Comparison of kits for SARS-CoV-2 extraction in liquid and passive samples.

Effective extraction and detection of viral nucleic acids from sewage are fundamental components of a successful SARS-CoV-2 sewage surveillance programme. As there is no standard method employed in sewage surveillance, understanding the performance of different extraction kits in the recovery of SARS-CoV-2 and the impact that PCR inhibitors have on quantification is essential to minimize data discrepancies caused by sample extraction. Three commercial nucleic acid extraction kits: the RNeasy PowerSoil Total RNA Kit (PS), the RNeasy PowerMicrobiome Kit (PMB), and the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (MM), with minor modifications, were evaluated. Their efficacy in recovering viral ribonucleic acid and removal of PCR inhibitors was assessed using two South Australian wastewater matrices-one from a major metropolitan site and one from a regional centre. Both had SARS-CoV-2 present due to active COVID-19 cases in these communities. Overall, the MM kit had a higher recovery of SARS-CoV-2 from the samples tested, followed by PMB and PS. The PMB kit performance was strongly influenced by the sample matrix when compared to the MM kit. It is recommended to assess the performance of extraction kits using different local wastewater matrices to ensure the accuracy and reliability of monitoring results to avoid false reporting.

The detection of Japanese encephalitis virus in municipal wastewater during an acute disease outbreak.

AIM: To demonstrate the capability of wastewater-based surveillance (WBS) as a tool for detecting potential cases of Japanese Encephalitis Virus (JEV) infection in the community. METHODS AND RESULTS: In this study, we explore the potential of WBS to detect cases of JEV infection by leveraging from an established SARS-CoV-2 wastewater surveillance program. We describe the use of two reverse transcriptase quantitative polymerase chain reaction (RTqPCR) assays targeting JEV to screen archived samples from two wastewater treatment plants (WWTPs). JEV was detected in wastewater samples collected during a timeframe coinciding with a cluster of acute human encephalitis cases, alongside concurrent evidence of JEV detection in mosquito surveillance and the sentinel chicken programs within South Australia's Riverland and Murraylands regions. CONCLUSIONS: Current surveillance measures for JEV encounter multiple constraints, which may miss the early stages of JEV circulation or fail to capture the full extent of transmission. The detection of JEV in wastewater during a disease outbreak highlights the potential WBS has as a complementary layer to existing monitoring efforts forming part of the One Health approach required for optimal disease response and control.

Recycling drinking water treatment sludge into eco-concrete blocks with CO2 curing: Durability and leachability.

Drinking water treatment sludge (DWTS) can be recycled into low-strength concrete blocks for construction use. The sodium sulfate resistance and leaching behaviours of the DWTS-derived blocks are investigated in this study. The experimental results show that the addition of DWTS degrades the sodium sulfate resistance of the concrete blocks, however CO2 curing compensates for such property, especially in the case of blocks incorporating 30% DWTS. The improvement can be attributed to the formation of crystalline CaCO3 during CO2 curing for microstructure refinement evidenced by X-ray Computed Tomography and Scanning Electron Microscopy. Leaching analyses show that Cu and Al concentrations increased with increasing DWTS content, and CO2 curing adversely increased the leachability of metals due to the decrease of pH, especially at early leaching stage. Nevertheless, the total leaching concentrations of Cu and Al after 60-day test is far below the prescribed limitations, regardless of samples subject to air curing or CO2 curing. In summary, sludge-derived blocks exposed to CO2 curing are safe and behave well in aggressive environments. Therefore, this study showcases a green technology that successfully recycling DWTS into value-added and durable concrete blocks with low environmental impacts.

Utilization of drinking water treatment sludge in concrete paving blocks: Microstructural analysis, durability and leaching properties.

The management of abundant drinking water treatment sludge (DWTS) in landfill remains an important issue. The reuse of DWTS as construction material could contribute to the development of greener concrete product and to mitigating the detrimental environment effect from excessive production of DWTS. This paper investigates the potential of using DWTS as sand replacement in Concrete Paving Blocks (CPB). Five CPB mixtures were designed and the replacement ratios of sand by DWTS were 0%, 5%, 10%, 15%, and 20%, by weight. Properties of CPB such as compressive strength, water absorption, abrasion resistance, sulfate attack and metal leachability were determined. The results indicated that above 10% of DWTS, the replacement was detrimental to such properties of the CPB. Microstructure analysis proved the addition of DWTS could result in ettringite formation and the interfacial transition zone (ITZ) between the cement matrix and DWTS was more porous than that of sand. In addition, the metal leachability test of CPB demonstrated that the addition of high-copper DWTS into CPB was safe.

