title: Quantification of cytomegalovirus DNA by a fully automated real-time PCR for early diagnosis and monitoring of active viral infection in solid organ transplant recipients.
abstract: BACKGROUND
  Quantification of cytomegalovirus (CMV) DNA by real-time PCR is currently considered an alternative diagnostic approach for the evaluation of active infection in transplant patients. The pp65 antigenemia assay has been used as reference test for monitoring active CMV infection and guiding preemptive therapy in transplant recipients. However, this assay suffers from some limitations: need for immediate processing of the samples, labour-intensive process, lack of standardization and subjective result interpretation.
  
  
  OBJECTIVE
  The aim of this study was to evaluate the performance of a new commercially available real-time PCR assay coupled with a fully automated DNA extraction system (COBAS Ampliprep/COBAS Taqman CMV Test, Roche Diagnostics) for the detection of CMV-DNA in plasma comparing it with pp65 antigenemia assay for monitoring active CMV infection in solid organ transplant recipients (SOTRs).
  
  
  METHODS
  A total of 266 consecutive samples from 45 SOTRs were monitored with pp65 antigenemia and in parallel with CMV-DNA quantitation by real-time PCR assay.
  
  
  RESULTS
  Fifty-eight samples resulted PCR-positive, 163 negative and for 45 samples the CMV-DNA values obtained were below the lower limit of quantification (<150 copies/ml); pp65 antigen was detected in 47 samples and resulted negative in 219 specimens. Concordance between the two evaluations was 76.7%; also a good correlation was observed (r=0.718). Considering the existing treatment criteria based on pp65 antigenemia evaluation corresponding to pp65 levels≥20 positive cells/200,000, preemptive therapy was administered to four asymptomatically infected patients. The corresponding cut-off value of CMV-DNA load calculated for discrimination between self-clearing infections and those requiring therapy was 2500 copies/ml (or 2275 IU/ml).
  
  
  CONCLUSIONS
  The fully automated real-time PCR from Roche provided specific and sensitive results and represented a rapid and simple assay for the evaluation and monitoring of CMV infection in SOTRs. Further studies are required to validate the threshold level for the initiation of preemptive therapy.
authors.affiliation: Department of Pathology and Diagnostics, Section of Microbiology, University of Verona, Verona, Italy. marzia.boaretti@univr.it
authors.forename: M
authors.initials: M
authors.lastname: Boaretti
authors.forename: A
authors.initials: A
authors.lastname: Sorrentino
authors.forename: C
authors.initials: C
authors.lastname: Zantedeschi
authors.forename: A
authors.initials: A
authors.lastname: Forni
authors.forename: L
authors.initials: L
authors.lastname: Boschiero
authors.forename: R
authors.initials: R
authors.lastname: Fontana
mesh_terms: D000328:Adult; D000368:Aged; D001331:Automation / Q000379:methods; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000821:virology; D004279:DNA, Viral / Q000302:isolation & purification; D042241:Early Diagnosis; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D014180:Transplantation; D019737:Transplants / Q000009:adverse effects; D019562:Viral Load / Q000379:methods*; D055815:Young Adult
chemical_list: D004279:DNA, Viral