Measles outbreaks remain a significant public health challenge, despite high vaccination coverage in many regions. Wastewater-based surveillance (WBS) offers a noninvasive and community-level approach to monitoring the circulation of pathogens, including the measles virus. Here, we retrospectively applied a duplex digital PCR assay to distinguish between wild-type and vaccine strains of the measles virus in wastewater samples available from an existing national WBS program. Samples originated from the wastewater treatment plant serving the Lausanne city catchment area, where an outbreak occurred before spreading to the broader Canton Vaud region. Despite high vaccination rates, viral loads of the measles wild type were detected during the first transmission event involving 21 cases identified within a week. However, viral loads were no longer detectable after the initial 21 cases, despite an additional 30 cases reported in the following 3 weeks, possibly due to lower incidence rate or location outside the catchment. Measles vaccine strain was not detected during the outbreak. Our results demonstrate the complementarity of WBS with clinical surveillance and suggest its potential as an early warning system for measles and other vaccine-preventable diseases. Further improvements in the assay sensitivity and integration with epidemiological data could enhance the utility of WBS for outbreak detection and control.

Measles, mumps, and rubella are highly contagious viral diseases that can cause severe health complications, particularly in children and the elderly. To reduce risks, vaccination with the Measles, Mumps, Rubella (MMR) vaccine is recommended, however outbreaks can still occur in communities, particularly those with lagging vaccination coverage. A recent example occurred from January to March 2024 in Lausanne when a measles outbreak emerged involving 51 identified cases. To help support Switzerland’s strategy to monitor and track these three pathogens, we have developed two duplex digital PCR (dPCR) assays for wastewater screening based on previously published assays. The assays targeting: i) measles wild type (WT) and measles vaccine (VA) strains, and ii) mumps and rubella. In this report, we describe the development and validation of the two dPCR assays. The measles WT/VA assay was further applied retrospectively on wastewater extracts collected during the 2024 outbreak in Lausanne. Our findings showed that wastewater viral loads were temporally associated with clinical case data. The developed assays can be implemented to complement clinical-based surveillance, supporting a rapid response to outbreaks, tracking of relative changes, and helping to inform mitigation strategies.

Poliovirus is the causative agent of poliomyelitis, a potentially debilitating disease that is a target for global eradication. Vaccination against poliomyelitis is an important tool in the eradication effort, but one of the available vaccines, the oral poliovirus vaccine, contains live, attenuated strains that can revert to virulent forms. Community transmission of so-called vaccine-derived polioviruses (VDPVs) can contribute to outbreaks, including in areas with no recently reported poliomyelitis cases. Wastewater-based surveillance (WBS) is a widely applied method to monitor communities for outbreaks through the detection and characterization of VDPVs in wastewater including sewage. Results of WBS can help to target interventions, such as increased vaccination efforts. Switzerland is a Polio-Free country, with no evidence of community circulation of poliomyelitis. However, Polio-Free countries remain at risk of importation of wild poliovirus and VDPVs. This document has two parts: Part I describes the development of a digital PCR pan-poliovirus assay to screen wastewater for the presence of poliovirus. Our pan-poliovirus assay has been sufficiently developed to allow application when required. In Part II, the need to further characterize detected poliovirus through strain-specific differentiation assays is discussed. A literature review of available differentiation assays complements this part. Together, the report documents wet-lab methodological approaches to implement poliovirus screening in wastewater within the scope of the ongoing WBS methods targeting respiratory viruses (Influenza A, Influenza B, SARS-CoV-2, and Respiratory Syncytial Virus) and offers a resource to help aid response and surveillance decisions.

Quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) are applied for quantifying molecular targets in disease diagnostics, pathogen detection, and ecological monitoring. Uptake of dPCR is increasing due to its higher quantification accuracy relative to qPCR, which stems from its independence from standard curves and its increased resistance to PCR inhibitors. Throughput can be increased through multiplexing, which allows simultaneous quantification of multiple targets. However, multiplexing with dPCR faces unique challenges relative to qPCR. Here, we describe the three-phase development process of non-competing multiplex dPCR assays using target-specific fluorescently labeled hydrolysis probes. We highlight common challenges encountered, along with recommended solutions. Phase 1: In silico assay design; target-specific primers and probes are selected or designed, potential issues with primer and probe interactions are identified, and fluorophores and quenchers are chosen based on dPCR instrumentation. Phase 2: Wet-lab validation; assays are benchmarked using positive controls. Insufficient performance leads to assay redesign, as needed. Phase 3: Assay implementation; assay specificity and sensitivity are validated on relevant sample matrices. Finally, we provide recommendations on the future design and standardization of multiplexed dPCR assays, highlighting the need for better in silico predictions of assay performance, standardizing positive controls, and automating partition classification systems.

Wastewater-based epidemiology offers a complementary approach to clinical case-based surveillance of emergent diseases and can help identify regions with infected people to prioritize clinical surveillance strategies. However, tracking emergent diseases in wastewater requires reliance on novel testing assays with uncertain sensitivity and specificity. Limited pathogen shedding may cause detection to be below the limit of quantification or bordering the limit of detection. Here, we investigated how the definition of limit of detection for quantitative polymerase chain reaction (qPCR) impacts epidemiological insights during an mpox outbreak in Switzerland. 365 wastewater samples from three wastewater treatment plants in Switzerland from 9 March through 31 October 2022 were analyzed for mpox DNA using qPCR. We detected mpox DNA in 22% (79 of 365) wastewater samples based on a liberal definition of qPCR detection as any exponentially increasing fluorescence above the threshold. Based on a more restrictive definition as the lowest concentration at which there is 95% likelihood of detection, detection was 1% (5 of 365). The liberal definition shows high specificity (90%) and accuracy (78%), but moderate sensitivity (64%) when benchmarked against available clinical case reporting, which contrasts with higher specificity (98%) but lower sensitivity (10%) and accuracy (56%) of the 95% likelihood definition. Wastewater-based epidemiology applied to an emergent pathogen will require optimizing public health trade-offs between reporting data with high degrees of uncertainty and delaying communication and associated action. Information sharing with relevant public health stakeholders could couple early results with clear descriptions of uncertainty. Impact Statement: When a novel pathogen threatens to enter a community, wastewater-based epidemiology offers an opportunity to track its emergence and spread. However, rapid deployment of methods for to detect a novel pathogen may rely on assays with uncertain sensitivity and specificity. Benchmarking the detection of mpox DNA in Swiss wastewaters with reported clinical cases in 2022, we demonstrate how definitions of detection of a qPCR assay influence epidemiological insights from wastewater. The results highlight the need for information sharing between public health stakeholders that couple early insights from wastewater with descriptions of methodological uncertainty to optimize public health actions.