Environmental Science & Technology Letters

Viruses of concern for quantitative wastewater monitoring are usually selected as a result of an outbreak and subsequent detection in wastewater. However, targeted metagenomics could proactively identify viruses of concern when used as an initial screening tool. To evaluate the utility of targeted metagenomics for wastewater screening, we used ViroCap, a panel of probes designed to target all known vertebrate viruses. Untreated wastewater was collected from wastewater treatment plants (WWTPs) and building-level manholes associated with vulnerable populations in Houston, TX. We evaluated differences in vertebrate virus detection between WWTP and building-level samples, classified human viruses in wastewater, and performed phylogenetic analysis on astrovirus sequencing reads to evaluate targeted metagenomics for subspecies level classification. Vertebrate viruses varied widely across building-level samples. Rarely detected and abundant viruses were identified in WWTP and building-level samples, including enteric, respiratory, and bloodborne viruses. Furthermore, full length genomes were assembled from astrovirus reads and two human astrovirus serotypes were classified in wastewater samples. This study demonstrates the utility of targeted metagenomics as an initial screening step for public health surveillance. SynopsisThis work demonstrates the utility of targeted metagenomic shotgun sequencing to screen for human viruses in centralized and building-level wastewater samples. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/23287251v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@15c4fd8org.highwire.dtl.DTLVardef@17e2a19org.highwire.dtl.DTLVardef@1fe10b3org.highwire.dtl.DTLVardef@fa726f_HPS_FORMAT_FIGEXP M_FIG C_FIG

With COVID-19 N95 respirator shortages, frontline medical personnel are forced to reuse this disposable - but sophisticated - multilayer textile respirator. Widely used for decontamination of nonporous surfaces, UV-C light has germicidal efficacy on porous, non-planar N95 respirators when [&ge;]1.0 J/cm2 dose is applied across all surfaces. Here, we address outstanding limitations of photochromic indicators (qualitative readout and insufficient dynamic range) and introduce a photochromic UV-C dose quantification technique for: (1) design of UV-C treatments and (2) in-process UV-C dose validation. Our methodology establishes that color-changing dosimetry can achieve the necessary accuracy (>90%), uncertainty (<10%), and UV-C specificity (>95%). Furthermore, we adapt consumer electronics for accessible quantitative readout and extend the dynamic range >10x using optical attenuators. In a measurement infeasible with radiometers, we observe striking 20x dose variation over 3D N95 facepieces. By transforming photochromic indicators into quantitative dosimeters, we illuminate critical design considerations for both photochromic indicators and UV-C decontamination.

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-two recent peer-reviewed studies employing capsid-integrity qPCR for viruses in the last decade (2009 to 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 {micro}M; incubation on ice or at room temperature (20 - 25{degrees}C) for 5 to 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/20095364v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@12f34b5org.highwire.dtl.DTLVardef@2d7a8forg.highwire.dtl.DTLVardef@1c951c3org.highwire.dtl.DTLVardef@16f7df_HPS_FORMAT_FIGEXP M_FIG C_FIG

Far-UVC (222 nm UV-C light) is a promising tool to mitigate aerosol transmission of pathogens indoors. However, recent studies have raised concerns related to ozone (O3) production and secondary chemistry. In this study, we measured indoor O3 and ultra-fine particle (UFP, 17.5-289 nm) concentrations with and without 222 nm far-UVC (average fluence rate 1.7-1.8 {micro}W/cm2) in a hotel quarantine facility in Baltimore (MD, USA). We obtained nearby outdoor O3 concentrations from the Environmental Protection Agency (EPA) website. In a sealed empty guest room, the average O3 concentrations were 3 ppb (UV off, 0.1-0.5 ACH), 16 ppb (UV on, 0.1 ACH) and 9 ppb (UV on, 0.5 ACH). In a standard guest room, the average O3 concentrations were 12 ppb (UV off, 1.4 ACH) and 14 ppb (UV on, 1.4 ACH), and correlated with outdoor concentrations ({rho} = 0.65 - 0.74, p = 2*10-12 - 2*10-29). A linear regression model, adjusted for outdoor O3, estimated that use of far-UVC lamps increased the O3 concentration by 5.7 ppb (95% confidence interval (CI) 4.9 - 6.5 ppb) in the standard hotel room. Indoor O3 concentrations increased with far-UVC usage, however, the concentrations remained 6-12 ppb lower, on average, than outdoors and well below EPA ambient limits. We did not find a clear relationship between indoor UFP concentrations and UV usage. Although our study was limited by absence of direct outdoor measurements of local O3 and UFPs, our findings do not support a major impact of far-UVC on UFP concentrations in the real-world environment that we studied.

