Comparison of nested PCR for detection of DNA in plasma with pp65 leukocytic antigenemia procedure for diagnosis of human cytomegalovirus infection.

A nested PCR was used for the detection of human cytomegalovirus (HCMV) DNA in plasma. The presence of HCMV DNA and its correlation to pp65 leukocytic antigenemia were investigated with 299 blood samples from 45 organ transplant recipients and 63 AIDS patients. Of the 53 samples positive by nested PCR, 52 (98%) were also positive for leukocytic antigenemia and 23 had high levels of antigenemia (> 50 positive cells per 2 x 10(5) leukocytes). Of the 246 samples negative in PCR, only 3 (1.2%) had highly positive antigenemia. For 15 patients having a high antigenemia level in the course of their disease, consecutive blood samples were studied and also assessed for viremia in culture. The extent to which HCMV DNA, detected by PCR, was present in plasma correlated with increased levels of HCMV leukocytic antigenemia for six of the eight AIDS patients and for all the organ transplant recipients. Positivity for HCMV DNA in PCR and for viremia in cell culture was usually restricted to the highest antigenemia levels. From a total of 69 blood samples, PCR and culture gave positive results, respectively, for 17 of 32 samples (53%) and 14 of 32 samples (43%) from transplant recipients and for 15 of 37 samples (40%) and 9 of 37 samples (24%) from AIDS patients. Our findings have shown a strong correlation between high levels of leukocytic antigenemia and HCMV DNA in plasma. The detection of HCMV DNA in plasma by this nested PCR can prove HCMV dissemination in blood, but it lacks the rapidity and simplicity of the leukocytic pp65 antigenemia procedure.     

Quantitation of human cytomegalovirus in recipients of solid organ transplants by real-time quantitative PCR and pp65 antigenemia

Human cytomegalovirus (HCMV) infections and anti-HCMV treatment are usually monitored by measuring pp65 antigenemia. This method is time-consuming, labour-intensive and requires skilled operators. We have compared results obtained using real-time Light Cycler quantitative PCR (QPCR) and the pp65 antigen assay on serial samples collected from recipients of solid organ transplants. We collected 198 blood samples from 14 solid organ transplant recipients and assayed them for pp65 antigen and with Light Cycler PCR. HCMV DNA was extracted from leukocytes and measured using primers and probe located in the UL83 region. The quantity of HCMV DNA was calculated using a standard curve prepared from a plasmid containing the target sequence. There was a good correlation between the number of pp65-positive cells and the DNA copy number (r = 0.57, P < 0.0001). A clinical threshold of 50 positive polymorphonuclear leukocytes/200,000 cells was equivalent to two log10 genome copies per capillary by Light Cycler PCR. HCMV DNA was detected before pp65 antigen in three patients at a mean time of 10 days, whereas the two tests were positive simultaneously for eight patients. Both the pp65 antigen data and DNA copy number decreased over time during antiviral treatment, although the QPCR was positive 28.2 days after the pp65 antigen assay had become negative. The real-time Light Cycler quantitative PCR assay is a rapid and labour-saving technique. This molecular method could be useful for monitoring infections and antiviral treatment in recipients of solid organ transplants.     

Monitoring of cytomegalovirus infection in solid-organ transplant recipients by an ultrasensitive plasma PCR assay

