Development and Validation of Three Automated High-Throughput Molecular Tests to Detect Monkeypox Virus Infections

Area of Science:

• Virology
• Infectious Diseases
• Diagnostic Development

Background:

• The 2022 global outbreak of the clade IIb monkeypox virus (MPXV) necessitated rapid development of diagnostic tools.
• Existing diagnostic methods required enhancement for high-throughput, sensitivity, and reproducibility.

Purpose of the Study:

• To develop and validate novel diagnostic assays for monkeypox virus detection.
• To create assays suitable for high-throughput screening during MPX outbreaks.

Main Methods:

• Developed three monkeypox virus detection assays: two for the m2000 RealTime system (MPXV+ and MPXV) and one for the Alinity m system (MPXV).
• Incorporated dual targets (E9L/B6R for MPXV+ and J2L/B7R for MPXV) to enhance resistance to viral mutations.
• Utilized in silico predictions to assess cross-reactivity and specificity.

Main Results:

• The m2000 RealTime MPXV+ and MPXV assays demonstrated a sensitivity of 3.2 plaque-forming units/mL.
• The Alinity m MPXV assay achieved a lower limit of detection of 200 copies/mL.
• Assays were validated using lesion swabs and saliva mixtures in universal transport medium (UTM), showing high agreement with known MPX-positive specimens.

Conclusions:

• The developed RealTime MPXV+, RealTime MPXV, and Alinity m MPXV assays are high-throughput and sensitive for monkeypox virus detection.
• These assays are valuable tools for managing and responding to MPX outbreaks.