Optimizing start-up strategies for the two-inflow nitritation/anammox process: Influence on biofilm microbial community composition.

Low-energy nitrogen removal from ammonium-rich wastewater is crucial in preserving the water environment. A one-stage nitritation/anammox process with two inflows treating ammonium-containing wastewater, supplied from inside and outside the wound filter, is expected to stably remove nitrogen. Laboratory-scale reactors were operated using different start-up strategies; the first involved adding nitritation inoculum after anammox biomass formation in the filter, which presented a relatively low nitrogen removal rate (0.171 kg N/m3 · d), at a nitrogen loading rate of 1.0 kg N/m3 · d. Conversely, the second involved the gradual cultivation of anammox and nitritation microorganisms, which increased the nitrogen removal rate (0.276 kg N/m3 · d). Furthermore, anammox (Candidatus Brocadia) and nitritation bacteria (Nitrosomonadaceae) coexisted in the biofilm formed on the filter surface. The abundance of nitritation bacteria (10.5%) in the reactor biofilm using the second start-up strategy was higher than that using the first (3.7%). Thus, the two-inflow nitritation/anammox process effectively induced habitat segregation using a suitable start-up strategy.

Real-Time On-Site Monitoring of Viruses in Wastewater Using Nanotrap® Particles and RICCA Technologies.

Wastewater-based epidemiology (WBE) is an effective and efficient tool for the early detection of infectious disease outbreaks in a community. However, currently available methods are laborious, costly, and time-consuming due to the low concentration of viruses and the presence of matrix chemicals in wastewater that may interfere with molecular analyses. In the present study, we designed a highly sensitive "Quick Poop (wastewater with fecal waste) Sensor" (termed, QPsor) using a joint approach of Nanotrap microbiome particles and RICCA (RNA Isothermal Co-Assisted and Coupled Amplification). Using QPsor, the WBE study showed a strong correlation with standard PEG concentrations and the qPCR technique. Using a closed format for a paper-based lateral flow assay, we were able to demonstrate the potential of our assay as a real-time, point-of-care test by detecting the heat-inactivated SARS-CoV-2 virus in wastewater at concentrations of 100 copies/mL and within one hour. As a proof-of-concept demonstration, we analyzed the presence of viral RNA of the SARS-CoV-2 virus and PMMoV in raw wastewater samples from wastewater treatment plants on-site and within 60 min. The results show that the QPsor method can be an effective tool for disease outbreak detection by combining an AI-enabled case detection model with real-time on-site viral RNA extraction and amplification, especially in the absence of intensive clinical laboratory facilities. The lab-free, lab-quality test capabilities of QPsor for viral prevalence and transmission in the community can contribute to the efficient management of pandemic situations.

On the Disproportionate Contribution of Membrane Electron Donor Functionality in Membrane Biofouling.

This study set out to uncover which interfacial properties have the greatest impact on membrane organic fouling, biofouling, and fouling resistance. A relatively simple manipulation of the basic equations used in determining Lifshitz-van der Waals (LW) and Lewis acid-base (AB) surface tensions for solid materials reveals that the high electron accepticity of water makes the electron donicity of membrane and biofouling materials the key component governing their interfacial free energy of adhesion (ΔG132), which defines the favorability (or unfavorability) of one material (1) adhering to another (2) when immersed in a liquid (3). Various biofoulant and membrane LW and AB surface tensions were systematically characterized. Static bacterial adhesion, alginic acid filtration, and wastewater filtration tests were conducted to determine the fouling propensities of three different polymeric membrane materials. Experimental results of microbial adhesion, alginate fouling, and biofouling tests all correlated well with membrane electron density, where higher electron density produced less organic fouling or biofouling. These combined theoretical and experimental results confirm the importance of surface electron donicity in determining the fouling propensity of polymeric membranes.

Evaluation of vehicle running performance on ash-covered roads.

