Raw Sewage Samples Research Articles

Seasonal Prevalence and Detection of Enteric and Respiratory Viruses in Wastewater and Hospitalized Children with Acute Gastroenteritis.

Human enteric and some respiratory viruses are identified to be involved with acute gastroenteritis that can be shed in feces of infected persons into the environment. Understanding the abundance of these viruses in wastewater is significant when assessing the public health impacts associated with discharge of wastewater into the environment. This study aimed to investigate the prevalence and seasonal variation of human enteric adenovirus (HAdV), Aichi virus (AiV-1), bocavirus (HBoV), and respiratory syndrome coronavirus 2 virus (SARS-CoV-2) in wastewater as well as their prevalence among hospitalized children with acute gastroenteritis. The viruses were detected and quantified with real-time PCR. HAdV was the most detected virus in raw sewage (88.5%), treated sewage (28%), and stool gastroenteritis (74%), followed by HBoV (45.8% for raw sewage, 14.6% for treated sewage, and 55.3% for stool samples). The detection rate of AiV-1 was 59.4%, 19.8%, and 62.6% in raw sewage, treated sewage, and stool samples, respectively. The rate of SARS-CoV-2 detection in raw sewage, treated sewage, and stool samples was 33.3%, 7.3%, and 20.6%, respectively. The viral concentrations ranged between 4.50 × 101 and 8.75 × 107 GC/ml in raw sewage samples, 1.20 × 101 and 5.43 × 106 GC/ml in treated sewage samples, and 4.80 × 101 and 9.88 × 108 GC/gram in stool samples. The overall log means of virus reduction during the wastewater treatment process ranged from 1.68 log10 (HAdV) to 3.31 log10 (AiV-1). The peak prevalence of the four viruses in raw sewage samples occurred during the winter season. This study showed the high detection rates of the four targeted viruses in wastewater and demonstrated that virological surveillance of wastewater in local wastewater treatment plants is a suitable model for epidemiological monitoring of diarrheal and respiratory diseases in middle- and low-resource countries.

Assessment of crAssphage as a biological variable for SARS-CoV-2 data normalization in wastewater surveillance.

This study aimed to assess the use of cross-assembled phage (crAssphage) as an endogenous control employing a multivariate normalization analysis and its application as a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) data normalizer. A total of 188 twelve-hour composite raw sewage samples were obtained from eight wastewater treatment plants (WWTP) during a 1-year monitoring period. Employing the N1 and N2 target regions, SARS-CoV-2 RNA was detected in 94% (177) and 90% (170) of the samples, respectively, with a global median of 5 log10 genomic copies per liter (GC l-1). CrAssphage was detected in 100% of the samples, ranging from 8.29 to 10.43 log10 GC l-1, with a median of 9.46±0.40 log10 GC l-1, presenting both spatial and temporal variabilities. Although SARS-CoV-2 data normalization employing crAssphage revealed a correlation with clinical cases occurring during the study period, crAssphage normalization by the flow per capita per day of each WWTP increased this correlation, corroborating the importance of normalizing wastewater surveillance data in disease trend monitoring.

Cold-chain free nucleic acid preservation using porous super-absorbent polymer (PSAP) beads to facilitate wastewater surveillance

The instability of viral targets including SARS-CoV-2 in sewage is an important challenge in wastewater monitoring projects. The unrecognized interruptions in the ‘cold-chain’ transport from the sample collection to RNA quantification in the laboratory may undermine the accurate quantification of the virus. In this study, bovine serum albumin (BSA)-modified porous superabsorbent polymer (PSAP) beads were applied to absorb raw sewage samples as a simple method for viral RNA preservation. The preservation efficiency for SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA were examined during storage for 14 days at 4 °C or room temperature against the control (no beads applied). While a non-significant difference was observed at 4 °C (∼80 % retention for both control and PSAP-treated sewage), the reduction of SARS-CoV-2 RNA concentrations was significantly lower in sewage retrieved from PSAP beads (25–40 % reduction) compared to control (>60 % reduction) at room temperature. On the other hand, the recovery of PMMoV, known for its high persistence in raw sewage, from PSAP beads or controls were consistently above 85 %, regardless of the storage temperature. Our results demonstrate the applicability of PSAP beads to wastewater-based epidemiology (WBE) projects for preservation of SARS-CoV-2 RNA in sewage, especially in remote settings with no refrigeration capabilities.

