Wilson, Jennie

ABSTRACT Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients.

ABSTRACT The availability of microbiologic methods that detect early replication of cytomegalovirus (CMV) posttransplantation will enhance the process of initiating preemptive antiviral therapy prior to the appearance of CMV disease. Using PCR techniques we sought to determine which region of the CMV genome present in peripheral blood leukocytes (PBLs) or serum provides the highest sensitivity for the detection of CMV posttransplantation. Blood samples were prospectively collected weekly for at least 8 weeks from a cohort of 21 consecutive liver transplant recipients not receiving anti-CMV prophylaxis. Results of PCR assays were correlated with recovery of CMV in cell cultures and histopathological findings from biopsy specimens of infected organs to assess clinical symptomatic infection. Of 148 specimens, primer pairs directed to the Hin dIII-X fragment region of CMV detected target DNA with a 94% sensitivity, compared to an 87% sensitivity with primer pairs directed to Eco RI fragment D, 32% sensitivity with primer pairs directed to the immediate-early antigen 1 gene (IEA1 gene), and 20% sensitivity with primer pairs directed to the major immediate-early (MIE) gene. The performance characteristics in terms of the sensitivity of primers for amplifying CMV DNA associated with symptomatic infection ranged from 100% ( Hin dIII-X) to 20% (MIE gene); however, specificity was inversely related ( Hin dIII-X, 45%; MIE gene, 91%) to primers directed to these gene targets. When Hin dIII-X and Eco RI-D primer sets were used, CMV DNA from PBLs was a more sensitive target than CMV DNA from serum for the early detection of symptomatic CMV infection (17 versus 12 days). Importantly, CMV DNA was not detected in five patients with no evidence of this viral infection. In conclusion, primers directed to the Hin dIII-X fragment region were the most optimal for the early detection of CMV DNA in PBLs and sera from symptomatic liver transplant recipients.

ABSTRACT Cytomegalovirus (CMV) DNA quantitation in clinical specimens is progressively becoming a cornerstone in the diagnosis and management of CMV infection in the immunocompromised host. We evaluated two automated and reproducible PCR tests, the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and the COBAS AMPLICOR CMV Monitor (Roche Diagnostics, Pleasanton, Calif.), for the detection of CMV DNA in blood samples from transplant recipients with CMV infection as determined by shell vial culture. Following a log transformation analysis, the mean CMV DNA in plasma (PL), whole blood (WB), peripheral blood leukocytes (PBL), and peripheral blood mononuclear cells (PBMC) using the LightCycler was 6.79 copies per ml, 7.23 copies per ml, 6.38 copies per 2 × 10 6 cells, and 6.27 copies per 2 × 10 6 cells, respectively. This compares to 7.86 copies per ml, 8.37 copies per ml, 7.59 copies per 2 × 10 6 cells, and 7.44 copies per 2 × 10 6 cells, respectively, using COBAS AMPLICOR CMV Monitor. While higher CMV DNA levels were observed for the various blood compartments analyzed using COBAS AMPLICOR CMV Monitor, a high degree of correlation was evident between the two automated systems (jackknife correlation r = PL 0.77 [95% confidence interval (CI); 0.64, 0.90], WB 0.77 [95% CI; 0.62, 0.92], PBL 0.77 [95% CI; 0.67, 0.88], and PBMC 0.81 [95% CI; 0.72, 0.89], all P < 0.001). Therefore, we conclude that either automated diagnostic system is accurate for CMV DNA quantitation.