Feasibility of Monkeypox virus sequencing from antigen rapid diagnostic tests as a potential tool to enhance genomic surveillance
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Background
Sequencing of monkeypox virus (MPXV) from antigen rapid diagnostic tests (Ag-RDTs) could expand genomic surveillance during outbreaks in decentralized settings where sequencing equipment and cold chain transportation are unavailable. We aimed to evaluate the efficacy of MPXV sequencing from MPXV antigen Ag-RDTs.
Methods
We tested MPXV Ag-RDTs from three different brands using serial dilutions of cultured MPXV subclade Ib. Positive Ag-RDTs with different intensities of the test band were stored for 19 days, either at room temperature or at +4°C, after which viral DNA was extracted from the pads of the test cassettes. Metagenomic and tiled amplicon-based Oxford Nanopore technology sequencing methods were then performed.
Results
Viral DNA extraction from MPXV Ag-RDTs showed a consistent decrease in viral load of 3 logs compared to the initial viral load of the applied viral dilution. Both sequencing methods were able to reach high coverage but the tiled amplicon-based demonstrated more consistent results with a coverage always above 85%.
Conclusion
This proof-of-concept supports the development of this approach in the field, with the aim of combining genomic surveillance with decentralized testing, including in remote areas.
Importance statement
As monkeypox virus (MPXV) continues to expand its circulation, genomic surveillance of MPXV is essential to monitor viral evolution, detect emerging variants, guide control measures and adapt public health responses. However, diagnostic and sequencing capacity is limited in many regions where mpox is endemic, and transporting clinical samples from remote testing sites to reference laboratories remains logistically challenging. Rapid antigen diagnostic tests (Ag-RDTs) now have the potential to be increasingly used for decentralized mpox testing and could represent an alternative source of viral genetic material for sequencing. In this study, we demonstrate that MPXV DNA can be recovered from used Ag-RDT devices and successfully sequenced using Oxford Nanopore technologies, even after 19 days of storage at room temperature. Importantly, in almost all tested conditions, genome coverage was sufficient for viral subclade identification and mutation analysis. These findings highlight the potential of Ag-RDTs not only as diagnostic tools but also as practical sampling devices for genomic surveillance, which could significantly expand sequencing capacity in resource-limited and remote settings.
