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.     

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.     

A multisite trial comparing two cytomegalovirus (CMV) pp65 antigenemia test kits, biotest CMV brite and Bartels/Argene CMV antigenemia

A total of 513 blood specimens, predominantly from organ transplant recipients, human immunodeficiency virus-positive patients, and bone marrow transplant recipients, were tested for cytomegalovirus (CMV) by culture and pp65 antigenemia across four test sites. Peripheral blood leukocytes were examined by using both the Biotest CMV Brite and the Bartels/Argene CMV Antigenemia kits. A total of 109 specimens were positive for CMV, 106 (97%) were positive by antigenemia, and 34 (31%) were positive by culture. According to the manufacturers' instructions, 150,000 cells were applied per slide for the Biotest kit and 200,000 cells per slide for the Bartels kit. A total of 93 specimens (88%) were positive by the Biotest kit, and 86 (81%) were positive by the Bartels kit. In specimens found to be positive by only one kit, the positive cell counts were low (median, 1; range, 1 to 7). When the data from all four sites were combined and analyzed, there was no statistical difference between the performance of the two kits; the Biotest and Bartels kits were found to be equivalent in sensitivity, specificity, and positive and negative predictive values for the detection of CMV pp65 antigenemia.     

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.     

Comparison of LightCycler-based PCR,COBAS amplicor CMV monitor,and pp65 antigenemia assays for quantitative measurement of cytomegalovirus viral load in peripheral blood specimens from patients after solid organ transplantation

In order to evaluate the LightCycler-based PCR (LC-PCR) as a diagnostic assay technique, a classical pp65 antigenemia assay and the commercially available COBAS Amplicor CMV Monitor (CACM) assay were compared to the LC-PCR assay for the detection and quantitation of cytomegalovirus (CMV) load in 404 parallel specimens of peripheral blood from 66 patients after solid organ transplantation. A good correlation existed among these three assays (r congruent with 0.6, P < 0.0001). The LC-PCR assay was the most sensitive (54% of specimens positive) compared to the CACM (48.6%) and the pp65 antigenemia (26%) assays. The LC-PCR assay detected all samples found positive by using both the CMV pp65 antigenemia assay and the CACM assay. The LC-PCR also had the widest dynamic range (from 250 to 10(7) DNA copies/ml of plasma). No cross-reactions were found among CMV and Epstein-Barr virus, varicella-zoster virus, or herpes simplex virus in the LC-PCR by using amplification with specifically designed primer pairs. Precision, expressed as the coefficient of variation, was <3% with standard DNA from cell cultures and between 6.55 and 14.1% with clinical specimens in repeat LC-PCR runs. One run of the LC-PCR took half of the time required for the semiautomated CACM procedure. Because of its sensitivity, specificity, cost-effectiveness, and simplicity, the LC-PCR assay could replace the pp65 antigenemia and the CACM assays as the preferred technique for the surveillance, diagnosis, and monitoring of response of CMV diseases in high-risk populations.     

Multicenter comparison of the digene hybrid capture CMV DNA assay (version 2.0), the pp65 antigenemia assay, and cell culture for detection of cytomegalovirus viremia

We compared the Digene Hybrid Capture CMV DNA Assay version 2.0, the pp65 antigenemia assay, traditional tube culture, and shell vial culture for the detection of cytomegalovirus (CMV) viremia in several patient populations at three centers. Of 561 blood specimens collected from 402 patients, complete clinical and laboratory data were available for 489. Using consensus definitions for true positives and true negatives, the sensitivities of the Hybrid Capture assay, antigenemia, shell vial, and tube culture were 95, 94, 43, and 46%, respectively. The specificities of the Hybrid Capture assay and antigenemia were 95 and 94%, respectively. At all three study sites, the detected level of CMV viremia was significantly higher with the Hybrid Capture assay or antigenemia than with shell vial and tube culture. In a group of 131 healthy nonimmunosuppressed volunteers, the Hybrid Capture assay demonstrated a specificity of over 99%. The Hybrid Capture assay is a standardized assay that is simple to perform and can utilize whole blood specimens that have been stored for up to 48 h. The high sensitivity and specificity of the Hybrid Capture assay along with its simplicity and flexibility make it a clinically useful assay for the detection of CMV viremia in immunocompromised or immunosuppressed patients. Further evaluation to determine its role in predicting CMV disease and for monitoring the therapeutic response to anti-CMV therapy is needed.     

