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

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 200,000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200,000 leukocytes, was equivalent to 3.4 log 10 genome copies in 200 l 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.Human cytomegalovirus (HCMV) is the most important opportunistic pathogen in immune-suppressed patients, especially those who have received solid organ or bone marrow transplants. While antiviral treatment reduces the mortality and morbidity rates, the development of sensitive, specific techniques for detecting HCMV infection has now made possible preemptive treatment.A wide variety of techniques have been developed, including both nonmolecular and molecular tests. Because of its great sensitivity, qualitative PCR was the first method used (8, 18), but this method cannot differentiate between infection and disease in transplants. In contrast, quantitative PCR methods have shown that immune-suppressed patients with a high virus load are at great risk of developing HCMV disease (3,9,16,(19)(20)(21). PCR tests developed in-house and the first commercially available tests are time consuming and labor intensive because they require a post-PCR detection step. The development of automated real-time PCR instruments that can quantify DNA has led to a significant change in the routine diagnosis of CMV infection (6, 10, 17).The virus loads in several blood compartments, such as peripheral blood leukocytes (PBL) and plasma, have been compared to pp65 antigenemia (4, 13). Virus was most frequently detected in the PBL, and the virus loads were significantly higher than in the plasma (1, 2, 12). More recently, real-time quantitative PCR using the Taqman PCR or Light Cycler PCR methods have been used to measure virus DNA in various blood compartments of immune-suppressed patients, and the results were compared with pp65 antigenemia (6, 7, 10) or other PCR techniques (14). One study fou...

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre‐emptive therapy

“…The threshold of 4 log 10 copies/ml has been proposed in the setting of SCT for initiation of pre-emptive therapy for CMV infections [Verkruyse et al, 2006], and thus corresponds to the threshold used in this study (3.6 log 10 copies/10 6 PBLs). The equivalence observed between the two ways of expressing viral load is in accordance with published studies on CMV viral loads in the setting of solid organ transplantation [Mengelle et al, 2003b;Garrigue et al, 2006]. In four leucopenic allogeneic stem cell transplanted patients, cellular normalization of CMV DNA loads permitted to initiate pre-emptive therapy earlier than if it had been expressed per ml of whole blood.…”

Prediction of Cytomegalovirus (CMV) Plasma Load from Evaluation of CMV Whole-Blood Load in Samples from Renal Transplant Recipients

“…Boeckh et al (3) concluded that even though the sensitivity of plasma PCR was significantly lower than that of PBL PCR, plasma PCR could be particularly useful when leukocyte counts were inadequate for the performance of cell-based assays. For others, the higher sensitivity of WB and its higher yield of CMV DNA make it an optimal sample for monitoring the CMV DNA load during CMV disease in immunocompromised patients (7,10,18,29). Moreover, the suitability of WB for bone marrow transplant recipients, who are often in aplasia or leukopenic, has been shown (18).…”

“…The question of which type of blood fraction (PBL, plasma, or whole blood [WB]) is best for monitoring CMV DNA in blood is still unresolved and may be context specific. CMV is highly cell associated, and viral loads (VL) have been shown to be higher in PBL and WB than in plasma (1,7,9,18,29). Plasma viral load monitoring is of modest clinical utility for prediction of CMV disease and delays the detection of CMV DNA, since a negative PCR result for CMV in plasma does not rule out active infection (2,4,5,12,24,26).…”

“…For others, the higher sensitivity of WB and its higher yield of CMV DNA make it an optimal sample for monitoring the CMV DNA load during CMV disease in immunocompromised patients (7,10,18,29). Moreover, the suitability of WB for bone marrow transplant recipients, who are often in aplasia or leukopenic, has been shown (18). However, if the WB assay is chosen as the only test for the monitoring of transplant recipients in routine care management, the information on early active replication provided by the plasma viral load could be missed.…”

Severe Steroid-resistant Thrombocytopenia Secondary to Cytomegalovirus Infection in an Immunocompetent Adult

“…However the high CMV-DNA level, as assessed by PCR assay of whole blood, was still sustained at that point, and we confirmed its improvement after 10 days. Although several studies have demonstrated that whole blood detection of CMV-DNA improves sensitivity compared with plasma detection of that (26)(27)(28). Very recently, a large prospective study comparing the use of whole blood versus plasma in monitoring therapeutic response to CMV infection revealed that enhanced detection of viremia using a whole blood real-time PCR does not necessarily predict recurrence of CMV disease (29).…”