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Arboviruses are viral pathogens transmitted by blood-borne vectors that impose a great social and economic burden globally. Most clinical surveillance of arbovirus outbreaks underestimates the true prevalence as a large proportion of cases exhibit no or only mild clinical symptoms (i.e., are subclinical). Reports of urinary shedding of several arboviruses such as Dengue (DENV), yellow fever (YFV), and Zika (ZIKV) viruses suggest the possibility of utilizing wastewater surveillance to assess the prevalence of arboviral outbreaks. To determine the feasibility of wastewater surveillance, we investigated the decay of representative arboviruses (including DENV-2, DENV-3, YFV, and ZIKV) along with murine hepatitis virus (MHV) as a surrogate for human coronavirus within a wastewater matrix at 6, 25, or 37 °C using RT-qPCR. DENV-2, DENV-3, YFV, ZIKV, and MHV experienced a one log10reduction within 3.95-6.21 days at 25 °C and within 2.60-5.12 days at 37 °C, while incubation at 6 °C did not indicate substantial decay within 21 days. Our work suggests that these arboviruses or their RNA could persist sufficiently in wastewater over a range of temperatures, supporting the potential for wastewater-based surveillance of arboviral outbreaks. Ministry of Education (MOE) National Research Foundation (NRF) Singapore-MIT Alliance for Research and Technology (SMART) This research was supported by the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program, through Intra-CREATE Thematic Grant (Cities) Grant NRF2019-THE001-0003 to J.T. and E.J.A., the Singapore-MIT Alliance for Research and Technology (SMART) Antimicrobial Resistance Interdisciplinary Research Group (AMR IRG), and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to the Singapore Centre for Environmental Life Sciences Engineering (SCELSE).

In recent decades, natural swimming pools (NSPs) have gained popularity in Europe, especially in Germany and Austria. NSPs differ from swimming pools in that they utilize biological treatment processes based on wetland processes with no disinfection residual. However, data are missing on the specific log-reduction performance of NSPs to address enteric virus, bacteria, and parasitic protozoa removal considered necessary to meet the North American risk-based benchmark (

Controlled struvite (NH4MgPO4·6H2O) precipitation has become a well-known process for nutrient recovery from wastewater treatment systems to alleviate the pressures of diminishing, finite rock phosphate reservoirs. Nonetheless, coprecipitation of potential microbial and chemical hazards is poorly understood. On the other hand, antimicrobial resistance (AMR) is a major global public health concern and wastewater is thought to disseminate resistance genes within bacteria. Fecal indicator bacteria (FIB) are typically used as measures of treatment quality, and with multiresistant E. coli and Enterococcus spp. rising in concern, the quantification of FIB can be used as a preliminary method to assess the risk of AMR. Focusing on struvite produced from full-scale operations, culture and qPCR methods were utilized to identify FIB, antibiotic resistance genes, and human enteric viruses in the final product. Detection of these hazards occurred in both wet and dry struvite samples indicating that there is a potential risk that needs further consideration. Chemical and biological analyses support the idea that the presence of other wastewater components can impact struvite formation through ion and microbial interference. While heavy metal concentrations met current fertilizer standards, the presence of K, Na, Ca, and Fe ions can impact struvite purity yet provide benefit for agricultural uses. Additionally, the quantified hazards detected varied among struvite samples produced from different methods and sources, thus indicating that production methods could be a large factor in the risk associated with wastewater-recovered struvite. In all, coprecipitation of metals, fecal indicator bacteria, antimicrobial resistance genes, and human enteric viruses with struvite was shown to be likely, and future engineered wastewater systems producing struvite may require additional step(s) to manage these newly identified public health risks.

