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. 2003 Aug;41(8):3840-5.
doi: 10.1128/JCM.41.8.3840-3845.2003.

Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay

C Mengelle  1 , K Sandres-Sauné, C Pasquier, L Rostaing, J-M Mansuy, M Marty, I Da Silva, M Attal, P Massip, J Izopet
Affiliations

Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay

C Mengelle et al. J Clin Microbiol. 2003 Aug.

Abstract

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 log(10) genome copies in 200000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200000 leukocytes, was equivalent to 3.4 log(10) genome copies in 200 microl 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.

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Figures

FIG 1.
FIG 1.
Correlation between quantitative PCR results in WB and in PBL. Results of quantitative PCR show close correlation between HCMV DNA log10 copy numbers in PBL and in WB.
FIG 2.
FIG 2.
Monitoring the HCMV load in WB and the PBL of five solid organ transplant recipients (SOT-1 to SOT-5) by using real-time Light Cycler PCR. The results of serial samples collected from five patients are shown, as are the periods of treatment with intravenous ganciclovir (IV GCV) and Zelitrex (Val ACV). Diamonds show HCMV DNA copy number in WB, and squares show HCMV DNA copy number in PBL. Qualitative results in plasma are also shown.
FIG 2.
FIG 2.
Monitoring the HCMV load in WB and the PBL of five solid organ transplant recipients (SOT-1 to SOT-5) by using real-time Light Cycler PCR. The results of serial samples collected from five patients are shown, as are the periods of treatment with intravenous ganciclovir (IV GCV) and Zelitrex (Val ACV). Diamonds show HCMV DNA copy number in WB, and squares show HCMV DNA copy number in PBL. Qualitative results in plasma are also shown.
FIG 2.
FIG 2.
Monitoring the HCMV load in WB and the PBL of five solid organ transplant recipients (SOT-1 to SOT-5) by using real-time Light Cycler PCR. The results of serial samples collected from five patients are shown, as are the periods of treatment with intravenous ganciclovir (IV GCV) and Zelitrex (Val ACV). Diamonds show HCMV DNA copy number in WB, and squares show HCMV DNA copy number in PBL. Qualitative results in plasma are also shown.
FIG 2.
FIG 2.
Monitoring the HCMV load in WB and the PBL of five solid organ transplant recipients (SOT-1 to SOT-5) by using real-time Light Cycler PCR. The results of serial samples collected from five patients are shown, as are the periods of treatment with intravenous ganciclovir (IV GCV) and Zelitrex (Val ACV). Diamonds show HCMV DNA copy number in WB, and squares show HCMV DNA copy number in PBL. Qualitative results in plasma are also shown.
FIG 2.
FIG 2.
Monitoring the HCMV load in WB and the PBL of five solid organ transplant recipients (SOT-1 to SOT-5) by using real-time Light Cycler PCR. The results of serial samples collected from five patients are shown, as are the periods of treatment with intravenous ganciclovir (IV GCV) and Zelitrex (Val ACV). Diamonds show HCMV DNA copy number in WB, and squares show HCMV DNA copy number in PBL. Qualitative results in plasma are also shown.
FIG 3.
FIG 3.
Monitoring of HCMV load in WB and the PBL of two BMT recipients, BMT-1 (A) and -2 (B), by using real-time Light Cycler PCR. The results of serial samples are shown, as are the periods of intravenous ganciclovir (IV GCV) and Foscavir (IV PFA) treatment. Diamonds shown HCMV DNA copy number in WB, and squares show HCMV DNA copy number in PBL. Qualitative results in plasma are also shown.
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