Advances in wastewater analysis revealing the co-circulating viral trends of noroviruses and Omicron subvariants.

Co-presence of enveloped and non-enveloped viruses is common both in community circulation and in wastewater. Community surveillance of infections requires robust methods enabling simultaneous quantification of multiple viruses in wastewater. Using enveloped SARS-CoV-2 Omicron subvariants and non-enveloped norovirus (NoV) as examples, this study reports a robust method that integrates electronegative membrane (EM) concentration, viral inactivation, and RNA preservation (VIP) with efficient capture and enrichment of the viral RNA on magnetic (Mag) beads, and direct detection of RNA on the beads. This method provided improved viral recoveries of 80 ± 4 % for SARS-CoV-2 and 72 ± 5 % for Murine NoV. Duplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays with newly designed degenerate primer-probe sets offered high PCR efficiencies (90-91 %) for NoV (GI and GII) targets and were able to detect as few as 15 copies of the viral RNA per PCR reaction. This technique, combined with duplex detection of NoV and multiplex detection of Omicron, successfully quantified NoV (GI and GII) and Omicron variants in the same sets of 94 influent wastewater samples collected from two large wastewater systems between July 2022 and June 2023. The wastewater viral RNA results showed temporal changes of both NoV and Omicron variants in the same wastewater systems and revealed an inverse relationship of their emergence. This study demonstrated the importance of a robust analytical platform for simultaneous surveillance of enveloped and non-enveloped viruses in wastewater. The ability to sensitively determine multiple viral pathogens in wastewater will advance applications of wastewater surveillance as a complementary public health tool.

ChloroDBPFinder: Machine Learning-Guided Recognition of Chlorinated Disinfection Byproducts from Nontargeted LC-HRMS Analysis.

High-resolution mass spectrometry (HRMS) is a prominent analytical tool that characterizes chlorinated disinfection byproducts (Cl-DBPs) in an unbiased manner. Due to the diversity of chemicals, complex background signals, and the inherent analytical fluctuations of HRMS, conventional isotopic pattern (37Cl/35Cl), mass defect, and direct molecular formula (MF) prediction are insufficient for accurate recognition of the diverse Cl-DBPs in real environmental samples. This work proposes a novel strategy to recognize Cl-containing chemicals based on machine learning. Our hierarchical machine learning framework has two random forest-based models: the first layer is a binary classifier to recognize Cl-containing chemicals, and the second layer is a multiclass classifier to annotate the number of Cl present. This model was trained using ∼1.4 million distinctive MFs from PubChem. Evaluated on over 14,000 unique MFs from NIST20, this machine learning model achieved 93.3% accuracy in recognizing Cl-containing MFs (Cl-MFs) and 92.9% accuracy in annotating the number of Cl for Cl-MFs. Furthermore, the trained model was integrated into ChloroDBPFinder, a standalone R package for the streamlined processing of LC-HRMS data and annotating both known and unknown Cl-containing compounds. Tested on existing Cl-DBP data sets related to aspartame chlorination in tap water, our ChloroDBPFinder efficiently extracted 159 Cl-containing DBP features and tentatively annotated the structures of 10 Cl-DBPs via molecular networking. In another application of a chlorinated humic substance, ChloroDBPFinder extracted 79 high-quality Cl-DBPs and tentatively annotated six compounds. In summary, our proposed machine learning strategy and the developed ChloroDBPFinder provide an advanced solution to identifying Cl-containing compounds in nontargeted analysis of water samples. It is freely available on GitHub (https://github.com/HuanLab/ChloroDBPFinder).

Quantification and Differentiation of SARS-CoV-2 Variants in Wastewater for Surveillance.

