Implementation and clinical evaluation of an Mpox virus laboratory-developed test on a fully automated random-access...

While Mpox virus (MPXV) diagnostics were performed in specialized laboratories only, the global emergence of Mpox cases in 2022 revealed the need for a more readily available diagnostic. Automated random-access platforms with fast nucleic acid extraction and PCR have become established in many laboratories, providing faster and more accessible testing. In this study, we adapted a previously published generic MPXV-PCR as a lab-developed test (LDT) on a NeuMoDx Molecular System and isolated MPXV clones from patient materials. To reduce the handling of infectious material, we evaluated a viral lysis buffer (VLB) for sample pretreatment. We further compared the MPXV-LDT-PCR to conventional real-time PCR, determined its sensitivity and specificity using positive swabs, and assessed its performance using external quality assessment samples. Pretreatment of samples with 50% VLB reduced MPXV infectivity by approximately 200-fold while maintaining PCR sensitivity. The assay demonstrated a sensitivity and specificity of 100% with no cross-reactivity in the samples tested and performed with a limit of detection of 262 GE/mL. In summary, the assay had a turnaround time of fewer than 2 h and can easily be transferred to other automated PCR platforms, providing a basis for developing rapid assays for upcoming pandemics.