While real-time monitoring of physicochemical parameters has widely been incorporated into drinking water treatment systems, real-time microbial monitoring has lagged behind, resulting in the use of surrogate parameters (disinfectant residual, applied dose, concentration x time [CT]) to assess disinfection system performance. Near real-time flow cytometry (NRT-FCM) allows for automated quantification of total and intact microbial cells but has not been widely implemented in full-scale systems. This study sought to investigate the feasibility of NRT-FCM for full-scale drinking water ozone disinfection system performance monitoring. A water treatment plant with high lime solids turbidity in the ozone contactor influent was selected to evaluate the NRT-FCM in challenging conditions. Total and intact cell counts were monitored for 40 days and compared to surrogate parameters (ozone residual, ozone dose, and CT) and grab sample assay results for cellular adenosine triphosphate (cATP), heterotrophic plate counts (HPC), impedance flow cytometry, and 16S rRNA gene sequencing. NRT-FCM provided insight into the dynamics of the full-scale ozone system, including offering early warning of increased contactor effluent cell concentrations, which was not observed using surrogate measures. A strong correlation between log intact cell removal and CT was also not observed (Kendalls tau= -0.09, p=0.04). Positive correlations were observed between intact cell counts and cATP levels (Kendalls tau=0.40, p<0.01), HPC (Kendalls tau=0.20, p<0.01), and impedance flow cytometry results (Kendalls tau=0.30, p<0.01). However, 16S rRNA gene sequencing results showed that passage through the ozone contactor significantly changed the microbial community (p<0.05), supporting the hypothesis that regrowth was occurring in the later chambers of the contactor. This study demonstrates the utility of direct, near real-time microbial analysis for monitoring full-scale disinfection systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/23300640v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@13f5e68org.highwire.dtl.DTLVardef@14f33d3org.highwire.dtl.DTLVardef@d3821dorg.highwire.dtl.DTLVardef@373af_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINear real-time flow cytometry (NRT-FCM) was effective for ozone system monitoring. C_LIO_LIIntact microbial cell counts were consistent with cellular ATP and HPC results. C_LIO_LINRT-FCM provided early detection of increased effluent cell concentrations. C_LIO_LIThe ozone contactor influent and effluent microbial communities were distinct. C_LI

