title: Analysis of a quantitative PCR assay for CMV infection in liver transplant recipients: an intent to find the optimal cut-off value.
abstract: BACKGROUND
  Preemptive therapy required highly predictive tests for CMV disease. CMV antigenemia assay (pp65 Ag) has been commonly used for rapid diagnosis of CMV infection. Amplification methods for early detection of CMV DNA are under analysis.
  
  
  OBJECTIVE
  To compare two diagnostic methods for CMV infection and disease in this population: quantitative PCR (qPCR) performed in two different samples, plasma and leukocytes (PMNs) and using a commercial diagnostic test (COBAS Amplicor Monitor Test) versus pp65 Ag.
  
  
  METHODS
  Prospective study conducted in liver transplant recipients from February 2000 to February 2001.
  
  
  RESULTS
  Analyses were performed on 164 samples collected weekly during early post-transplant period from 33 patients. Agreements higher than 78% were observed between the three assays. Optimal qPCR cut-off values were calculated using ROC curves for two specific antigenemia values. For antigenemia >or=10 positive cells, the optimal cut-off value for qPCR in plasma was 1330 copies/ml, with a sensitivity (S) of 58% and a specificity (E) of 98% and the optimal cut-off value for qPCR-cells was 713 copies/5x10(6) cells (S:91.7% and E:86%). Using a threshold of antigenemia >or=20 positive cells, the optimal cut-off values were 1330 copies/ml for qPCR-plasma (S 87%; E 98%) and 4755 copies/5x10(6) cells for qPCR-cells (S 87.5%; E 98%). Prediction values for the three assays were calculated in patients with CMV disease (9 pts; 27%). Considering the assays in a qualitative way, the most sensitive was CMV PCR in cells (S: 100%, E: 54%, PPV: 40%; NPV: 100%). Using specific cut-off values for disease detection the sensitivity, specificity, PPV and NPV for antigenemia >or=10 positive cells were: 89%; 83%; 67%; 95%, respectively. For qPCR-cells >or=713 copies/5x10(6) cells: 100%; 54%; 33% and 100% and for plasma-qPCR>or=1330 copies/ml: 78%, 77%, 47%, 89% respectively.
  
  
  CONCLUSIONS
  Optimal cut-off for viral load performed in plasma and cells can be obtained for the breakpoint antigenemia value recommended for initiating preemptive therapy with high specificities and sensitivities. Diagnostic assays like CMV pp65 Ag and quantitative PCR for CMV have similar efficiency and could be recommended as methods of choice for diagnosis and monitoring of active CMV infection after transplantation.
authors.affiliation: Department of Infectious Diseases, Hospital Ramón y Cajal, Madrid, Spain. pmdavila@terra.es
authors.forename: P
authors.initials: P
authors.lastname: Martín-Dávila
authors.forename: J
authors.initials: J
authors.lastname: Fortún
authors.forename: C
authors.initials: C
authors.lastname: Gutiérrez
authors.forename: P
authors.initials: P
authors.lastname: Martí-Belda
authors.forename: A
authors.initials: A
authors.lastname: Candelas
authors.forename: A
authors.initials: A
authors.lastname: Honrubia
authors.forename: R
authors.initials: R
authors.lastname: Barcena
authors.forename: A
authors.initials: A
authors.lastname: Martínez
authors.forename: A
authors.initials: A
authors.lastname: Puente
authors.forename: E
authors.initials: E
authors.lastname: de Vicente
authors.forename: S
authors.initials: S
authors.lastname: Moreno
mesh_terms: D000956:Antigens, Viral / Q000097:blood; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D003586:Cytomegalovirus Infections / Q000175:diagnosis*; D004279:DNA, Viral / Q000097:blood; D006801:Humans; D007962:Leukocytes / Q000821:virology; D016031:Liver Transplantation; D010949:Plasma / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D013030:Spain; D019562:Viral Load; D014779:Virus Replication
chemical_list: D000956:Antigens, Viral; D004279:DNA, Viral