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.

Assessing the effects of COVID-19 restrictions on alcohol consumption in Melbourne, Australia using high-resolution wastewater sampling.

The impact of the COVID-19 pandemic and related restrictions on alcohol consumption in Australia remains unclear. High-resolution daily samples from a wastewater treatment plant (WWTP) which served one of the largest cities in Australia, Melbourne, were analysed for temporal trends in alcohol consumption under extended periods of COVID-19 restrictions in 2020. Melbourne experienced two major lockdowns in 2020, which divided the year of 2020 into five periods (pre-lockdown, first lockdown, between lockdown, second lockdown and post second-lockdown). In this study, daily sampling identified shifts in alcohol consumption during different periods of restrictions. Alcohol consumption in the first lockdown period, when bars closed and social and sports events ceased, was lower than pre-lockdown period. However, alcohol consumption was higher in the second lockdown period than the previous lockdown period. There were spikes in alcohol consumption at the start and end of each lockdown (except for post lockdown). For most of 2020, the usual weekday-weekend variations in alcohol consumption were less evident but there was a significant difference in alcohol consumption between weekdays and weekends after the second lockdown. This suggests that drinking patterns eventually returned to normal after the end of the second lockdown. This study demonstrates the usefulness of high-resolution wastewater sampling in evaluating the effects on alcohol consumption of social interventions in specific temporal locations.

Diagnosing water treatment critical control points for cyanobacterial removal: Exploring benefits of combined microscopy, next-generation sequencing, and cell integrity methods.

A wide range of cyanobacterial species and their harmful metabolites are increasingly detected in water bodies worldwide, exacerbated by climate change and human activities. The resulting bloom conditions represent significant challenges to production of safe drinking water and cost effective water reuse, therefore their removal is a priority to ensure public safety. While current microscopic taxonomy identification methods provide valuable information about cell numbers during treatment, these methods are incapable of providing information about the fate of cells during treatment. The objectives of this study were to (1) identify the critical control points for breakthrough and accumulation of cells by investigating the fate of cells during treatment processes using a combination of taxonomy, cell integrity and next-generation sequencing (NGS), and (2) assess the impact of pre-treatment processes on breakthrough prevention at critical control points, and the benefits of cell integrity and NGS analysis for improved management purposes. This paper presents the results of an unprecedented cyanobacterial monitoring program conducted in four full scale water treatment plants located in three different climate zones. Cyanobacterial cell integrity and accumulation during operation process were assessed for the first time using next generation of gene sequencing methods. NGS analysis led to detection of cyanobacterial and melainabacteria orders in water samples that were not identified by microscopy. 80 ±â€¯5% of cells were completely lysed post pre-oxidation (for both ozone and potassium permanganate). However unlike pre-ozonation, the remaining cells were undamaged cells with the potential to accumulate and grow within the plants post-KMnO4 treatment, particularly in clarifier sludge. To effectively monitor water quality, this study presents a synergistic approach coupling new and traditional analytical methods and demonstrates the importance of identifying critical points for managing accumulation of cyanobacteria within plants.

Evaluation of Techniques for Measuring Microbial Hazards in Bathing Waters: A Comparative Study.

Recreational water quality is commonly monitored by means of culture based faecal indicator organism (FIOs) assays. However, these methods are costly and time-consuming; a serious disadvantage when combined with issues such as non-specificity and user bias. New culture and molecular methods have been developed to counter these drawbacks. This study compared industry-standard IDEXX methods (Colilert and Enterolert) with three alternative approaches: 1) TECTA™ system for E. coli and enterococci; 2) US EPA's 1611 method (qPCR based enterococci enumeration); and 3) Next Generation Sequencing (NGS). Water samples (233) were collected from riverine, estuarine and marine environments over the 2014-2015 summer period and analysed by the four methods. The results demonstrated that E. coli and coliform densities, inferred by the IDEXX system, correlated strongly with the TECTA™ system. The TECTA™ system had further advantages in faster turnaround times (~12 hrs from sample receipt to result compared to 24 hrs); no staff time required for interpretation and less user bias (results are automatically calculated, compared to subjective colorimetric decisions). The US EPA Method 1611 qPCR method also showed significant correlation with the IDEXX enterococci method; but had significant disadvantages such as highly technical analysis and higher operational costs (330% of IDEXX). The NGS method demonstrated statistically significant correlations between IDEXX and the proportions of sequences belonging to FIOs, Enterobacteriaceae, and Enterococcaceae. While costs (3,000% of IDEXX) and analysis time (300% of IDEXX) were found to be significant drawbacks of NGS, rapid technological advances in this field will soon see it widely adopted.

Into the deep: Evaluation of SourceTracker for assessment of faecal contamination of coastal waters.

Faecal contamination of recreational waters is an increasing global health concern. Tracing the source of the contaminant is a vital step towards mitigation and disease prevention. Total 16S rRNA amplicon data for a specific environment (faeces, water, soil) and computational tools such as the Markov-Chain Monte Carlo based SourceTracker can be applied to microbial source tracking (MST) and attribution studies. The current study applied artificial and in-laboratory derived bacterial communities to define the potential and limitations associated with the use of SourceTracker, prior to its application for faecal source tracking at three recreational beaches near Port Phillip Bay (Victoria, Australia). The results demonstrated that at minimum multiple model runs of the SourceTracker modelling tool (i.e. technical replicates) were required to identify potential false positive predictions. The calculation of relative standard deviations (RSDs) for each attributed source improved overall predictive confidence in the results. In general, default parameter settings provided high sensitivity, specificity, accuracy and precision. Application of SourceTracker to recreational beach samples identified treated effluent as major source of human-derived faecal contamination, present in 69% of samples. Site-specific sources, such as raw sewage, stormwater and bacterial populations associated with the Yarra River estuary were also identified. Rainfall and associated sand resuspension at each location correlated with observed human faecal indicators. The results of the optimised SourceTracker analysis suggests that local sources of contamination have the greatest effect on recreational coastal water quality.