Highly sensitive wastewater surveillance of SARS-CoV-2 variants by targeted next-generation amplicon sequencing provides early warning of incursion in Victoria, Australia.

The future of the COVID pandemic and its public health and societal impact will be determined by the profile and spread of emerging variants and the timely identification and response to them. Wastewater surveillance of SARS-CoV-2 has been widely adopted in many countries across the globe and has played an important role in tracking infection levels and providing useful epidemiological information that cannot be adequately captured by clinical testing alone. However, novel variants can emerge rapidly, spread globally, and markedly alter the trajectory of the pandemic, as exemplified by the Delta and Omicron variants. Most mutations linked to the emergence of new SARS-CoV-2 variants are found within variable regions of the SARS-CoV-2 Spike protein. We have developed a duplex hemi-nested PCR method that, coupled with short amplicon sequencing, allows simultaneous typing of two of the most highly variable and informative regions of the Spike gene: the N-terminal domain and the receptor binding motif. Using this method in an operationalized public health program, we identified the first known incursion of Omicron BA.1 into Victoria, Australia and demonstrated how sensitive amplicon sequencing methods can be combined with wastewater surveillance as a relatively low-cost solution for early warning of variant incursion and spread.IMPORTANCEThis study offers a rapid, cost-effective, and sensitive approach for monitoring SARS-CoV-2 variants in wastewater. The method's flexibility permits timely modifications, enabling the integration of emerging variants and adaptations to evolving SARS-CoV-2 genetics. Of particular significance for low- and middle-income regions with limited surveillance capabilities, this technique can potentially be utilized to study a range of pathogens or viruses that possess diverse genetic sequences, similar to influenza.

Passive Sampling of SARS-CoV-2 for Wastewater Surveillance.

The shedding of pathogens by infected humans enables the use of sewage monitoring to conduct wastewater-based epidemiology (WBE). Although most WBE studies use data from large sewage treatment plants, timely data from smaller catchments are needed for targeted public health action. Traditional sampling methods, like autosamplers or grab sampling, are not conducive to quick ad hoc deployments and high-resolution monitoring at these smaller scales. This study develops and validates a cheap and easily deployable passive sampler unit, made from readily available consumables, with relevance to the COVID-19 pandemic but with broader use for WBE. We provide the first evidence that passive samplers can be used to detect SARS-CoV-2 in wastewater from populations with low prevalence of active COVID-19 infections (0.034 to 0.34 per 10,000), demonstrating their ability for early detection of infections at three different scales (lot, suburb, and city). A side by side evaluation of passive samplers (n = 245) and traditionally collected wastewater samples (n = 183) verified that the passive samplers were sensitive at detecting SARS-CoV-2 in wastewater. On all 33 days where we directly compared traditional and passive sampling techniques, at least one passive sampler was positive when the average SARS-CoV-2 concentration in the wastewater equaled or exceeded the quantification limit of 1.8 gene copies per mL (n = 7). Moreover, on 13 occasions where wastewater SARS-CoV-2 concentrations were less than 1.8 gene copies per mL, one or more passive samplers were positive. Finally, there was a statistically significant (p < 0.001) positive relationship between the concentrations of SARS-CoV-2 in wastewater and the levels found on the passive samplers, indicating that with further evaluation, these devices could yield semi-quantitative results in the future. Passive samplers have the potential for wide use in WBE with attractive feasibility attributes of cost, ease of deployment at small-scale locations, and continuous sampling of the wastewater. Further research will focus on the optimization of laboratory methods including elution and extraction and continued parallel deployment and evaluations in a variety of settings to inform optimal use in wastewater surveillance.

First report of anatoxin-a producing cyanobacteria in Australia illustrates need to regularly up-date monitoring strategies in a shifting global distribution.

Routine monitoring of toxic cyanobacteria depends on up-to-date epidemiological information about their distribution. In Australia, anatoxin producing cyanobacteria are not regularly tested for and thought to be rare if not absent from the continent. Our study investigated the presence of anatoxin-a (ATX-a) producing cyanobacteria in surface water samples (n = 226 from 67 sampling locations) collected from 2010 to 2017 across the state of Victoria, Australia. We (1) detected the presence and distribution of anaC (anatoxin-a synthetase C) gene sequences previously associated with various cyanobacteria, including Cuspidothrix issatschenkoi, Aphanizomenon sp., D. circinale, Anabaena sp., and Oscillatoria sp., from 31 sampling locations, and (2) determined the concentration of ATX-a in samples tested using ELISA, in two instances detected at >4 µg · L-1. These data present the first confirmation of ATX-a producers in Australia. Our study indicates that ATX-a should be included in regular testing of cyanobacterial blooms in Australia and highlights the importance of regular investigation of the distributions of toxic cyanobacteria worldwide, particularly amid the known expanding distribution of many cyanobacterial taxa in a period of increased eutrophication and rising surface water temperatures.

An improved method for PCR-based detection and routine monitoring of geosmin-producing cyanobacterial blooms.

Production of taste and odour (T/O) compounds, principally geosmin, by complex cyanobacterial blooms is a major water quality issue globally. Control of these cyanobacteria imposes a significant cost on water producing and dependent industries, and requires routine monitoring and management. Classic monitoring methods, including microscopy and direct chemical analysis, lack sensitivity, are laborious, expensive or cannot reliably identify the source of geosmin production. Polymerase Chain Reaction (PCR) based tools targeting the geosmin synthase gene (geoA) provide a novel tool for routine monitoring. However, geoA is variable at the nucleotide level and potential geosmin producers represent a broad taxonomic distribution, such that multiple PCR primers with distinct amplification protocols are needed to target all potential sources of this important T/O compound. Development of novel primers is hindered by a lack of sequence data and limited field and laboratory data on geosmin producers prevents prioritizing taxa for PCR testing. Here we performed a genetic screen of 253 bloom samples from Victoria, Australia using each existing PCR protocol targeting geoA. We detected Dolichospermum ucrainicum as the major geosmin producer (87% of sequenced samples) along with 3 unknown geoA sequence types. Using these data, we designed a novel, short amplicon, PCR protocol utilising a single standardised primer pair, capable of amplifying all geoA positive samples in our study, as well as a Nostoc punctiforme positive control. This single protocol geoA PCR can further be tested on other geosmin producers and will simplify routine monitoring of T/O producing cyanobacteria.