Occurrence of Naegleria fowleri and faecal indicators in sediments from Lake Pontchartrain, Louisiana.

The occurrence of amoeba, Naegleria fowleri, in sediment samples from Lake Pontchartrain in Louisiana was investigated. This amoeba is pathogenic and can cause primary amoebic meningoencephalitis. In this study, quantitative polymerase chain reaction methods were used to test for the prevalence of Naegleria fowleri, HF183, and E. coli. N. fowleri was detected in 51.25% of our sediment samples. Illumina sequencing of sediment samples revealed ten different phyla, with Cyanobacteria being the most predominant at sites that generally presented with the highest median N. fowleri concentrations. N. fowleri was however strongly negatively correlated with HF183 (r = -0.859, p < 0.001). Whenever sediment E. coli concentrations were below 1.54 Log GC/g, there was only a 37.5% chance that N. fowleri would be detected in the same sample. When sediment E. coli concentrations exceeded 2.77 Log GC/g, the chances of detecting N. fowleri in the same sample increased to 90%, potentially suggesting predatory activity by the amoeba. The effect of temperature was observed to be different in relation to observed N. fowleri concentrations and detection rates. Although sediment samples collected during periods of higher temperatures had significantly lower mean N. fowleri concentrations (2.7 Log GC/g) compared to those collected at lower temperatures (3.7 Log GC/g, t(39) = 4.167, p < 0.001), higher N. fowleri detection rates in the overall samples were observed at higher temperatures (>19.1 °C) than at lower temperatures (<19.1 °C).

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.

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.

Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples.

Microbial source tracking (MST) methods measure fecal contamination levels and identify possible sources using quantitative PCR (qPCR) that targets host-associated fecal microorganisms. To date, most established MST assays for human sources, especially bacterial markers, have shown some nonhuman host cross-reactions. Recently developed assays, such as the crAssphage CPQ_056, Lachnospiraceae Lachno3, and Bacteroides BacV6-21, have more limited information on host sensitivity and host specificity for human or sewage sources, particularly in countries other than the United States. In this study, we rigorously evaluated six sewage-associated MST assays (i.e., Bacteroides HF183, human adenovirus [HAdV], human polyomavirus [HPyV], crAssphage CPQ_056, Lachno3, and BacV6-21) to show advantages and disadvantages of their applications for MST. A total of 29 human and 3 sewage samples and 360 nonhuman fecal samples across 14 hosts collected from a subtropical region of Australia were tested for marker host specificity, host sensitivity, and concentrations. All sewage samples were positive for all six marker genes tested in this study. Bacterial markers were more prevalent than viral markers in human feces. Testing against animal hosts showed human feces (or sewage)-associated marker gene specificity was HAdV (1.00) > HPyV (0.99) > crAssphage CPQ_056 (0.98) > HF183 (0.96) > Lachno3 (0.95) > BacV6-21 (0.90), with marker concentrations in some animal fecal samples being 3 to 5 orders of magnitude lower than those in sewage. When considering host specificity, sensitivity, and concentrations in source samples, the HF183, Lachno3, and crAssphage CPQ_056 tests were the most suitable assays in this study for sewage contamination tracking in subtropical waters of Australia.IMPORTANCE Large financial investments are required to remediate fecal contamination sources in waterways, and accurate results from field studies are crucial to build confidence in MST approaches. Host specificity and sensitivity are two main performance characteristics for consideration when choosing MST assays. Ongoing efforts for marker assay validation will improve interpretation of results and could shed light on patterns of occurrence in nontarget hosts that might explain the underlying drivers of cross-reaction of certain markers. For field applications, caution should be taken to choose appropriate MST marker genes and assays based on available host specificity and sensitivity data and background knowledge of the contaminating sources in the study area. Since many waterborne pathogens are viruses, employing both viral and bacterial markers in investigations could provide insight into contamination dynamics and ecological behavior in the environment. Therefore, combined usage of marker assays is recommended for more accurate and informative sewage contamination detection and fecal source resolution.

Quantification of hookworm ova from wastewater matrices using quantitative PCR.

