Optimization of Quantitative Detection of Cytomegalovirus DNA in Plasma by Real-Time PCR
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| Published in | Journal of Clinical Microbiology Vol. 42; no. 3; pp. 1142 - 1148 |
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| Main Authors | , , , , , , |
| Format | Journal Article |
| Language | English |
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Washington, DC
American Society for Microbiology
01.03.2004
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Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples ( P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log 10 . Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation ( R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens. Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens. Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens. Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive- antigenemia negative) could be confirmed at different time points during follow- up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV- seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log sub(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens. |
| Author | Terry Stevens-Ayers MeeiLi Huang James Ferrenberg Michael Boeckh Lawrence Corey W. Garrett Nichols Laurence Stensland |
| AuthorAffiliation | Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109-1024 |
| AuthorAffiliation_xml | – name: Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109-1024 |
| Author_xml | – sequence: 1 givenname: Michael surname: Boeckh fullname: Boeckh, Michael organization: Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109-1024 – sequence: 2 givenname: MeeiLi surname: Huang fullname: Huang, MeeiLi organization: Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109-1024 – sequence: 3 givenname: James surname: Ferrenberg fullname: Ferrenberg, James organization: Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109-1024 – sequence: 4 givenname: Terry surname: Stevens-Ayers fullname: Stevens-Ayers, Terry organization: Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109-1024 – sequence: 5 givenname: Laurence surname: Stensland fullname: Stensland, Laurence organization: Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109-1024 – sequence: 6 givenname: W. Garrett surname: Nichols fullname: Nichols, W. Garrett organization: Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109-1024 – sequence: 7 givenname: Lawrence surname: Corey fullname: Corey, Lawrence organization: Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109-1024 |
| BackLink | http://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=15595196$$DView record in Pascal Francis https://www.ncbi.nlm.nih.gov/pubmed/15004066$$D View this record in MEDLINE/PubMed |
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| ContentType | Journal Article |
| Copyright | 2004 INIST-CNRS Copyright © 2004, American Society for Microbiology 2004 |
| Copyright_xml | – notice: 2004 INIST-CNRS – notice: Copyright © 2004, American Society for Microbiology 2004 |
| DBID | AAYXX CITATION IQODW CGR CUY CVF ECM EIF NPM 7U9 H94 7X8 5PM |
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| Keywords | Virus Polymerase chain reaction Cytomegalovirus Microbiology Herpesviridae Detection Real time Betaherpesvirinae Optimization Quantitative analysis |
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| Notes | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, Program in Infectious Diseases, 1100 Fairview Ave., N., P.O. Box 19024, Seattle, WA 98109-1024. Phone: (206) 667-6702. Fax: (206) 667-4411. E-mail: mboeckh@FHCRC.org. |
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Mendeley... Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood.... |
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| SubjectTerms | Antigens, Viral - blood Antigens, Viral - genetics Antiviral Agents - therapeutic use Base Sequence Biological and medical sciences Bronchoalveolar Lavage Fluid - virology Cytomegalovirus - genetics Cytomegalovirus - isolation & purification Cytomegalovirus Infections - diagnosis Cytomegalovirus Infections - drug therapy Cytomegalovirus Infections - etiology DNA Primers DNA, Viral - blood DNA, Viral - genetics DNA, Viral - isolation & purification Enzyme-Linked Immunosorbent Assay Fundamental and applied biological sciences. Psychology Human cytomegalovirus Humans Infectious diseases Medical sciences Microbiology Miscellaneous Phosphoproteins - blood Phosphoproteins - genetics Polymerase Chain Reaction - methods Predictive Value of Tests Reproducibility of Results Sensitivity and Specificity Stem Cell Transplantation - adverse effects Viral Load Viral Matrix Proteins - blood Viral Matrix Proteins - genetics Virology |
| Title | Optimization of Quantitative Detection of Cytomegalovirus DNA in Plasma by Real-Time PCR |
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