Building Environmental and Sociological Predictive Intelligence to Understand the Seasonal Threat of SARS-CoV-2 in Human Populations.

Current modeling practices for environmental and sociological modulated infectious diseases remain inadequate to forecast the risk of outbreak(s) in human populations, partly due to a lack of integration of disciplinary knowledge, limited availability of disease surveillance datasets, and overreliance on compartmental epidemiological modeling methods. Harvesting data knowledge from virus transmission (aerosols) and detection (wastewater) of SARS-CoV-2, a heuristic score-based environmental predictive intelligence system was developed that calculates the risk of COVID-19 in the human population. Seasonal validation of the algorithm was uniquely associated with wastewater surveillance of the virus, providing a lead time of 7-14 days before a county-level outbreak. Using county-scale disease prevalence data from the United States, the algorithm could predict COVID-19 risk with an overall accuracy ranging between 81% and 98%. Similarly, using wastewater surveillance data from Illinois and Maryland, the SARS-CoV-2 detection rate was greater than 80% for 75% of the locations during the same time the risk was predicted to be high. Results suggest the importance of a holistic approach across disciplinary boundaries that can potentially allow anticipatory decision-making policies of saving lives and maximizing the use of available capacity and resources.

Portable, single nucleotide polymorphism-specific duplex assay for virus surveillance in wastewater.

The Argonaute protein from the archaeon Pyrococcus furiosus (PfAgo) is a DNA-guided nuclease that targets DNA with any sequence. We designed a virus detection assay in which the PfAgo enzyme cleaves the reporter probe, thus generating fluorescent signals when amplicons from a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay contain target sequences. We confirmed that the RT-LAMP-PfAgo assay for the SARS-CoV-2 Delta variant produced significantly higher fluorescent signals (p < 0.001) when a single nucleotide polymorphism (SNP), exclusive to the Delta variant, was present, compared to the samples without the SNP. Additionally, the duplex assay for Pepper mild mottle virus (PMMOV) and SARS-CoV-2 detection produced specific fluorescent signals (FAM or ROX) only when the corresponding sequences were present. Furthermore, the RT-LAMP-PfAgo assay does not require dilution to reduce the impact of environmental inhibitors. The limit of detection of the PMMOV assay, determined with 30 wastewater samples, was 28 gc/µL, with a 95 % confidence interval of [11,103]. Finally, using a point-of-use device, the RT-LAMP-PfAgo assay successfully detected PMMOV in wastewater samples. Based on our findings, we conclude that the RT-LAMP-PfAgo assay can be used as a portable, SNP-specific duplex assay, which will significantly improve virus surveillance in wastewater.

Improved performance of nucleic acid-based assays for genetically diverse norovirus surveillance.

Nucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of their genetic diversity. Here, we calculated the variability of nucleotides within the genomes of 10 human viral species in silico and found that endemic viruses exhibit a high percentage of variable nucleotides (e.g., 51.4% for norovirus genogroup II). This genetic diversity led to the variable probability of detection of PCR assays (the proportion of viral sequences that contain the assay's target sequences divided by the total number of viral sequences). We then experimentally confirmed that the probability of the target sequence detection is indicative of the number of mismatches between PCR assays and norovirus genomes. Next, we developed a degenerate PCR assay that detects 97% of known norovirus genogroup II genome sequences and recognized norovirus in eight clinical samples. By contrast, previously developed assays with 31% and 16% probability of detection had 1.1 and 2.5 mismatches on average, respectively, which negatively impacted RNA quantification. In addition, the two PCR assays with a lower probability of detection also resulted in false negatives for wastewater-based epidemiology. Our findings suggest that the probability of detection serves as a simple metric for evaluating nucleic acid-based assays for genetically diverse virus surveillance.IMPORTANCENucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are employed widely as a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of the rapid evolution and genetic variation of viruses. The study analyzed clinical and wastewater samples using multiple PCR assays and found significant performance variation among the PCR assays for genetically diverse norovirus surveillance. This finding suggests that some PCR assays may miss detecting certain virus strains, leading to a compromise in detection sensitivity. To address this issue, we propose a metric called the probability of detection, which can be simply calculated in silico using a code developed in this study, to evaluate nucleic acid-based assays for genetically diverse virus surveillance. This new approach can help improve the sensitivity and accuracy of virus detection, which is crucial for effective infectious disease surveillance and control.