Case study on water quality index for wastewater treatment in Mumbai

Abstract The traditional wastewater treatment process is laborious and demanding in terms of time, energy, and space. With rapid advancements, there's a quest for more efficient techniques that can achieve comparable treatment outcomes while minimizing these demands. This study proposes a solution by employing ozone as an Advanced Oxidation Process (AOP) to expedite the treatment while enhancing the water quality. The Water Quality Index (WQI) offers a consolidated representation of overall water quality by considering multiple quality parameters. Although typically used for assessing surface water quality, this study utilizes WQI to gauge the enhancement in parameters post-treatment. The research investigates the impact of varied ozone doses on raw sewage samples collected from four different sites in Mumbai, focusing on parameters such as dissolved oxygen, turbidity, hardness, chemical oxygen demand (COD), and WQI. The results demonstrate a notable percentage enhancement in WQI, ranging from 30 to 60% across various sites within a short 25-minute timeframe, attributed to the application of ozone.

Quantification and Potential Viability of Human Noroviruses in Final Effluent from Wastewater Treatment Works in Pretoria, South Africa

Growing global concerns over water scarcity, worsened by climate change, drive wastewater reclamation efforts. Inadequately treated wastewater presents significant public health risks. Previous studies in South Africa (SA) have reported high norovirus levels in final effluent and sewage-polluted surface water, indicating pathogen removal inefficiency. However, the viability of these virions was not explored. This study assessed human norovirus viability in final effluent from wastewater treatment works (WWTWs) in Pretoria, SA. Between June 2018 and August 2020, 200 samples were collected from two WWTWs, including raw sewage and final effluent. Norovirus concentrations were determined using in-house RNA standards. Viability of noroviruses in final effluent was assessed using viability RT-qPCR (vPCR) with PMAxx™-Triton X-100. There was no significant difference in GI concentrations between raw sewage (p = 0.5663) and final effluent (p = 0.4035) samples at WWTW1 and WWTW2. WWTW1 had significantly higher GII concentrations in raw sewage (p < 0.001) compared to WWTW2. No clear seasonal pattern was observed in norovirus concentrations. At WWTW1, 50% (7/14) of GI- and 64.9% (24/37) of GII-positive final effluent samples had no quantifiable RNA after vPCR. At WWTW2, the majority (92.6%, 25/27) of GII-positive final effluent samples showed a 100% RNA reduction post vPCR. PMAxx™-Triton X-100 vPCR provides a more accurate reflection of discharge of potentially viable noroviruses in the environment than standard RT-qPCR. Despite significant reductions in potentially viable noroviruses after wastewater treatment, the levels of potentially viable viruses in final effluent are still of concern due to the high initial load and low infectious dose of noroviruses.

One-Year Investigation of Hepatitis A Virus and Rotavirus in Two Waste Water Treatment Plants in Egypt

Background: Waterborne enteric viruses are evolving source of disease outbreaks and represent a major threat to global public health. The repetitive assessment of water environments is necessary to manage enteric virus-mediated fecal infection and the probable emergence of different variants. Methods: Here, we detected human rotavirus and hepatitis A virus circulating in two wastewater treatment plants, for one year in sewage and sludge samples from two largest wastewater treatment plant (El-Gabal El-Asfar and Zenin) located in Egypt. For this purpose, we examine predefined viruses' prevalence in 72 samples (24 raw sewage samples, 24 treated effluent samples, and 24 sludge) collected from August 2022 to July 2023. Viral RNA was extracted and detected by nested Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) in case of HAV and by multiplex semi nested RT-PCR In case of rotavirus with the Results: Percentage of detection rate 58.3%, 25% 41.6%, 50%, 16.6%58.6%, 41.6%, 8.3%, 41.6%41.6%, 8.3% and 41.6% for inlet, outlet, sludge in WWTP zenin and WWTP El-Gabal El-Asfar for HAV, RV respectively. Using generic primer result indicated G1P8 was the most common although G2P [4], G1, G2, G3P? G9P [8], G9P [11], G1 P [4] were detected with different percentage. Conclusion: These results demonstrated that HAV and RV still remains in the environment after sewage treatment and could play an important role in maintaining the endemicity of HAV and RV infection. Routine and continuous monitoring of waterborne viruses are most useful approach to understand disease occurrence in communities.