The scale of volcanic eruptions influences the effects of wide-ranging disasters involving ashfall, such as the disruption of lifelines and paralysis of urban functions. This highlights the importance of vehicle availability on ash-covered roads for disaster management personnel involved in rescue and recovery efforts and for citizens who must evacuate or continue their social lives. We conducted tests to scientifically verify the running ability of vehicles on ash-covered roads. Results revealed that all-wheel-drive vehicles showed better running performance than two-wheel-drive vehicles, which get stuck when ashfall thickness exceeds 10 cm. Most of the vehicle's drive power is consumed as energy to scrape ash grains from under the tires, hindering sufficient propulsion. In addition, the tires sink into the ash layer, which increases driving resistance and causes the vehicle to get stuck. Running ability on ash-covered roads is mainly determined by the relation between the "drive system" of the vehicle and the "thickness of ash" on the roads. In addition, road surface conditions, including ash thickness, could change in time and space because of traffic volume and weather conditions.

Important properties of anion exchange resins for efficient removal of PFOS and PFOA from groundwater.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) present in various water sources have raised a serious concern on their health risk worldwide. Anion exchange is known to be one of the effective treatment methods but the resin properties suitable for theses contaminants have not been fully understood. We examined four commercially available anion exchange resins with different properties (DIAION™ PA312, HPA25M, UBA120, and WA30) and one polymer-based adsorbent (HP20), for their PFOA and PFOS removal in the batch experiment. All or a part of the selected resins were further characterized for their functional group, surface morphology and pore size distribution. The 72 h batch experiment with the 100 mg/L PFOA or PFOS in the laboratory pure water matrix showed a superior capacity of the strong base anion exchange resins, the porous-type HPA25M and PA312, and the gel-type UBA120, for PFOA removal (92.6-97.9%). Among those resins, the high porous HPA25M was suggested most effective due to its remarkably high reaction rate and effectiveness to PFOS (99.9%). In the groundwater matrix, however, the performance of the those anion exchange resins was generally suppressed, causing up to 71% decrease in their removal rates. The least matrix impact was observed for PFOS removal by HPA25M, which indicated the resin's high selectivity to the contaminant. The physiochemical analysis indicated that the presence of relatively large pores (1 nm-10 nm) over HPA25M played an important role in the PFAS removal.

Response of wastewater-based epidemiology predictor for the second wave of COVID-19 in Ahmedabad, India: A long-term data Perspective.

In this work, we present an eight-month longitudinal study of wastewater-based epidemiology (WBE) in Ahmedabad, India, where wastewater surveillance was introduced in September 2020 after the successful containment of the first wave of COVID-19 to predict the resurge of the infection during the second wave of the pandemic. The study aims to elucidate the weekly resolution of the SARS-CoV-2 RNA data for eight months in wastewater samples to predict the COVID-19 situation and identify hotspots in Ahmedabad. A total of 287 samples were analyzed for SARS-CoV-2 RNA using RT-PCR, and Spearman's rank correlation was applied to depict the early warning potential of WBE. During September 2020 to April 2021, the increasing number of positive wastewater influent samples correlated with the growing number of confirmed clinical cases. It also showed clear evidence of early detection of the second wave of COVID-19 in Ahmedabad (March 2021). 258 out of a total 287 samples were detected positive with at least two out of three SARS-CoV-2 genes (N, ORF- 1 ab, and S). Monthly variation represented a significant decline in all three gene copies in October compared to September 2020, followed by an abrupt increase in November 2020. A similar increment in the gene copies was observed in March and April 2021, which would be an indicator of the second wave of COVID-19. A lead time of 1-2 weeks was observed in the change of gene concentrations compared with clinically confirmed cases. Measured wastewater ORF- 1 ab gene copies ranged from 6.1 x 102 (October 2020) to 1.4 x 104 (November 2020) copies/mL, and wastewater gene levels typically lead to confirmed cases by one to two weeks. The study highlights the value of WBE as a monitoring tool to predict waves within a pandemic, identify local disease hotspots within a city, and guide rapid management interventions.

Antimicrobial resistome and mobilome in the urban river affected by combined sewer overflows and wastewater treatment effluent.

