Quantitative Analysis of HCMV DNA Load in Whole Blood of Renal Transplant Patients Using Real-Time PCR Assay

J Clin Virol. 2004 Mar;29(3):194-201. doi: 10.1016/S1386-6532(03)00124-0.

Abstract

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.

Publication types

  • Evaluation Study

MeSH terms

  • Cytomegalovirus / growth & development
  • Cytomegalovirus / isolation & purification*
  • Cytomegalovirus Infections / microbiology*
  • DNA, Viral / blood*
  • DNA, Viral / isolation & purification
  • Humans
  • Kidney Transplantation / adverse effects*
  • Phosphoproteins / blood
  • Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Viral Load*
  • Viral Matrix Proteins / blood

Substances

  • DNA, Viral
  • Phosphoproteins
  • Viral Matrix Proteins
  • cytomegalovirus matrix protein 65kDa