one of whom also had high viral load of EBV and CMV and one of EBV only. These three patients died within 2 months after detection of ADV viremia. A low AdV viral load (<500copies/mL) was detected in three surviving patients and they did not have concomitant high viral load of neither CMV nor EBV. CONCLUSIONS: AdV viremia was present in 15% of the HSCT recipients and a high AdV viral load was associated with fatal outcome. Screening for AdV DNA with quantitative PCR in blood may be of clinical importance in allogeneic HSCT recipients in order to prevent severe clinical virological complications.     

Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients

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.     

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.     

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.     

Whole blood real-time quantitative PCR for cytomegalovirus infection follow-up in transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) remains a major opportunistic agent among transplant recipients. While detection of CMV pp65-lower matrix protein (pp65Ag) is still widely used for monitoring CMV infection, real-time PCR assays have been recently developed for routine quantitation of CMV DNA. However, correlations are lacking between results of pp65Ag and quantitative PCR assays and there is no consensus yet as to the more appropriate blood compartment (whole blood (WB), leukocytes, plasma) to be tested with PCR assays. OBJECTIVES: The aims of the study were to determine, in a population of transplant recipients: (i) the correlation between pp65Ag and CMV quantitative real-time PCR in our setting and (ii) the utility of plasma CMV DNA quantitation in comparison to WB quantitation. METHODS: In 170 blood samples (from 61 solid organ or bone marrow transplant recipients) with pp65Ag results, CMV quantitation was performed in WB and plasma using an in-house real-time quantitative PCR. RESULTS: Real-time PCR and pp65Ag results in WB were correlated: thresholds of 10 and 50(+) cells/200,000 cells were equivalent to 3.3 log(10)copies/mL (2,000 copies/mL) and 3.8 log(10)copies/mL (6,300 copies/mL), respectively. When WB viral load was >or=3.6 log(10)copies/mL, the risk to have a negative plasma CMV DNA result was 相似文献   

Evaluation of a quantitative plasma PCR plate assay for detecting cytomegalovirus infection in marrow transplant recipients.

A plasma PCR test, using a nonradioactive PCR plate assay, was evaluated for detection of human cytomegalovirus reactivation. This assay was compared to Southern blotting and found to perform well. As a noncompetitive method of quantitation, it was similar to a competitive method for detecting the number of genome copies per milliliter of plasma in marrow transplant recipients. This is a technically simplified assay with potential for adaptation to automation.     

Performance characteristics of an automated PCR assay for the quantification of cytomegalovirus DNA in plasma

The COBAS Amplicor CMV Monitor test (Roche Diagnostics), an automated polymerase chain reaction (PCR) assay for the quantification of cytomegalovirus (CMV) DNA in plasma samples, was evaluated in a routine diagnostic laboratory. Using cell culture-derived CMV and CMV-negative human plasma, the linear detection range of the assay as well as its intra-and inter-assay variabilities were assessed. The study design allowed distinguishing variations in results related to amplification and detection from those caused by differences in the efficiency of DNA extraction. The assay was able to identify the majority of samples correctly as positive with CMV DNA concentrations above the limit of detection. However, the reported values were often twofold or more different from the (theoretical) input, which could be explained partly by inefficient DNA extraction. The following values were computed for the coefficients of determination R(2): inter-assay variability excluding DNA extraction, R(2)=0.982; including DNA extraction, R(2)=0.977; intra-assay variability excluding DNA extraction, R(2)=0.992; including DNA extraction, R(2)=0.992. On balance, the test has acceptable within-run and between-run reproducibility. It therefore allows the comparison of results obtained at different time-points as well as in different laboratories, e.g. in multi-centre studies.     

Spontaneously-resolving episodes of cytomegalovirus DNAemia in allogeneic hematopoietic stem cell transplant recipients: Virological features and clinical outcomes

It has been reported that low-plasma cytomegalovirus (CMV) DNA loads are associated with an increased risk of overall mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Utilizing a conservative strategy for initiation of preemptive antiviral therapy (>1500 IU/mL), we characterized the virological features of spontaneously-resolving episodes of CMV DNAemia and assessed their impact on mortality through the first year after transplantation. We reviewed the CMV DNA polymerase chain reaction results and clinical charts of 230 consecutive adult patients who underwent T-cell replete allo-HSCT at our center. A total of 280 episodes of CMV DNAemia were registered in 164 patients, of which 144 episodes cleared spontaneously. Clearance of CMV DNAemia was significantly delayed in initial and recurrent self-resolving episodes featuring CMV DNA peak loads > 250 IU/mL compared with those displaying lower values. All-cause mortality in patients with self-resolving episodes of CMV DNAemia was comparable (P = 0.7) to that in patients with no CMV DNAemia and was not related to the CMV DNA peak load (≥250 IU/mL vs <250 IU/mL) (P = 0.6). In summary, in our setting, the magnitude of the CMV DNA peak load reached during self-resolving episodes of CMV DNAemia correlated directly with duration of episodes, but had no apparent impact on all-cause mortality taking patients with no documented CMV DNAemia as a reference.     

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.     