The dissemination of antimicrobial resistance in the environment is an emerging global health problem. Wastewater treatment effluent and combined sewer overflows (CSOs) are major sources of antimicrobial resistance in urban rivers. This study aimed to clarify the effect of municipal wastewater treatment effluent and CSO on antimicrobial resistance genes (ARGs), mobile gene elements, and the microbial community in an urban river. The ARG abundance per 16S-based microbial population in the target river was 0.37-0.54 and 0.030-0.097 during the CSO event and dry weather, respectively. During the CSO event, the antimicrobial resistome in the river shifted toward a higher abundance of ARGs to clinically important drug classes, including macrolide, fluoroquinolone, and ß-lactam, whereas ARGs to sulfonamide and multidrug by efflux pump were relatively abundant in dry weather. The abundance of intI1 and tnpA genes were highly associated with the total ARG abundance, suggesting their potential application as an indicator for estimating resistome contamination. Increase of prophage during the CSO event suggested that impact of CSO has a greater potential for horizontal gene transfer (HGT) via transduction. Consequently, CSO not only increases the abundance of ARGs to clinically important antimicrobials but also possibly enhances potential of HGT in urban rivers.

ARL-17477 is a dual inhibitor of NOS1 and the autophagic-lysosomal system that prevents tumor growth in vitro and in vivo.

ARL-17477 is a selective neuronal nitric oxide synthase (NOS1) inhibitor that has been used in many preclinical studies since its initial discovery in the 1990s. In the present study, we demonstrate that ARL-17477 exhibits a NOS1-independent pharmacological activity that involves inhibition of the autophagy-lysosomal system and prevents cancer growth in vitro and in vivo. Initially, we screened a chemical compound library for potential anticancer agents, and identified ARL-17477 with micromolar anticancer activity against a wide spectrum of cancers, preferentially affecting cancer stem-like cells and KRAS-mutant cancer cells. Interestingly, ARL-17477 also affected NOS1-knockout cells, suggesting the existence of a NOS1-independent anticancer mechanism. Analysis of cell signals and death markers revealed that LC3B-II, p62, and GABARAP-II protein levels were significantly increased by ARL-17477. Furthermore, ARL-17477 had a chemical structure similar to that of chloroquine, suggesting the inhibition of autophagic flux at the level of lysosomal fusion as an underlying anticancer mechanism. Consistently, ARL-17477 induced lysosomal membrane permeabilization, impaired protein aggregate clearance, and activated transcription factor EB and lysosomal biogenesis. Furthermore, in vivo ARL-17477 inhibited the tumor growth of KRAS-mutant cancer. Thus, ARL-17477 is a dual inhibitor of NOS1 and the autophagy-lysosomal system that could potentially be used as a cancer therapeutic.

A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes.

The method of spiking synthetic internal standard genes (ISGs) to samples for amplicon sequencing, generating sequences and converting absolute gene numbers from read counts has been used only for phylogenetic markers and has not been applied to functional markers. In this study, we developed ISGs, including gene sequences of the 16S rRNA, pmoA, encoding a subunit of particulate methane monooxygenase and amoA, encoding a subunit of ammonia monooxygenase. We added ISGs to the samples, amplified the target genes and performed amplicon sequencing. For the mock community, the copy numbers converted from read counts using ISGs were equivalent to those obtained by the quantitative real-time polymerase chain reaction (4.0 × 104 versus 4.1 × 104 and 3.0 × 103 versus 4.0 × 103 copies µL-DNA-1 for 16S rRNA and pmoA genes, respectively), but we also identified underestimation, possibly due to primer coverage (7.8 × 102 versus 3.7 × 103 µL-DNA-1 for amoA gene). We then applied this method to environmental samples and analysed phylogeny, functional diversity and absolute quantities. One Methylocystis population was most abundant in the sludge samples [16S rRNA gene (3.8 × 109 copies g-1 ) and the pmoA gene (2.3 × 109 copies g-1 )] and were potentially interrelated. This study demonstrates that ISG spiking is useful for evaluating sequencing data processing and quantifying functional markers.

Reduction of SARS-CoV-2 by biological nutrient removal and disinfection processes in full-scale wastewater treatment plants.