Early and accurate monitoring of cytomegalovirus (CMV) infection in solid-organ transplant recipients is of major importance. We have assessed the potential benefit of an ultrasensitive plasma-based PCR assay for renal transplant recipients. The pp65 CMV antigen (pp65 Ag) assay using leukocytes was employed as a routine test for the monitoring of CMV in 23 transplant recipients. We compared the pp65 antigenemia with the CMV load quantified by an ultrasensitive PCR (US-PCR) with a limit of detection of 20 CMV DNA copies/ml of plasma. CMV infection was detected in 215 (67%) of 321 plasma samples by the US-PCR compared with 124 (39%) of 321 samples by the pp65 Ag assay. The US-PCR assay permitted the detection of CMV infection episodes following transplantation a median of 12 days earlier than the pp65 Ag assay. Moreover, during CMV infection episodes, DNA detection by the US-PCR was consistently positive, whereas false negative results were frequently observed with the pp65 Ag assay. We found a good correlation between the two assays, and the peak viral loads were significantly higher in patients with CMV-related complications (median, 5000 DNA copies/ml) than in those without symptoms (1160 DNA copies/ml) (P = 0.048). In addition, patients that did not require preemptive therapy based on the results of the pp65 assay had CMV loads significantly lower (median, 36 DNA copies/ml) than those that needed treatment (median, 4703 DNA copies/ml) (P < 0.001). These observations provided cutoff levels that could be applied in clinical practice. The ultrasensitive plasma-based PCR detected CMV infection episodes earlier and provided more consistent results than the pp65 Ag assay. This test could improve the monitoring of CMV infection or reactivation in renal transplant recipients.     

Monitoring of patients for cytomegalovirus after organ transplantation by centrifugation culture and PCR.

A modified centrifugation culture technique and a polymerase chain reaction (PCR) is described for detection of early antigen and IE antigen DNA, respectively, for rapid and sensitive monitoring of active cytomegalovirus (HCMV) infection after organ transplantation. In a preliminary study, 541 clinical specimens (blood, urine, bronchoalveolar lavage, pharyngeal wash, sputum) from 59 organ recipients were assayed for HCMV antigen by centrifugation culture; 144 samples were tested by PCR simultaneously. Antigenemia detected by centrifugation culture correlated strongly with active HCMV infection and clinical symptoms and proved useful for monitoring the efficacy of antiviral therapy. PCR was more sensitive in an earlier phase of infection when centrifugation culture was still negative. The clinical usefulness of both methods is discussed.     

HCMVpp65抗原、HCMV mRNA和IgM抗体检测在诊断HCMV活动性感染中的比较

目的比较人巨细胞病毒（HCMV）pp65抗原、HCMV mRNA和血清HCMV—IgM抗体检测3种方法在诊断HCMV活动性感染中的实用意义。方法采集医院TORCH检查HCMV—IgM阳性的病人外周血（60份）。将标本分2份2ml和3ml,别用于HCMV mRNA和pp65检测。将三者结果进行比较。结果pp65抗原检测的结果与IgM抗体检测的阳性符合率为81．67％。与HCMV mRNA检测相比pp65抗原检测法的符合率、特异度和敏感度分别为81．67％,81．81％和81．63％。而且高pp65抗原血症与患者的临床症状密切相关。结论pp65抗原血症反映该病毒活动状况,可监测HCMV活动性感染,联合HCMV—IgM的检测可以提高临床的诊断率并可用于指导临床用药及监测药物疗效。     

Quantitative analysis of HCMV DNA load in whole blood of renal transplant patients using real-time PCR assay.

BACKGROUND: Preemptive antiviral treatment of Human Cytomegalovirus (HCMV) disease is a major goal in the management of organ transplant patients. It requires sensitive diagnostic methods. Automated real-time PCR systems have been recently proposed to monitor HCMV infection in such patients. OBJECTIVE: Objectives of this study was to compare a real-time quantitative PCR on whole blood with the HCMV pp65 antigenemia assay in renal transplant recipients, and also to evaluate two different DNA extraction methods. STUDY DESIGN: A total of 248 specimens from 21 patients were tested by quantitative pp65 antigenemia and quantitative real-time PCR. DNA was extracted from whole blood samples using two different methods: a conventional column manual assay and an automated system. RESULTS: Quantification of HCMV DNA using the two extraction methods showed highly similar results (Spearman rank test, r=0.863). We found a significant correlation between DNA quantification by real-time PCR in whole blood and pp65 antigenemia test (Spearman rank test, r=0.767). This correlation was not modified when the HCMV DNA results were normalized by quantification of the albumin cellular gene. In eight patients, HCMV infection was detected earlier with quantitative PCR than with the antigenemia test (mean delay of 11.25 days). HCMV DNA load equivalent of 50 pp65 positive cells/200,000 polymorphonuclear leukocytes (PMNLs) is log4.095 copies per ml of blood. CONCLUSIONS: Real-time PCR in whole blood is a sensitive method for estimating the HCMV genome load in renal transplant patients, and is more rapid and practicable than using PMNLs for pp65 antigenemia tests.     