Wastewater surveillance plays an important role in the monitoring of infections of SARS-CoV-2 at the community level. We report here the determination of SARS-CoV-2 and differentiation of its variants of concern in 294 wastewater samples collected from two major Canadian cities from May 2021 to March 2023. The overall method of analysis involved extraction of the virus and viral components using electronegative membranes, in situ stabilization and concentration of the viral RNA onto magnetic beads, and direct analysis of the viral RNA on the magnetic beads. Multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays, targeting specific and naturally selected mutations in SARS-CoV-2, enabled detection and differentiation of the Alpha, Beta, Gamma, Delta, and Omicron variants. An Omicron triplex RT-qPCR assay targeting three mutations, HV 69-70 deletion, K417N, and L452R, was able to detect and differentiate the Omicron BA.1/BA.3, BA.2/XBB, and BA.4/5. This assay had efficiencies of 90-104% for all three mutation targets and a limit of detection of 28 RNA copies per reaction. Analyses of 294 wastewater samples collected over a two-year span showed the concentrations and trends of Alpha, Beta, Gamma, Delta, and Omicron variants as they emerge in two major Canadian cities participating in the wastewater surveillance program. The trends of specific variants were consistent with clinical reports for the same period. At the beginning of each wave, the corresponding variants were detectable in wastewater. For example, RNA concentrations of the BA.2 variant were as high as 104 copies per 100 mL of wastewater collected in January 2022, when approximately only 50-60 clinical cases of BA.2 infection were reported in Canada. These results show that the strategy and highly sensitive assays for the variants of concern in wastewater are potentially useful for the detection of newly emerging SARS-CoV-2 variants and other viruses for future community biomonitoring.

Multiplex Assays Enable Simultaneous Detection and Identification of SARS-CoV-2 Variants of Concern in Clinical and Wastewater Samples.

The targeted screening and sequencing approaches for COVID-19 surveillance need to be adjusted to fit the evolving surveillance objectives which necessarily change over time. We present the development of variant screening assays that can be applied to new targets in a timely manner and enable multiplexing of targets for efficient implementation in the laboratory. By targeting the HV69/70 deletion for Alpha, K417N for Beta, K417T for Gamma, and HV69/70 deletion plus K417N for sub-variants BA.1, BA.3, BA.4, and BA.5 of Omicron, we achieved simultaneous detection and differentiation of Alpha, Beta, Gamma, and Omicron in a single assay. Targeting both T478K and P681R mutations enabled specific detection of the Delta variant. The multiplex assays used in combination, targeting K417N and T478K, specifically detected the Omicron sub-variant BA.2. The limits of detection for the five variants of concern were 4-16 copies of the viral RNA per reaction. Both assays achieved 100% clinical sensitivity and 100% specificity. Analyses of 377 clinical samples and 24 wastewater samples revealed the Delta variant in 100 clinical samples (nasopharyngeal and throat swab) collected in November 2021. Omicron BA.1 was detected in 79 nasopharyngeal swab samples collected in January 2022. Alpha, Beta, and Gamma variants were detected in 24 wastewater samples collected in May-June 2021 from two major cities of Alberta (Canada), and the results were consistent with the clinical cases of multiple variants reported in the community.

Amino Acids as Potential Precursors to Odorous Compounds in Tap Water during Spring Runoff Events.

The onset of spring runoff in northern climates and tap water odor events are difficult to predict because common water quality parameters cannot fully explain the intermittent odor events that occurred over past decades. Studies have shown that small polar water-soluble compounds, such as amino acids (AAs), leach first from ice/snowmelt. AAs are known to produce odorous compounds, such as aldehydes and chloroaldimines, upon chlorination. Therefore, we proposed that AAs may serve as markers for small and soluble organics that contribute to the odor of chlorinated tap water. Here, we studied the occurrence of AAs in source water collected at two water treatment plants and the odor profiles of tap water at >300 homes during the 2021 and 2022 spring runoff events. AA concentrations were at baseline levels (<100 ng/L) during the 2021 runoff but much higher (up to 5500 ng/L) in 2022 and associated with an escalation in odor complaints. AA concentrations peaked at the onset of the 2022 spring runoff and corresponded with the strongest reported odor intensities in tap water. We obtained high resolution MS and MS/MS spectra of chloroaldimines and confirmed the formation of chloroaldimines under chlorination of the six AAs detected in source water. The results indicate that AAs signal the onset of spring runoff and represent small polar water-soluble compounds that may contribute to tap water odor problems.

