TABLE 18.

Quantitative detection of cytomegalovirus DNA by real-time PCRa

PlatformReferenceSpecimenTargetQuantitative standardReporting unitsComparison to other systemsComments
ABI PrismMachida et al. 2000 (293)16 patients (bone marrow transplant); 136 blood samples from patients: 70 blood specimens from healthy volunteersUS17 geneRange: 10 to 107 CMV DNA copies/wellCopies of CMV DNA/500 mg of DNA (blood), copies CMV DNA/100 μl plasmaReal-time PCR compared with antigenemia assay and by shell vial cell culture.CMV DNA was not detected in blood specimens of the 55 healthy patients seropositive for CMV. The results of PCR (plasma) did not correlate well with those obtained by the antigenemia test; however, PCR (nucleated cells from blood) had high correlation with the antigenemia test results.
Nitsche et al. 2000 (347)Plasma; bone marrow transplant patientsMajor immediate-early geneAs a positive control, a plasmid containing the target sequence from the target gene was used with 101 to 107 plasmids/assayCopies CMV genome/ml plasmaCMV load was higher in CMV antigen-positive (antigenemia) patients than in antigen-negative patients.CMV DNA was detected from 5/27 healthy blood donors seronegative for CMV.
Tanaka et al. 2000 (473)Peripheral blood leukocytes, plasmaImmediate-early geneRange from 6 to over 106 copies of CMV DNA, Plasmid containing the IE gene used to develop a standard curve for quantitative results.Copies CMV DNA/106 cellsReal-time PCR compared with antigemia assay.The correlation between the CMV DNA copy number and the pp65-positive cell count (antigenemia test) was statistically significant (P = 0.01). CMV DNA copy level/106 cells was higher in symptomatic patients (blood, plasma specimens) than in asymptomatic patients.
Funato et al. 2001 (133)Whole-blood samples from infants with hepatitisMajor immediate-early geneA plasmid containing the target gene of CMV was constructed and used as a quantitative standard. Reference value for the CMV DNA copy numbers was determined as 109 molecules/3.7 ng/μl based on the molecular weight of the DNA inserted into the plasmid.Copies CMV DNA/μg DNAConventional PCR and real-time PCR were compared for the detection of CMV DNA in this patient population.No CMV DNA was detected in 97 healthy patients by conventional or by real-time PCR methods. Eighteen samples tested negative by conventional PCR: 14 samples were negative; four samples contained CMV DNA with low copy levels of CMV DNA (10-80).
Gault et al. 2001 (138)Blood (peripheral blood leukocytes) (46 samples)UL83 (pp65 gene)Plasmid containing one copy of the UL83 target sequence used as a quantitative standard plasmid containing human genomic DNA (albumin gene) coamplified with specimen DNA.Copies CMV DNA/2 × 105 leukocytesKnown pp65 antigenemia-positive (PBL samples) collected from solid organ transplant patients were amplified by real-time PCR.The results of the real-time PCR assay correlated with those of the antigenemia assay (P< 0.0001).
Griscelli et al. 2001 (149)Peripheral blood leukocytes of bone marrow transplant patientsUL83, pp65 gene (phosphorylated matrix protein)A plasmid containing both the CMV and the glyceraldehyde-3-phosphate dehydrogenase genes was used as a standard.Copies CMV DNA/200,000 leutkocytesReal-time PCR was compared to the antigenemia test.Study quantified CMV DNA in the glyceraldehyde-3-phosphate dehydrogenase gene using a plasmid containing both sequences of 16 patients, CMV DNA was detected by PCR in 13/16 (91.3%), a mean of 15 days prior to the appearance of antigenemia.
Guiver et al. 2001 (156)Blood, 362 samples from 25 patients; 12 single lung, 13 heart transplantGlycoprotein B genePlasmid standard of CMV DNA diluted to contain 10 to 106 copies/PCRCopies CMV DNA/ml (limit of sensitivity was 500 copies/ml blood based on sampling volume of 2μ l of EDTA treated blood following extraction.Real-time PCR was compared to CMV antigenemia test.Viral loads of real-time PCR showed a highly significant linear correlation with antigemia levels.
Limaye et al. 2001 (271)Plasma samples from stem cell transplant patientsImmediate-early geneCopies CMV DNA/mlCMV DNA was detected a median of 13 days before onset of CMV disease (range, 0-35 days). Monitoring plasma CMV DNA loads may be useful in the prevention of pre-engraftment CMV disease.
