Evaluation of the ReSSQ assay in relation to the COBAS AMPLICOR CMV MONITOR test and an in-house nested PCR method for detection of cytomegalovirus DNA

The ReSSQ CMV assay is a novel commercially available kit for quantification of cytomegalovirus (CMV), based on real-time PCR with a peptide nucleic acid probe coupled with a single dye. In combination with the LightCycler, the ReSSQ CMV assay was evaluated with respect to specificity, PCR inhibition, linearity, reproducibility, and sensitivity. All nontested CMV materials were negative, and the assay was not inhibited by the use of different anticoagulants or other factors that may influence blood samples. The dynamic range was between 10 and 5 x 10(8) copies/PCR, and intra- and interassay variabilities were below 0.10 and 0.12 log10 standard deviations, respectively. Assay sensitivity was validated by analysis of 24 samples from a proficiency panel and by comparison to a nested in-house CMV PCR and the COBAS AMPLICOR CMV MONITOR test, using 159 clinical samples. Results from the proficiency panel were well in accordance with input values over the entire range of viral concentrations tested (50 to 31,250 copies/ml). The association between the ReSSQ CMV assay and the in-house PCR was in agreement in 90% of the clinical samples, and discordant results were found for all types of sample materials analyzed. The ReSSQ CMV and COBAS AMPLICOR assays showed no significant differences for samples containing >1,000 CMV copies/ml, but results differed to a greater extent at lower viral concentrations. The results demonstrate that the ReSSQ CMV assay is a CMV-specific, robust, and reproducible method and hence is well suited for routine use in clinical virology laboratories.     

Comparison of quantitative and qualitative PCR assays for cytomegalovirus DNA in plasma

We analyzed the performance characteristics of the qualitative AMPLICOR CMV Test (Roche Molecular Systems, Pleasanton, Calif.) and quantitative COBAS AMPLICOR CMV MONITOR Test (Roche Molecular Systems) assays and compared the performance of the AMPLICOR quantitative assay with an in-house-developed cytomegalovirus (CMV) DNA PCR assay. The quantitative AMPLICOR assay was found to be more sensitive than the qualitative AMPLICOR assay. The quantitative AMPLICOR assay has a lower limit of sensitivity of 400 CMV DNA copies/ml of plasma and is linear to 50,000 CMV DNA copies/ml of plasma. Compared to the in-house PCR assay, the AMPLICOR quantitative assay gave lower viral load values at all concentrations tested, but the difference between the two assays was not consistent across the entire dynamic range of the AMPLICOR quantitative assay. At the lower end of the assay, the viral load values obtained with the in-house PCR assay were three- to fivefold (0.5 to 0.7 log units) higher than those measured with the AMPLICOR assay. At higher input concentrations, the differences between the two assays approached 10-fold. This direct comparison of the in-house assay and the quantitative AMPLICOR assay provides the ability to compare previously published in-house data with an assay widely available for future research and clinical monitoring of patients with CMV infections.     

Quantitation of cytomegalovirus DNA by real-time polymerase chain reaction in peripheral blood specimens of patients with solid organ transplants: comparison with end-point PCR and pp65 antigen test