Recent advances in RNA sample preparation techniques for the detection of SARS-CoV-2 in saliva and gargle.

Molecular detection of SARS-CoV-2 in gargle and saliva complements the standard analysis of nasopharyngeal swabs (NPS) specimens. Although gargle and saliva specimens can be readily obtained non-invasively, appropriate collection and processing of gargle and saliva specimens are critical to the accuracy and sensitivity of the overall analytical method. This review highlights challenges and recent advances in the treatment of gargle and saliva samples for subsequent analysis using reverse transcription polymerase chain reaction (RT-PCR) and isothermal amplification techniques. Important considerations include appropriate collection of gargle and saliva samples, on-site inactivation of viruses in the sample, preservation of viral RNA, extraction and concentration of viral RNA, removal of substances that inhibit nucleic acid amplification reactions, and the compatibility of sample treatment protocols with the subsequent nucleic acid amplification and detection techniques. The principles and approaches discussed in this review are applicable to molecular detection of other microbial pathogens.

An efficient method to enhance recovery and detection of SARS-CoV-2 RNA in wastewater.

Wastewater surveillance (WS) of SARS-CoV-2 currently requires multiple steps and suffers low recoveries and poor sensitivity. Here, we report an improved analytical method with high sensitivity and recovery to quantify SARS-CoV-2 RNA in wastewater. To improve the recovery, we concentrated SARS-CoV-2 viral particles and RNA from both the solid and aqueous phases of wastewater using an electronegative membrane (EM). The captured viral particles and RNA on the EM were incubated in our newly developed viral inactivation and RNA preservation (VIP) buffer. Subsequently, the RNA was concentrated on magnetic beads and inhibitors removed by washing. Without eluting, the RNA on the magnetic beads was directly detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Analysis of SARS-CoV-2 pseudovirus (SARS-CoV-2 RNA in a noninfectious viral coat) spiked to wastewater samples showed an improved recovery of 80%. Analysis of 120 wastewater samples collected twice weekly between May 2021 and February 2022 from two wastewater treatment plants showed 100% positive detection, which agreed with the results independently obtained by a provincial public health laboratory. The concentrations of SARS-CoV-2 RNA in these wastewater samples ranged from 2.4×102 to 2.9×106 copies per 100 mL of wastewater. Our method's capability of detecting trace and diverse concentrations of SARS-CoV-2 in complex wastewater samples is attributed to the enhanced recovery of SARS-CoV-2 RNA and efficient removal of PCR inhibitors. The improved method for the recovery and detection of viral RNA in wastewater is important for wastewater surveillance, complementing clinical diagnostic tests for public health protection.

Aspartame-Sweetened Tap Water: Transformation Products and 2,6-Dichloro-1,4-Benzoquinone Formation.

Aspartame (APM), a dipeptide of aspartic acid (ASP) and phenylalanine (PHE), is a widely used artificial sweetener in beverages. It is unclear whether residual chlorine in tap water can react with APM to form disinfection byproducts (DBPs). Therefore, we investigated the formation of DBPs from the reaction of APM with residual chlorine in authentic tap water. APM and a commercial sweetener (CS) packet containing APM were studied under authentic and simulated tap water conditions. Eight chlorinated products of APM were detected using solid-phase extraction (SPE) and high performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). These new chloro-products were tentatively identified based on accurate masses, isotopic patterns of 35,37Cl, and MS/MS spectra. Furthermore, we identified APM as a precursor to 2,6-dichloro-1,4-benzoquinone (DCBQ). DCBQ significantly increased to 2.3-12 ng/L with the addition of APM or CS in tap waters collected from different locations compared to 1.4-1.8 ng/L in the same tap water samples without sweetener. DCBQ and two of the chlorinated transformation products were identified in cold prepared tea containing APM. DCBQ formation was eliminated when the residual chlorine in tap water was reduced by ascorbic acid or boiling prior to the addition of APM or CS. This study found that eight new DBPs and DCBQ were produced by the reactions of residual chlorine with APM and CS. These findings show an unintended exposure source of emerging DBPs via APM sweetened beverages.