Chlorine inactivation of coxsackievirus B5 in recycled water destined for non-potable reuse.

Currently guidelines for disinfection of water with free chlorine, while primarily developed for potable water, are often used for virus disinfection of nitrified recycled water of >1 NTU (Nephelometric Turbidity Unit). More information is needed on the disinfection efficacy of free chlorine for viruses in waters of varying turbidity and pH due to significant reuse of treated wastewater of varying quality. In this study, disinfection efficacy in nitrified/denitrified activated sludge treated wastewater was investigated for coxsackievirus B5 (CB5), an enterovirus known to be highly resistant to free chlorine. The required chlorine contact times (CT) values (mg.min/L) for inactivation of CB5 were established in treated wastewater at 10 °C and of varying turbidity (0.2, 2, 5 and 20 NTU) and pH (7, 8 and 9). CTs were calculated to achieve 1 to 4 log10 inactivation. Robust data is presented in support of the chlorine CT values required to inactivate a chlorine-resistant virus in a range of turbidities and pHs in treated wastewaters. The testing method used a conservative approach and the data presented have been used to develop the free chlorine virus inactivation guildelines for recycled water in Victoria and South Australia, Australia.

Virus removal by ultrafiltration: Understanding long-term performance change by application of Bayesian analysis.

Ultrafiltration is an effective barrier to waterborne pathogens including viruses. Challenge testing is commonly used to test the inherent reliability of such systems. Performance validation seeks to demonstrate the adequate reliability of the treatment system. Appropriate and rigorous data analysis is an essential aspect of validation testing. In this study we used Bayesian analysis to assess the performance of a full-scale ultrafiltration system which was validated and revalidated after five years of operation. A hierarchical Bayesian model was used to analyse a number of similar ultrafiltration membrane skids working in parallel during the two validation periods. This approach enhanced our ability to obtain accurate estimations of performance variability, especially when the sample size of some system skids was limited. This methodology enabled the quantitative estimation of uncertainty in the performance parameters and generation of predictive distributions incorporating those uncertainties. The results indicated that there was a decrease in the mean skid performance after five years of operation of approximately 1 log reduction value (LRV). Interestingly, variability in the LRV also reduced, with standard deviations from the revalidation data being decreased by a mean 0.37 LRV compared with the original validation data. The model was also useful in comparing the operating performance of the various parallel skids within the same year. Evidence of differences was obtained in 2015 for one of the membrane skids. A hierarchical Bayesian analysis of validation data provides robust estimations of performance and the incorporation of probabilistic analysis which is increasingly important for comprehensive quantitative risk assessment purposes.

Bayesian belief network modelling of chlorine disinfection for human pathogenic viruses in municipal wastewater.

Chlorine disinfection of biologically treated wastewater is practiced in many locations prior to environmental discharge or beneficial reuse. The effectiveness of chlorine disinfection processes may be influenced by several factors, such as pH, temperature, ionic strength, organic carbon concentration, and suspended solids. We investigated the use of Bayesian multilayer perceptron (BMLP) models as efficient and practical tools for compiling and analysing free chlorine and monochloramine virus disinfection performance as a multivariate problem. Corresponding to their relative susceptibility, Adenovirus 2 was used to assess disinfection by monochloramine and Coxsackievirus B5 was used for free chlorine. A BMLP model was constructed to relate key disinfection conditions (CT, pH, turbidity) to observed Log Reduction Values (LRVs) for these viruses at constant temperature. The models proved to be valuable for incorporating uncertainty in the chlor(am)ination performance estimation and interpolating between operating conditions. Various types of queries could be performed with this model including the identification of target CT for a particular combination of LRV, pH and turbidity. Similarly, it was possible to derive achievable LRVs for combinations of CT, pH and turbidity. These queries yielded probability density functions for the target variable reflecting the uncertainty in the model parameters and variability of the input variables. The disinfection efficacy was greatly impacted by pH and to a lesser extent by turbidity for both types of disinfections. Non-linear relationships were observed between pH and target CT, and turbidity and target CT, with compound effects on target CT also evidenced. This work demonstrated that the use of BMLP models had considerable ability to improve the resolution and understanding of the multivariate relationships between operational parameters and disinfection outcomes for wastewater treatment.