A quantitative PCR (qPCR) assay was used to quantify Ancylostoma caninum ova in wastewater and sludge samples. We estimated the average gene copy numbers for a single ovum using a mixed population of ova. The average gene copy numbers derived from the mixed population were used to estimate numbers of hookworm ova in A. caninum seeded and unseeded wastewater and sludge samples. The newly developed qPCR assay estimated an average of 3.7×103 gene copies per ovum, which was then validated by seeding known numbers of hookworm ova into treated wastewater. The qPCR estimated an average of (1.1±0.1), (8.6±2.9) and (67.3±10.4) ova for treated wastewater that was seeded with (1±0), (10±2) and (100±21) ova, respectively. The further application of the qPCR assay for the quantification of A. caninum ova was determined by seeding a known numbers of ova into the wastewater matrices. The qPCR results indicated that 50%, 90% and 67% of treated wastewater (1L), raw wastewater (1L) and sludge (~4g) samples had variable numbers of A. caninum gene copies. After conversion of the qPCR estimated gene copy numbers to ova for treated wastewater, raw wastewater, and sludge samples, had an average of 0.02, 1.24 and 67 ova, respectively. The result of this study indicated that qPCR can be used for the quantification of hookworm ova from wastewater and sludge samples; however, caution is advised in interpreting qPCR generated data for health risk assessment.

Assessment of Genetic Markers for Tracking the Sources of Human Wastewater Associated Escherichia coli in Environmental Waters.

In this study, we have evaluated the performance characteristics (host-specificity and -sensitivity) of four human wastewater-associated Escherichia coli (E. coli) genetic markers (H8, H12, H14, and H24) in 10 target (human) and nontarget (cat, cattle, deer, dog, emu, goat, horse, kangaroo, and possum) host groups in Southeast Queensland, Australia. The overall host-sensitivity values of the tested markers in human wastewater samples were 1.0 (all human wastewater samples contained the E. coli genetic markers). The overall host-specificity values of these markers to differentiate between human and animal host groups were 0.94, 0.85, 0.72, and 0.57 for H8, H12, H24, and H14, respectively. Based on the higher host-specificity values, H8 and H12 markers were chosen for a validation environmental study. The prevalence of the H8 and H12 markers was determined among human wastewater E. coli isolates collected from a wastewater treatment plant (WWTP). Among the 97 isolates tested, 44 (45%) and 14 (14%) were positive for the H8 and H12 markers, respectively. A total of 307 E. coli isolates were tested from environmental water samples collected in Brisbane, of which 7% and 20% were also positive for the H8 and H12 markers, respectively. Based on our results, we recommend that these markers could be useful when it is important to identify the source(s) of E. coli (whether they originated from human wastewater or not) in environmental waters.

Study of Shellfish Growing Area During Normal Harvesting Periods and Following Wastewater Overflows in an Urban Estuary With Complex Hydrography.

Viral testing combined with hydrographic studies is considered standard good practice in determining microbiological impacts on shellfish growing areas following wastewater overflows. In this study, norovirus genogroup I and II, indicators of viral contamination (F-RNA bacteriophage genogroup II (F-RNA GII), crAssphage, pepper mild mottle virus) and Escherichia coli were monitored during periods of normal harvesting and following overflows in two commercial shellfish growing areas in Otago Harbour (Aotearoa New Zealand). Dye tracing, drogue tracking and analysis of particle tracking modelling were also undertaken to assess the dispersion, dilution and time of travel of wastewater discharged from a pump station discharge that impacts the growing areas. Norovirus was not detected in any of the 218 shellfish samples tested. PMMoV and crAssphage were more prevalent than F-RNA GII as determined by RT-qPCR. The dye study indicated long residence time of the waters (≥5 days) in the embayment impacted by the discharge. No relationships were found between the concentrations of viral indicators or E. coli and wastewater dilution, distance between the discharge and the growing areas or time since the last overflow. For the three spills studied (≤327 m3), there was little evidence of microbiological impact on the growing areas. This was likely associated with a deep shipping channel that enhances water flushing in the harbour and reduces contaminant transport to the growing areas. We recommend flexibility in the approach for closure/reopening growing areas impacted by spills, particularly for small duration/volume spills and when norovirus is not present in the community.

Black soldier fly-based bioconversion of biosolids: Microbial community dynamics and fate of antibiotic resistance genes.

