Detection of cytomegalovirus DNA in dried urine on filter paper by polymerase chain reaction for the diagnosis of congenital cytomegalovirus infection: Method validation

Objective: This study aimed to contribute to national screening programs by developing a standardized and validated method based on the evaluation of dried urine samples on filter paper using the polymerase chain reaction (PCR) technique for the diagnosis of congenital cytomegalovirus (cCMV) infection. Method: The urine of a newborn diagnosed with congenital CMV infection was used as the CMV DNA–positive sample, and Guthrie cards were used as the filter paper. Serial dilutions (10⁴, 10³, 10² copies/mL) were prepared from urine containing 10⁵ copies/mL CMV DNA, spotted onto filter papers, dried, and stored under standard laboratory conditions. DNA extraction was performed on predetermined days (1st, 2nd, 4th, 8th, and 16th days) following drying, and subsequently, PCR analysis was conducted. Data were evaluated through a confusion matrix, and diagnostic performance parameters such as accuracy, sensitivity, specificity, precision, and ROC analysis were calculated. Results: The method demonstrated high analytical performance. A 100% positivity rate was obtained at 10³, 10⁴, and 10⁵ copies/mL concentrations, whereas a lower positivity rate was observed at 10² copies/mL. The test achieved 100% accuracy, sensitivity, specificity, and precision. ROC analysis revealed an AUC value of 1.0, indicating perfect discrimination between positive and negative groups. The optimal cut-off value was determined as 10³ copies/mL. Consistent results across different test days confirmed the high stability of the dried urine samples. Conclusion: The analysis of dried urine samples on filter paper using the PCR method demonstrated high accuracy and practicality in the diagnosis of congenital CMV infection. The method provides advantages in terms of sample collection, stability, and portability, making it applicable under both laboratory and field conditions. Furthermore, it may contribute to the prevention of CMV-related complications when integrated into neonatal screening programs. Key words: Cytomegalovirus (CMV), congenital CMV diagnosis, polymerase chain reaction (PCR), dried urine on filter paper, validation