Multiplex polymerase chain reaction for the evaluation of cytomegalovirus DNA load in organ transplant recipients

Because of the considerable impact of human cytomegalovirus (HCMV) infection, sensitive, specific, and standardized methods are required for rapid and accurate evaluation of viral load in monitoring transplant recipients. The aim of the present study was to evaluate the usefulness of a multiplex polymerase chain reaction (PCR) for the coamplification of HCMV-DNA and beta-globin genomic sequence in polymorphonuclear leukocytes (PMNL). Analysis and quantification of PCR products were carried out by a DNA enzyme immunoassay (DEIA), which is based on the hybridization of amplified DNA with a single-stranded DNA probe, which coats microtitre wells. Colorimetric detection of the DNA-antibody complex was carried out and optical density (O.D.) was recorded at 450/630 nm. To quantify HCMV/DNA load, a standard curve to which samples O.D. refer was obtained by amplifying serial dilutions of recombinant PGEM-3Z plasmid DNA containing a genomic fragment of glycoprotein B. 340 PMNL specimens from 102 solid organ recipients were tested for the detection of pp65 antigen and HCMV-DNA. The results showed a good correlation between viral load and clinical symptoms of HCMV infection; high specificity and predictive values for HCMV disease were found by PCR, using a cut-off limit of 10(3) genomic copies per 2 x 10(5) PMNL. These findings indicate that the system described is an efficient and reproducible diagnostic method easy to apply for routine diagnosis and therapeutic monitoring of transplanted patients.     

A novel pattern of pp65-positive cytomegalic endothelial cells circulating in peripheral blood from a renal transplant recipient

The present study reports a novel pattern of cytomegalic endothelial cells (CEC) in peripheral blood from a female renal transplant recipient infected with human cytomegalovirus (HCMV), which has not been reported previously. Localization of specific early antigen of HCMV, pp65 antigen, was examined by immunohistochemistry. Staining of an endothelial cell marker (CD34) was used to characterize endothelial cells. It is demonstrated that many leukocytes surrounded and adhered to a protein-like material, in which pp65-positive CEC were detected. The composition and function of this protein-like material are yet unknown. The patient lacked clinical symptoms of HCMV disease. Furthermore, similar localization patterns were found in other renal transplant recipients suffering from HCMV infections as determined by real-time PCR to detect HCMV DNA in blood. These patients showed no or only minor clinical symptoms of HCMV infection. It is suggested that these novel localization patterns of CEC may play a role in the host defense in patients infected with HCMV, but the exact relation between HCMV infection and CEC formation needs further investigation.     

Monitoring for cytomegalovirus infection in organ transplant recipients: Analysis of pp65 antigen,DNA and late mRNA in peripheral blood leukocytes

The use of sensitive and specific methods for rapid and reliable diagnosis is required due to the considerable impact of human cytomegalovirus (HCMV) in organ transplant recipients. For this purpose the demonstration of the presence of viral antigens in peripheral blood leukocytes (PMNLs) and of viral nucleic acids in the same cells or in sera would seem to be of valid support. The present study was designed to test pp65 antigen, HCMV DNA and HCMV late mRNA in order to provide clinical information for the management of the infection. Fifty solid organ recipients were monitored for six months after transplant. The data obtained from the various tests were analysed from the first evidence of HCMV infection revealed by positive antigenaemia and/or DNA-polymerase chain reaction (PCR). In 3 asymptomatic and in 7 symptomatic patients, PCR became positive 1–2 weeks before antigenaemia but PCR did not discriminate the clinical evolution of HCMV infection. The antigenaemia test well correlated to the development of viral infection being positive in all symptomatics and in 31, 2% of asymptomatics. The antigenic load >100/2 × 10⁵ positive cells was always associated with clinical signs of illness. The detection of late mRNA was more indicative of the virus replicative status in the follow-up of patients treated with ganciclovir. In some cases there was evidence, prior to the other two tests, the block of viral replication due to the antiviral therapy and in others the onset of HCMV infection relapse. J. Med. Virol. 53:189–195, 1997. © 1997 Wiley-Liss, Inc.     