Detection of SARS-CoV-2 RNA in wastewater poses people's concerns regarding the potential risk in water bodies receiving wastewater treatment effluent, despite the infectious risk of SARS-CoV-2 in wastewater being speculated to be low. Unlike well-studied nonenveloped viruses, SARS-CoV-2 in wastewater is present abundantly in both solid and liquid fractions of wastewater. Reduction of SARS-CoV-2 in past studies were likely underestimated, as SARS-CoV-2 in influent wastewater were quantified in either solid or liquid fraction only. The objectives of this study were (i) to clarify the reduction in SARS-CoV-2 RNA during biological nutrient removal and disinfection processes in full-scale WWTPs, considering the SARS-CoV-2 present in both solid and liquid fractions of wastewater, and (ii) to evaluate applicability of pepper mild mottle virus (PMMoV) as a performance indicator for reduction of SARS-CoV-2 in WWTPs. Accordingly, large amount of SARS-CoV-2 RNA were partitioned in the solid fraction of influent wastewater for composite sampling than grab sampling. When SARS-CoV-2 RNA in the both solid and liquid fractions were considered, log reduction values (LRVs) of SARS-CoV-2 during step-feed multistage biological nitrogen removal (SM-BNR) and enhanced biological phosphorus removal (EBPR) processes ranged between>2.1-4.4 log and did not differ significantly from those in conventional activated sludge (CAS). The LRVs of SARS-CoV-2 RNA in disinfection processes by ozonation and chlorination did not differ significantly. PMMoV is a promising performance indicator to secure reduction of SARS-CoV-2 in WWTPs, because of its higher persistence in wastewater treatment processes and abundance at a detectable concentration even in the final effluent after disinfection.

Moving forward with COVID-19: Future research prospects of wastewater-based epidemiology methodologies and applications.

Wastewater-based epidemiology (WBE) has been demonstrated for its great potential in tracking of coronavirus disease 2019 (COVID-19) transmission among populations despite some inherent methodological limitations. These include non-optimized sampling approaches and analytical methods; stability of viruses in sewer systems; partitioning/retention in biofilms; and the singular and inaccurate back-calculation step to predict the number of infected individuals in the community. Future research is expected to (1) standardize best practices in wastewater sampling, analysis and data reporting protocols for the sensitive and reproducible detection of viruses in wastewater; (2) understand the in-sewer viral stability and partitioning under the impacts of dynamic wastewater flow, properties, chemicals, biofilms and sediments; and (3) achieve smart wastewater surveillance with artificial intelligence and big data models. Further specific research is essential in the monitoring of other viral pathogens with pandemic potential and subcatchment applications to maximize the benefits of WBE beyond COVID-19.

Biological treatment of perfluorooctanesulfonic acid (PFOS) using microbial capsules of a polysulfone membrane.

Perfluorooctanesulfonic acid (PFOS) is a persistent organic substance that has been extensively applied in many industries and causes severe, widespread adverse health impacts on humans and the environment. The development of an effective PFOS treatment method with affordable operational costs has been expected. This study proposes the biological treatment of PFOS using microbial capsules enclosing a PFOS-reducing microbial consortium. The objective of this study was to evaluate the performance of the polymeric membrane encapsulation technique for the biological removal of PFOS. First, a PFOS-reducing bacterial consortium, composed of Paracoccus (72%), Hyphomicrobium (24%), and Micromonosporaceae (4%), was enriched from activated sludge by acclimation and subsequent subculturing with PFOS containing media. The bacterial consortium was first immobilized in alginate gel beads, then enclosed in membrane capsules by coating the gel beads with a 5% or 10% polysulfone (PSf) membrane. The introduction of microbial membrane capsules could increase PFOS reduction to between 52% and 74% compared with free cell suspension, which reduced by 14% over three weeks. Microbial capsules coated with 10% PSf membrane demonstrated the highest PFOS reduction at 80% and physical stability for six weeks. Candidate metabolites including perfluorobutanoic acid (PFBA) and 3,3,3- trifluoropropionic acid were detected by FTMS, suggesting the possible biological degradation of PFOS. In microbial membrane capsules, the initial adsorption of PFOS on the shell membrane layer enhanced subsequent biosorption and biological degradation by PFOS-reducing bacteria immobilized in the core alginate gel beads. The 10%-PSf microbial capsules exhibited a thicker membrane layer with the fabric structure of a polymer network, which maintained longer physical stability than 5%-PSf microbial capsules. This outcome suggests the potential application of microbial membrane capsules to PFOS-contaminated water treatment.

