Cytomegalovirus DNA Load In Plasma Research Articles

Performance evaluation of the Alinity m system for quantifying cytomegalovirus DNA in samples of the respiratory, gastrointestinal, and urinary tract.

Quantitation of cytomegalovirus (CMV) DNA load in specimens other than blood such as bronchoalveolar lavages, intestinal biopsies, or urine has become a common practice as an ancillary tool for the diagnosis of CMV pneumonitis, intestinal disease, or congenital infection, respectively. Nevertheless, most commercially available CMV PCR platforms have not been validated for CMV DNA detection in these specimen types. In this study, a laboratory-developed test based on Alinity m CMV ("Alinity LDT") was evaluated. Reproducibility assessment using spiked bronchial aspirate (BAS) or urine samples showed low standard deviations of 0.08 and 0.27 Log IU/mL, respectively. Evaluating the clinical performance of Alinity LDT in comparison to a laboratory-developed test based on RealTime CMV ("RealTime LDT") showed good concordance across 200 clinical specimens including respiratory specimens, intestinal biopsies, urine, and stool. A high Pearson's correlation coefficient of r = 0.92, a low mean bias of -0.12 Log IU/mL, a good qualitative agreement of 90%, and a Cohen's kappa value of 0.76 (substantial agreement) were observed. In separate analyses of the sample types BAS, tracheal aspirates, bronchoalveolar lavage, biopsies, and urine, the assay results correlated well between the two platforms with r values between 0.88 and 0.99 and a bias <0.5 Log IU/mL. Overall, the fully automated, continuous, random access Alinity LDT yielded good reproducibility, high concordance, and good correlation to RealTime LDT in respiratory, gastrointestinal, and urine samples and may enhance patient management with rapid result reporting.IMPORTANCEIn transplant recipients, a major cause for morbidity and mortality is end-organ disease by primary or secondary CMV infection of the respiratory or gastrointestinal tract. In addition, sensorineural hearing loss and neurodevelopmental abnormalities are frequent sequelae of congenital CMV infections in newborns. Standard of care for highly sensitive detection and quantitation of the CMV DNA load in plasma and whole blood specimens is real-time PCR testing. Beyond that, there is a need for quantitative determination of CMV DNA levels in respiratory, gastrointestinal, and urinary tract specimens using a highly automated, random access CMV PCR assay with a short turnaround time to enable early diagnosis and treatment. In the present study, clinical performance of the fully automated Alinity m analyzer in comparison to the current RealTime LDT assay was evaluated in eight different off-label sample types.

Letermovir use may impact on the Cytomegalovirus DNA fragmentation profile in plasma from allogeneic hematopoietic stem cell transplant recipients.

Cytomegalovirus (CMV) DNA in plasma is mainly unprotected and highly fragmented. The size of the amplicon largely explains the variation in CMV DNA loads quantified across PCR platforms. In this proof-of-concept study, we assessed whether the CMV DNA fragmentation profile may vary across allogeneic hematopoietic stem cell transplant recipients (allo-SCT), within the same patient over time, or is affected by letermovir (LMV) use. A total of 52 plasma specimens from 14 nonconsecutive allo-SCT recipients were included. The RealTime CMV PCR (Abbott Molecular), was used to monitor CMV DNA load in plasma, and fragmentation was assessed with a laboratory-designed PCR generating overlapping amplicons (around 90-110 bp) within the CMV UL34, UL80.5, and UL54 genes. Intrapatient, inter-patient, and LMV-associated qualitative and quantitative variations in seven amplicons were observed. These variations were seemingly unrelated to the CMV DNA loads measured by the Abbott PCR assay. CMV DNA loads quantified by UL34_4, UL54.5, and UL80.5_1 PCR assays discriminate between LMV and non-LMV patients. Our observations may have relevant implications in the management of active CMV infection in allo-SCT recipients, either treated or not with LMV, although the data need further validation.

