Wastewater to clinical case (WC) ratio of COVID-19 identifies insufficient clinical testing, onset of new variants of concern and population immunity in urban communities (preprint)

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the extant and anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) is likely to have greater value as an important diagnostic tool to inform public health. As the widespread adoption of WWS is relatively new at the scale employed for COVID-19, interpretation of data, including the relationship to clinical cases, has yet to be standardized. An in-depth analysis of the metrics derived from WWS is required for public health units/agencies to interpret and utilize WWS-acquired data effectively and efficiently. In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven different cities in Canada over periods ranging from 8 to 21 months. Significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing in these communities. The WC ratio decreased significantly during the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized communitys wastewater (40-60% allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized communitys wastewater (40-60% allelic proportion). Finally, a rapid and significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variants greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when vaccine-induced community immunity was high. The WC ratio, used as an additional monitoring metric, complements clinical case counts and wastewater signals as individual metrics in its ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.

Quantitative detection of SARS-CoV-2 Omicron variant in wastewater through allele-specific RT-qPCR (preprint)

On November 26, 2021, the B.1.1.529 COVID-19 variant was named as the Omicron variant of concern. Reports of higher transmissibility and potential immune evasion triggered flight bans and heightened health control measures across the world to stem its distribution. Wastewater-based surveillance has demonstrated to be a useful complement for community-based tracking of SARS-CoV-2 variants. Using design principles of our previous assays that detect SARS-CoV-2 variants (Alpha and Delta), here we report an allele-specific RT-qPCR assay that simultaneously targets mutations Q493R, G496S and Q498R for quantitative detection of the Omicron variant in wastewater. This method is open-sourced and can be implemented using commercially available RT-qPCR protocols, and would be an important tool for tracking the spread and introduction of the Omicron variant in communities for informed public health responses.

Quantitative SARS-CoV-2 tracking of variants Delta, Delta plus, Kappa and Beta in wastewater by allele-specific RT-qPCR (preprint)

The Delta (B.1.617.2) variant has caused major devastation in India and other countries around the world. First detected in October 2020, it has now spread to more than 100 countries, prompting WHO to declare it as a global variant of concern (VOC). The Delta (B.1.617.2), Delta plus (B.1.617.2.1) and Kappa (B.1.617.1) variants are all sub-lineages of the original B.1.617 variant. Prior to the inception of B.1.617, vaccine rollout, safe-distancing and timely lockdowns greatly reduced COVID-19 hospitalizations and deaths. However, the Delta variant, allegedly more infectious and for which existing vaccines seemed less effective, has catalyzed the resurgence of cases. Therefore, there is an imperative need for increased surveillance of the B.1.617 variants. While the Beta variant is increasingly outpaced by the Delta variant, the spread of the Beta variant remains of concern due to its vaccine resistance. Efforts have been made to utilize wastewater-based surveillance for community-based tracking of SARS-CoV-2 variants, however wastewater with its low SARS-CoV-2 viral titers and mixtures of viral variants, requires assays to be variant-specific yet accurately quantitative for meaningful interpretation. Following on the design principles of our previous assays for the Alpha variant, here we report allele-specific and multiplex-compatible RT-qPCR assays targeting mutations T19R, D80A, K417N, T478K and E484Q, for quantitative detection and discrimination of the Delta, Delta plus, Kappa and Beta variants in wastewater. This method is open-sourced and can be implemented using commercially available RT-qPCR protocols, and would be an important tool for tracking the spread of B.1.617 and the Beta variants in communities.

Quantitative detection of SARS-CoV-2 B.1.1.7 variant in wastewater by allele-specific RT-qPCR (preprint)

Wastewater-based epidemiology (WBE) has emerged as a critical public health tool in tracking the SARS-CoV-2 epidemic. Monitoring SARS-CoV-2 variants of concern in wastewater has to-date relied on genomic sequencing, which lacks sensitivity necessary to detect low variant abundances in diluted and mixed wastewater samples. Here, we develop and present an open-source method based on allele specific RT-qPCR (AS RT-qPCR) that detects and quantifies the B.1.1.7 variant, targeting spike protein mutations at three independent genomic loci highly predictive of B.1.1.7 (HV69/70del, Y144del, and A570D). Our assays can reliably detect and quantify low levels of B.1.1.7 with low cross-reactivity, and at variant proportions between 0.1% and 1% in a background of mixed SARS-CoV-2. Applying our method to wastewater samples from the United States, we track B.1.1.7 occurrence over time in 19 communities. AS RT-qPCR results align with clinical trends, and summation of B.1.1.7 and wild-type sequences quantified by our assays strongly correlate with SARS-CoV-2 levels indicated by the US CDC N1/N2 assay. This work paves the path for rapid inexpensive surveillance of B.1.1.7 and other SARS-CoV-2 variants in wastewater.

Wastewater Surveillance of SARS-CoV-2 across 40 U.S. states (preprint)

Wastewater-based disease surveillance is a promising approach for monitoring community outbreaks. Here we describe a nationwide campaign to monitor SARS-CoV-2 in the wastewater of 159 counties in 40 U.S. states, covering 13% of the U.S. population from February 18 to June 2, 2020. Out of 1,751 total samples analyzed, 846 samples were positive for SARS-CoV-2 RNA, with overall viral concentrations declining from April to May. Wastewater viral titers were consistent with, and appeared to precede, clinical COVID-19 surveillance indicators, including daily new cases. Wastewater surveillance had a high detection rate (>80%) of SARS-CoV-2 when the daily incidence exceeded 13 per 100,000 people. Detection rates were positively associated with wastewater treatment plant catchment size. To our knowledge, this work represents the largest-scale wastewater-based SARS-CoV-2 monitoring campaign to date, encompassing a wide diversity of wastewater treatment facilities and geographic locations. Our findings demonstrate that a national wastewater-based approach to disease surveillance may be feasible and effective.

SARS-CoV-2 titers in wastewater foreshadow dynamics and clinical presentation of new COVID-19 cases (preprint)

Current estimates of COVID-19 prevalence are largely based on symptomatic, clinically diagnosed cases. The existence of a large number of undiagnosed infections hampers population-wide investigation of viral circulation. Here, we use longitudinal wastewater analysis to track SARS-CoV-2 dynamics in wastewater at a major urban wastewater treatment facility in Massachusetts, between early January and May 2020. SARS-CoV-2 was first detected in wastewater on March 3. Viral titers in wastewater increased exponentially from mid-March to mid-April, after which they began to decline. Viral titers in wastewater correlated with clinically diagnosed new COVID-19 cases, with the trends appearing 4-10 days earlier in wastewater than in clinical data. We inferred viral shedding dynamics by modeling wastewater viral titers as a convolution of back-dated new clinical cases with the viral shedding function of an individual. The inferred viral shedding function showed an early peak, likely before symptom onset and clinical diagnosis, consistent with emerging clinical and experimental evidence. Finally, we found that wastewater viral titers at the neighborhood level correlate better with demographic variables than with population size. This work suggests that longitudinal wastewater analysis can be used to identify trends in disease transmission in advance of clinical case reporting, and may shed light on infection characteristics that are difficult to capture in clinical investigations, such as early viral shedding dynamics.