Biosolids as by-products of wastewater treatment can contain a large spectrum of pathogens and antibiotic resistance genes (ARGs). Insect-based bioconversion using black soldier fly larvae (BSFL) is an emerging technology that has shown to reduce significant amounts of biosolids quickly and produce larvae biomass containing low heavy metal concentrations. However, to the best of our knowledge, this is the first study investigating the transfer of pathogens and ARGs from biosolids into the process' end-products, BSFL and frass. We hypothesized that BSF-based bioconversion can decrease the abundance of pathogenic bacteria and ARGs in biosolids. In this study, we performed BSFL feeding trials with biosolids blended or not blended with wheat bran, and wheat bran alone as a low bioburden diet (control). We conducted 16S rRNA amplicon sequencing to monitor changes of the BSFL-associated microbial community and the fate of biosolids-associated pathogens. A diverse set of ARGs (ermB, intl1, sul1, tetA, tetQ, tetW, and blaCTX-M-32) were quantified by qPCR and were linked to changes in substrate- and BSFL-associated microbiomes. BSF-based bioconversion of biosolids-containing substrates led to a significant reduction of the microbial diversity, the abundance of several pathogenic bacteria and the investigated ARGs (< 99 %). Feeding with a high bioburden biosolid diet resulted in a higher microbial diversity, and the accumulation of pathogenic bacteria and ARGs in the BSFL. Results of this study demonstrated that BSF-based bioconversion can be a suitable waste management technology to (1) reduce significant amounts of biosolids and (2) reduce the presence of pathogens and ARGs. However, the resulting larvae biomass would need to undergo further post-treatment to reduce the pathogenic load to allow them as animal feed.

PMAxx-RT-qPCR to Determine Human Norovirus Inactivation Following High-Pressure Processing of Oysters.

Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity of norovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450 MPa at 15 °C, and 300, 450 and 600 MPa at 20 °C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300 MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15 °C compared to 1.1 [0.9, 1.8] at 20 °C. At 450 MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15 °C compared to 1.3 [1.0, 1.6] at 20 °C. At 600 MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20 °C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600 MPa at both 15 °C and 20 °C, with the median [95% CI] RNI ratio value at 300 MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies.

Seroprevalence of SARS-CoV-2 infection in the general population of Nepal during the first and second generalized waves of the COVID-19 pandemic-2020-2021.

Few seroprevalence studies have been conducted on coronavirus disease (COVID-19) in Nepal. Here, we aimed to estimate seroprevalence and assess risk factors for infection in the general population of Nepal by conducting two rounds of sampling. The first round was in October 2020, at the peak of the first generalized wave of COVID-19, and the second round in July-August 2021, following the peak of the wave caused by the delta variant of SARS-CoV-2. We used cross-sectional probability-to-size (PPS)-based multistage cluster sampling to estimate the seroprevalence in the general population of Nepal at the national and provincial levels. We tested for anti-SARS-CoV-2 total antibody using the WANTAI SARS-CoV-2 Ab ELISA kit. In Round 1, the overall national seroprevalence was 14.4%, with provincial estimates ranging from 5.3% in Sudurpaschim to 27.3% in Madhesh Province. In Round 2, the estimated national seroprevalence was 70.7%, with the highest in the Madhesh Province (84.8%) and the lowest in the Gandaki Province (62.9%). Seroprevalence was comparable between males and females (Round 1, 15.8% vs. 12.2% and Round 2, 72.3% vs. 68.7%). The seroprevalence in the ecozones-Terai, hills, and mountains-was 76.3%, 65.3%, and 60.5% in Round 2 and 17.7%, 11.7%, and 4.6% in Round 1, respectively. In Nepal, COVID-19 vaccination was introduced in January 2021. At the peak of the first generalized wave of COVID-19, most of the population of Nepal remained unexposed to SARS-CoV-2. Towards the end of the second generalized wave in April 2021, two thirds of the population was exposed.

Incidence of human associated HF183 Bacteroides marker and E. coli levels in New Orleans Canals.