BackgroundPer- and polyfluoroalkyl substances (PFAS) have been reported in human milk. However, prior U.S. studies have not included novel PFAS alternatives of emerging concern or infants Estimated Daily Intake (EDI) of PFAS. MethodsHuman milk was collected between 2019 and 2020 at 6 weeks after delivery from 100 Cincinnati, Ohio, nursing women participants in the IMPRINT study; 29 PFAS congeners were measured using ultrahigh performance liquid chromatography-mass spectrometry. We performed descriptive exposure analyses and assessed infants PFAS EDI from human milk. ResultsAll human milk samples contained PFAS. Of the 19 PFAS detected, 5 congeners were concurrently found in [&ge;] 50% of the samples. Legacy PFAS had the highest detection frequencies and concentrations: 97.7% for perfluorooctanesulfonic acid (PFOS) (median concentration: 14.5 ng/L) and 89.8% for perfluorooctanoic acid (PFOA) (median: 17.4 ng/L), 71.6% for perfluorohexanesulfonic acid (PFHxS) (median: 3.7 ng/L), and 70.0% for perfluorohexanoic acid (PFHxA) (median: 10.4 ng/L). An emerging PFAS, dodecafluoro-3H-4,8-dioxanonanoate (ADONA), was detected in 68.0% of samples (median: 3.5 ng/L). The PFAS with the highest EDI included PFOA (median: 8.6 ng), PFOS (median: 7.1 ng), and PFHxA (median: 5.8 ng). About 98% of samples had PFAS levels above the European Food Safety Authority (EFSA) tolerable weekly intake of 4.4 ng/kg body weight/week for the sum of PFOA, PFOS, PFHxS and perfluorononanoate (PFNA). ConclusionsHuman milk from women in Cincinnati, Ohio, contained both legacy and emerging PFAS and infants PFAS consumption through breastfeeding exceeded EFSA tolerable weekly intakes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an elusive and highly pathogenic agent, has resulted in the ongoing COVID-19 pandemic affecting numerous populations worldwide. New studies investigating the tenacity of SARS-CoV-2 have highlighted its ability to persist on a myriad of surfaces for several days, including gowns and shoes. As a result, there is a global need for sterilization of a variety of potentially-contaminated items, ranging from clothing to personal protective equipment like face coverings. To this end, we have designed and constructed a cost-effective, scalable, and sustainable sterilization system that uses ozone gas to inactivate viral particles. We sought to determine the efficacy of the system in the sterilization of viral particles as well as its ability to sterilize N95 respirators for reuse. N95 respirators inoculated with P22 bacteriophage and sterilized in the ozone system showed a 6-log10 reduction in viral load when treated at 25 ppm for 150 minutes. Further, N95 respirators treated with five 150-minute cycles at 35 ppm for a total concentration-time product (CT) of 26,250 ppm min in the ozone system showed comparable filtration efficiency to untreated N95 respirators in a 50 to 200 nmr particulate challenge filtration test. Interestingly, the surgical N95 respirators tested showed complete inactivation of fluid resistance and degradation of the elasticity of polyisoprene straps after five cycles in the sterilization system. Taken together, these data suggest that while our ozone system may negatively affect certain protective aspects of surgical N95 respirators, it does effectively sterilize viral particles and can be utilized for a multitude of other use cases, including sterilizing polypropylene face coverings after potential SARS-CoV-2 contamination. In addition to providing long-term environmental benefits, deployment of this system during the ongoing pandemic reduces the risk of COVID-19 community transmission while conserving monetary resources otherwise spent on the continuous purchase of disposable face coverings.

The booming development of commercial products containing graphene nanoplatelets (GNPs) triggers growing concerns over their release into the air. Precise prediction of human respiratory system deposition of airborne GNPs, especially in alveolar region, is very important for inhalation exposure assessment. In this study, the pulmonary deposition of airborne GNPs was predicted by the multiple-path particle dosimetry (MPPD) model with consideration of GNPs plate-like shape and folded structure effect. Different equivalent diameters of GNPs were derived and utilized to describe different deposition mechanisms in the MPPD model. Both of small GNPs (geometric lateral size dg < 0.1 m) and large GNPs (dg > 10 m) had high deposition fractions in human respiratory system. The total deposition fractions for 0.1 m and 30 m GNPs were 41.6% and 75.6%, respectively. Most of the small GNPs deposited in the alveolar region, while the large GNPs deposited in the head airways. The aerodynamic diameter of GNPs was much smaller than the geometric lateral dimension due to the nanoscale thickness. For GNPs with geometric lateral size of 30 m, the aerodynamic diameter was 2.98 m. The small aerodynamic diameter of plate-like GNPs enabled deposition in the alveolar region, and folded GNPs had higher alveolar deposition than planar GNPs. Heavy breathing led to higher GNPs deposition fraction in head airways and lower deposition fractions in the alveolar region than resting breathing. Our results reveal that large GNPs can have small enough aerodynamic diameters to be respirable and deposit beyond the ciliated airways. The plate-like morphology and folded structure of GNPs resulted in higher alveolar deposition compared to spherical particles.