The polymerase chain reaction (PCR) for cytomegalovirus (CMV) DNA quantitation provides sensitive and specific data for detecting CMV as well as monitoring the infection and determining the appropriate antiviral strategy. A recently introduced real-time PCR assay for CMV DNA quantitation was applied on 158 peripheral blood leukocytes (PBLs) from 32 liver-transplanted patients with CMV asymptomatic infection and correlated with a commercial quantitative end-point PCR (COBAS AMPLICOR CMV Monitor) and CMV pp65 antigenemia. A good correlation was found between real-time PCR and pp65 antigen test (r2 = 0.691) and the two PCR assays (r2 = 0.761). Real-time PCR data were higher in pre-emptive treated patients (>20 pp65 + positive cells, median CMV DNA value: 3.8 log(10) copies/500,000 PBLs) than in not-treated ones (2.9 logs). According to pp65 levels of 0, 1-10, 11-20, 21-50, 51-100, and >100 positive cells/200,000 PBLs, median CMV DNA by real-time PCR was 2.6, 3.0, 3.6, 4.0, 4.2, and 4.8 logs, respectively, (CMV DNA levels by COBAS AMPLICOR: 2.8, 2.9, 3.8, 3.7, 3.9, and 4.0 logs). For samples with >20 pp65 + cells, real-time PCR gave significantly higher values than in groups with <20 pp65 + cells, whereas the COBAS AMPLICOR results showed a slower progression rate. Dilutions of CMV AD169 strain were used to probe real-time PCR reproducibility (between and intra-assay variability <2%) and sensitivity (100% detection rate at 10 copies/reaction, 28.5% with end-point PCR). In conclusion, real-time PCR significantly improves the study of CMV DNA dynamics due to a more reliable quantitation of CMV DNA for moderate and high DNA level compared to end-point PCR with better sensitivity and specificity. Real-time PCR provides more precise information for evaluating infection progress and assessing antiviral response, simplifying and accelerating the process of producing a reliable quantitation of CMV DNA for clinical purposes.     

Evaluation of the AMPLICOR Cytomegalovirus Test with Specimens from Human Immunodeficiency Virus-Infected Subjects

The AMPLICOR cytomegalovirus (CMV) test, a new qualitative assay for the detection of CMV DNA in plasma, was compared to conventional methods and quantitative PCR (Q-PCR) assays by using leukocytes and plasma from 179 blood samples from subjects with AIDS. For the diagnosis of CMV disease, cell-based assays such as a Q-PCR with polymorphonuclear leukocytes (Q-PCR-PMNL) and a pp65 antigenemia assay had the highest sensitivities but suffered from a lack of specificity. The best agreement between the results of the Q-PCR-PMNL assay and those of the AMPLICOR test was found when a threshold diagnostic value of 690 copies per 10⁵ cells was selected for the Q-PCR-PMNL assay. In that context, the AMPLICOR CMV test had a sensitivity of 96.4% and a specificity of 95.3% when results were compared to results of the cell-based PCR assay. This threshold was close to the one described as associated with the best sensitivity and specificity for the diagnosis of CMV disease in a recently published study (4). Blood samples that tested positive by the Q-PCR-PMNL assay but negative by the AMPLICOR CMV test were associated with viral loads (mean, 785 copies, median, 96 copies per 10⁵ leukocytes) lower than the viral loads of blood samples that tested positive by both assays (mean, 21,452 copies; median, 9,784 copies per 10⁵ leukocytes) (P = 0.003). The AMPLICOR CMV test gave positive results at least 48 days before the development of symptomatic CMV disease in a longitudinal analysis of a limited subset of patients (n = 6) from whom sequential specimens were available for testing. In conclusion, the AMPLICOR CMV test is a very convenient assay combining rapidity, simplicity, and the possibility of batch testing. A positive result by this test seems particularly important since this implies, in most instances, the presence or the imminence of CMV disease, although a negative test result does not rule out disease.     

Quantitative analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay and the COBAS AMPLICOR CMV MONITOR PCR test after blood and marrow allogeneic transplantation

