Surveillance of Naegleria fowleri in Louisiana's public water systems.

The free-living amoeba Naegleria fowleri (Nf) inhabits soil and natural waters worldwide: it is thermophilic and thrives at temperatures up to 45 °C and in a multitude of environments. Three deaths in Louisiana were attributed to primary amoebic meningoencephalitis (PAM) caused by Nf infection in 2011 and 2013. Following these incidents, public water systems are now monitored for the presence of Nf in Louisiana. From 2014 to 2018, 29% (27/93) of samples collected showed positive for Nf and 68% (63/93) showed all thermophilic amoeba culture. Ten raw water sources and 17 distribution water systems tested positive. The year 2017 showed the highest number of samples with Nf (n = 10) followed by nine samples in 2015. As climate change increases surface water temperatures, continued testing for Nf prevalence will be an important facet of water monitoring and will need to extend into locations farther north than the current most common range.

Classifying Building Roof Damage Using High Resolution Imagery for Disaster Recovery.

Post-hurricane damage assessments are often costly and time-consuming. Remotely sensed data provides a complementary method of data collection that can be completed comparatively quickly and at relatively low cost. This study focuses on 15 Florida counties impacted by Hurricane Michael (2018), which had category 5 strength winds at landfall. The present study evaluates the ability of aerial imagery collected to cost-effectively measure blue tarps on buildings for disaster impact and recovery. A support vector machine model classified blue tarp, and parcels received a damage indicator based on the model's prediction. The model had an overall accuracy of 85.3% with a sensitivity of 74% and a specificity of 96.7%. The model results indicated approximately 7% of all parcels (27 926 residential and 4431 commercial parcels) in the study area as having blue tarp present. The study results may benefit jurisdictions that lacked financial resources to conduct on-the-ground damage assessments.

The importance of accessory protein variants in the pathogenicity of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS- CoV-2) with an estimated fatality rate of less than 1%. The SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 possess putative functions to manipulate host immune mechanisms. These involve interferons, which appear as a consensus function, immune signaling receptor NLRP3 (NLR family pyrin domain-containing 3) inflammasome, and inflammatory cytokines such as interleukin 1ß (IL-1ß) and are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins were observed across six continents of all complete SARS-CoV-2 proteomes based on the data reported before November 2020. A decreasing order of percentage of unique variations in the accessory proteins was determined as ORF3a > ORF8 > ORF7a > ORF6 > ORF10 > ORF7b across all continents. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. These findings suggest that the wide variations in accessory proteins seem to affect the pathogenicity of SARS-CoV-2.

Periodically aperiodic pattern of SARS-CoV-2 mutations underpins the uncertainty of its origin and evolution.

Various lineages of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have contributed to prolongation of the Coronavirus Disease 2019 (COVID-19) pandemic. Several non-synonymous mutations in SARS-CoV-2 proteins have generated multiple SARS-CoV-2 variants. In our previous report, we have shown that an evenly uneven distribution of unique protein variants of SARS-CoV-2 is geo-location or demography-specific. However, the correlation between the demographic transmutability of the SARS-CoV-2 infection and mutations in various proteins remains unknown due to hidden symmetry/asymmetry in the occurrence of mutations. This study tracked how these mutations are emerging in SARS-CoV-2 proteins in six model countries and globally. In a geo-location, considering the mutations having a frequency of detection of at least 500 in each SARS-CoV-2 protein, we studied the country-wise percentage of invariant residues. Our data revealed that since October 2020, highly frequent mutations in SARS-CoV-2 have been observed mostly in the Open Reading Frame (ORF) 7b and ORF8, worldwide. No such highly frequent mutations in any of the SARS-CoV-2 proteins were found in the UK, India, and Brazil, which does not correlate with the degree of transmissibility of the virus in India and Brazil. However, we have found a signature that SARS-CoV-2 proteins were evolving at a higher rate, and considering global data, mutations are detected in the majority of the available amino acid locations. Fractal analysis of each protein's normalized factor time series showed a periodically aperiodic emergence of dominant variants for SARS-CoV-2 protein mutations across different countries. It was noticed that certain high-frequency variants have emerged in the last couple of months, and thus the emerging SARS-CoV-2 strains are expected to contain prevalent mutations in the ORF3a, membrane, and ORF8 proteins. In contrast to other beta-coronaviruses, SARS-CoV-2 variants have rapidly emerged based on demographically dependent mutations. Characterization of the periodically aperiodic nature of the demographic spread of SARS-CoV-2 variants in various countries can contribute to the identification of the origin of SARS-CoV-2.