The Delta (B.1.617.2) variant has caused major devastation in India and other countries around the world. First detected in October 2020, it has now spread to more than 100 countries, prompting WHO to declare it as a global variant of concern (VOC). The Delta (B.1.617.2), Delta plus (B.1.617.2.1) and Kappa (B.1.617.1) variants are all sub-lineages of the original B.1.617 variant. Prior to the inception of B.1.617, vaccine rollout, safe-distancing and timely lockdowns greatly reduced COVID-19 hospitalizations and deaths. However, the Delta variant, allegedly more infectious and for which existing vaccines seemed less effective, has catalyzed the resurgence of cases. Therefore, there is an imperative need for increased surveillance of the B.1.617 variants. While the Beta variant is increasingly outpaced by the Delta variant, the spread of the Beta variant remains of concern due to its vaccine resistance. Efforts have been made to utilize wastewater-based surveillance for community-based tracking of SARS-CoV-2 variants, however wastewater with its low SARS-CoV-2 viral titers and mixtures of viral variants, requires assays to be variant-specific yet accurately quantitative for meaningful interpretation. Following on the design principles of our previous assays for the Alpha variant, here we report allele-specific and multiplex-compatible RT-qPCR assays targeting mutations T19R, D80A, K417N, T478K and E484Q, for quantitative detection and discrimination of the Delta, Delta plus, Kappa and Beta variants in wastewater. This method is open-sourced and can be implemented using commercially available RT-qPCR protocols, and would be an important tool for tracking the spread of B.1.617 and the Beta variants in communities.

Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-one recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009–2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 μM; incubation on ice or at room temperature (20 - 25 °C) for 5–120 min; and dye activation using LED or high light (500–800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever., Graphical abstract Image 1, Highlights • PMA/PMAxx have higher efficiency removing false negatives from qPCR for DNA/RNA viruses than EMA. • One size fits all pretreatment approaches are possible but lead to lower virus signal reduction. • Capsid integrity qPCR is a valuable tool to adapt existent workflows for improving risk assessment. • Azo dye pretreatment can help refine significance of qPCR during virus outbreaks.

Wastewater-based epidemiology (WBE) has emerged as a critical public health tool in tracking the SARS-CoV-2 epidemic. Monitoring SARS-CoV-2 variants of concern in wastewater has to-date relied on genomic sequencing, which lacks sensitivity necessary to detect low variant abundances in diluted and mixed wastewater samples. Here, we develop and present an open-source method based on allele specific RT-qPCR (AS RT-qPCR) that detects and quantifies the B.1.1.7 variant, targeting spike protein mutations at three independent genomic loci highly predictive of B.1.1.7 (HV69/70del, Y144del, and A570D). Our assays can reliably detect and quantify low levels of B.1.1.7 with low cross-reactivity, and at variant proportions between 0.1% and 1% in a background of mixed SARS-CoV-2. Applying our method to wastewater samples from the United States, we track B.1.1.7 occurrence over time in 19 communities. AS RT-qPCR results align with clinical trends, and summation of B.1.1.7 and wild-type sequences quantified by our assays strongly correlate with SARS-CoV-2 levels indicated by the US CDC N1/N2 assay. This work paves the path for rapid inexpensive surveillance of B.1.1.7 and other SARS-CoV-2 variants in wastewater.

The presence of biofilms in drinking water distribution systems (DWDS) can affect both water quality and system integrity; yet these systems remain poorly studied due to lack of accessibility. We established two independent full-scale DWDS Testbeds (A and B) on two different campuses situated in a tropical urban environment and equipped them with online sensors. Testbed B experienced higher levels of monochloramine and lower water age than Testbed A within the campus. Based on long amplicon-sequencing of bacterial 16S rRNA genes extracted from the mature biofilms (MPB) growing on pipes and young biofilms (YSB) growing on the sensors, a core community was identified in the two testbeds. The relative abundances of operational taxonomic units at the family level, including Mycobacteriaceae, Methylobacteriaceae, Rhodospirillaceae, Nitrosomonadaceae, and Moraxellaceae, were consistent for MPB and YSB on each campus. The MPB community was found to be influenced by conductivity, sample age, and pipe diameter as determined by both canonical correlation analysis and fuzzy set ordination. MPB displayed higher α-diversity based on Hill numbers than YSB; in general, second order Hill numbers correlated positively with conductivity and sample age, but negatively with ORP and nitrite. Pseudomonas spp. together with Bacillus spp. likely initiated biofilm formation of YSB on Testbed A under conditions of reduced monochloramine and high water age. Significant levels of orthophosphate were detected in YSB samples at two stations and associated with higher levels of stagnation based on long-term differential turbidity measurement (DTM). Orthophosphate and DTM may act as indicators of the biofilm growth potential within DWDS.Highlights- Established two testbeds to study biofilms in full-scale distribution system- Biofilms on pipes and sensors had core community- Temporal effect and higher α-diversity for biofilms on pipes- Water chemistry was related to biofilm community differencesGraphical Abstract