With a focus on five sites in an impaired, densely populated area in the New Orleans area, we investigated the temporal and spatial variability of standard FIB and a marker of human-associated pollution (Bacteroides HF183). With all sites combined, only a weak positive correlation (r = 0.345; p = 0.001) was observed between E. coli and HF183. Also, specific conductivity (r = - 0.374; p < 0.0001) and dissolved oxygen (r = - 0.390; p < 0.0001) were observed to show a weak moderate correlation with E. coli. These correlations increased to moderately negative when HF183 was correlated with specific conductivity (r = - 0.448; p < 0.0001) and dissolved oxygen (r = - 0.455; p < 0.0001). E. coli contamination was generally highest at the sites in the canal that are situated in the most densely populated part of the watershed while HF183 was frequently detected across all sites. E. coli concentrations were significantly higher (p < 0.05) when HF183 was present. HF183 was detected at significantly higher concentrations in samples that exceeded the EPA water quality standard (WQS) than those that did not (p < 0.05). Dissolved oxygen and specific conductivity were significantly lower when E. coli WQS was exceeded or when HF183 was present (p < 0.05). Rainfall impacted E. coli concentrations and HF183 differently at the study sites. While HF183 and E. coli concentrations levels were significantly higher (p < 0.05) if the days prior to sampling had been wet, the frequency of detection of HF183 was unimpacted, as comparable detection rates were recorded during wet and dry weather conditions. Without testing for HF183, it would have been assumed, based on testing for E. coli alone, that human fecal pollution was only associated with densely populated areas and rainfall events. E. coli alone may not be an effective indicator of sewage pollution at the study sites across all weather conditions and may need to be complemented with HF183 enumeration to optimize human fecal pollution identification and management at the watershed level.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater.

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2-5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Quantitative microbial risk assessment (QMRA) of occupational exposure to SARS-CoV-2 in wastewater treatment plants.

The current global Severe Acute Respiratory Syndrome- Coronavirus-2 (SARS-CoV-2) epidemic has heightened calls for studies to evaluate respiratory exposure for wastewater treatment workers. In this global first study, we assess occupational health risks to wastewater treatment plant (WWTP) operators from inhalation of aerosolized SARS-CoV-2 using a Quantitative Microbiological Risk Assessment (QMRA) framework. The following considerations were used to develop the QMRA and assess the illness risks to workers: a) the proportion of the population who are infected and thus responsible for shedding SARS-CoV-2 into raw wastewater; b) the concentration of SARS-CoV-2 in raw and treated wastewater; c) the volume of aerosolized water inhaled by a WWTP operator during work; d) humidity and temperature-dependent viability of coronaviruses in aerosolized waste water; e) estimation of the amount, frequency, and duration of exposure; and f) exposure doses. The variables were then fed into an exponential dose response model to estimate the risks in three scenarios representing low-grade, moderate and aggressive outbreaks. These scenarios were designed on the assumption of 0.03%, 0.3% and 3% of the wastewater-generating population being infected with SARS-CoV-2. In terms of averaged-out illness risk profiles, the individual illness risks for low grade, moderate and aggressive outbreak scenarios respectively are 0.036, 0.32 and 3.21 illness cases per 1000 exposed WWTP operators. Our study suggests that the risk of accidental occupational exposure to SARS-CoV-2 in raw wastewater, via inhalation at the WWTP environment, is negligible, particularly when less than 0.3% of the population served by the plant are actively infected.

Application of crAssphage, F-RNA phage and pepper mild mottle virus as indicators of human faecal and norovirus contamination in shellfish.

Shellfish growing waters contaminated with inadequately treated human wastewater is a major source of norovirus in shellfish and poses a significant human health risk to consumers. Microbial source tracking (MST) markers have been widely used to identify the source (s) of faecal contamination in water but data are limited on their use for shellfish safety. This study evaluated the source specificity, sensitivity, occurrence and concentration of three viral MST markers i.e. cross-assembly phage (crAssphage), F-specific RNA bacteriophage genogroup II (F-RNA phage GII) and pepper mild mottle virus (PMMoV) using animal faeces (n = 119; 16 animal groups), influent wastewater (n = 12), effluent wastewater (n = 16) and shellfish (n = 33). CrAssphage, F-RNA phage GII and PMMoV had source specific values of 0.97, 0.99 and 0.91, respectively. The sensitivity of MST markers was confirmed by their 100% detection frequency in influent wastewaters. The frequency of detection in effluent wastewater ranged from 81.3% (F-RNA phage GII) to 100% (PMMoV). Concentration of F-RNA phage GII was one log10 (influent wastewater) and 2-3 log10 (effluent wastewater) lower than crAssphage and PMMoV, respectively. Despite lower prevalence of F-RNA phage GII in oysters and mussels compared to crAssphage and PMMoV, concentrations of the three MST markers were similar in mussels. As an indicator of norovirus contamination in shellfish, crAssphage and PMMoV had greater predictive sensitivity (100%; [95% CI; 81.5%-100%)]) and F-RNA phage GII had greater predictive specificity (93.3%; [95% CI; 68.1%-99.8%]). In contrast, crAssphage and F-RNA phage GII have similar accuracy for predicting norovirus in shellfish, however, PMMoV significantly overestimated its presence. Therefore, a combination of crAssphage and F-RNA phage GII analysis of shellfish could provide a robust estimation of the presence of human faecal and norovirus contamination.