Remotely sensed measures of Hurricane Michael damage and adverse perinatal outcomes and access to prenatal care services in the Florida panhandle.

BACKGROUND: Studies of effects of hurricanes on perinatal outcomes often rely on approximate measures of exposure. This study aims to use observed damage from aerial imagery to refine residential building damage estimates, evaluate the population changes post landfall, and assess the associations between the extent of residential building damage and adverse perinatal outcomes and access to prenatal care (PNC) services.  METHODS: Vital statistics data from the Florida Department of Health's Office of Vital Statistics were used to align maternal geocoded address data to high-resolution imagery (0.5-foot resolution, true color with red, blue, and green bands) aerial photographs. Machine learning (support vector machines) classified residential roof damage across the study area. Perinatal outcomes were compared with the presence or absence of damage to the mother's home. Log-binomial regression models were used to compare the populations living in and outside of high-risk/damage areas, to assess the population changes after Hurricane Michael, and to estimate the associations between damage after Hurricane Michael and adverse perinatal outcomes/access to PNC services. A semi-parametric linear model was used to model time of first PNC visit and increase in damage. RESULTS: We included 8,965 women in analysis. Women with lower education and/or of Black or other non-White race/ethnicity were more likely to live in areas that would see high damage than other groups. Moreover, there was a greater proportion of births delivered by women living in the high-risk/damage area (> 25% damaged parcels after Michael) in the year before Michael than the year after Michael. Lastly, living in the area with relatively high damage increased the risk of having intermediate or inadequate PNC (adjusted Risk Ratio = 1.21, 95% CI: 1.03, 1.43), but not other adverse perinatal outcomes. CONCLUSIONS: Aerially observed damage data enable us to evaluate the impact of natural disasters on perinatal outcomes and access to PNC services based on residential building damage immediately surrounding a household. The association between the extent of damage and adverse perinatal outcomes should be further investigated in future studies.

Harmful algal bloom-related 311 calls, Cape Coral, Florida 2018-2019.

Harmful algal blooms (HABs) can adversely impact water quality and threaten human and animal health. People working or living along waterways with prolonged HAB contamination may face elevated toxin exposures and breathing complications. Monitoring HABs and potential adverse human health effects is notoriously difficult due to routes and levels of exposure that vary widely across time and space. This study examines the utility of 311 calls to enhance HAB surveillance and monitoring. The study focuses on Cape Coral, FL, USA, located along the banks of the Caloosahatchee River and Estuary and the Gulf of Mexico. The wider study area experienced a prolonged cyanobacteria bloom in 2018. The present study examines the relationship between weekly water quality characteristics (temperature, dissolved oxygen, pH, microcystin-LR) and municipal requests for information or services (algal 311 calls). Each 1 µg/L increase in waterborne microcystin-LR concentrations corresponded with 9% more algal 311 calls (95% confidence interval: 1.03-1.15, p = 0.002). The results suggest water quality monitoring and the 311 dispatch systems may be further integrated to improve public health surveillance.

Intraday variability of indicator and pathogenic viruses in 1-h and 24-h composite wastewater samples: Implications for wastewater-based epidemiology.