Environmental surveillance for COVID-19 using SARS-CoV-2 RNA concentration in wastewater – a study in District East, Karachi, Pakistan

Wastewater-based surveillance is used to track the temporal patterns of the SARS-CoV-2 virus in communities. Viral RNA particle detection in wastewater samples can indicate an outbreak within a catchment area. We describe the feasibility of using a sewage network to monitor SARS-CoV-2 trend and use of genomic sequencing to describe the viral variant abundance in an urban district in Karachi, Pakistan. This was among the first studies from Pakistan to demonstrate the surveillance for SARS-CoV-2 from a semi-formal sewage system. Four sites draining into the Lyari River in District East, Karachi, were identified and included in the current study. Raw sewage samples were collected early morning twice weekly from each site between June 10, 2021 and January 17, 2022, using Bag Mediated Filtration System (BMFS). Secondary concentration of filtered samples was achieved by ultracentrifugation and skim milk flocculation. SARS-CoV-2 RNA concentrations in the samples were estimated using PCR (Qiagen ProMega kits for N1 & N2 genes). A distributed-lag negative binomial regression model within a hierarchical Bayesian framework was used to describe the relationship between wastewater RNA concentration and COVID-19 cases from the catchment area. Genomic sequencing was performed using Illumina iSeq100. Among the 151 raw sewage samples included in the study, 123 samples (81.5%) tested positive for N1 or N2 genes. The average SARS-CoV-2 RNA concentrations in the sewage samples at each lag (1-14 days prior) were associated with the cases reported for the respective days, with a peak association observed on lag day 10 (RR: 1.15; 95% Credible Interval: 1.10-1.21). Genomic sequencing showed that the delta variant dominated till September 2022, while the omicron variant was identified in November2022. Wastewater-based surveillance, together with genomic sequencing provides valuable information for monitoring the community temporal trend of SARS-CoV-2. PATH, Bill & Melinda Gates Foundation, and Global Innovation Fund.

Evaluating the aeration efficiency of hydraulic flume on photo-oxidative disinfection of wastewater

Photosensitized oxidation has been considered to offer better disinfection mechanism under natural sunlight and the efficiency of its inactivation pathway depends on the concentration of dissolving oxygen, the solar irradiance, and the reactive nature of the liquid. This study aimed to evaluate the hydraulic mixer's aeration performance during photo-oxidation treatment. A hydraulic jump was created to aerate the sewage prior to its passage into a reactor, and the amount of dissolved oxygen generated was measured. The effect of mixing intensity on sunlight inactivation of faecal coliforms in a reactor was investigated for 60 min using velocity gradients of 520 s−1, 640 s−1, 720 s−1 and 960 s−1 produced under flow rates of 3 L min−1, 5 L min−1 and 7 L min−1 and 10 L min−1 respectively. The photo-oxidation kinetics of faecal coliform contained in settled and raw sewage samples were also studied under two dark control tests (static and dynamic tests) and a light test. The system achieved a 2.0–2.34 log reduction of faecal coliforms and dissolved oxygen was within the range of 1.9 mg L−1 - 3.0 mg L−1. In the dynamic control test, the inactivation rate constant of bacteria indicated a reduction of 1.11 log (92%) for settled sewage and 1.0 log (90%) for raw sewage. The coliform inactivation rate constant, due to the combined effect of turbulent mixing speed and dark inactivation rate constant, was 2.3 h−1, and the solar irradiance inactivation rate constant was 2.4 h−1. These results have demonstrated that induced aeration is an economically friendly approach to enhance wastewater treatment in developing nations with long sunshine hours.

