Wastewater surveillance of vaccine-preventable diseases may provide early warning of outbreaks and identify areas to target for immunization. To advance wastewater monitoring of measles, mumps, and rubella viruses, we developed and validated a multiplexed RT-ddPCR assay for the detection of their RNA. Because the measles-mumps-rubella (MMR) vaccine is an attenuated live virus vaccine, we also developed an assay that distinguishes between wild-type and vaccine strains of measles in wastewater and validated it using a wastewater sample collected from a facility with an active measles outbreak. We also evaluated the partitioning behavior the viruses in between the liquid and solid fractions of influent wastewater. We found that assaying the liquid fraction of the wastewater resulted in more sensitive detection of the viruses despite the fact that the viral RNA was enriched in the solid fraction due to the low solids content of the influent wastewater. Finally, we investigated the stability of measles, mumps, and rubella RNA in wastewater samples spiked with viruses over 28 days at two different concentrations and two temperatures (4C and room temperature) and observed limited viral decay. Our study supports the feasibility of wastewater monitoring for measles, mumps, and rubella viruses for population-level surveillance.

The authors have declared no competing interest.

This study was funded by the Centers of Disease Control and Prevention (NI50CK000557) and the Houston Health Department.

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All data produced in the present study are available upon reasonable request to the authors and the sequencing data has been submitted to NCBI.