Najioullah et al. 2001 (329)Blood (serum) (positive for CMV in cell cultures) from transplant patientsHXFL4 genePlasmid dilutions from 5× 102 to 2 × 108 copies of CMV DNA/ml were tested. (four dilutions were used to develop a standard curve in the quantitative test). CMV DNA obtained from Advanced Biotechnologies used as a positive control for real-time PCR.Copies CMV DNA/μg DNAReal-time PCR and conventional PCR compared with positive cell culture results obtained with blood specimens.The qualitative PCR was positive in 48 samples and the quantitative real-time assay was positive in 46 samples from a total of 50 cell culture-positive specimens.
Sanchez et al. 2001 (426)Blood specimens from lung transplant recipientsDNA polymerase gene, human apoprotein B geneCopies CMV DNA/μg human DNARisk of CMV pneumonitis after lung transplantation is related to the level of CMV DNA in blood.
Satou et al. 2001 (429)Whole bloodMajor immediate-early geneTarget gene inserted into a plasmid.Copies CMV DNA/μg DNAResults of real-time PCR correlated with conventional PCR and with serology in several specimens from one liver transplant patient.Forty specimens from normal subjects were negative for CMV DNA. Real-time PCR was useful for the quantitative detection of CMV DNA from serial samples of blood from a patient post-liver transplantation.
Dworkin et al. 2002 (103)Aqueous vitreous specimens from patients with posterior uveitisNot providedCMV DNA obtained from Advanced Biotechnologies, Inc., Columbia, MO; SYBR Green I dye fluorescein detection systemCMV genome/μl vitreousReal-time PCR may be applied to infectious agents responsible for posterior uveitis; the technique will likely be useful for the diagnosis of this entity and the linkage of viral pathogens to the disease.
Greenlee et al. 2002 (146)Blood (RBC, WBC-reduced), 100 blood donors seroprositive for CMV; 93 blood donors seronegative for CMVImmediate-early gene of CMVABI standard at stock concentration of 104 copies/ml. The standard was used as a template for the TaqMan assay at 5, 50, 500, and 50,000 copies/50 μl PCR.Copies CMV DNA/mlCMV DNA was not detected in any of the RBC samples.
PlatformReferenceSpecimenTargetQuantitative standardReporting unitsComparison to other systemsComments
Mori et al. 2002 (321)Plasma allogeneic hematopoietic stem cell transplant patientsUS17 geneA standard ranging 101 to 107 copies/well was used for a standard curve for quantitation of CMV DNACopies CMV DNA/100. μlQualitative results of the real-time PCR were compared with the determinations obtained with the antigenemia test.Authors used real-time PCR results to guide preemptive therapy (ganciclovir) for CMV disease. Qualitative results of real-time PCR were detected earlier than antigenemia in the posttransplantation period in 18/30 (60%) patients. Antigenemia was detected before PCR results in only 3/30 (10%) patients.
Sanchez et al. 2002 (427)Plasma and leukocyte lysate preparationsDNA polymerase gene. Primers and probes for amplification of the human apoprotein gene were included in the assay.CMV quantitation was linear over a range of 101 to 106 copies.Copies CMV DNA/mlAuthors created a multiplex, quantitative real-time PCR assay that amplifies CMV DNA and human DNA in the same reaction tube.Applied to whole blood, the assay provides a measurement of CMV DNA in reaction to cellular content without a need for cell-counting procedures.
Yakushiji et al. 2002 (549)Plasma from stem cell transplant patientsUS17CMV DNA copies/mlCMV antigenemia and quantitative real-time PCR were compared for monitoring CMV reactivation after allogenic stem cell transplantation.The number of CMV antigen-positive cells by the antigenemia assay and the level of CMV DNA by real-time PCR correlated well.
Hänfler et al. 2003 (162)Buffy coat leukocytespp150 IL-32 geneLinear range 101 to 107 copies standard curve data prepared from a plasmid with target insert diluted from 0.5 × 101 to 0.5 × 107 copies/μlCMV DNA copies/2-μl sampleAuthors developed a duplex real-time PCR that was capable of quantifying CMV DNA and β-actin DNA as internal control simultaneously within one reaction.A high degree of conformity was attained between PCR viral load and antigenemia tests using leukocyte specimens.