Antibiotic Resistance and Sewage-Associated Marker Genes in Untreated Sewage and a River Characterized During Baseflow and Stormflow.

Since sewage is a hotspot for antibiotic resistance genes (ARGs), the identification of ARGs in environmental waters impacted by sewage, and their correlation to fecal indicators, is necessary to implement management strategies. In this study, sewage treatment plant (STP) influent samples were collected and analyzed using quantitative polymerase chain reaction (qPCR) to investigate the abundance and correlations between sewage-associated markers (i.e., Bacteroides HF183, Lachnospiraceae Lachno3, crAssphage) and ARGs indicating resistance to nine antibiotics (belonging to aminoglycosides, beta-lactams, sulfonamides, macrolides, and tetracyclines). All ARGs, except bla VIM, and sewage-associated marker genes were always detected in untreated sewage, and ermF and sul1 were detected in the greatest abundances. intl1 was also highly abundant in untreated sewage samples. Significant correlations were identified between sewage-associated marker genes, ARGs and the intl1 in untreated sewage (τ = 0.488, p = 0.0125). Of the three sewage-associated marker genes, the BIO-ENV procedure identified that HF183 alone best maximized correlations to ARGs and intl1 (τ = 0.590). Additionally, grab samples were collected from peri-urban and urban sites along the Brisbane River system during base and stormflow conditions, and analyzed for Escherichia coli, ARGs, the intl1, and sewage-associated marker genes using quantitative polymerase chain reaction (qPCR). Significant correlations were identified between E. coli, ARGs, and intl1 (τ = 0.0893, p = 0.0032), as well as with sewage-associated marker genes in water samples from the Brisbane River system (τ = 0.3229, p = 0.0001). Of the sewage-associated marker genes and E. coli, the BIO-ENV procedure identified that crAssphage alone maximized correlations with ARGs and intl1 in river samples (τ = 0.4148). Significant differences in E. coli, ARGs, intl1, and sewage-associated marker genes, and by flow condition (i.e., base vs. storm), and site types (peri-urban vs. urban) combined were identified (R = 0.3668, p = 0.0001), where percent dissimilarities between the multi-factorial groups ranged between 20.8 and 11.2%. Results from this study suggest increased levels of certain ARGs and sewage-associated marker genes in stormflow river water samples compared to base flow conditions. E. coli, HF183 and crAssphage may serve as potential indicators of sewage-derived ARGs under stormflow conditions, and this merits further investigation. Data presented in this study will be valuable to water quality managers to understand the links between sewage pollution and ARGs in urban environments.

A multi-platform metabolomics approach to identify possible biomarkers for human faecal contamination in Greenshell™ mussels (Perna canaliculus).

Bivalve molluscs have the potential to bioaccumulate microbial pathogens including noroviruses from aquatic environments and as such, there is a need for a rapid and cheap in-situ method for their detection. Here, we characterise the tissue-specific response of New Zealand Greenshell™ mussels (Perna canaliculus) to faecal contamination from two different sources (municipal sewage and human faeces). This is done with the view to identify potential biomarkers that could be further developed into low cost, rapid and sensitive in-situ biosensors for human faecal contamination detection of mussels in growing areas. Tissue-specific metabolic profiles from gills, haemolymph and digestive glands were analysed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Clear differentiation of metabolic profiles was observed among treatments in each tissue type. Overall, energy pathways such as glycolysis, citrate cycle and oxidative phosphorylation were downregulated across the three mussel tissues studied following simulated contamination events. Conversely, considerable sterol upregulation in the gills was observed after exposure to contamination. Additionally, free pools of nucleotide phosphates and the antioxidant glutathione declined considerably post-exposure to contamination in gills. These results provide important insights into the tissue-specific metabolic effects of human faecal contamination in mussels. This study demonstrates the utility of metabolomics as a tool for identifying potential biomarkers in mussels.