Application of neighborhood-scale wastewater-based epidemiology in low COVID-19 incidence situations.

Wastewater-based epidemiology (WBE), an emerging approach for community-wide COVID-19 surveillance, was primarily characterized at large sewersheds such as wastewater treatment plants serving a large population. Although informed public health measures can be better implemented for a small population, WBE for neighborhood-scale sewersheds is less studied and not fully understood. This study applied WBE to seven neighborhood-scale sewersheds (average population of 1471) from January to November 2021. Community testing data showed an average of 0.004 % incidence rate in these sewersheds (97 % of monitoring periods reported two or fewer daily infections). In 92 % of sewage samples, SARS-CoV-2 N gene fragments were below the limit of quantification. We statistically determined 10-2.6 as the threshold of the SARS-CoV-2 N gene concentration normalized to pepper mild mottle virus (N/PMMOV) to alert high COVID-19 incidence rate in the studied sewershed. This threshold of N/PMMOV identified neighborhood-scale outbreaks (COVID-19 incidence rate higher than 0.2 %) with 82 % sensitivity and 51 % specificity. Importantly, neighborhood-scale WBE can discern local outbreaks that would not otherwise be identified by city-scale WBE. Our findings suggest that neighborhood-scale WBE is an effective community-wide disease surveillance tool when COVID-19 incidence is maintained at a low level.

Inactivation Mechanism and Efficacy of Grape Seed Extract for Human Norovirus Surrogate.

Proper disinfection of harvested food and water is critical to minimize infectious disease. Grape seed extract (GSE), a commonly used health supplement, is a mixture of plant-derived polyphenols. Polyphenols possess antimicrobial and antifungal properties, but antiviral effects are not well-known. Here we show that GSE outperformed chemical disinfectants (e.g., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. GSE induced virus aggregation, a process that correlated with a decrease in virus titers. This aggregation and disinfection were not reversible. Molecular docking simulations indicate that polyphenols potentially formed hydrogen bonds and strong hydrophobic interactions with specific residues in viral capsid proteins. Together, these data suggest that polyphenols physically associate with viral capsid proteins to aggregate viruses as a means to inhibit virus entry into the host cell. Plant-based polyphenols like GSE are an attractive alternative to chemical disinfectants to remove infectious viruses from water or food. IMPORTANCE Human noroviruses are major food- and waterborne pathogens, causing approximately 20% of all cases of acute gastroenteritis cases in developing and developed countries. Proper sanitation or disinfection are critical strategies to minimize human norovirus-caused disease until a reliable vaccine is created. Grape seed extract (GSE) is a mixture of plant-derived polyphenols used as a health supplement. Polyphenols are known for antimicrobial, antifungal, and antibiofilm activities, but antiviral effects are not well-known. In studies presented here, plant-derived polyphenols outperformed chemical disinfectants (i.e., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. Based on data from molecular assays and molecular docking simulations, the current model is that the polyphenols in GSE bind to the Tulane virus capsid, an event that triggers virion aggregation. It is thought that this aggregation prevents Tulane virus from entering host cells.

Design of SARS-CoV-2 Variant-Specific PCR Assays Considering Regional and Temporal Characteristics.