A Prospective Study of Plasma and Bronchoalveolar Lavage Fluid CMV DNA Load Quantification for the Diagnosis and Outcome of CMV Pneumonitis in Immunocompromised Hosts.

Molecular testing has been utilized for cytomegalovirus (CMV) pneumonitis (CMVP) diagnosis, although its validity and optimal cut-off values remain limited. A prospective study of CMVP diagnosis among immunocompromised patients was conducted by measuring quantitative CMV DNA polymerase chain reaction in plasma and bronchoalveolar lavage fluid (BALF). Forty-five adult immunocompromised patients were investigated. Thirty-two patients (71%) received immunosuppressive therapy. Eleven patients (24%) were confirmed to have CMVP. Of those, three and eight patients were classified as proven and probable CMVP, respectively. Median (IQR) plasma CMV DNA loads in CMVP and non-CMVP were 41,939 (4,424-122,608) and 0 (0-44) IU/mL, respectively (p<0.001). Median (IQR) BALF CMV DNA loads in CMVP and non-CMVP were 379,652 (163,800-1,254,000) and 0 (0-1,348) IU/mL, respectively (p<0.001). A significant correlation was observed between plasma and BALF CMV DNA loads (r=0.887, p<0.001). Plasma CMV DNA load of 831 IU/mL was established as a cut-off value for diagnosing CMVP (AUC 0.9987, sensitivity 100%, specificity 94.1%, positive predictive value 84.5%, negative predictive value 100%). A strongly positive correlation was observed between CMV DNA loads measured in plasma and BALF. CMV DNA load quantification could potentially assist in diagnosing CMVP in immunocompromised patients, although bronchoscopy remains encouraged for a definitive diagnosis.

Role of highly sensitive nucleic acid amplification testing for plasma cytomegalovirus DNA load in diagnosis of cytomegalovirus gastrointestinal disease among kidney transplant recipients.

High plasma cytomegalovirus (CMV) DNA load is generally associated with CMV tissue-invasive disease in solid organ transplant recipients. However, some tissue-invasive diseases, especially CMV gastrointestinal (GI) disease, have undetectable to very low plasma CMV DNA loads. Highly sensitive nucleic acid amplification testing (NAAT) has been increasingly used to quantify low-level CMV DNA loads. Our primary objective was to investigate the epidemiology of CMV GI disease and evaluate the validity of plasma CMV NAAT for the diagnosis of CMV GI disease in kidney transplant (KT) recipients. We conducted a retrospective study of all KT recipients who developed CMV GI disease from January 2016 to December 2018. Plasma CMV DNA load was measured using real-time PCR. The cut-off value of plasma CMV DNA load for diagnosing and risk factors of CMV GI disease were analyzed. A total 17 (3.4%) cases of CMV GI disease occurred in 494 KT recipients. Fifteen (88%) patients had CMV D + /R + serostatus. Fourteen (82%) patients developed CMV GI disease within 6months after KT. Plasma CMV DNA loads were detectable in all (100%) patients with a median load 11,102 (IQR 2,935-107,160) IU/ml. A plasma CMV DNA load of 4,063IU/ml was established as al cut-off for diagnosing CMV GI disease (AUC 0.74, sensitivity 76.5%, specificity 70%, PPV 68, NPV 78). In multivariate analysis, prolonged cold ischemic time (HR 1.14, 95% CI 1.05-1.23, P=.002) and CMV D + /R - serostatus (HR 9.31, 95% CI 2.12-40.74, P=.003) were associated with CMV GI disease. Using highly sensitive NAAT could potentially assist in the diagnosis of CMV GI disease in a CMV D + /R + serostatus setting. KT recipients with CMV seromismatch and prolonged cold ischemic time are at higher risk of CMV GI disease.

A Prospective Study of Cytomegalovirus-Specific Cell-Mediated Immune Monitoring and Cytomegalovirus Infection in Patients With Active Systemic Lupus Erythematosus Receiving Immunosuppressants.