Relationship of cytomegalovirus load assessed by real-time PCR to pp65 antigenemia in organ transplant recipients

BACKGROUND: Cytomegalovirus (CMV) infection in immunocompromised patients can lead to viremia associated with morbidity and mortality. Monitoring of viral loads in blood is critical for initiating and monitoring antiviral treatment. OBJECTIVES: Validate quantitative real-time PCR assay targeting the US17 and UL54 regions of the CMV genome for automated DNA and extraction and amplification. STUDY DESIGN: 3422 blood specimens from organ transplant recipients, including longitudinal specimens from 12 organ transplant recipients, were tested by CMV PCR and pp65 antigenemia. RESULTS: CMV PCR for both US17 and UL54, was more sensitive and detected CMV DNA earlier and for longer than the CMV pp65 antigenemia test. Using antigenemia results as a reference standard, an optimal cutoff of 500 normalized copies was calculated for both US17 and UL54 PCR targets based on high sensitivity, specificity, and positive and negative predictive values. CMV DNA levels tracked well with clinical symptoms, response to treatment, and antigenemia. CONCLUSIONS: Detection of persistent increases in CMV DNA levels above 500 normalized copies by this real-time PCR assay is indicative of symptomatic CMV disease in organ transplant recipients. Quantitative real-time PCR for CMV DNA can be used in lieu of antigenemia for monitoring CMV infection and determining when to initiate preemptive treatment.     

Detection of human cytomegalovirus (HCMV) pp67-mRNA and pp65 antigenemia in relation to development of clinical HCMV disease in renal transplant recipients

Objective   To evaluate the performance of the recently introduced method based on detection of human cytomegalovirus (HCMV) pp67 mRNA in blood by the nucleic acid sequence-based amplification (NucliSens), in comparison to semiquantitative detection of pp65 HCMV antigen in white blood cells, in relation to development of clinical HCMV disease.
Methods   Thirty patients, recipients of renal transplants, were monitored prospectively for the presence of pp67 mRNA, the presence and level of pp65 antigenemia, IgG and IgM antibodies, and the development of clinical HCMV disease. A total of 148 samples were examined during the observation period.
Results   Twenty-five samples were positive for pp67-mRNA and 45 samples contained at least one pp65 positive cell, with 68% agreement between the two assays. Both assays predicted correctly the development of clinical disease in five patients, giving a sensitivity of 100%. However, the specificity of the pp67-mRNA test was 72%, and of the pp65 antigenemia test from 20 to 64%, depending on the level of antigenemia chosen for cut-off. pp67-RNA appeared somewhat earlier than pp65 antigenemia, and responded earlier to treatment. Sero-conversion and appearance of IgM antibodies were of very little clinical value.
Conclusion   Both the pp67-mRNA and the pp65 antigenemia assay predicted correctly the development of clinical HCMV disease in renal transplant recipients. However, the specificity of both tests with respect to development of HCMV disease, especially the pp65 antigen test was moderate. Significantly positive tests not necessarily prove the development of clinical disease. Testing for pp67-mRNA may improve the diagnosis and management of HCMV disease in renal transplant patients.     

