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.     

Going beyond serology for stratifying the risk of CMV infection in transplant recipients

Knowledge of donor and recipient (D/R) cytomegalovirus (CMV) serostatus is critical for risk stratification of CMV infection and disease in transplant recipients, particularly in the solid organ transplantation (SOT) setting. Despite its broad availability and the success of it use, the risk stratification based on the D/R serostatus is not free of limitations since there are a nondepreciable number of patients that are not accurately categorized by this approach. In fact, up to 20% of seropositive SOT recipients, classically considered at intermediate risk, develop episodes of CMV infection and disease after transplantation. Here, we provide an overview of additional donor and recipient factors that may have utility in identifying patients at risk for post‐transplant CMV infection. Specifically, we summarize our current understanding regarding the potential use of use CMV‐specific T‐cell‐mediated immunity, neutralizing antibodies and host genetics that may influence the risk of CMV infection and disease. We provide an overview of the benefits and limitations associated with using these immunological factors in risk stratification and propose specific variables that could be analyzed at the pretransplant evaluation to improve the identification of patients with increased individual susceptibility.     

Immune response to CMV in solid organ transplant recipients: current concepts and future directions

Despite advances in immunosuppression and antiviral therapy, CMV continues to be a significant opportunistic pathogen adversely affecting the outcome of solid organ transplantation (SOT) recipients. While a significant proportion of CMV disease is caused by reactivation of latent virus, the risk is highest among CMV donor+ and recipient- SOT patients. CMV is responsible for both direct (e.g., pneumonitis, colitis) and indirect (e.g., rejection, atherosclerosis) morbidity and mortality. Healthy CMV-seropositive individuals have a high frequency of CMV-specific CD4(+) and CD8(+) T cells that provide immune protection by limiting CMV reactivation and replication. Changes to the innate and adaptive immune system from immunosuppressive therapy following SOT contribute to CMV disease pathogenesis. CMV disease after SOT is associated with poorer outcomes, thus novel strategies to prevent it are an area of active research. In this article, we review the current state of knowledge on the immune response to CMV following SOT.     

Immune response to CMV in solid organ transplant recipients: current concepts and future directions

Despite advances in immunosuppression and antiviral therapy, CMV continues to be a significant opportunistic pathogen adversely affecting the outcome of solid organ transplantation (SOT) recipients. While a significant proportion of CMV disease is caused by reactivation of latent virus, the risk is highest among CMV donor+ and recipient- SOT patients. CMV is responsible for both direct (e.g., pneumonitis, colitis) and indirect (e.g., rejection, atherosclerosis) morbidity and mortality. Healthy CMV-seropositive individuals have a high frequency of CMV-specific CD4⁺ and CD8⁺ T cells that provide immune protection by limiting CMV reactivation and replication. Changes to the innate and adaptive immune system from immunosuppressive therapy following SOT contribute to CMV disease pathogenesis. CMV disease after SOT is associated with poorer outcomes, thus novel strategies to prevent it are an area of active research. In this article, we review the current state of knowledge on the immune response to CMV following SOT.     

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.     

Prophylaxis and treatment for invasive fungal infections in solid organ transplant recipients]

Solid organ transplantation is becoming increasingly common in Japan. Despite invasive fungal infections being less common than bacterial and viral infections, fungal infections still result in a higher mortality rate. Empiric and pre-emptive therapy plays an important role in management of invasive fungal infections, because successful treatment is difficult after a definite diagnosis of an invasive disease, especially invasive aspergillosis. Given this situation, to improve outcome, high risk patients need to be identified and antifungal prophylaxis is mandatory in preventing the development of the disease. However, antifungal prophylaxis for solid organ transplant recipients remains controversial. New antifungal agents might change the choice of fungal prevention and treatment.     

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.     

Cytomegalovirus infection in solid organ transplant recipients: new challenges and their implications for preventive strategies.