BackgroundThe effects of cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) on CMV infection in patients with autoimmune diseases receiving immunosuppressants have not been explored.MethodsPatients with active systemic lupus erythematosus (SLE) were preemptively monitored for clinically significant CMV infection (CsCMVI; defined as plasma CMV DNA loads >3 log10 IU/mL). CMV-specific CMI was assessed using an enzyme-linked immunosorbent assay (QuantiFERON-CMV [QF]) before as well as 1 and 3 months after intense immunosuppressive therapy.ResultsThe study included 55 patients with active SLE; patients were a mean age (SD) of 34 (13) years and had a median SLE Disease Activity Index 2000 score (SD) of 14 (8), and 93% were female. Most patients had renal involvement (67%), received methylprednisolone (93%), and were CMV-seropositive (95%). Thirteen (23.6%) patients developed CsCMVI. Among patients with active SLE who were QF-negative (QF–) and QF-positive (QF+) before receiving immunosuppressive therapy, 28.6% and 25% developed CsCMVI, respectively (P = .69). However, 1 month postimmunosuppression, more QF– than QF+ patients developed CsCMVI (44.4% vs 11.8%; P = .03; adjusted hazard ratio, 4.97; 95% CI, 1.07–23.10; P = .04).ConclusionsPatients with active SLE and low CMV-specific T-cell responses could develop CMV infection after receiving immunosuppressants. Further studies should focus on CMV-specific CMI among patients with autoimmune diseases.

Reconstitution of cytomegalovirus-specific T-cell immunity following unmanipulated haploidentical allogeneic hematopoietic stem cell transplantation with posttransplant cyclophosphamide.

Cytomegalovirus (CMV) DNAemia and CMV disease have been reported as more frequent in patients undergoing haploidentical allogeneic hematopoietic stem cell transplantation (Haplo-HSCT) than in those receiving HLA-matched allografts. This could be due to impaired CMV-specific T-cell reconstitution. Here, we conducted a multicenter observational study to assess CMV pp65 and IE-1-specific T cells kinetics in patients undergoing unmanipulated Haplo-HSCT with posttransplant cyclophosphamide (PT/Cy-haplo) and compared it with patients allografted with HLA-matched donors. Plasma CMV DNA load was monitored by real-time PCR and enumeration of CMV-specific IFN-γ-producing CD8+ and CD4+ T cells was performed by flow cytometry for intracellular cytokine staining at days +30, +60, +90, and +180 after transplantation. CMV DNAemia developed in 62 patients, occurring with comparable frequency in PT/Cy-haplo and MRD/MUD recipients (P = 0.14). There were no significant differences across groups in the number of patients either displaying detectable CMV-specific CD8+ and CD4+ T-cell responses or acquiring CMV-specific T-cell levels conferring protection against subsequent infection. CMV-specific T-cell counts were comparable between groups at most time points examined, irrespective of whether CMV DNAemia occurred or not prior to monitoring. Collectively the data suggest that PT/Cy-haplo recipients may reconstitute CMV-specific T-cell immunity to the same extent as patients undergoing HLA-matched allo-HSCT.

Features of Cytomegalovirus DNAemia Blips in Allogeneic Hematopoietic Stem Cell Transplant Recipients: Implications for Optimization of Preemptive Antiviral Therapy Strategies.