Ammonia tolerance and microbial community in thermophilic co-digestion of sewage sludge initiated with lignocellulosic biomass.

Rice straw is a useful lignocellulosic biomass for controlling ammonia inhibition in the thermophilic anaerobic digestion of sewage sludge. However, it is challenging to procure rice straw throughout the year because of its seasonal production. This study investigated methane production in a laboratory-scale digester by gradually decreasing rice straw addition to solid thermophilic sewage sludge digestion. The decrease in rice straw did not accumulate volatile fatty acids and stabilized methane production. Even with increased sludge concentration without rice straw, methane production continued under high ammonia conditions. Ammonia tolerance of the digested sludge of the experimental digester was higher than that of conventionally digested sludge. The cellulose-degrading bacteria Clostridia and high ammonia-resistant archaea Methanosarcina were dominant in the experimentally digested sludge. The community was maintained for over 200 days after discontinuing the rice straw supply. These findings suggest that anaerobic digestion initiation with rice straw is appropriate to facilitate ammonia-tolerant communities.

Carbon/nitrogen ratios determine biofilm formation and characteristics in model microbial cultures.

The influence of growth medium water chemistry, specifically carbon/nitrogen (C/N) molar ratios, on the characteristics and development of biofilms of the model microorganism Pseudomonas aeruginosa was investigated. C/N = 9 had a unique effect on biofilm composition as well as quorum sensing (QS) pathways, with higher concentrations of carbohydrates and proteins in the biofilm and a significant upregulation of the QS gene lasI in planktonic cells. The effect of C/N ratio on total attached biomass was negligible. Principal component analysis revealed a different behavior of most outputs such as carbohydrates and QS chemicals at C/N = 9, and pointed to correlations between parameters of biofilm formation and steady state distribution of cells and extracellular components. C/N ratio was also shown to influence organic compound utilization by both planktonic and sessile organisms, with a maximum chemical oxygen demand (COD) removal of 83% achieved by biofilms at C/N = 21. Planktonic cells achieved higher COD removal rates, but greater overall rates after six days occurred in biofilms. The development of a dual-species biofilm of P. aeruginosa and Nitrobacter winogradskyi was also influenced by C/N, with increase in the relative abundance of the slower-growing N. winogradskyi above C/N = 9. These results indicate that altering operational parameters related to C/N would be relevant for mitigating or promoting biofilm formation and function depending on the desired industrial application or treatment configuration.

Correlation between SARS-CoV-2 RNA concentration in wastewater and COVID-19 cases in community: A systematic review and meta-analysis.

Wastewater-based epidemiology (WBE) has been considered as a promising approach for population-wide surveillance of coronavirus disease 2019 (COVID-19). Many studies have successfully quantified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentration in wastewater (CRNA). However, the correlation between the CRNA and the COVID-19 clinically confirmed cases in the corresponding wastewater catchments varies and the impacts of environmental and other factors remain unclear. A systematic review and meta-analysis were conducted to identify the correlation between CRNA and various types of clinically confirmed case numbers, including prevalence and incidence rates. The impacts of environmental factors, WBE sampling design, and epidemiological conditions on the correlation were assessed for the same datasets. The systematic review identified 133 correlation coefficients, ranging from -0.38 to 0.99. The correlation between CRNA and new cases (either daily new, weekly new, or future cases) was stronger than that of active cases and cumulative cases. These correlation coefficients were potentially affected by environmental and epidemiological conditions and WBE sampling design. Larger variations of air temperature and clinical testing coverage, and the increase of catchment size showed strong negative impacts on the correlation between CRNA and COVID-19 case numbers. Interestingly, the sampling technique had negligible impact although increasing the sampling frequency improved the correlation. These findings highlight the importance of viral shedding dynamics, in-sewer decay, WBE sampling design and clinical testing on the accurate back-estimation of COVID-19 case numbers through the WBE approach.