We monitored the concentration of indicator viruses crAssphage and pepper mild mottle virus (PMMoV) and human pathogen adenovirus (HAdV) in influent from a wastewater treatment plant in Brisbane, Australia in 1-h and 24-h composite samples. Over three days of sampling, the mean concentration of crAssphage gene copies (GC)/mL in 24-h composite samples did not differ significantly (p = 0.72-0.92), while for PMMoV GC/mL (p value range: 0.0002-0.0321) and HAdV GC/mL (p value range: 0.0028-0.0068) significant differences in concentrations were observed on one day of sampling compared to the other two. For all three viruses, the variation observed in 1-h composite samples was greater than the variation observed in 24-h composite samples. For crAssphage, in 54.1% of 1-h composite samples, the concentration was less than that observed in 24-h composite samples; whereas for PMMoV and HAdV the concentration was less in 79.2 and 70.9% of 1-h composite samples, respectively, compared to the relevant 24-h composite samples. Similarly, the concentration of crAssphage in 1-h compared to 24-h composite samples did not differ (p = 0.1082) while the concentrations of PMMoV (p < 0.0001) and HAdV (p < 0.0001) in 1-h composite samples were significantly different from 24-h composite samples. These results suggest that 24-h composite samples offer increased analytical sensitivity and decreased variability compared to 1-h composite samples when monitoring wastewater, especially for pathogenic viruses with low infection rates within a community. Thus, for wastewater-based epidemiology applications, 24-h composite samples are less likely to produce false negative results and erroneous public health information.

Molecular detection of opportunistic premise plumbing pathogens in rural Louisiana's drinking water distribution system.

Opportunistic premise plumbing pathogens (OPPPs) in drinking water distribution systems are responsible for causing numerous infections such as Legionnaires' disease and pneumonia through the consumption of contaminated drinking water. The incidence of opportunistic pathogens and the number of individuals at risk of contracting infections caused by these OPPPs in drinking water has risen drastically in the past decade. Preflush and postflush water samples were collected from 64 houses in a rural town in northeast Louisiana to determine drinking water quality in terms of understanding abiotic and biotic factors on potential proliferation of OPPPs. Physical and chemical water quality parameters, such as pH, temperature, dissolved oxygen, salinity, and specific conductance were also measured. The quantitative polymerase chain reaction (qPCR) results indicated that Legionella spp. had the highest prevalence and was found in 46/64 samples (72%), followed by Mycobacterium spp. which was found in 43/64 samples (67%), E. coli in 31/64 samples (48%) and, Naegleria fowleri in 4/64 samples (6%) respectively. The results indicate the persistence of Legionella spp. DNA marker in these water samples.

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T90 (time required for 1-log10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58-43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.

Molecular detection of opportunistic pathogens and insights into microbial diversity in private well water and premise plumbing.

Private well water systems in rural areas that are improperly maintained will result in poor drinking water quality, loss of water supply, and pose human health risk. The purpose of this study was to investigate the occurrence of fecal indicator bacteria (FIB) and opportunistic pathogens in private well water in rural areas surrounding New Orleans, Louisiana. Our results confirmed the ubiquitous nature of Legionella (86.7%) and mycobacteria (68.1%) in private well water in the study area, with gene concentration ranged from 0.60 to 5.53 and 0.67 to 5.95 Log10 of GC/100 mL, respectively. Naegleria fowleri target sequence was detected in 16.8% and Escherichia coli was detected in 43.4% of the water samples. Total coliform, as well as Legionella and mycobacteria genetic markers' concentrations were significantly reduced by 3-minute flushing. Next-generation sequencing (NGS) data indicated that the abundance of bacterial species was significantly increased in water collected in kitchens compared with samples from wells directly. This study provided integrated knowledge on the persistence of pathogenic organisms in private well water. Further study is needed to explore the presence of clinical species of those opportunistic pathogens in private well water systems to elucidate the health risk.

Possible Transmission Flow of SARS-CoV-2 Based on ACE2 Features.

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is engendering the severe coronavirus disease 2019 (COVID-19) pandemic. The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 binds to the three sub-domains viz. amino acids (aa) 22-42, aa 79-84, and aa 330-393 of ACE2 on human cells to initiate entry. It was reported earlier that the receptor utilization capacity of ACE2 proteins from different species, such as cats, chimpanzees, dogs, and cattle, are different. A comprehensive analysis of ACE2 receptors of nineteen species was carried out in this study, and the findings propose a possible SARS-CoV-2 transmission flow across these nineteen species.