Cytomegalovirus (CMV) DNAemia occurs frequently in allogeneic hematopoietic stem cell transplant recipients (allo-HSCT). There is limited information about the incidence, features, and clinical impact of CMV DNAemia blips (episodes defined by an isolated positive PCR result) in this setting. In this retrospective study, 225 consecutive adult patients undergoing any modality of allo-HSCT at our center between May 2012 and July 2019 were included. Plasma CMV DNA load was monitored using a highly sensitive real-time PCR assay. In all, 187 of 225 patients had CMV DNAemia through day 365 after allo-HSCT (total number of episodes, n=379). Eighty-three of the 187 patients had 1 or more blips (n=104). Blips occurred as a first episode of CMV DNAemia as opposed to prolonged CMV DNAemia (≥2 consecutive positive PCR results) in 47 patients; in 20 of these patients, blips represented the only documented episode throughout the study period, and in 27 patients, blips preceded a prolonged CMV DNAemia episode. In the remaining 36 patients, blips developed as recurrences. Blips presenting as initial episodes occurred more frequently (P < .001) in patients receiving an allograft from a CMV-seropositive donor. The cumulative incidence of recurrent CMV DNAemia following initial blips, self-resolving prolonged CMV DNAemia episodes, or CMV DNAemia episodes treated preemptively with antivirals was not significantly different (P=.34). Receiver operating characteristic curve analysis indicated that a CMV DNA load cutoff of 48 IU/mL yielded the highest combined sensitivity (66%) and specificity (70.2%) for predicting a prolonged CMV DNAemia episode. The practical implications of our data in the optimization of preemptive antiviral therapy strategies are discussed.

An investigation of the utility of plasma Cytomegalovirus (CMV) microRNA detection to predict CMV DNAemia in allogeneic hematopoietic stem cell transplant recipients.

Precise identification of patients at highest risk for developing Cytomegalovirus (CMV) DNAemia may improve CMV infection management in the allogeneic hematopoietic stem cell transplantation (allo-HSCT) setting. Here, we studied the potential use of detecting free CMV micro(mi)RNAs circulating in plasma for predicting CMV DNAemia in this clinical scenario. A total of 62 adult allo-HSCT recipients were included in this prospective observational multicenter study. Plasma CMV DNA load was monitored using the CMV RealTime CMV PCR (Abbott Molecular, Des Plaines, IL, USA). Detection of mature CMV miRNAs in plasma drawn by days + 7, + 14 and + 30 after allo-HSCT was performed using the miScript PCR System (Qiagen, Hilden, Germany). Assays could be optimized for five out of the seven targeted CMV miRNAs: UL36-5p, US33-5p, UL148D, UL22A-5p and UL112-3p. Of the 62 patients included in the study, 42 developed a first episode of CMV DNAemia at a median of 35days after allo-HSCT. All targeted CMV miRNA were detected early after transplantation, with CMV miRNA US33-5p and UL112-3p the most commonly found species at any time point; nevertheless, neither the detection rate of CMV miRNAs nor their abundance allowed discrimination between patients with subsequent CMV DNAemia and those with no CMV DNAemia. The data presented herein do not support any predictive utility of these CMV miRNAs for first episodes of CMV DNAemia in a cohort consisting primarily of allo-HSCT patients receiving haploidentical allografts.

A Nationwide Survey of Cytomegalovirus Prevention Strategies in Kidney Transplant Recipients in a Resource-Limited Setting.

ObjectiveStrategies to prevent cytomegalovirus (CMV) infection in resource-limited settings have been under-explored. We investigated CMV prevention strategies utilized among transplant centers in Thailand.MethodA questionnaire on CMV prevention strategies for kidney transplant (KT) recipients was developed using a web-based electronic survey website (www.surveymonkey.com). The survey was delivered to 31 transplant centers in Thailand. One infectious disease physician (ID) and 1 nephrologist (NP) from each center were included.ResultsThere were 43 respondents from 26 of the 31 transplant centers (84%), including 26 (60%) IDs and 17 (40%) NPs. Forty-one 95% (41/43) physicians agreed on the necessity of CMV prevention. Of these, 77% (33/43) physicians implemented prevention strategies for their patients. Interventions included preemptive approaches (48%), prophylaxis (45%), hybrid approaches; surveillance after prophylaxis (3%), and CMV-specific immunity-guided approaches (3%). For CMV-seropositive KT recipients, use of preemptive approaches (84%) exceeded prophylaxis (12%). However, 81% of the former preferred targeted prophylaxis in patients receiving antithymocyte globulin therapy. Sixty-five percent and 93% of physicians started preemptive therapy when plasma CMV DNA loads reached 2000 and 3000 copies/mL (1820 and 2730 IU/mL), respectively. A significantly greater percentage of NPs initiated preemptive therapy at a plasma CMV DNA load of 1820 IU/mL compared with IDs (88% vs 50%; P = .02). The most common barrier to prevention strategy implementation was financial inaccessibility of oral valganciclovir (67%) and quantitative CMV DNA testing (12%).ConclusionsMost physicians agreed on a need for preemptive approaches, although prophylaxis was targeted in those receiving intense immunosuppression. The financial implication is the main barrier for CMV prevention in Thailand.