Effects of high-strength landfill leachate effluent on stress-induced microalgae lipid production and post-treatment micropollutant degradation.

This research investigates the effects of landfill leachate effluent concentrations from moving bed biofilm reactor (MBBR) on stress-induced Chlorella vulgaris and Scenedesmus armatus lipid production and post-treatment micropollutant degradation. The effluent concentrations were varied between 25%, 50%, 75%, and 100% (v/v). The landfill leachate influent was treated using two-stage moving bed biofilm reactor under 24 h and 18 h hydraulic retention time (HRT). The results indicated that the effluent concentration was positively correlated with the stress-induced microalgae lipid production in the post-treatment of residual micropollutants. C. vulgaris and S. armatus completely remove residual micropollutants in the effluent. The superoxide dismutase and peroxidase activity were positively correlated with the cellular lipid content. The lipid content of C. vulgaris and S. armatus cultivated in the 18 h HRT effluent were 31-51% and 51-64%, while those in the 24 h HRT effluent were 15-16% and 5-19%. The optimal condition of microalgae cultivation for the post-treatment of residual micropollutants was 50-75% (v/v) effluent concentrations under 18 h HRT, achieving the highest lipid production of 113-116 mg/L for C. vulgaris and 74-75 mg/L for S. armatus. Essentially, the MBBR landfill leachate effluent holds promising potential as a substrate for microalgae lipid production.

Removal performance of SARS-CoV-2 in wastewater treatment by membrane bioreactor, anaerobic-anoxic-oxic, and conventional activated sludge processes.

The potential risk of SARS-CoV-2 in treated effluent from a wastewater treatment plant (WWTP) is concerned since SARS-CoV-2 is contained in wastewater during the COVID-19 outbreak. However, the removal of SARS-CoV-2 in WWTP has not been well investigated. The objectives of this study were (i) to clarify the removal performance of SARS-CoV-2 during wastewater treatment, (ii) to compare the removal performance of different secondary treatment processes, and (iii) to evaluate applicability of pepper mild mottle of virus (PMMoV) as a performance indicator for the reduction of SARS-CoV-2 RNA in wastewater treatment. Influent wastewater, secondary-treatment effluent (before chlorination), and final effluent (after chlorination) samples were collected from a WWTP from May 28 to September 24, 2020, during the COVID-19 outbreak in Japan. The target WWTP had three parallel treatment systems employing conventional activated sludge (CAS), anaerobic-anoxic -oxic (A2O), and membrane bioreactor (MBR) processes. SARS-CoV-2 in both the liquid and solid fractions of the influent wastewater was concentrated and quantified using RT-qPCR. SARS-CoV-2 in treated effluent was concentrated from 10 L samples to achieve a detection limit as low as 10 copies/L. The log reduction value (LRV) of SARS-CoV-2 was 2.7 ± 0.86 log10 in CAS, 1.6 ± 0.50 log10 in A2O, and 3.6 ± 0.62 log10 in MBR. The lowest LRV observed during the sampling period was 2.8 log10 in MBR, 1.2 log10 in CAS, and 1.0 log10 in A2O process, indicating that the MBR had the most stable reduction performance. PMMoV was found to be a good indicator virus to evaluate reduction performance of SARS-CoV-2 independent of the process configuration because the LRV of PMMoV was significantly lower than that of SARS-CoV-2 in the CAS, A2O and MBR processes.

CrAssphage as an indicator of human-fecal contamination in water environment and virus reduction in wastewater treatment.