Candida palmioleophila: A New Emerging Threat in Brazil?

Human activity directly or indirectly causes climate change, promoting changes in the composition of the atmosphere. This change is beyond the variation of the natural climate. In this manner, climate change could create an environmental pressure which is enough to trigger new fungal diseases. In addition to climate alterations, the onset of the COVID-19 pandemic has also been associated with the emergence of fungal pathogens. Fungi showed that an inability to grow at high temperatures limits the capacity of fungi to infect mammals. However, fungi can develop thermotolerance, gradually adapting to rising temperatures due to climate change, and generating a greater number of disease-causing organisms. In the present study, we reported the detection and identification of Candida palmioleophila isolates recovered from raw sewage samples in Niteroi city, Rio de Janeiro State, Brazil, during a monitoring program for measuring SARS-CoV-2 presence and concentration. Using polyphasic taxonomy to identify the species and evaluating some virulence aspects of this species, such as biofilm formation and extracellular enzyme production, our data highlight this species as a possible emerging pathogen in Brazil, especially in the pandemic context.

High prevalence of hepatitis A and E viruses in environmental and clinical samples from West Argentina.

Environmental surveillance of water sources is important to monitoring viral hepatitis transmission in clinical settings. This study investigated the circulation of hepatitis A (HAV) and E (HEV) viruses in sewage and clinical samples from Argentina. Between 2016 and 2017, 80 raw sewage samples and 86 clinical samples (stool and serum) from suspected cases of hepatitis A and hepatitis E were obtained. HAV and HEV were tested by both real-time and nested PCR. Positive samples were sequenced for genotype determination and phylogenetic analysis. Overall, HAV was recovered in 39% of sewage samples and 61.1% of clinical samples. HEV was detected in 22.5% of sewage samples and 15.9% of clinical samples. HAV was found more frequently in sewage during the winter and in clinical samples in spring; HEV was more prevalent in sewage during summer and in clinical samples in autumn. All HAV isolates belonged to genotype IA and HEV isolates belonged to genotype 3, the most prevalent genotypes in South America. High prevalence of HAV and HEV in environmental and clinical samples in Mendoza, Argentina was observed. These findings reinforce the importance of environmental surveillance and implementation of health strategies to control the spread of HAV and HEV in developing countries.

Antibiotic resistance profile of wastewater treatment plants in Brazil reveals different patterns of resistance and multi resistant bacteria in final effluents.

Wastewater treatment plants (WWTPs) are recognized as important sources of Antibiotic Resistant Bacteria (ARBs) and Antibiotic Resistant Genes (ARGs), and might play a role in the removal and dissemination of antimicrobial resistance (AMR) in the environment. Detailed information about AMR removal by the different treatment technologies commonly applied in urban WWTPs is needed. This study investigated the occurrence, removal and characterization of ARBs in WWTPs employing different technologies: WWTP-A (conventional activated sludge-CAS), WWTP-B (UASB reactor followed by biological trickling filter) and WWTP-C (modified activated sludge followed by UV disinfection-MAS/UV). Samples of raw sewage (RI) and treated effluent (TE) were collected and, through the cultivation-based method using 11 antibiotics, the antibiotic resistance profiles were characterized in a one-year period. MAS was effective in reducing ARB counts (2 to 3 log units), compared to CAS (1 log unit) and UASB/BTF (0.5 log unit). The composition of cultivable ARB differed between RI and TE samples. Escherichia was predominant in RI (56/118); whilst in TE Escherichia (31/118) was followed by Bacillus (22/118), Shigella (14/118) and Enterococcus (14/118). Most of the isolates identified (370/394) harboured at least two ARGs and in over 80 % of the isolates, 4 or more ARG (int1, blaTEM, TetA, sul1 and qnrB) were detected. A reduction in the resistance prevalence was observed in effluents after CAS and MAS processes; whilst a slight increase was observed in treated effluents from UASB/BTF and after UV disinfection stage. The multi-drug resistance (MDR) phenotype was attributed to 84.3 % of the isolates from RI (27/32) and 63.6 % from TE (21/33) samples and 52.3 % of the isolates (34/65) were resistant to carbapenems (imipenem, meropenem, ertapenem). The results indicate that treated effluents are still a source for MDR bacteria and ARGs dissemination to aquatic environments. The importance of biological sewage treatment was reinforced by the significant reductions in ARB counts observed. However, implementation of additional treatments is needed to mitigate MDR bacteria release into the environment.

