Enhanced detection of mpox virus in wastewater using a pre-amplification approach: A pilot study informing population-level monitoring of low-titer pathogens

A recent outbreak of the mpox virus (MPXV) occurred in non-endemic regions of the world beginning in May 2022. Pathogen surveillance systems faced pressure to quickly establish response protocols, offering an opportunity to employ wastewater-based epidemiology (WBE) for population-level monitoring. The pilot study reported herein aimed to: (i) develop a reliable protocol for MPXV DNA detection in wastewater which would reduce false negative reporting, (ii) test this protocol on wastewater from various regions across the United States, and (iii) conduct a state of the science review of the current literature reporting on experimental methods for MPXV detection using WBE. Twenty-four-hour composite samples of untreated municipal wastewater were collected from the states of New Jersey, Georgia, Illinois, Texas, Arizona, and Washington beginning July 3rd, 2022 through October 16th, 2022 (n = 60). Samples underwent vacuum filtration, DNA extraction from captured solids, MPXV DNA pre-amplification, and qPCR analysis. Of the 60 samples analyzed, a total of eight (13%) tested positive for MPXV in the states of Washington, Texas, New Jersey, and Illinois. The presence of clade IIb MPXV DNA in these samples was confirmed via Sanger sequencing and integration of pre-amplification prior to qPCR decreased the rate of false negative detections by 87% as compared to qPCR analysis alone. Wastewater-derived detections of MPXV were compared to clinical datasets, with 50% of detections occurring as clinical cases were increasing/peaking and 50% occurring as clinical cases waned. Results from the literature review (n = 9 studies) revealed successful strategies for the detection of MPXV DNA in wastewater, however also emphasized a need for further method optimization and standardization. Overall, this work highlights the use of pre-amplification prior to qPCR detection as a means to capture the presence of MPXV DNA in community wastewater and offers guidance for monitoring low-titer pathogens via WBE.