Occurrence of SARS-CoV-2 RNA in Six Municipal Wastewater Treatment Plants at the Early Stage of COVID-19 Pandemic in The United States.

In this study, we investigated the occurrence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) RNA in primary influent (n = 42), secondary effluent (n = 24) and tertiary treated effluent (n = 34) collected from six wastewater treatment plants (WWTPs A-F) in Virginia (WWTP A), Florida (WWTPs B, C, and D), and Georgia (WWTPs E and F) in the United States during April-July 2020. Of the 100 wastewater samples analyzed, eight (19%) untreated wastewater samples collected from the primary influents contained SARS-CoV-2 RNA as measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. SARS-CoV-2 RNA were detected in influent wastewater samples collected from WWTP A (Virginia), WWTPs E and F (Georgia) and WWTP D (Florida). Secondary and tertiary effluent samples were not positive for SARS-CoV-2 RNA indicating the treatment processes in these WWTPs potentially removed SARS-CoV-2 RNA during the secondary and tertiary treatment processes. However, further studies are needed to understand the log removal values (LRVs) and transmission risks of SARS-CoV-2 RNA through analyzing wastewater samples from a wider range of WWTPs.

Identification of reliable marker genes for the detection of canine fecal contamination in sub-tropical Australia.

Animal fecal contamination in aquatic environments is a major source of zoonotic diseases in humans. While concerns are focused on livestock, companion animals such as dogs can also be a source of a wide range of zoonotic pathogens. Therefore, detection of dog or canine fecal contamination in aquatic environments is important for mitigating risks. In this study, host-sensitivity and specificity of four canine fecal-associated marker genes were evaluated by analyzing 30 canine and 240 non-canine fecal samples. The application of these markers was also tested in water from an urban river under dry weather conditions. The host sensitivity values of the Bacteroides BacCan-UCD, DogBact, DF113 and DF418 were 1.00, 0.90, 0.83, and 0.90, respectively. The host specificity value of the BacCan-UCD, DogBact, DF113 and DF418 were 0.87, 0.98, 0.83, and 0.41, respectively. The mean concentrations of DF418 were highest (7.82 ± 1.13 log10 gene copies (GC)/g of feces) followed by BacCan-UCD (7.61 ± 1.06 log10 GC/g) and DogBact (7.15 ± 0.92 log10 GC/g). The mean concentration of DF113 (5.80 ± 1.25 log10 GC/g) was 1.5 to 2.5 orders of magnitude lower than the other marker genes. The DogBact marker gene was not detected in any other animal feces other than a small number of untreated sewage samples. The BacCan-UCD marker gene cross-reacted with cat, chicken, and pig fecal samples, while the DF113 marker gene cross-reacted with cat, chicken, cattle fecal and untreated sewage samples. The DF418 marker gene was detected in all sewage and animal feces and deemed not suitable for canine fecal contamination tracking in sub-tropical Australia. Canine fecal contamination was infrequently detected in environmental water samples. Based on the results obtained in this study, we recommend that at least two canine feces-associated marker genes should be used in field studies.

Surveillance of SARS-CoV-2 RNA in wastewater: Methods optimisation and quality control are crucial for generating reliable public health information.

Monitoring for SARS-CoV-2 RNA in wastewater through the process of wastewater-based epidemiology (WBE) provides an additional surveillance tool, contributing to community-based screening and prevention efforts as these measurements have preceded disease cases in some instances. Numerous detections of SARS-CoV-2 RNA have been reported globally using various methods, demonstrating the technical feasibility of routine monitoring. However, in order to reliably interpret data produced from these efforts for informing public health interventions, additional quality control information and standardization in sampling design, sample processing, and data interpretation and reporting is needed. This review summarizes published studies of SARS-CoV-2 RNA detection in wastewater as well as available information regarding concentration, extraction, and detection methods. The review highlights areas for potential standardization including considerations related to sampling timing and frequency relative to peak fecal loading times; inclusion of appropriate information on sample volume collected; sample collection points; transport and storage conditions; sample concentration and processing; RNA extraction process and performance; effective volumes; PCR inhibition; process controls throughout sample collection and processing; PCR standard curve performance; and recovery efficiency testing. Researchers are recommended to follow the Minimum Information for Publication of Quantitative Real-Time PCR (MIQE) guidelines. Adhering to these recommendations will enable robust interpretation of wastewater monitoring results and improved inferences regarding the relationship between monitoring results and disease cases.