Antiviral drugs for cytomegalovirus in transplant recipients: advantages of preemptive therapy

Currently available, potent antiviral agents have proven highly effective for the prevention of CMV disease during the time that prophylaxis is employed. However, late-onset CMV disease, occurring in 5-18% of the patients, has emerged as a significant complication in organ transplant recipients receiving prophylaxis. Complete suppression of the virus with long-term use of a potent antiviral agent may be less conducive to antigen-induced priming of host responses, which may explain why there is a greater likelihood of late-onset CMV disease. On the other hand, allowing controlled low-level viral replication, implicit in the strategy of preemptive therapy, may facilitate CMV-specific immune reconstitution and have a mitigating effect on the risk of late-onset CMV disease. Preemptive therapy with valganciclovir appears less likely to be associated with CMV disease, largely due to a lower incidence of late-onset CMV disease. Emerging data, documenting that CMV disease in the era of prophylaxis with potent antiviral agents is an independent contributor to mortality, particularly infection-associated mortality, stands to challenge the notion that prophylaxis is more likely to have a beneficial effect on CMV-related secondary outcomes. Preemptive therapy is a more logical and theoretically more appealing approach for the prevention of CMV disease in transplant recipients.     

Breakthrough disseminated Aspergillus ustus infection in allogeneic hematopoietic stem cell transplant recipients receiving voriconazole or caspofungin prophylaxis

Aspergillus ustus is an uncommon clinical species which is poorly susceptible to antifungals. We report two cases of A. ustus infections that occurred in allogeneic stem cell transplant recipients while they were receiving either voriconazole or caspofungin. Prolonged use of these new antifungal agents may increase the risk of the emergence of resistant organisms.     

Diagnostic yield of the cytomegalovirus (CMV) antigenemia assay and clinical features in solid organ transplant recipients and hematopoietic stem cell transplant recipients with CMV pneumonia

Data are limited on the value of non-invasive diagnostic methods, such as the cytomegalovirus (CMV) antigenemia assay, and the clinical features of CMV pneumonia in patients who have undergone solid organ transplant (SOT) compared with those who have had hematopoietic stem cell transplant (HSCT). All adult patients with suspected CMV pneumonia, who had received SOT or HSCT in a tertiary hospital during a 5-year period, were retrospectively enrolled. CMV pneumonia was defined as clinical and radiographic evidence of pneumonia in association with the isolation of CMV in viral cultures of bronchoalveolar lavage or lung tissue specimens, or with the identification of CMV in lung tissue. In total, 36 patients with CMV pneumonia were identified. Of these, 29 (80%) had received SOT and 7 (20%) had received HSCT. The incidence of CMV pneumonia in the patients with SOT (3.0 per 1000 person-years [95% confidence interval {CI} 0.6-8.7]) was lower than in those with HSCT (17.0 per 1000 person-years [95% CI 9.9-27.2], P?=?0.003) and CMV-related mortality showed a tendency to have lower mortality in patients with SOT (10% [3/29]) than with HSCT (43% [3/7], P?=?0.07). The overall sensitivity of the CMV assay (≥?1/200,000 leukocytes) in patients with CMV pneumonia was 69% (95% CI 52-84%). The CMV antigenemia test is of limited value in diagnosing CMV pneumonia, given the high cost of false-negative diagnoses of CMV pneumonia.     

Extended lamivudine therapy against hepatitis B virus infection in hematopoietic stem cell transplant recipients.

Lamivudine has demonstrated efficacy in the treatment and prevention of hepatitis B virus (HBV) reactivation after hematopoietic stem cell transplantation (HSCT). However, most of these studies involved short durations of prophylaxis, so there is significant concern regarding lamivudine resistance in these patients. Between March 1984 and November 2002, 71 HBV surface antigen-positive HSCT recipients, including a subgroup of 16 who received pretransplantation lamivudine therapy, which was continued into the posttransplantation period to prevent reactivation hepatitis, were enrolled onto our study. The efficacy of lamivudine therapy was first evaluated for the subgroup of 16 patients in terms of treatment response, lamivudine resistance, and viral recurrence after discontinuation by using virologic assays. Efficacy was then evaluated for all patients in terms of the hazards of lamivudine therapy for reactivation hepatitis after transplantation. During a median lamivudine therapy period of 73 weeks (range, 19-153 weeks), the initial response showed a median reduction of 2.54 log10 in serum HBV DNA (-0.28 to 6.72 range). Lamivudine-resistant mutations were detected in 10 (63%) of 16 patients during therapy, and 1 (12%) of 16 patients finally developed a viral breakthrough. At a median follow-up of 30 months after discontinuation, 3 (27%) of 11 cases had recurrence of HBV infection. Despite the emergence of the mutations, no deaths were due to HBV reactivation or severe cases of hepatitis. In the Cox proportion regression model regarding reactivation hepatitis after transplantation of all enrolled patients, lamivudine therapy was found to be the only favorable factor for the event, with a hazard ratio of 0.122 (95% confidence interval, 0.016-0.908; P = .040). In conclusion, extended lamivudine therapy is safe and effective for the prevention of HBV reactivation in an HSCT setting and significantly decreases reactivation hepatitis after transplantation.