Ikewaki et al. 2003 (193)PlasmaUS17 genePlasmid containing the target sequence was diluted (seven 10-fold dilutions) corresponding to 101 to 107 plasmid copies/reaction. Dynamic range: 5 × 102 to 5 × 108 copies/mlCMV DNA/mlStudy compared real-time PCR assay for detection of CMV DNA with nested conventional PCR and antigenemia tests.Real-time PCR was more sensitive than the antigenemia and nested PCR assays. In addition, real-time PCR was able to detect CMV reactivation earlier in the clinical course than the antigenemia and nested PCR. CMV viral loads of 5,000 copies/ml were proposed as the cutoff for initiating therapy in adult T-cell leukemia-lymphoma patients.
Li et al. 2003 (269)Whole bloodGlycoprotein BPlasmid standard concentration calibrated by spectrophotometry at 260 nm.Copies CMV DNA/mlA laboratory-developed PCR assay and a semiquantitative pp65 antigenemia assay were compared to a quantitative real-time assay for the detection of CMV.High correlation between antigenemia value and CMV DNA loads. Antigenemia values of 11 to 100, 101 to 1,000, and over 1,000 positive cells/2 × 105 leukocytes corresponded to median CMV DNA levels of 1,000, 4,000,    and 10,000 copies/ml and are proposed as cutoff points for initiating antiviral therapy in patient groups with high, intermediate, and low risk of CMV disease, respectively.
Nitsche et al. 2003 (346)Blood leukocytesMajor immediate-early geneAuthors compared real-time PCR with antigenemia test using blood specimens from patients who underwent stem cell transplantation.The PCR and antigenemia tests were positive in 19 of 77 patients. An additional 26% (22 patients) were positive exclusively by PCR. in 5/20 patients. Febrile episodes in two patients with fever of unknown origin may have been caused by a reactivation of CMV. These results imply that CMV infection can be expected not only in transplant patients but also in chemotherapy treated neutropenic patients.
Persson et al. 2003 (373)Neutropenic patients following chemotherapyUL65 (pp67 protein)CMV DNA genomes/mlCMV DNA but not HHV-6 (or HHV-7) was detected
Boeckh et al. 2004 (35)A double primer assay was designed consisting of two sets of primers and probes to amplify a portion of the gB region (UL55) and UL123-exon 4 regionCopies CMV DNA/mlThe double-primer (UL55/UL123-exon 4) was superior to pp65 antigenemia and viremia by culture with regard to sensitivity, specificity, and predictive values.
Nesbitt et al. 2004 (335)Plasma, 78 EDTA-anticoagulated whole blood samplesJunction between glycoprotein B and UL123Copies CMV DNA/mlTested the effects of storing whole blood for 24 h before separating plasmaStudy indicated that valid results can be obtained from EDTA-anticoagulated blood stored at room temperature for at least 24 h before separation of plasma.
Visconti et al. 2004 (520)Peripheral blood mononuclear cells experimentally infected with CMVImmediate earlyLinear range over 4 log10CMV DNA genome copies/mlAuthors experimentally determined the efficiency of removal of CMV using leukocyte depletion filters in a model system of PBMC's infected with the virus.CMV DNA load reduced but was not eliminated in whole blood and platelets after treatment with leukocyte depletion filters.
LightCyclerNitsche et al. 1999 (348)Major immediate-early geneLinear range: 101 to 107 CMV DNA genome equivalent/assay. MIE gene inserted into plasmid vector; DNA concentration determined byCMV DNA genome equivalent/assayLightCycler and ABI Prism 7700 were compared for the detection of CMV DNA. The ABI Prism 7700 appears to be useful for the processing of
PlatformReferenceSpecimenTargetQuantitative standardReporting unitsComparison to other systemsComments
a spectrophotometer at 260 nm and the corresponding copy number calculated.large numbers of samples under standard conditions, whereas the LightCycler has its strength in smaller sample numbers and the use of various reaction conditions.
Schaade et al. 2000 (433)Plasma, urineGlycoprotein B geneAmplicon cloned in plasmid. Concentration of DNA calibrated by spectrometry at 260 nm. Dynamic range, 102 to 108 plasmid copies of CMV DNA/mlDNA copies/mlLightCycler detected higher levels of CMV DNA than the COBAS instrument; however, both tests had comparable sensitivities for detecting CMV DNA in clinical specimens.