Monitoring the prevalence of SARS-CoV-2 variants is necessary to make informed public health decisions during the COVID-19 pandemic. PCR assays have received global attention, facilitating a rapid understanding of variant dynamics because they are more accessible and scalable than genome sequencing. However, as PCR assays target only a few mutations, their accuracy could be reduced when these mutations are not exclusive to the target variants. Here we introduce PRIMES, an algorithm that evaluates the sensitivity and specificity of SARS-CoV-2 variant-specific PCR assays across different geographical regions by incorporating sequences deposited in the GISAID database. Using PRIMES, we determined that the accuracy of several PCR assays decreased when applied beyond the geographic scope of the study in which the assays were developed. Subsequently, we used this tool to design Alpha and Delta variant-specific PCR assays for samples from Illinois, USA. In silico analysis using PRIMES determined the sensitivity/specificity to be 0.99/0.99 for the Alpha variant-specific PCR assay and 0.98/1.00 for the Delta variant-specific PCR assay in Illinois, respectively. We applied these two variant-specific PCR assays to six local sewage samples and determined the dominant SARS-CoV-2 variant of either the wild type, the Alpha variant, or the Delta variant. Using next-generation sequencing (NGS) of the spike (S) gene amplicons of the Delta variant-dominant samples, we found six mutations exclusive to the Delta variant (S:T19R, S:Δ156/157, S:L452R, S:T478K, S:P681R, and S:D950N). The consistency between the variant-specific PCR assays and the NGS results supports the applicability of PRIMES. IMPORTANCE Monitoring the introduction and prevalence of variants of concern (VOCs) and variants of interest (VOIs) in a community can help the local authorities make informed public health decisions. PCR assays can be designed to keep track of SARS-CoV-2 variants by measuring unique mutation markers that are exclusive to the target variants. However, the mutation markers may not be exclusive to the target variants because of regional and temporal differences in variant dynamics. We introduce PRIMES, an algorithm that enables the design of reliable PCR assays for variant detection. Because PCR is more accessible, scalable, and robust for sewage samples than sequencing technology, our findings will contribute to improving global SARS-CoV-2 variant surveillance.

A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads.

Viruses are present at low concentrations in wastewater; therefore, an effective method for concentrating virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species, including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the final solution to that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency was significantly higher when calculated with virus titers than it was with genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk to humans who are inadvertently exposed to wastewater contaminated with infectious viruses. Furthermore, PCR inhibitors were not concentrated by PGM-MBs, suggesting that this tool will be successful for use with environmental samples. In addition, the PGM-MBs method is cost-effective (0.5 USD/sample) and has a fast turnaround time (3 h from virus concentration to genome quantification). Thus, this method can be implemented in high throughput facilities. Because of its strong performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, the PGM-MBs method can be successfully applied to WBE and ultimately provides valuable public health information.

Label-free SARS-CoV-2 detection and classification using phase imaging with computational specificity.

Efforts to mitigate the COVID-19 crisis revealed that fast, accurate, and scalable testing is crucial for curbing the current impact and that of future pandemics. We propose an optical method for directly imaging unlabeled viral particles and using deep learning for detection and classification. An ultrasensitive interferometric method was used to image four virus types with nanoscale optical path-length sensitivity. Pairing these data with fluorescence images for ground truth, we trained semantic segmentation models based on U-Net, a particular type of convolutional neural network. The trained network was applied to classify the viruses from the interferometric images only, containing simultaneously SARS-CoV-2, H1N1 (influenza-A virus), HAdV (adenovirus), and ZIKV (Zika virus). Remarkably, due to the nanoscale sensitivity in the input data, the neural network was able to identify SARS-CoV-2 vs. the other viruses with 96% accuracy. The inference time for each image is 60 ms, on a common graphic-processing unit. This approach of directly imaging unlabeled viral particles may provide an extremely fast test, of less than a minute per patient. As the imaging instrument operates on regular glass slides, we envision this method as potentially testing on patient breath condensates. The necessary high throughput can be achieved by translating concepts from digital pathology, where a microscope can scan hundreds of slides automatically.