The critical need for surveillance of SARS-CoV-2 variants of concern has prompted the development of methods that can track variants in wastewater. Here, we develop and present an open-source method based on allele-specific RT-qPCR (AS RT-qPCR) that detects and quantifies the B.1.1.7 variant, targeting spike protein mutations at three independent genomic loci that are highly predictive of B.1.1.7 (HV69/70del, Y144del, and A570D). Our assays can reliably detect and quantify low levels of B.1.1.7 with low cross-reactivity, and at variant proportions down to 1% in a background of mixed SARS-CoV-2. Applying our method to wastewater samples from the United States, we track the occurrence of B.1.1.7 over time in 19 communities. AS RT-qPCR results align with clinical trends, and summation of B.1.1.7 and wild-Type sequences quantified by our assays matches SARS-CoV-2 levels indicated by the U.S. CDC N1 and N2 assays. This work paves the way for AS RT-qPCR as a method for rapid inexpensive surveillance of SARS-CoV-2 variants in wastewater. Ministry of Education (MOE) National Research Foundation (NRF) Published version This research is supported by the National Research Foundation, Prime Minister's Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program, Intra-CREATE Thematic Grant (Cities) Grant NRF2019-THE001-0003a to J.T. and E. J.A., and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to the Singapore Centre for Environmental Life Sciences Engineering (SCELSE) to J.T. This work was also supported by funds from the Massachusetts Consortium on Pathogen Readiness and China Evergrande Group (M.M. and E.J.A.).

Fecal contamination of surface water is commonly evaluated by quantification of bacterial or viral indicators such as Escherichia coli and coliphages, or by direct testing for pathogens such as enteric viruses. Retention of fecally derived organisms in biofilms and sediments is less frequently considered. In this study, we assessed the distribution of E. coli, somatic coliphages, and enteric viruses including human adenovirus (HAdV), enterovirus (EV), norovirus genogroup GII (NoV GII) and group A rotavirus (RoV) in an urban river environment in Germany. 24 samples each of water, epilithic biofilms and sediments were examined. E. coli and somatic coliphages were prevalent not only in the flowing water, but also in epilithic biofilms and sediments, where they were accumulated compared to the overlying water. During enhanced rainfall, E. coli and coliphage concentrations increased by approximately 2.5 and 1 log unit, respectively, in the flowing water, whereas concentrations did not change significantly in epilithic biofilms and sediments. The occurrence of human enteric viruses detected by qPCR was higher in water than in biofilms and sediments. 87.5% of all water samples were positive for HAdV. Enteric viruses found less frequently were EV, RoV and NoV GII in 20.8%, 16.7% and 8.3% of the water samples, respectively. In epilithic biofilms and sediments, HAdV was found in 54.2% and 50.0% of the samples, respectively, and EV was found in 4.2% of both biofilm and sediment samples. RoV and NoV GII were not detected in any of the biofilms and sediments. Overall, the prevalence of enteric viruses was in the order of HAdV > EV > RoV ≥ NoV GII. In conclusion, epilithic biofilms and sediments can be reservoirs for fecal indicators and enteric viruses and thus should be taken into consideration when assessing microbial pollution of surface water environments.