Kearns et al. 2001 (213)Glycoprotein B geneRange: 2 × 103 to 5 × 108 CMV DNA copies/ml (copies/reaction converted to copies/ml)LightCycler assay compared to TaqMan real-time PCR. Of 50 samples, the majority of results (36/50, 72%) were within 0.5-1 log10 and 3 (14%) by log10.Data confirm and extend those of Schaade (433). The LightCycler assay was shown to be more sensitive than the detection of early antigent fluorescent foci testing for urine and respiratory specimens.
Kearns et al. 2001 (215)BloodGlycoprotein B geneRange: 10 to >2 × 105 CMV DNA copies EcoRI plasmid quantified and linearized and used as quantitative standard. A series of five log10 dilutions corresponding to 2 × 101 to 2 × 105 copies/2 μl were prepared and run as external standards.DNA copies/μlLightCycler results comparable to TaqMan (blood). TaqMan could reproducibly detect down to 20 plasmid copies. Detection level of LC, ≤10 copies CMV DNA. Of 51 positive samples (blood) the quantitative results using TaqMan ranged from 102 to 3 × 106 copies/ml. LightCycler results ranged from 1.1 × 102 to 1.7 × 106 copies/ml.PCR product identity confirmed by melting curve analysis. Runs acceptable if the external quantitative standard within 0.5 log10 of the target value and the standard curve gave a mean squared value of >10−2.
Ando et al. 2002 (9)Aqueous humor; 6 patients with clinically diagnosed CMV retinitisGlycoprotein B geneRange: 101 to 104 copies/μl. Plasmid with target insert used to develop standard for quantitationSYBR Green I dye CMV DNA was detected at levels up to 1.6 × 104 copies/μl of aqueous humor obtained from patients with retinitis. Study revealed correlation between levels of CMV DNA and the extent of the area affected by CMV retinitis before antiviral treatment and the prolonged retention of CMV genome after antiviral treatment.
Kearns et al. 2002 (216)Urine and respiratory samplesGlycoprotein B geneRange: 2 × 103 to 5 × 108 CMV DNA copies/ml. Note: copies/reaction (215) converted to copies/ml in the publicationDNA copies/mlLightCycler PCR provided a 3-fold increase in sensitivity compared to detection of CMV early antigen in cell culture (urine samples). Urine
viral loads were higher in congenitally CMV-infected infants (median, 1.6× 105 copies/ml) compared with 15 transplant recipients (median, 9 × 103 copies/ml). Urine samples did not require extraction prior to PCR testing.
Stöcher et al. 2002 (465)US17 gene of CMVLinear range: 500 to 50,000 CMV DNA copies/mlCMV DNA copies/capillaryAuthors developed a normalized quantitative competitive real-time PCR assay using a PCR competitor as heterologous DNA. The results obtained with conventional real-time quantification on the LightCycler instrument were almost identical to those obtained with the normalized based quantification assay.
Cortez et al. 2003 (79)Whole blood from 51 stem cell transplant recipientsPlasmid with target insert used to develop standard for quantitation (5000, 500, 50, 5 copies/reaction). Conversion to copies/ml: DNA extracted from 200 μl whole blood and then suspended in 50 μl water; 5 μl was used in each of two reactions. The sum of these two reactions represented 50 μl of whole blood for which the sum was multiplied by 25 to obtain copies/ml.CMV DNA copies/mlReal-time PCR results were positive earlier than antigenemia results in 30/39 (77%) episodes of CMV infection detected by antigenemia. Real-time PCR remained positive after treatment was discontinued in 14/39 (36%) episodes and predicted the return of CMV reactivation in 4/13 (31%) episodes.
Mengelle et al. 2003 (309)14 patients (solid organ transplant), 198 blood specimens (leukocytes)UL83 gene (codes for lower matrix protein pp65)Plasmid with CMV target sequence used to develop standard for quantification. Detection limit was 1 log10 genome copy/capillary Range: 1 log10-5 log10 copies/reaction.CMV DNA copies/mlCMV DNA was detected before pp65 antigen in three patients, whereas the two tests were positive simultaneously for eight patients. Molecular method of real-time PCR could be useful for monitoring infections and antiviral treatment in recipients of solid organ transplants.