The performance of a commercially available qualitative PCR test for plasma (AMPLICOR CMV Test; Roche Diagnostics) and a quantitative PCR test for plasma and leukocytes (COBAS AMPLICOR CMV MONITOR Test; Roche Diagnostics) was evaluated with samples from 50 blood or marrow allogeneic transplant recipients who received short courses of sequential ganciclovir therapy (2 weeks intravenously followed by 2 weeks orally) based on a positive cytomegalovirus (CMV) pp65 antigenemia (AG) assay. The number of persons with a positive CMV test was significantly higher for leukocyte-based assays (AG, 67.5%; PCR, 62.5%) compared to both quantitative and qualitative PCR tests of plasma (42.5 and 35%, respectively). One person developed CMV disease during the study despite a negative AG assay; in this particular case, all PCR assays were found to be positive 10 days before his death. There was a trend for earlier positivity after transplantation and more rapid negativity after initiation of ganciclovir for the tests performed on leukocytes. The mean number of CMV copies as assessed by PCR was significantly higher in leukocytes than in plasma (P = 0.02). Overall, excellent agreement (kappa coefficient, >0.75) was found only between the two PCR assays (qualitative and quantitative) based on plasma. These results suggest that either the pp65 AG assay or the COBAS AMPLICOR CMV MONITOR Test using leukocytes could be used to safely monitor CMV viremia in related allogeneic blood or marrow transplant recipients. Such a strategy will result in preemptive treatment for about two-thirds of the persons with a relatively low rate (<33%) of secondary viremic episodes following short courses of ganciclovir therapy.     

Comparison of three nucleic acid amplification assays of cerebrospinal fluid for diagnosis of cytomegalovirus encephalitis

The diagnostic reliabilities of three cytomegalovirus (CMV) nucleic acid amplification assays of cerebrospinal fluid (CSF) were compared by using CSF samples from human immunodeficiency virus-infected patients with a postmortem histopathological diagnosis of CMV encephalitis (n = 15) or other central nervous system conditions (n = 16). By using a nested PCR assay, the quantitative COBAS AMPLICOR CMV MONITOR PCR, and the NucliSens CMV pp67 nucleic acid sequence-based amplification assay, sensitivities were 93.3, 86.6, and 93.3%, respectively, and specificities were 93.7, 93.7, and 87.5%, respectively. The COBAS AMPLICOR assay revealed significantly higher CMV DNA levels in patients with diffuse ventriculoencephalitis than in patients with focal periventricular lesions.     

Detection of Cytomegalovirus in Plasma and Cerebrospinal Fluid Specimens from Human Immunodeficiency Virus-Infected Patients by the AMPLICOR CMV Test

We have developed the AMPLICOR CMV Test, which is rapid and sensitive for the detection of cytomegalovirus (CMV) in plasma and cerebrospinal fluid (CSF) specimens. The test incorporated an internal control in the reaction mixture to monitor the amplification efficiency and the presence of inhibitors. The AMPLICOR CMV Test was very specific in detecting 12 clinical CMV isolates and four laboratory CMV strains tested. Cross-reactivity with 26 non-CMV pathogens was not observed. The AMPLICOR CMV Test requires only 50 μl of specimen (plasma or CSF) for processing. The performance of the AMPLICOR CMV Test was compared to those of the CMV antigenemia assay and the conventional tube culture method. Among 112 plasma specimens from 43 human immunodeficiency virus-infected patients, CMV was detected in 20 (18%) of the specimens by the AMPLICOR CMV Test, 21 (19%) of the specimens by the CMV antigenemia assay, and 10 (9%) of the specimens by culture. In CSF specimens from AIDS patients, CMV was detected in 10 of 58 (17%) specimens tested by the AMPLICOR CMV Test, 5 of 28 (18%) specimens tested by the antigen assay, and none of the 25 specimens tested by culture. While the performance of the AMPLICOR CMV Test in this study was comparable to that of the CMV antigen assay, processing of specimens by the AMPLICOR CMV Test was much simpler than that by the antigen assay; in addition, the antigen assay requires greater than 10⁵ leukocytes from blood or 1 ml of CSF to perform the assay. Our study suggested that the AMPLICOR CMV Test could provide a rapid and sensitive assay for the detection of CMV in plasma and CSF specimens.     

Evaluation of the AMPLICOR CMV test for direct detection of cytomegalovirus in plasma specimens.