Measurement of Enterococcus spp. levels with qPCR allows for same-day advisory notification of recreational water quality conditions, representing a major advance over traditional culture-based methods that require 18 or more hours to obtain results. In 2015, the United States Environmental Protection Agency released an Enterococcus qPCR protocol for recreational water quality testing. Over the past decade, there have been multiple advances in qPCR-based environmental testing, affording the opportunity to update the current methodology. A streamlined Enterococcus qPCR protocol is introduced that simplifies the mathematical model to estimate target sequence concentrations (TSC), reduces sample testing time by 20 min, incorporates a certified control material for standard curve generation, and introduces an inactivated E. faecalis whole cell DNA standard (WCDS) control material. A series of experiments were conducted 1) to compare results of the two Enterococcus qPCR protocols in analysis of marine, estuarine, and freshwater samples (n = 60), 2) to investigate alternative practices to adjust results due to potential water sample matrix interference, control material degradation, and/or analyst inconsistencies, and 3) to evaluate the performance, homogeneity, and stability of an inactivated E. faecalis cell preparation as a WCDS control material. Findings indicate a strong correlation between water sample mean log10 TSC per reaction results (R2 = 0.980) and 100% agreement in amplification and sample processing control tests. A Bayesian approach that accounts for uncertainty in qPCR measurements confirmed statistical equivalence for all water samples yielding paired measurements in the range of quantification, with 72.7% of samples exhibiting reduced error with the new streamlined protocol. Evaluation of three alternative practices to adjust for variation in Enterococcus qPCR measurements indicated no significant difference in water sample log10 TSC per reaction results with varying concentrations of treated sewage influent. Systematic testing of an inactivated WCDS control material yielded statistically equivalent performance compared to viable E. faecalis cell preparations. Homogeneity and stability experiments indicated that Enterococcus qPCR measurements of inactivated WCDS are reproducible across multiple preparations and that the material is stable at -20{degrees}C for at least 38 weeks. Together, experiments demonstrate that the streamlined protocol and alternative practices should make Enterococcus qPCR faster, easier to implement, safer, and more reproducible.

Extended-spectrum-beta-lactamases (ESBLs) are a growing group of antimicrobial resistance (AMR) enzymes that can result in severe clinical outcomes. The CTX-M gene, which encodes for ESBLs in bacteria, confers resistance to third generation cephalosporins and is of high clinical concern. We developed a targeted, long-read sequencing method utilizing unique molecular identifiers to generate accurate, full length CTX-M gene sequences from wastewater. We characterized CTX-M in 36 samples from three Seattle area wastewater treatment plants from April 2020 to March 2021. We identified a core community of alleles that persisted across time and treatment plant. The CTX-M-15 containing protein variant (CTX-M-15/216/28) was detected in all but three samples and made up, at most, 30% of detected CTX-M alleles. We observed significant diversity across the CTX-M gene at the nucleic acid level, although most nucleotide mutations were synonymous - resulting in two to three amino acid variants across 19 loci. By average relative abundance, 23% of protein variants were novel, defined as those not represented in the CARD database. This method provides information (full length gene sequences) that cannot be obtained through other culture-independent methods. This flexible approach can be expanded to additional targets and implemented in settings where AMR surveillance is a priority, such as hospital wastewater.

Measles outbreaks continue to pose significant public health challenges globally despite the availability of effective vaccines. In this study, we evaluated wastewater-based surveillance for detection of measles virus during an ongoing outbreak in Texas. Weekly wastewater samples collected from two Texas cities between January 2 and March 17, 2025 were analyzed using multiple RT-PCR assays targeting the nucleoprotein and matrix genes of the measles virus. Viral RNA was detected in multiple days from both cities, with City A showing positives from January 13 and City B from January 6, both predating the first confirmed case in the state on January 23. Sequencing of PCR amplicons confirmed the specificity of detection and phylogenetic analysis using global and U.S. measles genome databases further validated that the viral RNA belonged to the currently circulating genotype D8. Our findings demonstrate that wastewater surveillance can provide early evidence of measles virus circulation in communities before clinical cases are recognized and can support public health responses to these re-emerging infectious diseases.