Emerging Disinfection Byproduct 2,6-Dichlorobenzoquinone-Induced Cardiovascular Developmental Toxicity of Embryonic Zebrafish and Larvae: Imaging and Transcriptome Analysis.

Epidemiological studies have observed the potential association of water disinfection byproduct (DBP) exposure with cardiac defects. Aromatic DBPs represent a significant portion of total DBPs, but their effects on cardiovascular development are unclear. In this study, we examined the effects of an aromatic DBP, 2,6-dichlorobenzoquinone (DCBQ), on the cardiovascular development of zebrafish embryos. After exposure to 2, 4, and 8 µM DCBQ, morphological images of growing zebrafish embryos clearly showed cardiovascular malformation. Fluorescent images of transgenic zebrafish strains with fluorescently labeled heart and blood vessels show that DCBQ exposure resulted in deformed atrium-ventricle looping, degenerated abdomen and trunk vessels, pericardial edema, and decreased blood flow. Furthermore, the expression of the marker gene myl7 (essential for the differentiation and motility of cardiomyocytes) was inhibited in a dose-dependent manner by DCBQ exposure. Finally, transcriptome analysis found that in the 4 µM DCBQ exposure group, the numbers of differentially expressed genes (DEGs) were 113 (50 upregulated and 63 downregulated) at 24 hpf, 2123 (762 upregulated and 1361 downregulated) at 48 hpf, and 61 (11 upregulated and 50 downregulated) at 120 hpf; in the 8 µM DCBQ exposure group, the number of DEGs was 1407 (647 upregulated and 760 downregulated) at 120 hpf. The FoxO signaling pathway was significantly altered. The in vivo results demonstrate the effects of 2,6-DCBQ (0-8 µM) on cardiovascular development, contributing to the understanding of the developmental toxicity of aromatic DBP halobenzoquinones (HBQs).

Neurotoxicity and transcriptome changes in embryonic zebrafish induced by halobenzoquinone exposure.

Halobenzoquinones (HBQs) are emerging disinfection byproducts (DBPs) with a widespread presence in drinking water that exhibit much higher cytotoxicity than regulated DBPs. However, the developmental neurotoxicity of HBQs has not been studied in vivo. In this work, we studied the neurotoxicity of HBQs on zebrafish embryos, after exposure to varying concentrations (0-8 µmol/L) of three HBQs, 2,5-dichloro-1,4-benzoquinone (2,5-DCBQ), 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), and 2,5-dibromo-1,4-benzoquinone (2,5-DBBQ) for 4 to 120 hr post fertilization (hpf). HBQ exposure significantly decreased the locomotor activity of larvae, accompanied by significant reduction of neurotransmitters (dopamine and γ-aminobutyric acid) and acetylcholinesterase activity. Furthermore, the expression of genes involved in neuronal morphogenesis (gfap, α1-tubulin, mbp, and syn-2α) were downregulated by 4.4-, 5.2-, 3.0-, and 4.5-fold in the 5 µmol/L 2,5-DCBQ group and 2.0-, 1.6-, 2.1-, and 2.3-fold in the 5 µmol/L 2,5-DBBQ group, respectively. Transcriptomic analysis revealed that HBQ exposure affected the signaling pathways of neural development. This study demonstrates the significant neurotoxicity of HBQs in embryonic zebrafish and provides molecular evidence for understanding the potential mechanisms of HBQ neurotoxicity.

Integration of solid phase extraction with HILIC-MS/MS for analysis of free amino acids in source water.