Modelling pathogen log10 reduction values achieved by activated sludge treatment using naïve and semi naïve Bayes network models.

Risk management for wastewater treatment and reuse have led to growing interest in understanding and optimising pathogen reduction during biological treatment processes. However, modelling pathogen reduction is often limited by poor characterization of the relationships between variables and incomplete knowledge of removal mechanisms. The aim of this paper was to assess the applicability of Bayesian belief network models to represent associations between pathogen reduction, and operating conditions and monitoring parameters and predict AS performance. Naïve Bayes and semi-naïve Bayes networks were constructed from an activated sludge dataset including operating and monitoring parameters, and removal efficiencies for two pathogens (native Giardia lamblia and seeded Cryptosporidium parvum) and five native microbial indicators (F-RNA bacteriophage, Clostridium perfringens, Escherichia coli, coliforms and enterococci). First we defined the Bayesian network structures for the two pathogen log10 reduction values (LRVs) class nodes discretized into two states (< and ≥ 1 LRV) using two different learning algorithms. Eight metrics, such as Prediction Accuracy (PA) and Area Under the receiver operating Curve (AUC), provided a comparison of model prediction performance, certainty and goodness of fit. This comparison was used to select the optimum models. The optimum Tree Augmented naïve models predicted removal efficiency with high AUC when all system parameters were used simultaneously (AUCs for C. parvum and G. lamblia LRVs of 0.95 and 0.87 respectively). However, metrics for individual system parameters showed only the C. parvum model was reliable. By contrast individual parameters for G. lamblia LRV prediction typically obtained low AUC scores (AUC < 0.81). Useful predictors for C. parvum LRV included solids retention time, turbidity and total coliform LRV. The methodology developed appears applicable for predicting pathogen removal efficiency in water treatment systems generally.

Uncertainties associated with assessing the public health risk from Legionella.

Legionella is an opportunistic pathogen of public health concern. Current regulatory and management guidelines for the control of this organism are informed by risk assessments. However, there are many unanswered questions and uncertainties regarding Legionella epidemiology, strain infectivity, infectious dose, and detection methods. This review follows the EnHealth Risk Assessment Framework, to examine the current information available regarding Legionella risk and discuss the uncertainties and assumptions. This review can be used as a tool for understanding the uncertainties associated with Legionella risk assessment. It also serves to highlight the areas of Legionella research that require future focus. Improvement of these uncertainties will provide information to enhance risk management practices for Legionella, potentially improving public health protection and reducing the economic costs by streamlining current management practices.

Detection of Legionella, L. pneumophila and Mycobacterium avium complex (MAC) along potable water distribution pipelines.

Inhalation of potable water presents a potential route of exposure to opportunistic pathogens and hence warrants significant public health concern. This study used qPCR to detect opportunistic pathogens Legionella spp., L. pneumophila and MAC at multiple points along two potable water distribution pipelines. One used chlorine disinfection and the other chloramine disinfection. Samples were collected four times over the year to provide seasonal variation and the chlorine or chloramine residual was measured during collection. Legionella spp., L. pneumophila and MAC were detected in both distribution systems throughout the year and were all detected at a maximum concentration of 103 copies/mL in the chlorine disinfected system and 106, 103 and 104 copies/mL respectively in the chloramine disinfected system. The concentrations of these opportunistic pathogens were primarily controlled throughout the distribution network through the maintenance of disinfection residuals. At a dead-end and when the disinfection residual was not maintained significant (p < 0.05) increases in concentration were observed when compared to the concentration measured closest to the processing plant in the same pipeline and sampling period. Total coliforms were not present in any water sample collected. This study demonstrates the ability of Legionella spp., L. pneumophila and MAC to survive the potable water disinfection process and highlights the need for greater measures to control these organisms along the distribution pipeline and at point of use.

Fate of pathogenic microorganisms and indicators in secondary activated sludge wastewater treatment plants.