We evaluated the AMPLICOR CMV test (PCR) for the direct detection of cytomegalovirus in plasma. Sixty-eight specimens were involved for the comparison between the AMPLICOR test and the antigenemia assay. The sensitivities, specificities, and positive and negative predictive values were 97.1, 100, 100, and 97.1%, respectively, for the AMPLICOR test and 79.4, 100, 100, and 82.9%, respectively, for the antigenemia assay.     

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.     

Comparative Quantitation of Cytomegalovirus (CMV) DNA in Solid Organ Transplant Recipients with CMV Infection by Using Two High-Throughput Automated Systems

Cytomegalovirus (CMV) DNA quantitation in clinical specimens is progressively becoming a cornerstone in the diagnosis and management of CMV infection in the immunocompromised host. We evaluated two automated and reproducible PCR tests, the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and the COBAS AMPLICOR CMV Monitor (Roche Diagnostics, Pleasanton, Calif.), for the detection of CMV DNA in blood samples from transplant recipients with CMV infection as determined by shell vial culture. Following a log transformation analysis, the mean CMV DNA in plasma (PL), whole blood (WB), peripheral blood leukocytes (PBL), and peripheral blood mononuclear cells (PBMC) using the LightCycler was 6.79 copies per ml, 7.23 copies per ml, 6.38 copies per 2 x 10(6) cells, and 6.27 copies per 2 x 10(6) cells, respectively. This compares to 7.86 copies per ml, 8.37 copies per ml, 7.59 copies per 2 x 10(6) cells, and 7.44 copies per 2 x 10(6) cells, respectively, using COBAS AMPLICOR CMV Monitor. While higher CMV DNA levels were observed for the various blood compartments analyzed using COBAS AMPLICOR CMV Monitor, a high degree of correlation was evident between the two automated systems (jackknife correlation r = PL 0.77 [95% confidence interval (CI); 0.64, 0.90], WB 0.77 [95% CI; 0.62, 0.92], PBL 0.77 [95% CI; 0.67, 0.88], and PBMC 0.81 [95% CI; 0.72, 0.89], all P < 0.001). Therefore, we conclude that either automated diagnostic system is accurate for CMV DNA quantitation.     

Evaluation of the COBAS AMPLICOR CMV MONITOR test for detection of viral DNA in specimens taken from patients after liver transplantation

Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients.     

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.     

Comparison of the Roche AMPLICOR MYCOBACTERIUM assay and Digene SHARP Signal System with in-house PCR and culture for detection of Mycobacterium tuberculosis in respiratory specimens.

Two commercial primer kits and detection systems, the Roche AMPLICOR MYCOBACTERIUM test and the Digene primer-probe kit with the SHARP Signal System, were compared to in-house PCR as well as standard culture techniques. For the 27 culture-positive specimens, the Roche AMPLICOR MYCOBACTERIUM test detected 20 specimens, the Digene system detected 19, and in-house PCR detected 21. Of the 86 culture-negative specimens, 13 were positive by the Roche AMPLICOR MYCOBACTERIUM test, 16 were positive by the Digene system, and 21 were positive by in-house PCR. When clinical situations were evaluated, 11 of 13 culture-negative Roche AMPLICOR MYCOBACTERIUM test-positive specimens, 10 of 16 culture-negative Digene system-positive specimens, and 13 of 21 culture-negative-in-house PCR-positive specimens were diagnosed as true-positive specimens. The sensitivities of Roche AMPLICOR MYCOBACTERIUM test, the Digene system, and in-house PCR were 73.81, 69.05, and 80.95%, and the specificities were 97.18, 91.55 and 88.73%, respectively. The positive predictive values were 93.94, 82.86, and 80.95%, and the negative predictive values were 86.25, 83.33, and 88.73%, respectively. For the commercial kits, the Roche AMPLICOR MYCOBACTERIUM test seems to be more sensitive and specific than the Digene system. However, the Roche AMPLICOR MYCOBACTERIUM test cannot be used on nonrespiratory specimens.     

