Monitoring an Emergent Pathogen at Low Incidence in Wastewater Using qPCR: Mpox in Switzerland

Abstract

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.

Keywords: Limit of detection; Mpox; Wastewater-based epidemiology; qPCR.

Fig. 1

A Reported weekly mpox cases in cantons of Geneva (GE), Ticino (TI), and Zurich (ZH) from March through October 2022 and corresponding B maximum observed concentrations (gce per ml wastewater) and C maximum observed loads (gce per day) of mpox DNA measured in wastewater treatment plants within the catchment. Notably, concentrations and loads were determined based on extrapolation of the standard curve below the determined LOQ

Fig. 2

Logit regression for detection of mpox DNA in one or more samples during a calendar week as a function of the reported cases within the same calendar week for A STEP Aïre in canton Geneva (GE), B ARA Werdhölzli in canton Zurich (ZH), C CDA Lugano in canton Ticino (TI), and D all wastewater treatment plants. Detection, here, is defined as any exponentially-increasing qPCR fluorescence above the threshold. Dashed lines represent fit logistic regression models with shaded areas representing the 95% confidence interval