BACKGROUND: Late-onset CMV disease is being increasingly recognized as a significant post-transplantation complication. OBJECTIVES: To discern the impact of antiviral prophylactic strategies on the emerging syndrome of late-onset CMV disease in organ transplant recipients. STUDY DESIGN: Review of existing reports and published data relevant to antiviral prophylaxis in organ transplant recipients. RESULTS: Prevention of CMV using prophylaxis has proven effective and is widely employed in organ transplant recipients. However, late-onset CMV disease is increasingly being recognized as a significant complication in these patients. The more potent the activity of the antiviral drug and the longer duration of prophylaxis, the greater likelihood of late-onset CMV disease. CMV seronegative recipients of seropositive donor allografts appear to be at a uniquely high risk. A higher proportion of patients with late-onset CMV have tissue invasive disease. Late-onset CMV disease in liver transplant recipients conferred an independently higher risk of mortality in the first post-transplant year. Prolonged antiviral therapy may impair the recovery of CMV-specific T-cell responses. Preemptive therapy appears to be less likely to be associated with CMV disease. CONCLUSIONS: Discernment of the pathophysiologic basis of late-onset CMV warrants investigation. Preemptive therapy may be the preferable approach to CMV prophylaxis.     

Invasive fungal infection in solid organ transplantation: toward evidence-based prophylaxis and preemptive treatment]

Although the opportunity to discuss infectious complications in solid organ transplantation is increasing in Japan as elsewhere, the length of clinical experience in extra-renal transplantation is still short and even experience in living donor organ transplant is very limited except for those involving the kidney or liver transplantation. Risk of invasive fungal infection in organ transplant recipients is highly dependent on the immunocompromised status accompanying end-stage organ failure before transplant operation and on the resultant history of infectious complications. These factors as well as surgical and postoperative should be incorporated in a systematic and dynamic manner to evaluate risk of invasive fungal infection. In addition to prophylactic management based on such risk evaluation, it is desirable that preemptive treatment be started on quantification of clinical symptoms, imaging diagnosis, screening culture, and serological indices. Emergence of newer and more potent antifungal agents with lower toxicity potentially changes the concept of antifungal treatment. On the other hand, early and impression-oriented preemptive treatment has tended to increase. It is still questionable whether the knowledge obtained from Western experience can be directly applied to solid organ transplant medicine in Japan. Extensive and detailed clinical experience is mandatory to pursue diagnosis, epidemiology, and risk factors in Japan and establish our criteria for prophylactic and preemptive use of antifungal agents.     

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.     

Assessment of CMV load in solid organ transplant recipients by pp65 antigenemia and real-time quantitative DNA PCR assay: correlation with pp67 RNA detection

After bone marrow (BM) or solid-organ (SO) transplantation viremic Cytomegalovirus (CMV) infection is observed frequently. Quantitative assay of CMV in blood helps the management of this clinical condition. In the present report, 83 samples from 39 solid organ recipients, three CMV assays were compared simultaneously for the first time: the Nuclisens CMV pp67 assay (nucleic acid sequence-based amplification, NASBA), an "in-house" quantitative real-time PCR assay (TaqMan) for CMV DNA, and pp65 antigenemia. The relation between CMV DNA and pp65 antigenemia, the quantitative assays, was evaluated on a larger group including 251 blood samples from 118 solid organ recipients. Real-time PCR provided the best results; > or =130 CMV DNA copies/2 x 10(5) peripheral blood leukocytes (PBLs) predicted > or =1 pp65 antigen positive (Ag+) cell/2 x 10(5) PBLs. By taking pp65 antigenemia as the "gold standard," the sensitivity of CMV DNA quantitation and of the pp67 RNA assay were 0.95 and 0.20, respectively, while the corresponding specificity values were 0.50 and 0.93. When real-time PCR was considered as the "gold standard," the sensitivity and specificity of the pp65 antigenemia were 0.65 and 0.91, respectively. Among the three tests examined, the sensitivity of the pp67 RNA assay was the lowest. On the other hand, the pp67 RNA assay was highly specific and effective in pinpointing high viremia patients. The present report, by providing predictive values for all three diagnostic profiles, DNA load, antigenemia, and pp67RNA, is a contribution for validation of real-time PCR as a new standard for quantitative assessment of CMV viremia in clinical settings.     