Amino acids (AAs) are prevalent in source water, particularly during spring run-off. Monitoring of amino acids in source water is desirable for water treatment plants (WTP) to indicate changes in source water quality. The objective of this study was to establish analytical procedures for reliable monitoring of amino acids in source water. Therefore, we examined two different methods, large volume inject (LVI) and solid phase extraction (SPE), for sample preparation prior to HILIC-MS/MS. The LVI-HILIC-MS/MS method can provide fast and sensitive detection for clean samples, but suffers from matrix effects, resulting in irreproducible separation and shortening column lifetime. We have demonstrated that SPE was necessary prior to HILIC-MS/MS to achieve reproducible and reliable quantification of AAs in source water. A natural heterocyclic amine 1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (MTCCA) was also included in the method to indicate changes in other natural nitrogenous compounds in source water. The SPE-HILIC-MS/MS method was able to achieve limits of detection from 2.6-3400 ng/L for the amino acids and MTCCA with RSDs (n=3) of 1.1%-4.8%. As well, retention times (RT) of the analytes were reproducible with variation less than 0.01 min (n=3) through the entire project. We further applied the SPE-HILIC-MS/MS method to determine AAs in authentic source water samples collected from two drinking water treatment plants (WTPs) during the 2021 spring run-off season. The results support that the SPE-HILIC-MS/MS method does not require derivatization and can provide reliable, accurate, and robust analysis of AAs and MTCCA in source water, supporting future monitoring of source water quality.

Maternal and child biomonitoring strategies and levels of exposure in western Canada during the past seventeen years: The Alberta Biomonitoring Program: 2005-2021.

The Alberta Biomonitoring Program (ABP) was created in 2005 with the initial goal of establishing baseline levels of exposure to environmental chemicals in specific populations in the province of Alberta, Canada, and was later expanded to include multiple phases. The first two phases focused on evaluating exposure in pregnant women (Phase One, 2005) and children (Phase Two, 2004-2006) by analyzing residual serum specimens. Phase Three (2013-2016) employed active recruitment techniques to evaluate environmental exposures using a revised list of chemicals in paired serum pools from pregnant women and umbilical cord blood. These three phases of the program monitored a total of 226 chemicals in 285 pooled serum samples representing 31,529 individuals. Phase Four (2017-2020) of the ABP has taken a more targeted approach, focusing on the impact of the federal legalization of cannabis on the exposure of pregnant women in Alberta to cannabis, as well as tobacco and alcohol using residual prenatal screening serum specimens. Chemicals monitored in the first three phases include herbicides, neutral pesticides, metals, metalloids, and micronutrients, methylmercury, organochlorine pesticides, organophosphate pesticides, parabens, phthalate metabolites, perfluoroalkyl substances (PFAS), phenols, phytoestrogens, polybrominated compounds, polychlorinated biphenyls (PCBs), dioxins and furans, polycyclic aromatic hydrocarbons (PAHs), and tobacco biomarkers. Phase Four monitored six biomarkers of tobacco, alcohol, and cannabis. All serum samples were pooled. Mean concentrations and 95% confidence intervals (CIs) were calculated for the chemicals detected in ≥25% of the sample pools. cross the first three phases, the data from the ABP has provided baseline exposure levels for the chemicals in pregnant women, children, and newborns across the province. Comparison within and among the phases has highlighted differences in exposure levels with age, geography, seasonality, sample type, and time. The strategies employed throughout the program phases have been demonstrated to provide effective models for population biomonitoring.

Unique On-Site Spinning Sampling of Highly Water-Soluble Organics Using Functionalized Monolithic Sorbents.

