In 2025 Ontario experienced a large resurgent measles outbreak. Investigators implemented wastewater surveillance (WS) concurrently with clinical surveillance in southwestern Ontario to evaluate detection of measles virus (MeV) during sustained community transmission. WS detected MeV RNA that was positively associated with clinical cases by epidemiologic week but did not provide an earlier alert at that resolution. MeV RNA was present across wastewater fractions but concentrated in solids. A multiplex assay adapted to differentiate vaccine and wild-type genotypes identified vaccine-genotype MeV in wastewater following a targeted vaccination campaign. The outbreak provided an opportunity for WS method development in real time.
WS expanded in use during the COVID-19 pandemic as an unbiased tool to monitor community infections and pathogen variants. The method has since been applied to other respiratory and emerging pathogens and for tracking disease burden beyond reliance on clinical testing. Public health concerns in 2025 included decreased routine immunization coverage and the resulting risk of outbreaks of vaccine-preventable diseases such as measles. In 2025, Canada reported 5,461 measles cases nationwide, with about 2,400 confirmed cases in Ontario; after 12 months of sustained transmission, Canada lost measles elimination status in November 2025. Southwestern Ontario experienced the highest caseload in the country, motivating local WS to complement overwhelmed clinical surveillance resources.
Composite 24-hour wastewater influent samples were collected three times per week from the Leamington Pollution Control Centre (LPCC) in the Windsor-Essex region during February–November 2025. The LPCC serves the urban center of Leamington and accepts septage from surrounding rural residential and congregate facilities, representing an approximate population of 30,000.
Passive sampling targeted sewer laterals draining Windsor Regional Hospital (WRH). Passive samplers (tampons) were deployed one to two times per week and submerged in the hospital effluent stream for 17–24 hours before retrieval and immediate laboratory processing.
For wastewater influent, concentration methods included filtration or affinity capture; hospital effluent samples were concentrated by centrifugation. After concentration, total nucleic acids were extracted and viral concentrations measured by quantitative reverse transcription PCR (qRT-PCR). The qRT-PCR assay targeted the measles nucleoprotein (N) gene for quantification. A separate assay measured pepper mild mottle virus for process control. Sequencing was used to validate the identity of obtained N gene amplicons.
Details on assay limits of detection, exact qRT-PCR conditions, and quantitative calibration curves were provided in the study appendix; the main text notes these methods but the excerpted source did not reproduce full protocol parameters.
To investigate partitioning, a 4-L postgrit sample from LPCC was processed to separate solids, colloids, and supernatant. Fractionation used Amicon Ultra 10K filters and operational workflows to derive these components. Partitioning calculations were carried out under three assumptions: a whole-sample basis (accounting for wastewater composition), an equal-mass basis, and an operational-volume basis reflecting commonly used laboratory processing volumes. These approaches allowed assessment of how MeV RNA distributes across physical fractions and how operational choices influence measured concentrations.
Raw influent samples were shipped weekly to the National Microbiology Laboratory for supplementary analysis. Samples were processed with minor modifications to established protocols. Investigators adapted a multiplex assay originally developed for clinical detection to distinguish wild-type and vaccine genotype MeV in wastewater. The adapted assay was applied during the outbreak and used to assess the presence of vaccine-derived sequences in environmental samples.
Some specific assay design elements and performance metrics were described in supplementary materials; the provided source excerpt ends before full assay performance data are detailed.
MeV RNA was detected in LPCC influent during the outbreak period and the wastewater signal correlated positively with clinical case counts when data were summarized by epidemiologic week. However, at the weekly resolution used, WS did not provide a leading indicator that preceded increases or decreases in clinically reported incidence. The study emphasizes that WS captured community-level infections, including those who might not seek clinical testing, and therefore complements clinical surveillance data.
Partitioning experiments indicated MeV RNA was present broadly in the liquid phase but was most concentrated in the solids fraction. These findings have implications for sampling and concentration method selection because solids-enriched approaches may yield higher recovery of MeV RNA.
The team also sampled WRH sewer laterals with passive samplers to estimate MeV shedding into wastewater from a known clinical source. They used these data to estimate shedding rates; however, explicit numeric shedding estimates and methodological subtleties for those calculations were not fully reported in the excerpt provided.
This resurgent measles outbreak provided an opportunity to develop WS methods for MeV in a semirural setting and to evaluate operational considerations: the distribution of viral RNA across wastewater fractions, the need for solid-focused concentration to increase sensitivity, and the capacity to detect vaccine genotype sequences after targeted vaccination campaigns. The positive correlation between wastewater MeV signal and clinical cases supports WS as a complementary population-level surveillance tool, particularly where clinical testing is incomplete or delayed.
Limitations noted in the study include challenges that affect broader WS adoption: variable shedding dynamics among infected persons, partitioning behavior that affects recoveries, detection sensitivity related to assay and concentration methods, and the importance of proximity of cases to monitored sewersheds. Some methodological details and quantitative performance metrics are available in the article appendix; the excerpt provided to this summary did not include all protocol specifics or complete numeric results for shedding estimates.
WS detected MeV RNA during the 2025 Ontario outbreak and mirrored epidemiologic trends when aggregated by epidemiologic week. Solids were an important reservoir of MeV RNA in wastewater, and assays adapted to differentiate vaccine and wild-type genotypes could identify vaccine-derived signals following vaccination campaigns. The outbreak functioned as a real-world case study for WS method development for measles, highlighting both the utility of WS as a complement to clinical surveillance and the technical and interpretive challenges that remain.