Pang et al. 2003 (365)404 plasma specimens from 66 solid organ transplant patientsGlycoprotein B geneRange: 2.5× 102 to 107 DNA copies/ml plasma. Positive control was prepared (106 genome copies/tube) from CMV-infected cell cultures.CMV DNA copies/ml plasmaLightCycler PCR was most sensitive (54% of specimens positive) compared to the COBAS AmplicorThe costs per sample were highest with COBAS ($104.7) compared with LightCycler ($39.8) and antigenemia ($35.8) (Canadian dollars).
PlatformReferenceSpecimenTargetQuantitative standardReporting unitsComparison to other systemsComments
CMV monitor (48.6%) and the pp65 antigenemia (26%) assay.Because of its sensitivity, specificity, cost effectiveness, and simplicity, the LC-PCR assay would replace the antigenemia and COBAS assay as the preferred technique for the surveillance, diagnosis, and monitoring of response of CMV diseases in high-risk populations.
Gouarin et al. 2004 (145)Whole blood from 21 renal transplant patients; 248 specimensUL83 gene (codes for lower matrix protein pp65)Plasmid with target insert used to develop standard for quantitation. Range: 5 to 5 × 106 copies/reactionCMV DNA copies/mlCMV DNA was detected earlier than antigenemia in the posttransplantation period. Real-time PCR with whole blood can be used to monitor renal transplant patients at risk of developing CMV disease and to assess response to antiviral therapy. Study found that >4 log10 copies/mL of whole blood indicates extensive CMV infection and leads to initiation of antiviral treatment.
Hong et al. 2004 (186)147 plasma specimens from bone marrow and stem cell transplant patientsGlycoprotein B or EcoRID regionβ-Globulin gene was amplified in parallel to control for the efficiency of nucleic acid extraction and amplification steps. Range: 125 copies/ml to 5 × 109 copies/ml. A CMV gB plasmid containing the whole 2.7-kb gB gene subcloned into a pCR2.1 vector was used as a quantification standard for the assay.CMV DNA copies/ml plasmagB target provided a sensitivity of 96% and specificity of 100% compared to the EcoRID target (real-time) and conventional (gel and Southern blot)The combination of automated DNA preparation and real-time PCR detection allows a sensitive, precise, accurate high-throughput assay of CMV viral load that can be used as a laboratory trigger for preemptive antiviral therapy.
iCycler iQAberle et al. 2002 (3)CSFus 17 geneCopies CMV DNA/ml (corresponding to 2 copies of DNA per TaqMan PCR mixture).Virus load was 2.0 × 102 to 1.9 × 106 CMV DNA/ml in patients with encephalopathy in immunosuppressed patients. Quantitation of viral loads in CSF may be important regarding prognosis of disease and prediction of distinct CNS manifestation.
RotorGeneHerrmann et al. 2004 (176)138 plasma specimens from 44 patients with suspected CMV infectionDNA polymerase gene; glycoprotein B geneLinear range of 103 to 108 copies/ml. Target DNA used for standard curves was purified genomic DNA from CMV strain AD169.CMV DNA copies/mlOf 138 samples, 105 were positive by real-time PCR and 71 were positive by COBAS assay (34 samples exclusively positive by real-time PCR) (PCR was 48% more sensitive than COBAS).Two quantitative CMV assays with different gene targets were developed. The duplex real-time PCR system had a higher sensitivity than the COBAS Amplicor Monitor test system and the linear measure level was at least 3 orders of magnitude higher.
Kalpoe et al. 2004 (205)409 specimens from solid organ transplant (SOT) and stem cell transplant (SCT) patients; 295 corresponding whole-blood samples were selected to address the correlation between CMV DNA loads in plasma and whole bloodImmediate-early antigen geneClinical samples were spiked with a fixed amount of phocine herpesvirus DNA as an internal control.CMV DNA copies/ml plasmaBased on a comparison with the pp65 antigen assay, quantification of CMV DNA in plasma appeared to be capable of guiding the clinical management of transplant patients.CMV DNA load in whole blood tends to be slightly but not significantly higher than that in plasma. A CMV DNA level in plasma of 10,000 copies/ml provided a threshold of 81% sensitivity and 90% specificity for initiating treating in transplant patients (SOT and SCT) at risk for CMV disease.
  • a Abbreviations: IE, immediate-early; RBC, red blood cells; WBC, white blood cells; IL, interleukin; HHV, human herpevirus; MIE, major immediate-early; LC-PCR, Light Cycler PCR.