Simultaneous Detection of Monkeypox Virus and Varicella Zoster Virus Using a Multiplex qPCR Assay: Analytical Performance and Clinical Validation

Abstract

Objective: Monkeypox virus (MPXV) and varicella-zoster virus (VZV) cause clinically similar vesicular pustular rashes and systemic symptoms, often complicating the diagnosis. The 2022 multi-countries MPXV outbreak highlighted the importance of accurate diagnostics, while VZV continues to represent a global health burden. Misdiagnosis can delay treatment, hinder outbreak control, and trigger unnecessary public health measures. Most commercial polymerase chain reaction (PCR) kits detect only a single pathogen, which increases the cost and turnaround time when differential diagnosis is required. Materials and Methods: We developed a multiplex quantitative PCR (qPCR) assay targeting the F3L gene of MPXV and the ORF4 gene of VZV, incorporating human cyclophilin A (CYPA) gene as an internal control to monitor sample integrity and amplification efficiency. A total of 15 VZV samples (6 positive, 9 negative) and 6 MPXV samples (1 positive, 5 negative) were analyzed. Analytical performance was evaluated using ten-fold serial dilutions of viral DNA controls, whereas clinical validation was performed on vesicle swab samples previously confirmed by the QIAstat-Dx viral vesicular panel (Qiagen, Germany). Results: The assay demonstrated high efficiency (0.93 for MPXV, 1.01 for VZV) and strong linearity (R²=0.999 and 0.984, respectively). The 95% limits of detection were 39.37 copies/reaction for MPXV and 30.61 copies/reaction for VZV. Comparison with the QIAstat-Dx panel showed 100% concordance, confirming the accuracy and reliability. Conclusion: This multiplex qPCR assay provides sensitive, specific, and cost-effective simultaneous detection of MPXV and VZV. Its scalability and rapid turnaround time support the timely differential diagnosis of vesicular rash illnesses, particularly in outbreak-prone or resource-limited settings.

Keywords

• MPXV
• VZV
• Multiplex qPCR
• Simultaneous Detection

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