Detection of Mpox Virus Using Microbial Cell-Free DNA: The Potential of Pathogen-Agnostic Sequencing for Rapid Identification of Emerging Pathogens

Background. The 2022 global outbreak of Monkeypox virus (MPXV) highlighted challenges with polymerase chain reaction detection as divergent strains emerged and atypical presentations limited the applicability of swab sampling. Recommended testing in the United States requires a swab of lesions, which arise late in infection and may be unrecognized. We present MPXV detections using plasma microbial cell-free DNA (mcfDNA) sequencing. Methods. Fifteen plasma samples from 12 case-patients were characterized through mcfDNA sequencing. Assay performance was confirmed through in silico inclusivity and exclusivity assessments. MPXV isolates were genotyped using mcfDNA, and phylodynamic information was imputed using publicly available sequences. Results. MPXV mcfDNA was detected in 12 case-patients. Mpox was not suspected in 5, with 1 having documented resolution of mpox >6 months previously. Six had moderate to severe mpox, supported by high MPXV mcfDNA concentrations; 4 died. In 7 case-patients, mcfDNA sequencing detected coinfections. Genotyping by mcfDNA sequencing identified 22 MPXV mutations at 10 genomic loci in 9 case-patients. Consistent with variation observed in the 2022 outbreak, 21 of 22 variants were G > A/C > T. Phylogenetic analyses imputed isolates to sublineages arising at different time points and from different geographic locations. Conclusions. We demonstrate the potential of plasma mcfDNA sequencing to detect, quantify, and, for acute infections with high sequencing coverage, subtype MPXV using a single noninvasive test. Sequencing plasma mcfDNA may augment existing mpox testing in vulnerable patient populations or in patients with atypical symptoms or unrecognized mpox. Strain type information may supplement disease surveillance and facilitate tracking emerging pathogens.