Cytomegalovirus infection management in solid organ transplant recipients across European centers in the time of molecular diagnostics: An ESGICH survey.

Scant information is available about how transplant centers are managing their use of quantitative molecular testing (QNAT) assays for active cytomegalovirus (CMV) infection monitoring in solid organ transplant (SOT) recipients. The current study was aimed at gathering information on current practices in the management of CMV infection across European centers in the era of molecular testing assays. A questionnaire-based cross-sectional survey study was conducted by the European Study Group of Infections in Immunocompromised Hosts (ESGICH) of the Society of Clinical Microbiology and Infectious Diseases (ESCMID). The invitation and a weekly reminder with a personal link to an Internet service provider (https://es.surveymonkey.com/) was sent to transplant physicians, transplant infectious diseases specialists, and clinical virologists working at 340 European transplant centers. Of the 1181 specialists surveyed, a total of 173 responded (14.8%): 73 transplant physicians, 57 transplant infectious diseases specialists, and 43 virologists from 173 institutions located at 23 different countries. The majority of centers used QNAT assays for active CMV infection monitoring. Most centers preferred commercially available real-time polymerase chain reaction (RT-PCR) assays over laboratory-developed procedures for quantifying CMV DNA load in whole blood or plasma. Use of a wide variety of DNA extraction platforms and RT-PCR assays was reported. All programs used antiviral prophylaxis, preemptive therapy, or both, according to current guidelines. However, the centers used different criteria for starting preemptive antiviral treatment, for monitoring systemic CMV DNA load, and for requesting genotypic assays to detect emerging CMV-resistant variants. Significant variation in CMV infection management in SOT recipients still remains across European centers in the era of molecular testing. International multicenter studies are required to achieve commutability of CMV testing and antiviral management procedures.

The impact of virus population diversity on the dynamics of cytomegalovirus DNAemia in allogeneic stem cell transplant recipients.

Mixed cytomegalovirus (CMV) infections are associated with delayed viral clearance in solid organ transplant recipients. We investigated whether this could be extrapolated to allogeneic stem cell transplant (allo-SCT) recipients. A total of 48 plasma specimens, obtained during 29 episodes of active CMV infection in 25 non-consecutive allo-SCT patients, were analysed. Baseline blood specimens, drawn shortly prior to the inception of pre-emptive antiviral therapy (pre-treatment specimen; n=29), as well as follow-up samples obtained either after the initiation of antiviral therapy (post-treatment specimen; n=15) or during recurrent episodes (n=4) were analysed. Plasma CMV DNA loads were quantified by real-time PCR and the CMV genotyping was performed by ultra-deep sequencing of hypervariable regions in the genes coding for glycoproteins N (gN) and O (gO). A trend towards higher CMV DNA peak loads, longer CMV DNAemia episode durations and slower CMV DNAemia decay rates was observed for episodes with mixed CMV genotype populations compared to those caused by single CMV variants, although the differences did not reach statistical significance. The length of the treatment course required to clear DNAemia was significantly longer in these mixed episodes (P=0.002). Significant changes in the number or frequency of CMV gN or gO genetic variants were documented following the initiation of antiviral therapy or in recurrent episodes. CMV diversity may have a major impact on the kinetics of CMV DNAemia clearance during the treatment of active CMV infection episodes in allo-SCT recipients.

