Development and analytical validation of a multiplex qPCR assay for Toxoplasma gondii, Treponema pallidum, cytomegalovirus, and herpes simplex virus types 1 and 2 in neonates.

BACKGROUND: Congenital and perinatal infections caused by pathogens such as Toxoplasma gondii (T. gondii), Treponema pallidum (T. pallidum), cytomegalovirus (CMV), and herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are significant causes of neonatal morbidity. Traditional diagnostic methods, such as serological tests or viral cultures, are frequently limited by cross-reactivity, variable accuracy, and prolonged turnaround times. Therefore, this study aimed to design and analytically validate a rapid, highly sensitive, and specific multiplex quantitative real-time polymerase chain reaction (qPCR) assay for the detection of these critical neonatal pathogens. METHODS: The multiplex qPCR assay was optimized, and analytical performance, including sensitivity, specificity, and efficiency, was evaluated following Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. RESULTS: The assay demonstrated a limit of detection (LOD) of 10 attograms per microliter (ag/µL) for T. gondii, T. pallidum, and CMV, and 1 ag/µL for herpes simplex virus (HSV)-1 and HSV-2. Additionally, the assay showed 100% analytical specificity against 38 organisms. The PCR efficiency exhibited ranged between 95% and 109% for each organism, with a coefficient of variation (CV) of less than 3%. CONCLUSION: This multiplex qPCR assay provides highly sensitive and specific analytical performance for detecting key congenital pathogens. It represents a promising high-throughput tool with the potential to improve the turnaround time of neonatal molecular screening.