Clinical evaluation of a fully automated CMV PCR assay

BackgroundThere is a growing need for sensitive high-throughput cytomegalovirus (CMV) PCR tests due to the increasing number of immunocompromised patients requiring monitoring for active CMV infection.ObjectivesTo compare the fully automated COBAS^® AmpliPrep/COBAS^® TaqMan^® (CAP/CTM) CMV test (this test is currently under development and not commercially available) for EDTA–plasma to the reference method COBAS^® AMPLICOR CMV MONITOR.Study designA prospective feasibility study with parallel analysis of 433 EDTA–plasma samples from 277 patients on both systems was carried out after the analytical performance of the new system had been assessed.ResultsThe new system has a wide linear range from 2.0 to 7.3 log₁₀ CMV-DNA copies/ml EDTA–plasma and a detection limit of 46 copies/ml with excellent accuracy and precision. When testing clinical samples, the CAP/CTM CMV test compared extremely well with the COBAS^® AMPLICOR CMV MONITOR (R² = 0.93, p < 0.001) with increased sensitivity and linear range. Discrepant samples all contained low titers of CMV-DNA. In two of the study patients, CMV-DNAemia was detected by the CAP/CTM CMV test up to eight weeks earlier than by COBAS^® AMPLICOR CMV MONITOR.ConclusionAn IVD/CE marked version of the CAP/CTM CMV test will enable laboratories to provide a sensitive, fully automated high-throughput CMV PCR.     

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.     

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.     

Detection of beta-herpesviruses in allogenic stem cell recipients by quantitative real-time PCR

The aim of this study was to investigate the clinical impact of reactivation of human herpes virus-6 (HHV-6) and HHV-7 infections in stem cell transplantation recipients, and to examine a possible increase in virulence of the two roseoloviruses when a reactivation of CMV (HHV-5) simultaneously occurs. For this purpose, quantitative real-time PCR systems were developed to assess the viral load of CMV, HHV-6, or HHV-7 in the plasma of haematopoetic stem cell recipients. One hundred and ninety-eight plasma samples from 37 patients who underwent allogeneic stem cell transplantation were tested for CMV, HHV-6, and HHV-7 by a 5'-exonuclease (TaqMan) quantitative real-time PCR. The CMV load obtained by the real-time PCR assay was compared retrospectively with results generated previously with a commercially available test (COBAS AMPLICOR CMV MONITOR Test, Roche). The results suggest that CMV and HHV-6 may be associated with post-transplantation end-organ disease, while HHV-7 reactivation had no impact on the patients included in this study. No evidence for a potential interaction of the roseoloviruses and CMV infections was found.     

Systematic Evaluation of Different Nucleic Acid Amplification Assays for Cytomegalovirus Detection: Feasibility of Blood Donor Screening

Acute primary cytomegalovirus (CMV) infections, which commonly occur asymptomatically among blood donors, represent a significant risk for serious morbidity in immunocompromised patients (a major group of transfusion recipients). We implemented a routine CMV pool screening procedure for plasma for the identification of CMV DNA-positive donors, and we evaluated the sensitivities and performance of different CMV DNA amplification systems. Minipools (MPs) of samples from 18,405 individual donors (54,451 donations) were screened for CMV DNA using the RealStar CMV PCR assay (Altona Diagnostic Technologies), with a minimum detection limit of 11.14 IU/ml. DNA was extracted with a high-volume protocol (4.8 ml, Chemagic Viral 5K kit; PerkinElmer) for blood donor pool screening (MP-nucleic acid testing [NAT]) and with the Nuclisens easyMAG system (0.5 ml; bioMérieux) for individual donation (ID)-NAT. In total, six CMV DNA-positive donors (0.03%) were identified by routine CMV screening, with DNA concentrations ranging from 4.35 × 10² to 4.30 × 10³ IU/ml. Five donors already showed seroconversion and detectable IgA, IgM, and/or IgG antibody titers (IgA⁺/IgM⁺/IgG⁻ or IgA⁺/IgM⁺/IgG⁺), and one donor showed no CMV-specific antibodies. Comparison of three commercial assays, i.e., the RealStar CMV PCR kit, the Sentosa SA CMV quantitative PCR kit (Vela Diagnostics), and the CMV R-gene PCR kit (bioMérieux), for MP-NAT and ID-NAT showed comparably good analytical sensitivities, ranging from 10.23 to 11.14 IU/ml (MP-NAT) or from 37.66 to 57.94 IU/ml (ID-NAT). The clinical relevance of transfusion-associated CMV infections requires further investigation, and the evaluated methods present powerful basic tools providing sensitive possibilities for viral testing. The application of CMV MP-NAT facilitated the identification of one donor with a window-phase donation during acute primary CMV infection.     