Impact of cytomegalovirus DNAemia on overall and non-relapse mortality in allogeneic stem cell transplant recipients.

We conducted a retrospective single-center study to investigate the potential impact of cytomegalovirus (CMV) DNAemia on mortality in allogeneic stem cell transplant (allo-SCT) recipients. A total of 151 consecutive patients who underwent T-cell replete allo-SCT were included in the study. Patients with CMV DNAemia were treated preemptively with antivirals upon detection of plasma CMV DNA loads >1500IU/mL. At least one episode of CMV DNAemia occurred in 109 (72.2%) patients, and 67 of these patients (61.5%) required one or more courses of antiviral therapy. The cumulative incidence of 1-year overall and non-relapse mortality (NRM) was 28.5% (95% confidence interval [CI], 18.4%-39.5%) and 23.2% (95% CI 12.81%-35.4%), respectively. The occurrence of either CMV DNAemia or CMV recurrences had no apparent effect on 1-year overall mortality and NRM; nevertheless, a trend towards an increased risk of death was seen in patients with one or more episodes of CMV DNAemia requiring antiviral therapy (hazard ratio [HR], 2.10; 95% CI, 0.96-4.61; P=.06 for overall mortality, and HR, 2.36; 95% CI, 0.96-5.76; P=.06 for NRM) but not in those displaying one or more self-resolving episodes. Therefore, the data suggest that withholding preemptive antiviral therapy until the plasma CMV DNA load reaches 1500 IU/mL has no apparent detrimental effect on patient survival.

Early Kinetics of Plasma Cytomegalovirus DNA Load in Allogeneic Stem Cell Transplant Recipients in the Era of Highly Sensitive Real-Time PCR Assays: Does It Have Any Clinical Value?

We report that in a population of allogeneic stem cell transplant recipients, determination of the viral doubling time (dt) of the cytomegalovirus (CMV) DNA plasma load predicted the eventual need for inception of preemptive antiviral therapy, whereas the level of the initial plasma CMV DNA load did not. The data thus indicated that determination of the dt of CMV DNA may be useful in the therapeutic management of CMV infection in this clinical setting.

An evaluation of the role of NKG2C+ natural killer cells in protection from cytomegalovirus DNAemia early following allogeneic stem cell transplantation

The role of natural killer (NK) cells in affording protection against human cytomegalovirus (CMV) in allogeneic stem cell transplant recipients is largely unknown. The current study was aimed at determining whether NKG2C+ NK cells confer protection from CMV DNAemia early following transplantation in patients lacking mono and polyfunctional CMV pp65 and IE-1-specific CD4+ and CD8+ T-cell responses, as measured by flow cytometry for intracellular cytokine staining. Fourteen out of the 36 patients included in this study developed CMV DNAemia between days +30 and +60 after transplant. Three patients did so after day +60. Peripheral blood levels of CD56(bright) CD16(-/low) and CD56(dim) CD16+ NKG2C+ NK cells measured at day +30 and at day +60 in patients who had or had not subsequent CMV DNAemia did not differ significantly. In addition, no significant correlation was found between CD56(bright) CD16(-/low) (σ = -0.229; P = 0.39) and CD56(dim) CD16+ (σ = -0.285; P = 0.28) NKG2C+ NK-cell levels and initial plasma CMV DNA loads. In summary, the data presented do not support a direct implication of NKG2C+ NK cells in preventing the development of CMV DNAemia or modulating the magnitude of CMV replication at early stages during episodes of CMV DNAemia in allogeneic stem cell transplant patients with unreconstituted CMV-specific T-cell responses.