Field demonstration of solar-powered electrocoagulation water treatment system for purifying groundwater contaminated by both total coliforms and arsenic.

People who drink groundwater in rural areas of Southeast Asia are exposed to pathogens and arsenic (As)-related health problems. A water treatment system consisting of electrocoagulation reactors, using iron (Fe) electrodes and a filtration tank, was designed to treat complex contaminated groundwater for drinking. Its applicability was demonstrated near the Red River in Vietnam. The water treatment system reduced 10.3 CFU/mL of total coliform and 376 µg/L of As(III) in the groundwater to 0 CFU/mL and 6.68 µg/L, respectively. Total coliforms were attenuated by Fe(II) infiltration or enmeshed during Fe precipitate formation. Of the total As, 43% formed As(III) complexation with the Fe precipitates and the other 57% was oxidized to As(V) then adsorbed to Fe precipitates. The Fe precipitates, containing total coliforms and As, were separated from the discharge water in the filtration tank. The system required 49 W of power to operate, which equates to 423 kWh/year, to continuously purify 0.5 t water/day. This requirement was powered by a 380-750 W solar panel, without external energy supply, making the water treatment system an appropriate option for addressing drinking water problems in rural areas.

Mechanism and efficacy of virus inactivation by a microplasma UV lamp generating monochromatic UV irradiation at 222 nm.

This study evaluated the potential of a microplasma UV lamp as an alternative UV source to the current mercury-based (Hg-based) UV lamp for water disinfection. We developed a set of PCR-based molecular assays (long-range qPCR, DNase, and binding assay) to quantify the adenovirus genome, capsid, and fiber damage with a wide detection range (100.5-106.5 PFU/mL). We used these molecular assays to characterize adenovirus (AdV) inactivation kinetics by microplasma UV that produced monochromatic UV at 222 nm. We found that the inactivation rate constant (0.142 cm2/mJ) due to microplasma UV was 4.4 times higher than that of low-pressure Hg UV (0.032 cm2/mJ). This high efficacy was attributed to monochromatic UV wavelength at 222 nm damaging the AdV capsid protein. The results of these molecular assays also proved that microplasma UV and medium-pressure Hg UV with a bandpass filter at 223 nm (MPUV223nm) have a similar influence on AdV (p>0.05). We then estimated the relative energy efficiency of MPUV and microplasma UV to LPUV for 4 log reduction of the viruses. We found that the microplasma UV resulted in higher inactivation rate constants for viruses than the current Hg-based UV. Consequently, microplasma UV could be more energy efficient than low-pressure Hg UV for water disinfection if the wall-plug efficiency of the microplasma UV lamp improved to 8.4% (currently 1.5%). Therefore, the microplasma UV lamp is a promising option for water disinfection.

Dry Heat as a Decontamination Method for N95 Respirator Reuse.

A pandemic such as COVID-19 can cause a sudden depletion of the worldwide supply of respirators, forcing healthcare providers to reuse them. In this study, we systematically evaluated dry heat treatment as a viable option for the safe decontamination of N95 respirators (1860, 3M) before their reuse. We found that the dry heat generated by an electric cooker (100 °C, 5% relative humidity, 50 min) effectively inactivated Tulane virus (TV, >5.2-log10 reduction), rotavirus (RV, >6.6-log10 reduction), adenovirus (AdV, >4.0-log10 reduction), and transmissible gastroenteritis virus (TGEV, >4.7-log10 reduction). The respirator integrity (determined on the basis of the particle filtration efficiency and quantitative fit testing) was not compromised after 20 cycles of a 50 min dry heat treatment. On the basis of these results, dry heat decontamination generated by an electric cooker (e.g., rice cookers, instant pots, and ovens) could be an effective and accessible decontamination method for the safe reuse of N95 respirators. We recommend users measure the temperature during decontamination to ensure the respirator temperature can be maintained at 100 °C for 50 min.

Applicability of electrochemical wastewater treatment system powered by temperature difference energy.