Monitoring of renal allograft recipients by quantisation of human cytomegalovirus genomes in peripheral blood leukocytes

The ratio of human cytomegalovirus (HCMV) genomes per cellular genomes in serial peripheral blood leukocyte (PEL) extracts of renal allograft recipients was quantitated by competitive nested polymerase chain reaction (PCR). Patients were also monitored for the development of acute HCMV infection by detection of HCMV pp65 antigenemia, HCMV IgM antibodies, and viruria. Compared to qualitative nested HCMV PCR, the frequency of positive PCR results in renal allograft recipients without further evidence of acute HCMV infection was significantly reduced by quantitative HCMV PCR. HCMV DMA levels ≥1,000 copies HCMV/10⁶ copies β-globin were found to be highly indicative for the development of a clinically symptomatic HCMV infection following renal allograft transplantation. In patients treated with ganciclovir, quantitation of HCMV target sequences allowed the assessment of the efficacy of antiviral therapy. © 1994 Wiley-Liss, Inc.     

Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay

The measurement of human cytomegalovirus (HCMV) DNA in blood is becoming the standard method for monitoring HCMV infection in immune-suppressed and unsuppressed patients. As various blood compartments can be used, we have compared the HCMV DNA measured in whole blood (WB), peripheral blood leukocytes (PBL), and plasma by real-time PCR. We tested 286 samples: HCMV DNA was extracted automatically from WB and PBL with the MagNA Pure instrument (Roche Molecular Biochemicals) and manually from plasma samples. The HCMV DNA from WB, PBL, and plasma was measured by real-time Light Cycler PCR. Primers and probe were located in the UL 83 region. HCMV DNA was detected more frequently in WB (88.5%) than in the PBL (65.7%) (P < 0.0001) or the plasma (55.2%) (P < 0.0001). There was a good correlation between the positive results in WB and in PBL (r = 0.68; P < 0.0001), and 3.15 log(10) genome copies in 200000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200000 leukocytes, was equivalent to 3.4 log(10) genome copies in 200 microl of WB. WB was shown to be suitable for automated extraction and the quantitation of HCMV DNA by real-time Light Cycler PCR by analysis of serial samples from representative patients of various populations. This system may be very useful for monitoring of immune-suppressed and unsuppressed patients.     

活动性巨细胞病毒感染与反复自然流产的关系探讨

目的探讨活动性人巨细胞病毒（human cytomegalovirus,HCMV）感染与反复自然流产（recurrent spontaneous abortion,RSA）的关系.方法采集反复自然流产孕妇和正常产前体检孕妇外周血,分离外周血单个核细胞（PBMCs）和血浆,分别用免疫荧光法和实时定量PCR检测HCMV pp65抗原和HCMV DNA,并比较2种方法的一致性.结果 65例RSA患者HCMV pp65抗原有20例阳性,阳性率30.8%,50例正常体检孕妇 HCMV pp65抗原有4例阳性,阳性率8.0%,2组孕妇HCMV活动性感染率有显著性差异（χ^2=8.87,P＜0.01）.孕妇HCMV pp65抗原阳性率升高,孕妇流产几率增加（χ^2=7.53,P＜0.01）. 免疫荧光法和实时定量PCR有较好的一致性（92.3%）.结论反复自然流产孕妇 HCMV活动性感染率显著高于正常孕妇,HCMV pp65抗原检测也许可作为RSA早期诊断指标之一.     

Early detection of human cytomegalovirus viremia in bone marrow transplant recipients by DNA amplification.