High-level sensitivity of quantitative pp65 cytomegalovirus (CMV) antigenemia assay for diagnosis of CMV disease in AIDS patients and follow-up.

Cytomegalovirus (CMV) antigenemia was evaluated in 174 patients positive for human immunodeficiency virus. Antigenemia could be detected in 96.7% of patients with CMV disease, 76.9% of patients suffering from a relapse of the disease, and 11.4% of asymptomatic patients with CD4 levels of < 100 cells per microliter. No antigenemia was detected in patients with CD4 levels of 250 to 500 cells per microliter. Specificity and the positive predictive value for CMV disease were increased only if more than 5 positive cells per slide were considered. However, CMV disease may also occur in patients with low-grade antigenemia.     

2-Hour cytomegalovirus pp65 antigenemia assay for rapid quantitation of cytomegalovirus in blood samples

Of 109 blood samples tested for cytomegalovirus (CMV) antigenemia, 18 (16.5%) were positive. CMV Brite detected 13 and CMV Brite Turbo detected 16 of the 18 positives. There was no significant difference in the number of positive cells detected per sample. The seven discrepant samples contained a median of only one positive cell.     

Evaluation of the clinical usefulness of COBAS AMPLICOR HCV MONITOR assay (ver2.0): Comparison with AMPLICOR HCV MONITOR assay (ver1.0) and HCV core protein level

The quantitation of serum levels of hepatitis C virus (HCV) RNA in chronic hepatitis C has been regarded as one of the most important indicators for the outcome of interferon (IFN) therapy. The AMPLICOR HCV MONITOR version 1.0 (AMPLICOR v1.0) assay is widely used for the evaluation of the HCV level. A new generation assay called the COBAS AMPLICOR HCV MONITOR version 2.0 (COBAS v2.0) assay, which is semiautomated and modified to amplify all genotypes equally, has been developed. The aim of this study was to evaluate the clinical relevance of the COBAS v2.0 assay in comparison with the AMPLICOR v1.0 assay and HCV core protein assay in patients with chronic hepatitis C before IFN therapy. HCV RNA was detectable in 230 cases (97.5%) and undetectable in 6 cases (2.5%) by the COBAS v2.0 assay. The RNA levels measured by the AMPLICOR v1.0 assay correlated significantly with those measured by the COBAS v2.0 assay, and the sensitivity of the new version 2.0 assay was better than that of version 1.0, especially in serotype 2. In relation to the outcome of IFN therapy, HCV RNA levels from virologically sustained responders by the AMPLICOR v1.0 assay were 82.3 +/- 22.9 kcopies/ml in serotype 1 and 36.9 +/- 13.4 kcopies/ml in serotype 2, and those from virologically nonsustained responders were 525.2 +/- 48.6 kcopies/ml in serotype 1 and 76.7 +/- 19.5 kcopies/ml in serotype 2.The rates of sustained response to <100 kcopies/ml were 34/63 (54.0%) in serotype 1 and 24/48 (50.0%) in serotype 2. A statistically significant virological response was seen in serotype 1 (P < 0.0001), but not in serotype 2. In contrast, the levels in virologically sustained responders by the COBAS v2.0 assay were 88.2 +/- 20.5 KIU/ml in serotype 1 and 136.8 +/- 40.1 KIU/ml in serotype 2, and those in virologically nonsustained responders were 608.8 +/- 48.4 KIU/ml in serotype 1 and 328.3 +/- 62.8 KIU/ml in serotype 2. The rates of sustained response to <100 KIU/ml were 33/60 (55.0%) in serotype 1 and 21/35 (60.0%) in serotype 2. Statistical significance in virological response was seen in both serotype 1 (P < 0.0001) and serotype 2 (P < 0.05). Although the sensitivity of the HCV core protein assay was lower than that with the COBAS v2.0 assay, the HCV core protein levels also correlated well with the results of the COBAS v2.0 assay. The HCV core protein levels of virologically sustained responders were 37.6 +/- 12.0 pg/ml in serotype 1, 81.3 +/- 37.0 pg/ml in serotype 2, and those of virologically nonsustained responders were 289.9 +/- 23.5 pg/ml in serotype 1, 191.4 +/- 32.1 pg/ml in serotype 2. This assay could predict the outcome of IFN therapy in both serotype 1 (P < 0.0001) and serotype 2 (P < 0.05). Thus, both the COBAS v2.0 assay and the HCV core protein assay showed that the viral load was an indicator of virologically sustained response in serotype 2 and in serotype 1.     