In this research, an electrochemical wastewater treatment system, powered through the conversion of temperature difference into energy, was designed. The wastewater treatment system was applied to drainage flowing from two different mines, one contaminated by arsenic (As), the other ferrous iron (Fe2+). Arsenic was adsorbed on, or co-precipitated with, iron hydroxide generated from iron electrodes. A pseudo second order model well described the tendency for As removal. Ferrous iron oxidation occurred directly on graphite electrodes and followed a first order reaction model. The efficiency of As and Fe2+ removal was up to 99.7% and 97.9%, respectively. The rate constants for each model were proportional to given temperature differences, as the relationship between current generation and temperature difference was linear. Economic evaluation of the wastewater treatment system was performed by comparing the predicted cost of the thermocouples under particular environmental conditions. The thermocouple technology currently available could be applied to wastewater treatment for hot springs with high temperatures and high As concentrations. The applicability of the system to low temperature wastewaters will expand as energy production capacity per unit cost of thermocouples increases, as occurred with the photovoltaic and heat pump systems currently in use.

Reliability improvement for predicting acid-forming potential of rock samples using static tests.

In predicting the acid-forming potential of rock samples, a combination of acid-base accounting (ABA) and net acid generation (NAG) tests has been commonly used. While simple and economical, this method sometimes shows low reliability such as categorizing certain samples as uncertain (UC). ABA and NAG tests were modified to selectively recover valid minerals in nature and substituted for the original tests. ABA test overestimated acid-producing capacity (in the case of weathered samples) and acid-neutralizing capacity (in the case of plagioclase-including samples) compared to the modified ABA test. NAG test yielded lower NAG pH compared to modified NAG test for samples with high total C content and low total S content. By comparing the correlation coefficients between acid generation amounts by the two evaluation methods, it was confirmed that modified evaluation method (MEM) has a much higher reliability (R 2 = 0.9582) than existing evaluation method (EEM) (R 2 = 0.5873). It was also concluded that exploiting advantages of both EEM and MEM is recommended where EEM is initially applied for general classification and a supplemented static test of MEM is executed for the purpose of correcting the error of UC categorized samples.

Evaluation of net acid generation pH as a single indicator for acid forming potential of rocks using geochemical properties.

The main purpose of this research was to evaluate the geochemical properties of rocks for a single indicator of acid-forming potential. The indicators, such as net acid generation (NAG), NAG pH and total S, were applied to 312 rock samples of various geological characteristics. Additional indicators, such as a Modified NAG pH, paste pH and available acid neutralizing capacity (ANC), were applied to 22 selected samples. Among them, NAG pH was considered the most plausible single indicator in evaluating acid-forming potential, as it is simple to measure, widely applicable to various samples and can be used to estimate the NAG value. The acid-forming potential of 287 samples (92% of samples examined in this research) was classified as either non-acid forming (NAF) or potentially acid forming (PAF) by NAG pH, with an NAF criteria of <3.21 and PAF of >4.52. The NAG pH was also a good estimate of the risk of short-term acid release when combined with paste pH information. However, application of NAG pH to coal mine wastes, with high organic carbon contents, produced erroneous results due to the generation of organic acid during the NAG test. In this research, a Modified NAG pH was assessed as an alternative to NAG pH in such situations.

Bauxite residue neutralization with simultaneous mineral carbonation using atmospheric CO2.

Simultaneous carbon mineralization during neutralization of bauxite residue, a caustic alkaline by-product of alumina refining, was tested using laboratory batch and a field pilot study in contact with atmospheric CO2. Since CO2 sequestration is limited by the Ca concentration in the bauxite residue, extra Ca sources were added in a semi-soluble mineral and salt form (flue gas desulfurization gypsum or CaCl2) to verify whether this Ca addition accelerated and enlarged the CO2 sequestration obtained as a consequence of neutralization. The results of 55 days of batch and longer-term field tests were in good agreement, and the neutralization rate was accelerated through the addition of both Ca sources. Without the addition of the extra Ca source, atmospheric CO2 contributed to neutralization of pore water alkalinity alone, while Ca addition induced further neutralization through mineral carbonation of atmospheric CO2 to CaCO3. This simple addition of environmentally benign Ca to bauxite residue may provide a feasible bauxite residue management practice that is cost-effective and easy to apply in the field.