Climate change and urbanization are increasing the distribution of insect vectors of infectious diseases. Dengue virus is an arbovirus that causes nearly 100 million symptomatic infections per year and is endemic in 124 countries, and the range of its mosquito vectors continues to increase. Surveillance of dengue virus infections is complicated by the fact that infections can be asymptomatic and symptoms may not be readily recognizable to clinicians. Here we show that wastewater monitoring can be used to detect dengue virus RNA to yield information about circulation of dengue infections in a community. We collected three samples of wastewater solids per week from three different wastewater treatment plants in Miami-Dade County, Florida where dengue infections have been locally acquired. Using molecular methods, we tested wastewater solids for RNA from the 4 dengue virus serotypes and consistently detected dengue virus 3 RNA at all three wastewater plants, and did not detect the other 3 serotypes. According to publicly available data on dengue infections, the vast majority of infections were caused by serotype 3. Wastewater detection of dengue virus RNA is possible with as few as 4.23 laboratory confirmed dengue cases per 1 million people, based on publicly available infection data. SynopsisDengue virus RNA was detected in wastewater solids in a location with local- and travel-associated dengue infections.

Wastewater represents a composite biological sample from the entire contributing population. People infected with monkeypox excrete monkeypox virus DNA via skin lesions, saliva, feces and urine and these can enter the wastewater via toilets, sinks, and shower drains. To test whether monkeypox can be detected and monitored in wastewater during a period when publicly reported monkey cases in the region were increasing, we deployed digital PCR assays that target genomic DNA from the monkeypox virus in our routine, ongoing wastewater surveillance program in the Greater Bay Area of California, USA. We measured monkeypox virus DNA daily in settled solids samples from nine wastewater plants over the period of approximately 4 weeks. During that period, we detected monkeypox virus DNA in wastewater solids at nearly all the wastewater plants we routinely sample. Frequency of occurrence and concentrations were highest at plants serving San Francisco County. To confirm the presence of monkeypox DNA, we used two assays that target distinct sequences on the monkeypox genome on a subset of samples and results from both assays were in close agreement strongly suggesting true positives in the wastewater. Additionally, we show that concentrations of monkeypox DNA is 103 times higher in the solid fraction compared to the liquid fraction of wastewater on a mass-equivalent basis.

Military training centers may be high risk environments for the spread of disease such as COVID-19. Individuals arrive after traveling from many parts of the country, live in communal settings, and undergo high-interaction training. A pilot study of wastewater testing was initiated in February, 2021 to determine its feasibility as a sentinel surveillance tool in the U.S. Coast Guard for SARS-CoV-2. Wastewater was analyzed for the presence of two viral genes, N and E, and quantified relative to levels of a fecal indicator virus, Pepper Mild Mottle Virus (PMMoV). A stability control, Bovine Syncytial Respiratory Virus vaccine, was added to samples to assess sample stability and degradation. Wastewater data was validated by comparison with concomitant screening and surveillance programs that identified asymptomatic individuals infected with SARS-CoV-2 by diagnostic testing at on site medical clinics using PCR. Elevated levels of SARS-CoV-2 in wastewater were frequently associated with diagnosed cases, and in several instances, led to screenings of asymptomatic individuals that identified infected personnel, mitigating the risk of spread of disease. Wastewater screening also successfully indicated the presence of breakthrough cases in vaccinated individuals. A method for assessing blackwater from Coast Guard vessels was also developed, allowing detection of SARS-CoV-2 virus in shipboard populations. In one instance, virus was detected in the blackwater four weeks following the diagnosis of a single person on a Coast Guard cutter. These data show that wastewater testing is an effective tool for measuring the presence and prevalence of SARS-CoV-2 in military populations so that mitigation can occur and suggest other diseases may be assessed similarly. As a result, the Coast Guard has established three laboratories with wastewater testing capability at strategic locations and is actively continuing its wastewater testing program.