Immunological Monitoring for Guidance of Preemptive Antiviral Therapy for Active Cytomegalovirus Infection in Allogeneic Stem-Cell Transplant Recipients: A Pilot Experience

Immunological Monitoring for Guidance of Preemptive Antiviral Therapy for Active Cytomegalovirus Infection in Allogeneic Stem-Cell Transplant Recipients: A Pilot Experience

Dynamics of Cytomegalovirus (CMV) Plasma DNAemia in Initial and Recurrent Episodes of Active CMV Infection in the Allogeneic Stem Cell Transplantation Setting: Implications for Designing Preemptive Antiviral Therapy Strategies

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 CMVDNAemia. 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.

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.

Virological and immunological features of active cytomegalovirus infection in nonimmunosuppressed patients in a surgical and trauma intensive care unit

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-alpha 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-alpha 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.

Preemptive Versus Sequential Prophylactic-Preemptive Treatment Regimens for Cytomegalovirus in Renal Transplantation: Comparison of Treatment Failure and Antiviral Resistance

Cytomegalovirus (CMV) infections after transplantation are commonly treated using a prophylactic or preemptive regimen with (val)ganciclovir. It remains unclear, which approach is most effective in preventing CMV disease in D+R- patients. The aim of this retrospective study was to compare the treatment response and antiviral resistance in CMV infections between two treatment regimens in D+R- renal transplant recipients. Before 2006, a preemptive treatment regimen with valganciclovir was applied (42 patients). From 2006 onwards, patients first received prophylaxis with valganciclovir for 90 days, followed by a preemptive regimen (29 patients). CMV infections were monitored by regular determination of the CMV DNA load in plasma. Patient charts were reviewed for antiviral treatment data, and resistance was analyzed by nucleotide sequence analysis of the UL97 and UL54 genes in CMV DNA-positive samples. Treatment failure, defined as a CMV DNA load more than or equal to 1000 copies/mL after at least 2 weeks of treatment, occurred less frequently in the prophylaxis cohort than in the preemptive cohort (14% vs. 71%, P<0.001). No CMV end-organ disease occurred in either cohort. Resistant viral isolates were found during treatment in one patient in the prophylaxis cohort versus in three patients in the preemptive group. All CMV infections with resistant virus were cleared without switch of (val)ganciclovir treatment. Treatment failure of CMV infections occurred less frequently in D+R- renal transplant patients on a sequential prophylaxis-preemptive regimen than in patients on a purely preemptive regimen. Antiviral resistance was observed infrequently and apparently played a minor role in treatment failure.

Comparable incidence and severity of cytomegalovirus infections following T cell-depleted allogeneic stem cell transplantation preceded by reduced intensity or myeloablative conditioning

Reports on infectious complications following reduced intensity conditioning (RIC) before allogeneic stem cell transplantation (allo-SCT) are equivocal. This prospective follow-up study compared the impact of cytomegalovirus (CMV) infections following RIC with fludarabine, ATG and busulphan or conventional myeloablative conditioning (MAC). Forty-eight RIC and 59 MAC patients were enrolled. The occurrence and severity of CMV infections within 100 days following allo-SCT were assessed, using plasma CMV DNA load kinetics. CMV DNAemia was observed in 21 RIC (60%) and in 19 MAC (44%) patients at risk for CMV. The mean CMV DNAemia free survival time was comparable following RIC and MAC: 70 days (95% (confidence interval) CI: 59-80 days) and 77 days (95% CI: 68-86 days), respectively (P=0.24). Parameters indicative for the level of CMV reactivation, including the area under the curve of CMV DNA load over time as well as the onset, the peak values and duration of CMV infection episodes, the numbers and duration of CMV treatment episodes and recurrent infections, were not different in both groups. During follow-up, none of the patients developed CMV disease. RIC with fludarabine, ATG and busulphan demonstrated safety comparable to conventional MAC with regard to frequency and severity of CMV infections within 100 days following T cell-depleted allo-SCT.