Analytical Performance and Inter-Method Agreement of a Laboratory-Developed CMV qPCR Assay in Clinical Plasma Samples

Cytomegalovirus (CMV) viral load monitoring forms the basis of preemptive treatment strategies in patients undergoing solid organ and hematopoietic stem cell transplantation. This study aimed to evaluate the analytical performance and inter-method agreement of a laboratory-developed CMV real-time PCR (qPCR) test compared to a commercial reference method using plasma samples.

A total of 100 EDTA plasma samples were analyzed in parallel using a laboratory-developed CMV qPCR test and the reference method (Roche Cobas^® CMV). Analytical sensitivity was determined us-ing synthetic DNA cloned into the pUC57 plasmid backbone containing the US17 region of the CMV genome, and the limit of detection (LoD₉₅) was calculated using probit regression analysis. The relationship between the quantitative results obtained from clinical samples was evaluated using the Spearman rank correlation coefficient, while inter-method clinical agreement was assessed using the Bland-Altman method.

The limit of detection (LoD₉₅) of the laboratory-developed CMV qPCR test, as determined by probit regression analysis, was 63.8 copies/µL. A weak and statistically non-significant correlation was ob-served between the laboratory-developed CMV qPCR test and the reference method in Spearman rank correlation analysis of samples for which numerical quantitative results were available from both methods (ρ = 0.32; p = 0.22; n = 16). Bland-Altman analysis showed a mean difference of -0.48 log₁₀ units, with the vast majority of measurements falling within the 95% limits of agreement.

The assay demonstrated measurable analytical performance and inter-method agreement; however, its use for quantitative viral load monitoring, particularly at low CMV DNA levels, should be interpreted with caution.