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Journal of Clinical Microbiology, August 2003, p. 3840-3845, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3840-3845.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Automated Extraction and Quantification of Human Cytomegalovirus DNA in Whole Blood by Real-Time PCR Assay

C. Mengelle,1* K. Sandres-Sauné,1 C. Pasquier,1 L. Rostaing,2 J.-M. Mansuy,1 M. Marty,1 I. Da Silva,1 M. Attal,3 P. Massip,4 and J. Izopet1

Laboratoire de Virologie,1 Unité de Greffe de Moelle,3 Service de Maladies Infectieuses et Tropicales, Hôpital Purpan,4 Unité de Transplantation d'Organes, Hôpital Rangueil, CHU Toulouse, Toulouse, France2

Received 8 November 2002/ Returned for modification 13 January 2003/ Accepted 6 April 2003

The measurement of human cytomegalovirus (HCMV) DNA in blood is becoming the standard method for monitoring HCMV infection in immune-suppressed and unsuppressed patients. As various blood compartments can be used, we have compared the HCMV DNA measured in whole blood (WB), peripheral blood leukocytes (PBL), and plasma by real-time PCR. We tested 286 samples: HCMV DNA was extracted automatically from WB and PBL with the MagNA Pure instrument (Roche Molecular Biochemicals) and manually from plasma samples. The HCMV DNA from WB, PBL, and plasma was measured by real-time Light Cycler PCR. Primers and probe were located in the UL 83 region. HCMV DNA was detected more frequently in WB (88.5%) than in the PBL (65.7%) (P < 0.0001) or the plasma (55.2%) (P < 0.0001). There was a good correlation between the positive results in WB and in PBL (r = 0.68; P < 0.0001), and 3.15 log10 genome copies in 200,000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200,000 leukocytes, was equivalent to 3.4 log10 genome copies in 200 µl of WB. WB was shown to be suitable for automated extraction and the quantitation of HCMV DNA by real-time Light Cycler PCR by analysis of serial samples from representative patients of various populations. This system may be very useful for monitoring of immune-suppressed and unsuppressed patients.

* Corresponding author. Mailing address: Laboratoire de Virologie, Hôpital Purpan, CHU Toulouse, TSA 40031, 31059 Toulouse Cedex 9, France. Phone: 33 5 61 77 21 13. Fax: 33 5 61 77 25 42. E-mail: mengelle.c{at}chu-toulouse.fr.

Journal of Clinical Microbiology, August 2003, p. 3840-3845, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3840-3845.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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