Prevalence and associated risk factors of Giardia duodenalis infection among school-going children in Nepal.

This study aimed to determine the prevalence of intestinal parasites and its associated risk factors among school-going children in Kathmandu, Nepal. Between August and September 2016, a total of 333 stool samples were collected from children at five public schools. The collected samples were subjected to formol-ether concentration, followed by conventional microscopic examination for intestinal parasites. The overall prevalence of intestinal parasites was 24.3% (81/333), with Giardia spp. showing the highest prevalence of 18.9% (63/333). Samples positive for Giardia spp. by microscopy were further subjected to quantitative polymerase chain reaction (qPCR) for G. duodenalis, resulting in a positive ratio of 100%. The positive ratio of Giardia spp. was considerably high among children consuming tanker water (27.3%), jar water (21.0%), and tap water (17.5%). Our results demonstrated that G. duodenalis remains predominant in school-going children in Nepal.

Online monitoring of Escherichia coli and Bacillus thuringiensis spore inactivation after advanced oxidation treatment.

Various studies have shown that advanced oxidation processes (AOPs) such as UV light in combination with hydrogen peroxide is an efficient process for the removal of a large variety of emerging contaminants including microorganisms. The mechanism of destruction in the presence of hydrogen peroxide (H2O2) is the enhanced formation of hydroxyl (·OH) radicals, which have a high oxidation potential. The goal of this study was to utilize in-line advanced oxidation to inactivate microbes, and document the inactivation via an in-line, real-time sensor. Escherichia coli cells and Bacillus thuringiensis spores were exposed to UV/H2O2 treatment in DI water, and the online sensor BioSentry(®) was evaluated for its potential to monitor inactivation in real-time. B. thuringiensis was selected as a non-pathogenic surrogate for B. anthracis, the causative agent of anthrax and a proven biological weapon. UV radiation and UV/H2O2 exposure resulted in a >6 log10 reduction of the viable culturable counts of E. coli vegetative cells, and a 3 log10 reduction of B. thuringiensis spores. Scanning electron microscopy of the treated samples revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the morphology of the B. thuringiensis spores. Following AOP exposure, the BioSentry sensor showed an increase in the categories of unknown, rod and spores counts, but overall, did not correspond well with viable count assays. Data from this study show that advanced oxidation processes effectively inactivate E. coli vegetative cells, but not B. thuringiensis spores, which were more resistant to AOP. Further, the BioSentry in-line sensor was not successful in documenting destruction of the microbial cells in real-time.

Inactivation of MS2 coliphage by UV and hydrogen peroxide: comparison by cultural and molecular methodologies.

The use of advanced oxidation processes (AOP) are expected to increase for removal of emerging contaminants and pathogens from drinking water. In this study, the performance of a small community ultraviolet light reactor in combination with hydrogen peroxide (H2O2) for MS2 coliphage inactivation with two different flow rate conditions of 1 gal/min (gpm) and 2 gpm was evaluated. Following UV radiation, MS2 showed a reduction of 5.3-5.8 log10 when quantified with cultural plaque counts, whereas corresponding quantitative polymerase chain reaction (qPCR) data showed only a 1.7-2.8 log10 reduction in viral RNA copy number. When H2O2 was added at either 2.5 or 5 ppm with UV at both flow rate conditions, enhanced MS2 inactivation occurred with a more than 7 log10 reduction observed via plaque counts, indicating that all added MS2 had been inactivated, since no plaques were formed after incubation at 37 °C for 24 h. In contrast, qPCR only showed a corresponding 3-4 log10 reduction in viral RNA copy number. This research also sheds light on the inactivation of MS2 with ultraviolet light and in the presence of hydroxyl radicals and provides a practical use of qPCR to detect MS2 concentration following advanced oxidation relative to traditional plaque methodology; however qPCR detection overestimates the true number of infective virus.