Viral indicators of human-fecal contamination in wastewaters and environmental waters have been getting much attention in the past decade. Cross-assembly phage (crAssphage) is the most abundant DNA virus in human feces. Recently, the usefulness of crAssphage as a microbial source tracking and water quality monitoring tool for human-fecal contamination has been highlighted. Here, we conducted a comprehensive review on crAssphage in water, focusing on detection methodology, concentration range in various waters and wastewaters, specificity to human-fecal contamination, and reduction in wastewater treatment systems. This review highlights that crAssphage is globally distributed in wastewaters and various fecal-contaminated water bodies at high concentrations without seasonal fluctuations. CrAssphage is highly specific to human-fecal contamination and is rarely found in animal feces. It also has a good potential as a performance indicator to ensure virus reduction in wastewater treatment systems. Accordingly, crAssphage could be an effective tool for monitoring of human-fecal contamination and potential presence of fecal pathogenic microbes in environmental waters. Bridging the research gaps highlighted in this review would make crAssphage a powerful tool to support the control of water-related health risks.

Sewage surveillance for SARS-CoV-2: Molecular detection, quantification, and normalization factors.

The presence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in wastewater systems provides a primary indication of the coronavirus disease 2019 (COVID-19) spread throughout communities worldwide. Droplet digital polymerase chain reaction (dd-PCR) or reverse transcription-polymerase chain reaction (RT-PCR) administration of SARS-CoV-2 in wastewaters provides a reliable and efficient technology for gathering secondary local-level public health data. Often the accuracy of prevalence estimation is hampered by many methodological issues connected with wastewater surveillance. Still, more studies are needed to use and create efficient approaches for deciphering the actual SARS-CoV-2 indication from noise in the specimens/samples. Nearly 39-65% of positive patients and asymptomatic carriers expel the virus through their faeces however, only ∼6% of the infected hosts eject it through their urine. COVID-19 positive patients can shed the remnants of the SARS-CoV-2 RNA virus within the concentrations ∼103-108 copies/L. However, it can decrease up to 102 copies/L in wastewaters due to dilution. Environmental virology and microbiology laboratories play a significant role in the identification and analysis of SARS-CoV-2 ribonucleic acid (RNA) in waste and ambient waters worldwide. Virus extraction or recovery from the wastewater (However, due to lack of knowledge, established procedures, and integrated quality assurance/quality control (QA/QC) approaches, the novel coronavirus RNA investigation for estimating current illnesses and predicting future outbreaks is insufficient and/or conducted inadequately. The present manuscript is a technical review of the various methods and factors considered during the identification of SARS-CoV-2 genetic material in wastewaters and/or sludge, including tips and tricks to be taken care of during sampling, virus concentration, normalization, PCR inhibition, and trend line smoothening when compared with clinically active/positive cases.

Artificial neural network-based estimation of COVID-19 case numbers and effective reproduction rate using wastewater-based epidemiology.

As a cost-effective and objective population-wide surveillance tool, wastewater-based epidemiology (WBE) has been widely implemented worldwide to monitor the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentration in wastewater. However, viral concentrations or loads in wastewater often correlate poorly with clinical case numbers. To date, there is no reliable method to back-estimate the coronavirus disease 2019 (COVID-19) case numbers from SARS-CoV-2 concentrations in wastewater. This greatly limits WBE in achieving its full potential in monitoring the unfolding pandemic. The exponentially growing SARS-CoV-2 WBE dataset, on the other hand, offers an opportunity to develop data-driven models for the estimation of COVID-19 case numbers (both incidence and prevalence) and transmission dynamics (effective reproduction rate). This study developed artificial neural network (ANN) models by innovatively expanding a conventional WBE dataset to include catchment, weather, clinical testing coverage and vaccination rate. The ANN models were trained and evaluated with a comprehensive state-wide wastewater monitoring dataset from Utah, USA during May 2020 to December 2021. In diverse sewer catchments, ANN models were found to accurately estimate the COVID-19 prevalence and incidence rates, with excellent precision for prevalence rates. Also, an ANN model was developed to estimate the effective reproduction number from both wastewater data and other pertinent factors affecting viral transmission and pandemic dynamics. The established ANN model was successfully validated for its transferability to other states or countries using the WBE dataset from Wisconsin, USA.