Surveillance blood cultures for human cytomegalovirus (HCMV) are commonly used to identify the bone marrow transplant (BMT) recipients with the highest risk of serious HCMV disease and for whom early interventional ganciclovir therapy would be beneficial. We monitored 36 allogeneic BMT recipients weekly for the presence of HCMV in the blood from 0 to 100 days posttransplantation. Viable HCMV in leukocytes (WBC) was detected by shell vial and tube culture methods. HCMV DNA in WBC and plasma was detected by PCR and DNA hybridization using primers and a probe from the EcoRI fragment D region of HCMV AD169. A uracil-N-glycosylase-dUTP PCR protocol was used to prevent false-positive results due to amplicon carryover. Seventeen patients had multiple consecutive positive samples containing HCMV DNA in plasma or WBC. In 14 of 17 patients, HCMV was also detected by blood culture. HCMV DNA was detected sporadically in six patients, none of whom had positive cultures. One patient had HCMV viremia detected by WBC culture only. The remaining 12 patients had no positive PCR assays or blood cultures. For the patients with positive blood cultures, PCR detection of HCMV DNA in plasma preceded detection of HCMV in culture by a mean of 8 days and detection in WBC preceded detection in culture by 6 days. HCMV disease (interstitial pneumonia) was documented for two patients with viremia (blood culture and PCR positive) and one patient without viremia (blood culture and PCR negative). The earlier recognition of high-risk patients provided by detection of HCMV DNA in plasma or WBC may improve the efficacy of early interventional antiviral therapy.     

Comparison of polymerase chain reaction and pp65 antigen test for early detection of human cytomegalovirus in blood leukocytes of cardiac transplant recipients

Objective: To establish whether polymerase chain reaction (PCR) for cytomegalovirus deoxyribonucleic acid (DNA) can provide clinical information for the management of the infection.
Methods: Leukocytes in 30 heart transplant recipients were monitored by pp65 antigen testing and PCR for 82 to 365 days after transplantation.
Results: Of the 30 patients, 26 developed cytomegalovirus infection, nine of whom were symptomatic. Altogether, 300 leukocyte samples were examined. The concordance between PCR and pp65 antigen test was 82.6%. In symptomatic patients after surgery, PCR detected cytomegalovirus infection after 38 ± 16 days and the pp65 antigen test, after 48 ± 15 days. Symptomatic infection correlated with a higher number of pp65-positive leukocytes than did asymptomatic infection: 310 ± 356 vs 24 ± 35 ( p < 0.005)/200,000 examined, respectively. Clearance of virus was observed by PCR after 125 ± 73 days (range 29 to 225) in symptomatic, and after 82 ± 70 days (range 16 to 301) in asymptomatic, cases of infection.
Conclusions: The positive predictive value of PCR for symptomatic infection was 34.6%. Our findings correlate with previous reports and show that the qualitative detection of cytomegalovirus DNA is not associated with overt disease whereas quantitation of pp65-positive leukocytes closely correlate with symptom onset. Insofar as the results are not quantitative, PCR is not a marker of clinically apparent infection. Careful monitoring of cytomegalovirus infection based on quantitative pp65 antigen assay can fulfill all clinical needs for early diagnosis and proper management of the infection     

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

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

Monitoring low cytomegalovirus viremia in transplanted patients by a real-time PCR on plasma

Until recently, human cytomegalovirus (hCMV) infection and anti-CMV treatment in transplanted patients have been monitored essentially by pp65 antigenemia, which is time-consuming and requires experienced operators. For the last two years, pp65 antigenemia levels have tended to be lower than previously in our laboratory, which could be due to better monitoring of CMV-related risk. Results obtained by real-time PCR with a LightCycler instrument or by pp65 antigen assay were compared on 145 serial samples from bone marrow or kidney transplant recipients under the usual conditions of our laboratory. CMV DNA was extracted from plasma and quantified by using primers and probes directed to HXFL4 gene. The plasma CMV DNA load was measured by using a standard curve constructed with a commercially available quantified CMV DNA suspension. Among the 145 samples, 139 showed a pp65 antigen which was negative or lower than 20 positively stained cells per 200,000 leukocytes. In the patients with positive pp65 antigenemia, the corresponding values of CMV DNA copy number/ml were significantly higher than those observed in patients without antigenemia (P < 0.001). CMV DNA was detected from 4 up to 52 days before pp65 antigen. Elsewhere, between two dates at which pp65 antigen was positive, intermediate PCR results could be positive while the pp65 antigen was negative. This real-time quantitative PCR assay is a rapid technique adapted to monitor plasma CMV DNA in transplant setting, even for low viremia.     

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

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