Comparative analysis of membrane filter diameters for detection of selected viruses in wastewater samples.

Wastewater serves as a valuable source of information as it contains biological markers that have been shed by infected individuals and from other biological organisms such as plants and animals. Wastewater has been proven to indicate the presence of emerging pathogens in a community before the manifestation of clinical symptoms. Several methods of concentration and nucleic acid extraction have been employed all around the world without a unified method. One such method involves the use of the adsorption extraction method (AE-method), which involves the use of electronegative membrane filters of different pore sizes. The membrane filters also differ by diameter, but no study has been reported on the effect of diameter on capture efficiency. This study was aimed at evaluating the comparative capture efficiency of two different membrane filter diameters of 45 and 90 mm with pore sizes of 0.45 µm for the detection of indicator and pathogenic viruses. Primary influent samples were obtained from two wastewater treatment plants in Baltimore, Maryland, between April 27 and June 29, 2023. A total of twenty samples were processed using 45- and 90-mm membrane filters. Nucleic acids were extracted from the filters using the QIAmp Viral RNA Mini Kit and assayed for four different targets: PMMoV, Norovirus (GI and GII), and CrAssphage by RT-qPCR. The result showed that 45 mm membrane filters had a higher combined mean capture efficiency in log10 gene copies per liter (gc/l) for crAssphage (7.40) than 90 mm membrane filters (7.10). Similarly, the 45-mm filter had higher mean capture efficiency for Norovirus GI (4.67) than the 90-mm filter (1.84) and likewise for Norovirus GII (2.14, 1.04). On the contrary, 90-mm membrane filters were observed to have better capture of PMMoV (6.84) compared to 45-mm membrane filters (6.69). This result therefore implies that 45-mm membrane filters could be more efficient for wastewater surveillance studies through the AE method for indicator viruses like CrAssphage and human disease-causing viruses like Norovirus.

Multiple pathogen contamination of water, hands, and fomites in rural Nepal and the effect of WaSH interventions.

Water, Sanitation, and Hygiene (WaSH) interventions are the most effective in reducing diarrheal disease severity and prevalence. However, very few studies have investigated the effectiveness of WaSH intervention in reducing pathogen presence and concentration. In this study, we employed a microfluidic PCR approach to quantify twenty bacterial pathogens in water (n = 360), hands (n = 180), and fomite (n = 540) samples collected in rural households of Nepal to assess the pathogen exposures and the effect of WaSH intervention on contamination and exposure rates. The pathogen load and the exposure pathways for each pathogen in intervention and control villages were compared to understand the effects of WaSH intervention. Pathogens were detected in higher frequency and concentration from fomites samples, toilet handle (21.42%; 5.4,0 95%CI: mean log10 of 4.69, 5.96), utensils (23.5%; 5.47, 95%CI: mean log10 of 4.77, 6.77), and water vessels (22.42%; 5.53, 95%CI: mean log10 of 4.79, 6.60) as compared to cleaning water (14.36%; 5.05, 95%CI: mean log10 of 4.36, 5.89), drinking water (14.26%; 4.37, 85%CI: mean log10 of 4.37, 5.87), and hand rinse samples (16.92%; 5.49, 95%CI: mean log10 of 4.77, 6.39). There was no clear evidence that WaSH intervention reduced overall pathogen contamination in any tested pathway. However, we observed a significant reduction (p < 0.05) in the prevalence, but not concentration, of some target pathogens, including Enterococcus spp. in the intervention village compared to the control village for water and hands rinse samples. Conversely, no significant reduction in target pathogen concentration was observed for water and hand rinse samples. In swab samples, there was a reduction mostly in pathogen concentration rather than pathogen prevalence, highlighting that a reduction in pathogen prevalence was not always accompanied by a reduction in pathogen concentration. This study provides an understanding of WaSH intervention on microbe concentrations. Such data could help with better planning of intervention activities in the future.