Detection of cytomegalovirus in blood donors by PCR using the digene SHARP signal system assay: effects of sample preparation and detection methodology.

Cytomegalovirus (CMV) is an important cause of transfusion-associated morbidity and mortality; however, only 0.4 to 12% of the blood products obtained from seropositive blood donors transmit infection. The effects of three commercially available whole-blood sample preparation kits on the detection of CMV PCR products by a semiquantitative adaptation of the Digene SHARP Signal System Assay (DSSSA) in samples from volunteer blood donors was assessed. Of 101 samples from seropositive blood donors, CMV was detected in 0 (0%) of the samples extracted with a QIAamp blood kit (QIAGEN), 1 (1%) of the samples extracted with an Amplicor whole-blood specimen preparation kit (Roche), and 8 (8%) of the samples extracted with an Isoquick nucleic acid extraction kit (modified by the addition of carrier tRNA) (Microprobe). CMV DNA was not detected in samples from seronegative blood donors (n = 13). Nested PCR of selected samples confirmed the detection of CMV in the sane eight samples extracted with the modified Isoquick nucleic acid extraction kit and detected an additional nine CMV-positive samples (n = 50). Samples from volunteer blood donors contain low copy numbers of CMV DNA. PCR amplification of such specimens can result in analytical sampling errors, giving results similar to the variations in titers recognized during determinations of the 50% tissue culture infective dose. The detection of CMV in blood samples from volunteer blood donors by PCR is a function of sample preparation, amplification conditions, and detection methodology. Accurate assessments of the clinical utility of CMV DNA detection by nucleic acid amplification for blood product screening and patients will require highly standardized and quantitative methodology.     

Detection of cytomegalovirus DNA in human specimens by LightCycler PCR

Detection of human cytomegalovirus (CMV) DNA in clinical specimens is considered a cornerstone in the diagnosis of CMV disease. The aim of this study was to evaluate a newly designed LightCycler-based quantitative CMV PCR. Specimens of human origin (n = 200) were tested using the LightCycler PCR, the quantitative COBAS AMPLICOR CMV MONITOR (CACM) assay, and a qualitative in-house PCR assay for the presence of CMV DNA. Samples that were reactive in at least two of the three assays were considered CMV DNA positive (n = 95 [47. 5%]), while samples that were nonreactive in two of the three assays were considered CMV DNA negative (n = 105 [52.5%]). Using the LightCycler assay, CMV DNA was detected in 91 of the 95 CMV DNA-positive human specimens (sensitivity, 95.8%; 95% confidence interval [CI], 89.6 to 98.8) and in 1 of the CMV DNA-negative specimens (specificity, 99%; 95% CI, 94.8 to 99.8). Results of CMV load determination as assessed by both quantitative test systems were correlated (r = 0.73; P < 0.0001; 95% CI, 0.61 to 0.81). Results for undiluted samples containing a high CMV load were more accurate with the LightCycler test than were results obtained with the CACM test, which underestimated the viral load of samples containing high DNA copy numbers. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler technology are favorable for the use of this system in the detection of CMV DNA in clinical specimens.