Wastewater-based surveillance in Italy leading to the first detection of mcr-10-positive Klebsiella quasipneumoniae

Wastewater-based surveillance enabled the first detection of the mobile colistin resistance gene mcr-10 in Italy. This plasmid-borne resistance gene was found in strains of Klebsiella quasipneumoniae isolated from samples of human raw sewage collected over several months. Although the isolates were phenotypically susceptible to colistin, the emergence of mcr-10 is concerning due to the highly variable expression of the gene and the potential for horizontal transfer to other species. In addition, the strains also carried an extended-spectrum β-lactamase gene and were phenotypically resistant to several beta-lactams. This study highlights the value of wastewater-based surveillance as an effective tool to monitor the emergence of antimicrobial resistance in strains circulating in the community and the environment.

Multiple Novel Human Norovirus Recombinants Identified in Wastewater in Pretoria, South Africa by Next-Generation Sequencing.

The genogroup II genotype 4 (GII.4) noroviruses are a major cause of viral gastroenteritis. Since the emergence of the Sydney_2012 variant, no novel norovirus GII.4 variants have been reported. The high diversity of noroviruses and periodic emergence of novel strains necessitates continuous global surveillance. The aim of this study was to assess the diversity of noroviruses in selected wastewater samples from Pretoria, South Africa (SA) using amplicon-based next-generation sequencing (NGS). Between June 2018 and August 2020, 200 raw sewage and final effluent samples were collected fortnightly from two wastewater treatment plants in Pretoria. Viruses were recovered using skimmed milk flocculation and glass wool adsorption-elution virus recovery methods and screened for noroviruses using a one-step real-time reverse-transcription PCR (RT-PCR). The norovirus BC genotyping region (570-579 bp) was amplified from detected norovirus strains and subjected to Illumina MiSeq NGS. Noroviruses were detected in 81% (162/200) of samples. The majority (89%, 89/100) of raw sewage samples were positive for at least one norovirus, compared with 73% (73/100) of final effluent samples. Overall, a total of 89 different GI and GII RdRp-capsid combinations were identified, including 51 putative novel recombinants, 34 previously reported RdRp-capsid combinations, one emerging novel recombinant and three Sanger-sequencing confirmed novel recombinants.

High occurrence of viable forms of Cryptosporidium and Giardia in domestic sewage from an agricultural region of Brazil.

Cryptosporidium and Giardia are the main etiologies of waterborne outbreaks caused by protozoa. These parasites are commonly detected in wastewater; however, there is little knowledge about the concentration of viable forms in treated sewage, mainly in small communities. To understand more about the presence of viable oocysts and cysts in domestic sewage, we monitored the affluent and effluent of a wastewater treatment plant (WWTP) in inner-city Brazil. Ten samplings and seven follow-ups were performed in 2020. Samples were concentrated by centrifugation, filtration and purified by fluctuation. Viability was accessed by propidium-monoazide (PMA) associated with nPCR and qPCR. Both viable protozoa were detected in all raw sewage samples (average: 438.5 viable oocysts/L). Regarding treated sewage, Cryptosporidium was detected in all of the samples (average: 92.8 viable oocysts/L) and Giardia was detected in 70% with viable cysts in 30%. Considering the follow-ups, 31.17% of Cryptosporidium viable oocysts remained in the effluent after the treatment. High amounts of Cryptosporidium and a high frequency of Giardia were detected, therefore both arrived at WWTP and were discharged into the river. These alert the presence of agro-industrial effluents into domestic sewage and demonstrated the effectiveness of the concentration technique for monitoring protozoa in wastewater.