BackgroundWastewater represents a composite biological sample from the entire contributing population. People infected with monkeypox virus (MPXV)1 may excrete viral DNA into wastewater via multiple ways such as in feces, urine, skin lesions, and/or saliva. We describe results from rapid establishment of wastewater surveillance in selected regions in California within a month of the first reported case of monkeypox in the United States. MethodsPCR assays targeting genomic DNA from MPXV were deployed in an ongoing wastewater surveillance program in California. MPXV DNA concentrations were measured daily in settled solids samples from nine wastewater plants. Results over a four-week period were validated across different MPXV assays, compared using influent and solids samples, and correlated using non-parametric methods (Kendalls tau) with the number of monkeypox cases reported from each sewershed. ResultsMPXV DNA was detected at all nine sites between June 19 and August 1, 2022; 5 of 9 sites detected MPXV DNA prior to or within a day of the first case identified in the source sewershed. At the four sites with >10 positive detections, we observed a positive, statistically significant correlation (p <0.001) between MPXV DNA in wastewater solids and incidence rate of reported cases. ConclusionsOur findings suggest wastewater can be used to effectively detect the introduction of MPXV and monitor its circulation in the community to inform public health and clinical response. This flexible wastewater surveillance infrastructure may be rapidly leveraged to monitor other pathogens of public health importance that are shed into wastewater.

Ventilation is one of the most critical components in a layered approach toward reducing the spread of airborne infectious diseases in indoor spaces. However, building ventilation systems act together with natural ventilation, local filtration systems and other aerosol removal processes to remove infectious aerosols from an occupied space. Airflow-based determinations of ACH do not account for the full range of aerosol removal processes; however understanding the effective aerosol removal rate is critical to providing airborne infection control. In this study, we investigated the relationship between the calculated air change rate of a space (i.e. volumetric airflow based) and the effective air change rate for aerosol particle removal within the breathing zone based on direct measurements of the rate of change in tracer particle concentrations at representative occupant locations in a room. Further, we examined positional effects under well mixed and non-well mixed conditions. Our results demonstrate that tracer particles combined with real-time sensors can be used to make rapid, accurate measurements of the effective air change rate (eACH) for respiratory aerosols within the breathing zone of non-well mixed rooms. We used two experimental test beds for these analyses. First, numerical simulation (computational fluid dynamic simulation, CFD) was conducted to visualize airflow and particle removal paths within a realistic large room. Here, simulated sensors were placed in concentric zones around a nebulizer providing test-particle releases. This CFD model allowed a direct comparison of the differences between eACH and airflow ACH values under varying levels of mixing and airflow, in a fully controlled system. We then recapitulated this system in physical space to validate the CFD results under real-world conditions that include all mechanisms of particle removal that contribute to true aerosol clearance rates, including deposition and leakage. Here, we measured eACH using the decay of DNA tracer aerosols nebulized and monitored in real-time. We find that a standard sampling time of 15 minutes from the end of nebulization is sufficient to produce an accurate eACH value under non-well mixed conditions. The availability of a rapid direct test for eACH will enable empirical optimization of a wide range of ventilation and filtration mechanisms to reach and maintain target aerosol clearance rates that deliver reliable airborne infection control in typical indoor environments.