Comparative evaluation of semiautomated COBAS AMPLICOR hepatitis B virus (HBV) monitor test and manual microwell plate-based AMPLICOR HBV MONITOR test

Comparative evaluation of the semiautomated COBAS AMPLICOR hepatitis B virus (HBV) MONITOR Test (COBAS-HBV) and manual AMPLICOR HBV MONITOR Test (AMPLICOR-HBV) on 208 serum samples revealed no significant difference in the sensitivities of the two assays. Twenty samples tested HBV DNA negative and 183 samples tested HBV DNA positive by both assays. Three samples tested positive by COBAS-HBV only and two samples tested positive by AMPLICOR-HBV only. HBV DNA concentrations determined by the two assays were significantly related (n = 183, r = 0.97, P < 0.0001), which indicates that COBAS-HBV could replace AMPLICOR-HBV. The major inconvenience of COBAS-HBV is the required performance of appropriate predilutions of high-titer samples in order to extend the narrow dynamic range of the assay.     

Surveillance of cytomegalovirus after solid-organ transplantation: comparison of pp65 antigenemia assay with a quantitative DNA hybridization assay.

In a multicenter study, 113 blood samples from 19 organ transplant patients were analyzed for cytomegalovirus by the pp65 antigenemia assay and a quantitative DNA hybridization assay. Overall, there was 84% agreement among the results obtained by the two tests. Fifteen of 16 episodes of active infection were detected by both assays. One episode was missed by the pp65 assay, and one patient showed significant DNA-emia but only low-level antigenemia.     

Comparison of a duplex quantitative real-time PCR assay and the COBAS Amplicor CMV Monitor test for detection of cytomegalovirus

A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.     

Comparison of the Murex Hybrid Capture CMV DNA (v2.0) assay and the pp65 CMV antigenemia test for the detection and quantitation of CMV in blood samples from immunocompromised patients.

BACKGROUND: Presently the semiquantitative pp65 cytomegalovirus (CMV) antigenemia test on white blood cells is often used for monitoring transplant patients for the appearance of active CMV infections. However, the need for immediate processing of the specimens and the lack of interlaboratory standardization of the test may sometimes be a disadvantage. OBJECTIVE: The aim of this study was to investigate the value of the recently developed second version of the Murex Hybrid Capture (MHC) CMV-DNA assay (v 2.0) in comparison with the CMV-pp65 test. The MHC CMV-DNA assay is a quantitative solution hybridization antibody capture assay, using alkaline phosphatase conjugated antibodies and a chemiluminescent substrate. STUDY DESIGN: 248 EDTA blood samples from 33 renal transplant patients and 32 samples from 22 other immunocompromised patients were tested by both the MHC CMV-DNA assay and the CMV-pp65 test. RESULTS: The qualitative ( + or -) results of both tests showed an overall concordance of 81.4%. Calculations on the basis of discordancy analyses showed that the sensitivity, the specificity, and the positive and negative predictive values were 87.7, 98.3, 98.6, 85.2% for the MHC CMV-DNA assay and 76.6, 100, 100, 75.5% for the CMV-pp65 test. Comparison of the quantitative results of both tests systems showed a correlation coefficient of 0.837. In addition, retesting of 50 samples with the MHC-CMV-DNA assay showed an excellent reproducibility with a correlation coefficient of 0.992. All patients which were tested regularly (at least five samples) became either positive with both tests or with none of them. Neither test system detected CMV significantly earlier than the other one. Both tests became strongly positive in all transplant patients with symptomatic CMV infections, and both tests showed a rapid decline of CMV during subsequent antiviral treatment. CONCLUSION: The quantitative Murex Hybrid Capture CMV-DNA assay (v 2.0) may become a valuable additional tool in CMV diagnosis. Further studies will be needed to support this preliminary judgement.     

Evaluation of CMV Brite kit for detection of cytomegalovirus pp65 antigenemia in peripheral blood leukocytes by immunofluorescence.