Full-Length 16S rRNA Gene Sequences from Raw Sewage Samples Spanning Geographic and Seasonal Gradients in Conveyance Systems across the United States.

ABSTRACTWastewater microbiome research often relies on sequencing of hypervariable regions of 16S rRNA genes, which are difficult to classify at refined taxonomic levels. Here, we introduce a data set of near-full-length 16S rRNA genes from samples designed to capture known geographic and seasonal variations in municipal wastewater microbial communities.

Quantification of infectious Human mastadenovirus in environmental matrices using PMAxx-qPCR.

Molecular methodologies providing data on viral concentration and infectivity have been successfully used in environmental virology, supporting quantitative risk assessment studies. The present study aimed to assess human mastadenovirus (HAdV) intact particles using a derivative of propidium monoazide associated with qPCR (PMAxx-qPCR) in aquatic matrices. Initially, different concentrations of PMAxx were evaluated to establish an optimal protocol for treating different naturally contaminated matrices, using 10 min incubation in the dark at 200 rpm at room temperature and 15 min of photoactivation in the PMA-Lite™ LED photolysis device. There was no significant reduction in the quantification of infectious HAdV with increasing concentration of PMAxx used (20 μM, 50 μM, and 100 μM), except for sewage samples. In this matrix, a reduction of 5.01 log of genomic copies (GC)/L was observed from the concentration of 50 μM and revealed 100% HAdV particles with damaged capsids. On the other hand, the mean reduction of 0.51 log in stool samples using the same concentration mentioned above demonstrated 83% of damaged particles eliminated in the stool. Following, 50 μM PMAxx-qPCR protocol revealed a log reduction of 0.91, 0.67, and 1.05 in other samples of raw sewage, brackish, and seawater where HAdV concentration reached 1.47 × 104, 6.81 × 102, and 2.33 × 102 GC/L, respectively. Fifty micrometers of PMAxx protocol helped screen intact viruses from different matrices, including sea and brackish water.

Wastewater based epidemiology as a silent sentinel of the trend of SARS-CoV-2 circulation in the community in central Argentina

Monitoring wastewater for the traces of viruses allows effective surveillance of entire communities, including symptomatic and asymptomatic infected individuals, providing information on whether a specific pathogen is circulating in a population. In the context of the COVID-19 pandemic, 261 wastewater samples from six communities of the province of Córdoba, Argentina were analyzed. From mid-May 2020 to the end of August 2021, raw sewage samples were collected from the central network pipe that enters into the Wastewater Treatment Plants (WWTP) in Córdoba city and five communities in the Punilla Valley. SARS-CoV-2 was concentrated by using the polyethylene glycol-6000 precipitation method. Viral genomes were extracted from concentrated samples, and N- and E-SARS-CoV-2 genes were detected by using real time RT-PCR. Wastewater samples that resulted positive for SARS-CoV-2 genome detection were subjected to viral variants of concern (VOCs) identification by real time RT-PCR. Overall, just by using the identification of the N gene or E gene, the rates of viral genome detection were 43.4% (86/198) and 51.5% (102/198) respectively, and by using both methodologies (positivity criterion: detection of N and / or E gene), the detection rate was 71.2% (141/198). Thereby, the optimal strategy to study the SARS-CoV-2 genome in wastewater would be the use of the combined detection of both genes. Detection of SARS-CoV-2 variants in wastewater reflected their circulation in the community, showing no VOCs detection in the first COVID-19 wave and their co-circulation with Gamma, Alpha and Delta VOCs during 2021. Therefore, SARS-CoV-2 Wastewater Based Epidemiology (WBE) described the introduction, permanence and/or the co-circulation of viral variants in the community. In geographical areas with a stable population, SARS-CoV-2 WBE could be used as an early warning sign of new COVID-19 cases, whereas in localities with a low number of inhabitants and high tourist influx, WBE may only be useful to reflect the circulation of the virus in the community. Overall, the monitoring of SARS-CoV-2 in wastewater can become a silent sentinel of the trend of viral circulation in the community, providing supplementary information for clinical surveillance to support public health measures.