Field application of selective precipitation for recovering Cu and Zn in drainage discharged from an operating mine.

Acid mine drainage (AMD) generated from mining activities has been recognized as a serious problem due to its increased acidity and high concentration of heavy metals. In this research, a feasibility test of the selective precipitation (SP) process was performed using AMD discharged from a currently operating mine in Korea for the purpose of minimizing the environmental impact of AMD. For the SP process, a pilot scale equipment (100L reaction tank) was used in field and among various metals, Cu and Zn were the target metals. Through the research, it was confirmed that AMD from an operating mine has two disadvantages of being applied to the SP: altering water quality and unexpected inclusion of clay debris. Despite unfavorable conditions, Cu and Zn precipitate of 80% purity with 90% precipitation rate was able to be obtained from 1.4L/min (2.0tons/day) AMD. The recovered precipitates were identified as amorphous CuS and ZnS with small amounts of impurities (Si minerals, CuFeS2, and Fe/Al hydroxide). The strategies to reduce these impurities were also discussed. Recovery rate, which is the amount of precipitate collected per unit volume of AMD, was proposed as an indicator to evaluate the working efficiency of the SP process. It was confirmed that the recovery rate was strongly dependent on flow rate and dose of coagulant. The results of this study may be helpful in reducing the potential complications which occurs when SP is applied on field.

Evaluation of design factors for a cascade aerator to enhance the efficiency of an oxidation pond for ferruginous mine drainage.

This research focused on the optimum design of a cascade aerator to enhance the efficiency of an oxidation pond in a passive treatment system for remediating ferruginous mine drainage. For this purpose, various aeration experiments with aerators of different drop heights (0-4 m) and formations (types A and B) were executed on mine drainage. Type A simply drops the mine drainage into the oxidation pond while type B sprays the mine drainage and retains it for 8 min in each step. The efficiency enhancement of the oxidation pond was strongly dependent on the increase in pH and DO of the mine drainage discharged into the pond. The water quality improved with the increase in drop height but especially showed better effect with type B. The reasons for this result were attributed to the increase of contact surface and retention time of the mine drainage. The cascade aerator, therefore, should be designed to be as high as possible with the assistance of spraying form and retention time of the mine drainage to maximize the efficiency of the oxidation pond. These effects could be evaluated by calculating required areas of the oxidation pond for 95% of Fe(2+) oxidation.

Removal characteristics of As(III) and As(V) from acidic aqueous solution by steel making slag.

This study focused on the environmental risk of steel making slag itself, arsenic removal mechanism and re-leaching possibility of arsenic to aqueous state after the adsorption. The purpose of the study is to promote the use of steel making slag as a low-cost adsorbent for arsenic in aqueous system. Calcium was easily dissolved out from the slag and become the dominant substance in the leachate. Some of the calcium could form amorphous calcium carbonate in alkaline condition, and arsenic in the aqueous solution would be removed by being co-precipitated with or adsorbed onto the amorphous calcium carbonate. Most of the amorphous calcium carbonate containing arsenic would be bound to amorphous iron oxide of the slag. When the slag was used as an adsorbent for arsenic removal, a little amount of toxic chemicals were leached from the slag itself under pH 0.8 to 13.6. Also, 70-80% of arsenic laden on the slag was bound to amorphous iron oxide which would not easily desorb unless given a reducing and complexing condition. Showing 95-100% removal efficiency near initial pH 2, the slag, therefore, could be used as an appropriate adsorbent for eliminating arsenic in acidic aqueous solution.