BackgroundSurveillance is a key component of the WHO 2030 measles elimination strategy. Wastewater and environmental surveillance (WES) has successfully supported polio surveillance, however, it has not been applied to measles control. AimWe developed a measles virus (MeV) digital-PCR (dPCR) assay using WHO recommended clinical primer-probes and applied it to retained wastewater samples from national SARS-CoV-2 wastewater sentinel surveillance sites. We compared results with national clinical measles surveillance data. SettingWastewater concentrates were collected from 47 SARS-CoV-2 wastewater surveillance sites across South Africa. MethodsWe determined the limit of detection using the assay on serial dilutions of MeV positive controls. Subsequently we conducted batch testing on wastewater concentrates retained at -20{degrees}C for up to 15 months and compared wastewater results with national laboratory-confirmed measles case data by district and epidemiological week. ResultsOur assay identified 43/2149 (2%) samples containing MeV RNA in concentrations ranging from 1.97 - 165.8 genome copies/mL. Amongst 27 week-district instances where at least one MeV positive wastewater sample was detected, no clinical cases were detected in 13 (48%) of these. ConclusionDespite likely RNA degradation, it was possible to detect MeV in wastewater samples in districts where clinical surveillance failed to identify cases. ContributionWES has evident potential to strengthen surveillance in support of the measles elimination agenda. With immediate processing and improved wastewater RNA concentration methods, WES sensitivity will likely increase. FundingThis work was funded by BMGF (INV-049271)

Legionella is an opportunistic waterborne pathogen that is difficult to eradicate in colonized drinking water pipes. Legionella control is further challenged by aging water infrastructure and lack of evidence-based guidance for building treatment. This study assessed multiple premise water remediation approaches designed to reduce Legionella pneumophila (Lp) within a residential building located in an aging, urban drinking water system over a two-year period. Samples (n=745) were collected from hot and cold-water lines and quantified via most probable number culture. Building-level treatment approaches included three single heat shocks (HS), three single chemical shocks (CS), and continuous low-level chemical disinfection (CCD) in the potable water system. The building was highly colonized with Lp with 71% Lp positivity. Single HS had a statistically significant Lp reduction one day post treatment but no significant Lp reduction one, two, and four weeks post treatment. The first two CS resulted in statistically significant Lp reduction at two days and four weeks post treatment, but there was a significant Lp increase at four weeks following the third CS. CCD resulted in statistically significant Lp reduction ten weeks post treatment implementation. This demonstrates that in a building highly colonized with Lp, sustained remediation is best achieved using CCD. SYNOPSISLong-term Legionella control is difficult to maintain within aging premise plumbing. This study supports continuous low-level building treatment as an effective long-term remediation of a building highly colonized with Legionella. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/23292444v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14d8188org.highwire.dtl.DTLVardef@17313aborg.highwire.dtl.DTLVardef@107188org.highwire.dtl.DTLVardef@18dbdb4_HPS_FORMAT_FIGEXP M_FIG C_FIG

In this retrospective study, enterovirus D68 (EVD68) genomic RNA in wastewater solids was measured longitudinally at two California wastewater treatment plants twice per week for 26 months. EVD68 RNA was undetectable except when concentrations increased between mid-July and mid-December 2022, during which time EVD68 cases were confirmed in the state.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths in the United States. Individual screening is typically done with either a clinical stool-based test or direct clinical examination such as a colonoscopy. Given the low compliance with current screening recommendations and the high morbidity and mortality observed in areas with health disparities, we consider whether population-based testing using human RNA biomarkers in wastewater might effectively track the presence of CRC at the neighborhood level might be feasible. Wastewater samples were collected from four clusters in Louisville, KY: three representing cancer hotspots and one serving as a control neighborhood for feasibility data. Three wastewater replicates were obtained from each cluster. Human RNA biomarkers were isolated, quantified, and their RNA concentration levels were compared to clinical correlates. All replicates showed detectable levels of human cancer-associated RNA, including CDH1, which is a colorectal neoplasia-associated biomarker. Among CRC cluster sewershed samples, 8 of 9 replicate samples (89%) had a ratio of CDH1/GAPDH >=1 while the control sewershed sample showed ratio <1 for 2 of 3 samples. These preliminary data indicate that human RNA biomarkers can be detected in pooled community wastewater samples. While we have successfully identified the presence of these markers, further investigation with additional samples and closer alignment with documented case activity is necessary.