Application of wastewater-based epidemiology for monitoring COVID-19 in hospital and housing wastewaters.

An alternative and complementary diagnostic method of surveillance is provided by wastewater-based surveillance (WBS), particularly in low-income nations like Nepal with scant wastewater treatment facilities and clinical testing infrastructure. In this study, a total of 146 water samples collected from two hospitals (n = 63) and three housing wastewaters (n = 83) from the Kathmandu Valley over the period of March 2021-Febraury 2022 were investigated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using quantitative reverse transcription TaqMan PCR assays targeting the N and E genes. Of the total, 67 % (98/146) samples were positive for SARS-CoV-2 RNA either by using N- or E-gene assay, with concentrations ranging from 3.6 to 9.1 log10 copies/L. There was a significant difference found between positive ratio (Chi-square test, p < 0.05) and concentration (t-test, p = 0.009) of SARS-CoV-2 RNA detected from hospital wastewater and housing waters. Wastewater data are correlated with COVID-19 active cases, indicating significance in specific areas like the Hospital (APFH) (p < 0.05). According to the application of a bivariate linear regression model (p < 0.05), the concentrations of N gene may be used to predict the COVID-19 cases in the APFH. Remarkably, SARS-CoV-2 RNA was detected prior to, during, and following clinical case surges, implying that wastewater surveillance could serve as an early warning system for public health decisions. The significance of WBS in tracking and managing pandemics is emphasized by this study, especially in resource-constrained settings.

Application of Skimmed-Milk Flocculation Method for Wastewater Surveillance of COVID-19 in Kathmandu, Nepal.

Wastewater surveillance (WS) has been used globally as a complementary tool to monitor the spread of coronavirus disease 2019 (COVID-19) throughout the pandemic. However, a concern about the appropriateness of WS in low- and middle-income countries (LMICs) exists due to low sewer coverage and expensive viral concentration methods. In this study, influent wastewater samples (n = 63) collected from two wastewater treatment plants (WWTPs) of the Kathmandu Valley between March 2021 and February 2022 were concentrated using the economical skimmed-milk flocculation method (SMFM). The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was tested by qPCR using assays that target the nucleocapsid (N) and envelope (E) genes. Overall, 84% (53/63) of the total samples were positive for SARS-CoV-2 according to at least one of the tested assays, with concentrations ranging from 3.5 to 8.3 log10 gene copies/L, indicating the effectiveness of the SMFM. No correlation was observed between the total number of COVID-19 cases and SARS-CoV-2 RNA concentrations in wastewater collected from the two WWTPs (p > 0.05). This finding cautions the prediction of future COVID-19 waves and the estimation of the number of COVID-19 cases based on wastewater concentration in settings with low sewer coverage by WWTPs. Future studies on WS in LMICs are recommended to be conducted by downscaling to sewer drainage, targeting a limited number of houses. Overall, this study supports the notion that SMFM can be an excellent economical virus-concentrating method for WS of COVID-19 in LMICs.

First quantitative detection of tomato brown rugose fruit virus in wastewater in Louisiana.

We investigated the occurrence of tomato brown rugose fruit virus (ToBRFV) at a conventional wastewater treatment plant in Louisiana over a 13-month period, from March 2017 to March 2018. Influent, secondary effluent, and final effluent wastewater samples were collected monthly, and viruses were concentrated by the adsorption-elution method using an electronegative filter, followed by the detection using quantitative polymerase chain reaction. ToBRFV was detected in 10 (77 %) of 13 influent samples, 9 (69 %) of 13 in secondary effluent, and 6 (50 %) of 12 final effluents. The concentrations of ToBRFV in the influent samples ranged from 3.5 to 6.1 log10 copies/L and it was always higher than those in secondary or final effluents. Wastewater samples showed a high positive ratio of ToBRFV during fall and winter months. The findings highlight that routine monitoring of new viral indicator such as ToBRFV is necessary to understand its environmental distribution and correlation with pathogenic viruses. This is the first study providing quantitative data on the occurrence of ToBRFV in wastewater.