Abstract
Background and Objectives: Herpes simplex virus (HSV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV) infections pose significant challenges in managing transplant patients and necessitate rapid and precise diagnostic methods due to their immunosuppressed state. This study designed and evaluated the performance of an in-house multiplex real-time PCR for simultaneous detection of these viruses. Materials and Methods: Plasma samples from 270 transplant patients were tested using an in-house multiplex real-time PCR assay specifically designed for HSV, VZV, and EBV. Analytical specificity and the assay's limit of detection (LOD) were determined. Statistical analyses were performed to evaluate the agreement between the in-house assay and the reference kit. Results: The method had a specificity of 98% for HSV, 97% for VZV, and 95% for EBV, alongside 100% sensitivity for all three viruses. No cross-reactivity was observed with other viral or bacterial DNA. The LOD for the in-house assay was determined to be 6.25, 25, and 25 copies/mL for HSV, VZV, and EBV, respectively. Additionally, precision analysis showed low CV values in both intra-assay and interassay evaluations (HSV: 1.5%-1.8%; VZV: 2.3%-2.6%; and EBV: 3.7%-3.9%), confirming the assay's robust analytical precision. Bland-Altman analysis showed mean differences of 1.35, -3.29, and 1.75 for HSV, VZV, and EBV, respectively. This multiplex real-time PCR method enables detection at lower concentrations. Cross-reactivity testing confirmed no interaction with DNA from other viruses or nontarget microorganisms. Bland-Altman and linear regression analyses also showed a strong agreement between commercial and in-house methods. Conclusion: These findings, compared to Altona diagnostic kits, highlight the value of designing and applying advanced diagnostic assays in managing viral infections in transplant patients.