Complete Genome Sequences of Five Bacteriophages That Infect Enterobacteriales Hosts.

ABSTRACTFull genome sequences of five bacteriophages that were isolated from raw sewage samples and infect Enterobacteriales hosts are presented. Brookers is a P22-like Proteus phage, OddieOddie is a 9g-like Escherichia coli phage, Diencephelon is a Kp3-like Klebsiella phage, and Rgz1 and Lilpapawes are classic T4-like and T7-like virulent Proteus phages, respectively.

Quantification of human enteric viruses as alternative indicators of fecal pollution to evaluate wastewater treatment processes.

We investigated the potential use and quantification of human enteric viruses in municipal wastewater samples of Winnipeg (Manitoba, Canada) as alternative indicators of contamination and evaluated the processing stages of the wastewater treatment plant. During the fall 2019 and winter 2020 seasons, samples of raw sewage, activated sludge, effluents, and biosolids (sludge cake) were collected from the North End Sewage Treatment Plant (NESTP), which is the largest wastewater treatment plant in the City of Winnipeg. DNA (Adenovirus and crAssphage) and RNA enteric viruses (Pepper mild mottle virus, Norovirus genogroups GI and GII, Rotavirus Astrovirus, and Sapovirus) as well as the uidA gene found in Escherichia coli were targeted in the samples collected from the NESTP. Total nucleic acids from each wastewater treatment sample were extracted using a commercial spin-column kit. Enteric viruses were quantified in the extracted samples via quantitative PCR using TaqMan assays. Overall, the average gene copies assessed in the raw sewage were not significantly different (p-values ranged between 0.1023 and 0.9921) than the average gene copies assessed in the effluents for DNA and RNA viruses and uidA in terms of both volume and biomass. A significant reduction (p-value ≤ 0.0438) of Adenovirus and Noroviruses genogroups GI and GII was observed in activated sludge samples compared with those for raw sewage per volume. Higher GCNs of enteric viruses were observed in dewatered sludge samples compared to liquid samples in terms of volume (g of sample) and biomass (ng of nucleic acids). Enteric viruses found in gene copy numbers were at least one order of magnitude higher than the E. coli marker uidA, indicating that enteric viruses may survive the wastewater treatment process and viral-like particles are being released into the aquatic environment. Viruses such as Noroviruses genogroups GI and GII, and Rotavirus were detected during colder months. Our results suggest that Adenovirus, crAssphage, and Pepper mild mottle virus can be used confidently as complementary viral indicators of human fecal pollution.

SARS-CoV-2 variant detection from wastewater: rapid spread of B.1.1.7 lineage in Hungary.

Wastewater-based epidemiology (WBE) is a recognised tool for tracking community transmission of COVID-19. From the second half of 2020, the emergence of new, highly infective, more pathogenic or vaccine-escape SARS-CoV-2 variants is the major public health concern. Variant analysis in sewage might assist the early detection of new mutations. Weekly raw sewage samples from 22 wastewater treatment plants (WWTPs) in Hungary (representing 40% of the population) were analysed between December 2020 and March 2021 for signature mutations N501Y and del H69/V70 of B.1.1.7 lineage by melting point genotyping and RT-digital droplet PCR (RT-ddPCR). The latter method proved to be more efficient in parallel detection of different variants and also provides quantitative information. Wastewater surveillance indicated that the B.1.1.7 variant first emerged in Budapest in early January 2021 and rapidly became dominant in the entire country. Results are in close agreement with the available clinical data (Pearson's correlation coefficient, R = 0.9153). RT-ddPCR was confirmed to be a reliable tool for tracking emerging variant ratios in wastewaters. It is a rapid and cost-effective method compared to whole-genome sequencing, but only applicable for the detection of known mutations. Efficient variant surveillance might require the combination of multiple methods.