This study was undertaken to investigate the removal of pathogenic microorganisms and their indicators in a laboratory scale biological treatment system that simulated the secondary treatment process of a wastewater treatment plant (WWTP). Four groups of microorganisms including bacteria, viruses, protozoa and helminths as well as the selected indicators were employed in the investigation. The results demonstrated that approximately 2-3 log10 removal of the microbial indicators was achieved in the treatment process. The log removal of Clostridium perfringens spores was low due to their irreversible adsorption to sludge flocs. The laboratory treatment system demonstrated a similar removal capability for Escherichia coli and the bacterial indicators (total coliforms, enterococci and particles <2.73 microm/L). The MS-2 bacteriophage, measured as a viral indicator, showed a lower removal than poliovirus, which may be considered as a worst case scenario for virus removal. The results of using particle profiling as an indicator for protozoa and helminths appeared to be inaccurate. The removal performance for bacterial and protozoan pathogens and their indicators in a full scale WWTP and the laboratory treatment system was compared.

Effect of water treatment processes on Cryptosporidium infectivity.

Conventional water treatment processes have the ability to remove Cryptosporidium oocysts through coagulation, flocculation, sedimentation and filtration, provided there is efficient management of plant performance. The potential exists for the breakthrough of oocysts through the treatment train. The effect of the water treatment chemical aluminium sulphate (alum) on Cryptosporidium oocyst infectivity has been assessed using an assay that combines cell culture and real-time polymerase chain reaction techniques. The infectivity of fresh and temperature-aged oocysts (stored up to 6 months at 4 or 15 degrees C) was unaffected by exposure to a range of doses of alum in standard jar test procedures and dissolved air flotation processes and subsequent exposure to chlorine or chloramine. Removal efficiencies and infectivity measures are important in determining risk to public health and will reflect the ability of water treatment plants to act as a barrier to these pathogens.

Bacterial degradation of microcystin toxins within a biologically active sand filter.

Microcystin toxins are a problem for water authorities as they are recalcitrant to conventional water treatment. In this study, biological sand filtration was assessed in laboratory column experiments for its ability to remove two microcystin analogues, microcystin-LR and microcystin-LA. A lag period of 3 days was evident prior to the commencement of degradation. Contact times were varied during the experiment; however, no microcystin was detected in the effluent after 4 days, even under conditions similar to those of a rapid sand filter. Removals of microcystin through the sand filters were shown to be primarily through biological degradation processes. Using polymerase chain reaction (PCR), biofilm, extracted from one of the sand filters that had effectively removed the microcystins, was shown to contain bacteria with the mlrA gene. Detection of this gene provided additional evidence that biological degradation of microcystin was the primary removal mechanism.

Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infectivity.

Cryptosporidium is a significant cause of water-borne enteric disease throughout the world and represents a challenge to the water industry and a threat to public health. In this study we report the use of a cell culture-TaqMan PCR assay to measure oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. While oocysts incubated at 4 degrees C and 15 degrees C remained infective over the 12-week holding period, we observed a 4 log(10) reduction in infectivity for both 20 and 25 degrees C incubation treatments at 12 and 8 weeks, respectively, for all water types examined, a faster rate of inactivation for oocysts than previously reported. This temperature-dependent inactivation was further investigated using a simple and rapid ATP assay described herein. Time course experiments performed in reagent-grade water at incubation temperatures of 4, 15, 20, 25, 30, and 37 degrees C identified a close relationship between oocyst infectivity and oocyst ATP content, demonstrating that temperature inactivation at higher temperatures is a function of increased oocyst metabolic activity. While water quality did not affect oocyst inactivation, biological antagonism appears to be a key factor affecting oocyst removal from environmental waters. Both the cell culture-TaqMan PCR assay and the ATP assay provide a sensitive and quantitative method for the determination of environmental oocyst inactivation, providing an alternative to the more costly and time-consuming mouse infection assay. The findings presented here relating temperature to oocyst inactivation provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in water.

Emerging technologies for the detection and genetic characterization of protozoan parasites.

The development and adaptation of new technologies for the genetic characterization and identification of parasites continue to accelerate, providing an increasing number of research and analytical tools. We review emerging technologies that have applications in this area, including real-time PCR and microarrays, and discuss the fundamental principles of some of these technologies and how they are applied to characterize parasites. We give special consideration to the application of genetic data to biological questions, where selection of the most appropriate technique depends on the biological question posed by the investigator.

Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection.

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log(10) unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.