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

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.

Denitrification kinetics indicates nitrous oxide uptake is unaffected by electron competition in Accumulibacter.

Denitrifying phosphorus removal is a cost and energy efficient treatment technology that relies on polyphosphate accumulating organisms (DPAOs) utilizing nitrate or nitrite as terminal electron acceptor. Denitrification is a multistep process, but many organisms do not possess the complete pathway, leading to the accumulation of intermediates such as nitrous oxide (N2O), a potent greenhouse gas and ozone depleting substance. Candidatus Accumulibacter organisms are prevalent in denitrifying phosphorus removal processes and, according to genomic analyses, appear to vary in their denitrification abilities based on their lineage. Denitrification kinetics and nitrous oxide accumulation in the absence of inhibition from free nitrous acid is a strong indicator of denitrification capabilities of Accumulibacter exposed long-term to nitrate or nitrite as electron acceptor. Thus, we investigated the preferential use of the nitrogen oxides involved in denitrification and nitrous oxide accumulation in two enrichments of Accumulibacter and a competitor - the glycogen accumulating organism Candidatus Competibacter. We modified a metabolic model to predict phosphorus removal and denitrification rates when nitrate, nitrite or N2O were added as electron acceptors in different combinations. Unlike previous studies, no N2O accumulation was observed for Accumulibacter in the presence of multiple electron acceptors. Electron competition did not limit denitrification kinetics or lead to N2O accumulation in Accumulibacter or Competibacter. Despite the presence of sufficient internal storage polymers (polyhydroxyalkanoates, or PHA) as energy source for each denitrification step, the extent of denitrification observed was dependent on the dominant organism in the enrichment. Accumulibacter showed complete denitrification, whereas Competibacter denitrification was limited to reduction of nitrate to nitrite. These findings indicate that DPAOs can contribute to lowering N2O emissions in the presence of multiple electron acceptors under partial nitritation conditions.

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

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.

Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications; a systematic review

Capsid-integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo-dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-two recent peer-reviewed studies employing capsid-integrity qPCR for viruses in the last decade (2009 to 2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 {micro}M; incubation on ice or at room temperature (20 - 25{degrees}C) for 5 to 120 min; and dye activation using LED or high light (500 - 800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/20095364v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@12f34b5org.highwire.dtl.DTLVardef@2d7a8forg.highwire.dtl.DTLVardef@1c951c3org.highwire.dtl.DTLVardef@16f7df_HPS_FORMAT_FIGEXP M_FIG C_FIG