The CMV Brite antigenemia kit was compared with culture and an established cytomegalovirus pp65 antigenemia assay (CMV AG). Of 300 clinical specimens tested, 92 were positive by CMV Brite, 83 were positive by CMV AG, and 34 were positive by culture. Discrepancies could be attributed to anticytomegalovirus therapy or low-level antigenemia.     

Prognosis value of the pp65 antigenemia and semi-quantitative PCR in the detection of the CMV reactivation in bone marrow grafted patients

In this article a Cytomegalovirus (CMV) antigenemia and semiquantitative PCR retrospective evaluation of 26 bone marrow allo-grafted patients for different haematological disease is reported. Eighteen patients had a CMV reactivation despite a prophylactic treatment, seven of those patients had both positive antigenemia pp65 and positive semi-quantitative CMV PCR. During CMV reactivation, 3 patients developed a CMV disease despite a pre-emptive therapy. The follow up of the antigenemia was performed since D21 until D100 post transplantation, the antigenemia positivity occurred at D53 and was preceded about 7 days by CMV PCR positivity The CMV disease wasn't associated with a high viral load. All patients that had CMV reactivation had a positive CMV serology before the graft, whereas only 37.5% of the patients who did not reactivate had a positive CMV serology. Respectively half patients who reactivated and only 12.5% of those who didn't had a Graft versus host disease (GVHD), witch preceded the reactivation about 21 days in six of the formers. Clinical and biological signs presented by our patients in this cases report, seems to be associated more with the GVHD than with CMV reactivation.     

Significance of cytomegalovirus (CMV)-pp65 antigenemia in the diagnosis of CMV disease in human immunodeficiency virus-infected patients

To establish the diagnostic value of the cytomegalovirus (CMV)-pp65 antigenemia in CMV disease occurring in human immunodeficiency (HIV)-infected patients, CMV-pp65 antigen in polymorphonuclear leukocytes (PMNLs) was assayed in 373 samples from 138 randomly included patients followed up for symptomatic HIV-1 infection and the correlation between CMV-pp65 antigenemia and diagnosis of CMV disease was investigated. Thirty-seven CMV disease episodes were observed in 30 patients and 89.2% of these episodes were associated with a positive CMV-pp65 antigenemia. In contrast, 94% of the patients negative for CMV-pp65 antigenemia remained free of CMV disease. Patients with CMV disease had significantly higher levels of CMV-pp65 antigenemia than CMV disease-free patients (695 positive cells/2 × 10⁵ PMNLs vs. 28 positive cells/2 × 10⁵ PMNLs). The positive and negative predictive values of the test were 45% and 94%, respectively, but were 93% and 80%, respectively, when a CMV-pp65 antigenemia level of >100 positive cells/2 × 10⁵ PMNLs was taken into consideration. These results indicate that the CMV-pp65 antigenemia assay is useful for the diagnosis and monitoring of CMV disease in HIV-infected patients. © 1996 Wiley-Liss, Inc.     

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

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

Quantitative analysis of cytomegalovirus load using a real-time PCR assay

A novel real-time PCR assay system was developed to quantify the cytomegalovirus (CMV) genome load. The real-time PCR assay could detect from 6 to over 10(6) copies of CMV-DNA with a wide linear range. The virus load of immunocompromised patients with symptomatic CMV infections was quantified and compared to that of asymptomatic ones. In symptomatic patients, all 17 peripheral blood leukocytes were positive for CMV DNA, and its mean value was 10(3.3) copies/10(6) cells. On the other hand, only 9 of 38 samples (24%) were positive in the asymptomatic patients, and its mean titer was lower (10(2.0) copies/10(6) cells) than that of the symptomatic group (P = 0.002). In plasma, the virus genome was detected in 13 out of 17 samples from symptomatic patients (76%), and its mean value was 10(4.0) copies/ml. In contrast, for the asymptomatic group, only one out of 36 samples were positive (3%). Finally, this system was used to monitor two patients with CMV infections serially. The CMV DNA copy number changed with their clinical symptoms and anti-CMV therapy, and virtually paralleled the result of the pp65 antigenemia assay in both cases. In one patient with the cord blood transplantation, however, the CMV DNA became positive faster than the antigenemia assay. These results indicate that this assay is sensitive and useful for estimating the CMV genome load not only in peripheral blood leukocytes but also in plasma. It can be very helpful for diagnosing CMV-related diseases and monitoring the virus load in patients with CMV infections.