Systematic review of ganciclovir pharmacodynamics during the prevention of cytomegalovirus infection in adult solid organ transplant recipients

Human cytomegalovirus (CMV) represents the most common infection among recipients of solid organ transplants (SOTs). Previous meta‐analysis showed 0.8% of SOT recipients developed CMV disease whilst receiving valganciclovir (ValGCV) prophylaxis. However, the clinical utility of monitoring ganciclovir (GCV) blood concentrations is unclear. We systematically reviewed the association between GCV concentrations during prophylaxis and the incidence of CMV. MEDLINE and EMBASE databases were searched for studies between 1946 and 2018, where GCV pharmacokinetics and incidence of CMV viraemia or disease in SOT were available. Research designs included randomised trials, comparative, prospective cohort, retrospective, or case report studies. Only human adult studies were included, with English language restriction. The 11 studies that met the eligibility criteria included 610 participants receiving GCV or ValGCV prophylaxis. Quality assessment showed 2/4 randomised trials, 4/6 cohort studies, and 1/1 case report were of high quality. Despite dose adjustments for renal impairment, mean GCV exposures for patients were heterogeneous and ranged between 28 and 53.7 μg·h/mL across three randomised trials. The incidence of CMV infection and disease ranged from 0% to 50% and 0% to 3.1%, respectively, with follow up between 3 to 9 months. One study showed statistical power in determining relationship, where GCV exposure at 40 to 50 μg·h/mL in high‐risk SOT recipients was associated with a reduced risk of viraemia. Clinical monitoring for GCV exposure can be applied to high‐risk SOT recipients during ValGCV prophylaxis; however, further studies are needed to determine the utility of monitoring in all SOT recipients.     

Evaluation of the AmpliSensor PCR and the SHARP signal detection system for the early prediction of symptomatic CMV infection in solid transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) is associated with high morbidity and mortality in transplant patients. Specific antiviral treatment at an early stage of CMV infection may effectively ameliorate, but not eliminate CMV disease in these patients. Presently, the pp65 antigenemia test on peripheral leukocytes is the method most widely used for predicting and monitoring transplant patients for active CMV infection. Nucleic acid amplification methods are less well defined since they lack standardisation. OBJECTIVE: A seminested fluorometric PCR assay (AmpliSensor-CMV, BAG, Germany) and a one-step PCR with a signal-amplification step (SHARP, Abbott, Germany) specific for the fragments of the CMV UL 122 and UL 123 genes, respectively, were evaluated for the early diagnosis of CMV infection. DESIGN: A total of 26 recipients of heterogeneous solid organs were monitored prospectively for a median of 99 days after transplantation. By testing 371 clinical samples parallel with the pp65-antigen assay and IgM and IgG EIA assays the sensitivity, specificity, correlation and quantitation potential of both PCRs was evaluated. RESULTS: Eight out of 26 patients developed active CMV infection. A total of 48 samples of these patients exceeded a CMV-DNA load threshold of 15 genome equivalents/10(5) leukocytes (AmpliSensor-CMV) and 41 samples exceeded the critical cut-off for the SHARP system. The AmpliSensor PCR exceeded its threshold consistently before the clinical onset of CMV disease (median 8 days). There was very good agreement between symptomatic CMV infection in patients and AmpliSensor-PCR, SHARP PCR, and pp65-antigen results (kappa-coefficient > 0.900). IgM and IgG EIA showed moderate agreement (kappa-coefficient = 0.591 and 0.552, respectively). CONCLUSION: Both PCRs and pp65 antigen assay correlated significantly better with CMV disease than serodiagnosis. The AmpliSensor PCR allowed more precisely than the SHARP system a quantitative determination of viral load and an early and reliable prediction of active CMV infection. The use of AmpliSensor PCR may improve the diagnosis and management of active CMV infection in organ transplant recipients.