Water utilities encounter unpredictable odor issues that cannot be explained by routine water parameters during spring runoff, even in the summer and fall. Highly water-soluble organics (e.g., amino acids and saccharides) have been reported to form odorous disinfection byproducts during disinfection, but the lack of simple and practical on-site sampling techniques hampers their routine monitoring at trace levels in source water. Therefore, we have created two functionalized nested-in-sponge silica monoliths (NiS-SMs) using a one-pot synthesis method and demonstrated their application for extracting highly soluble organics in water. The NiS-SMs functionalized with the sulfonic group and phenylboronic moiety selectively extracted amino acids and monosaccharides, respectively. We further developed a spinning sampling technique using the composites and evaluated its robust performance under varying water conditions. The spinning sampling coupled to high-performance liquid chromatography tandem mass spectrometry analysis provided limits of detection for amino acids at 0.038-0.092 ng L-1 and monosaccharides at 0.036-0.14 ng L-1. Using the pre-equilibrium sampling-rate calibration, we demonstrated the applicability of the spinning sampling technique for on-site sampling and monitoring of amino acids and monosaccharides in river water. The new composite materials and rapid on-site sampling technique are unique and efficient tools for monitoring highly soluble organics in water sources.

Formation Mechanism of Iodinated Aromatic Disinfection Byproducts: Acid Catalysis with H2OI.

Iodinated aromatic disinfection byproducts (I-DBPs) are a group of nonregulated but highly toxic DBPs. The formation of I-DBPs is attributed mainly to HOI because it is the most abundant reactive iodine species in chloraminated water. In this study, we used computational modeling of thermodynamics to examine the mechanism of iodination of aromatic contaminants, e.g., dipeptides and phenols. Computational prediction of the energy barriers of the formation of iodinated tyrosylglycine (I-Tyr-Gly) (66.9 kcal mol-1) and hydroxylated Tyr-Gly (OH-Tyr-Gly) (46.0 kcal mol-1) via iodination with HOI favors the formation of OH-Tyr-Gly over I-Tyr-Gly. Unexpectedly, mass spectrometry experiments detected I-Tyr-Gly but not OH-Tyr-Gly, suggesting that I-Tyr-Gly formation cannot be attributed to HOI alone. To clarify this result, we examined the thermodynamic role of the most reactive iodine species H2OI+ in the formation of aromatic I-DBPs under chloramination. Computational modeling of thermodynamic results shows that the formation of a loosely bonded complex of aromatic compounds with H2OI+ is the key step to initiate the iodination process. When H2OI+ serves as an acid catalyst and an iodinating agent, with HOI or H2O acting as a proton acceptor, the energy barrier of I-DBP formation was significantly lower (10.8-13.1 kcal mol-1). Therefore, even with its low concentration, H2OI+ can be involved in the formation of I-DBPs. These results provide insight into the mechanisms of aromatic I-DBP formation and important information for guiding research toward controlling I-DBPs in drinking water.

On-Site Viral Inactivation and RNA Preservation of Gargle and Saliva Samples Combined with Direct Analysis of SARS-CoV-2 RNA on Magnetic Beads.

Samples of nasopharyngeal swabs (NPS) are commonly used for the detection of SARS-CoV-2 and diagnosis of COVID-19. As an alternative, self-collection of saliva and gargle samples minimizes transmission to healthcare workers and relieves the pressure of resources and healthcare personnel during the pandemic. This study aimed to develop an enhanced method enabling simultaneous viral inactivation and RNA preservation during on-site self-collection of saliva and gargle samples. Our method involves the addition of saliva or gargle samples to a newly formulated viral inactivation and RNA preservation (VIP) buffer, concentration of the viral RNA on magnetic beads, and detection of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction directly from the magnetic beads. This method has a limit of detection of 25 RNA copies per 200 µL of gargle or saliva sample and 9-111 times higher sensitivity than the viral RNA preparation kit recommended by the United States Centers for Disease Control and Prevention. The integrated method was successfully used to analyze more than 200 gargle and saliva samples, including the detection of SARS-CoV-2 in 123 gargle and saliva samples collected daily from two NPS-confirmed positive SARS-CoV-2 patients throughout the course of their infection and recovery. The VIP buffer is stable at room temperature for at least 6 months. SARS-CoV-2 RNA (65 copies/200 µL sample) is stable in the VIP buffer at room temperature for at least 3 weeks. The on-site inactivation of SARS-CoV-2 and preservation of the viral RNA enables self-collection of samples, reduces risks associated with SARS-CoV-2 transmission, and maintains the stability of the target analyte.