Pathogenic free-living amoebae (FLA), such as Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba species isolated from aquatic environments have been implicated in central nervous system, eye and skin human infections. They also allow the survival, growth and transmission of bacteria such as Legionella, Mycobacteria and Vibrio species in water systems. The purpose of this study was to investigate the co-occurrence of potentially pathogenic FLA and their associated bacteria in hospital water networks in Johannesburg, South Africa. A total of 178 water (n = 95) and swab (n = 83) samples were collected from two hospital water distribution systems. FLA were isolated using the amoebal enrichment technique and identified using PCR and 18S rDNA sequencing. Amoebae potentially containing intra-amoebal bacteria were lysed and cultured on blood agar plates. Bacterial isolates were characterized using the VITEK®2 compact System. Free-living amoebae were isolated from 77 (43.3 %) of the samples. Using microscopy, PCR and 18S rRNA sequencing, Acanthamoeba spp. (T3 and T20 genotypes), Vermamoeba vermiformis and Naegleria gruberi specie were identified. The Acanthamoeba T3 and T20 genotypes have been implicated in eye and central nervous system infections. The most commonly detected bacterial species were Serratia marcescens, Stenotrophomonas maltophilia, Delftia acidovorans, Sphingomonas paucimobilis and Comamonas testosteroni. These nosocomial pathogenic bacteria are associated with systematic blood, respiratory tract, the urinary tract, surgical wounds and soft tissues infections. The detection of FLA and their associated opportunistic bacteria in the hospital water systems point out to a potential health risk to immune-compromised individuals. Electronic supplementary material The online version of this article (doi:10.1007/s00436-016-5271-3) contains supplementary material, which is available to authorized users.

This study investigated the occurrence of free-living amoebae (FLA) in a public hospital in South Africa. A total of 97 water and biofilm samples from the municipal water inlet of the hospital, theatres, theatre sterilization service unit, central sterilization service unit, endoscopy/gastroscopy unit, intensive care unit and the renal unit were collected and examined for the presence of FLA using an amoebal co-culture and molecular techniques. Of the 97 samples, 77 (79.4%), 40 (52%) water and 37 (48.1%) biofilm, contained FLA. The genera Acanthamoeba, Vermamoeba (formerly Hartmanella) and Naegleria were detected by morphology, 18S rRNA PCR (polymerase chain reaction) and sequence analyses. Further sequence analysis of the five Acanthamoeba-positive isolates revealed a close resemblance with the potentially pathogenic T20 genotype. These results show a potential health risk to immuno-compromised patients and health care workers as some of the species detected are pathogenic and may harbor potential intracellular bacteria responsible for nosocomial infections. To date, this is the first report on the detection of potentially pathogenic amoebae from South African hospital water systems.

Despite the great sensitivity of PCR in monitoring enteric viruses in an aquatic environment, PCR detects viral nucleic acids of both infectious and noninfectious viruses, limiting the conclusions regarding significance for public health. Ethidium monoazide (EMA) and propidium monoazide (PMA) are closely related membrane impermeant dyes that selectively penetrate cells with compromised membranes. Inside the cells, the dye can intercalate into nucleic acids and inhibit PCR amplification. To assess whether EMA and PMA pretreatment is a suitable approach to inhibit DNA amplification from noninfectious viruses upon heat treatment, UV exposure or chlorine treatment, viruses were measured by qPCR, EMA-qPCR, PMA-qPCR and cell culture titration. EMA/PMA-qPCR of UV- and heat-treated viruses did not correlate with the results of the cell culture assay. However, the data from EMA/PMA-qPCR of chlorine-inactivated viruses was consistent with the cell culture infectivity assay. Therefore, a dye treatment approach could be a rapid and inexpensive tool to screen the efficacy of chlorine disinfection, but it is not able to distinguish between infectious and noninfectious viruses inactivated via heat treatment or UV irradiation. Indeed, different viruses may have different trends and mechanisms of inactivation; thus, the assay must be evaluated for each virus separately.