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.

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.

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.

Comparative decay of sewage-associated marker genes in beach water and sediment in a subtropical region.

There is a growing move towards using the quantitative polymerase chain (qPCR)-based sewage-associated marker genes to assess surface water quality. However, a lack of understanding about the persistence of many sewage-associated markers creates uncertainty for those tasked with investigating microbial water quality. In this study, we investigated the decay of two qPCR FIB [E. coli (EC), and Enterococcus spp. (ENT) 23S rRNA genes] and four sewage-associated microbial source tracking (MST) marker genes [human Bacteroides HF183 16S rRNA, adenovirus (HAdV), and polyomavirus (HPyV), and crAssphage, a recently described bacteriophage in feces], in outdoor mesocosms containing fresh and marine waters and their corresponding sediments. Decay rates of EC 23S rRNA, ENT 23S rRNA, and HF183 16S rRNA were significantly (p < 0.05) faster than the HAdV, HPyV and crAssphage markers in water samples from all mesocosms. In general, decay rates of bacterial targets were similar in the water columns of the studied mesocosms. Similarly, decay rates of viral targets were also alike in mesocosm water columns in relation to each other. The decay rates of FIB and sewage-associated markers were significantly faster in water samples compared to sediments in all three mesocosms. In the event of resuspension, FIB and marker genes from sediments can potentially recontaminate overlying waters. Thus, care should be taken when interpreting the occurrence of FIB and sewage-associated MST markers in water, which may have originated from sediments. The differential decay of these targets may also influence health outcomes and need to be considered in risk assessment models.

Preliminary evaluation of BioFire FilmArray® Gastrointestinal Panel for the detection of noroviruses and other enteric viruses from wastewater and shellfish.

The BioFire FilmArray® Gastrointestinal Panel was evaluated for the rapid detection of adenovirus, astrovirus, norovirus, rotavirus and sapovirus from influent and effluent wastewater and shellfish. The multiplex BioFire FilmArray® Gastrointestinal Panel compared well to singleplex qPCR/RT-qPCR methods for the detection of adenovirus, astrovirus, rotavirus and sapovirus from influent and effluent wastewater samples. However, the BioFire FilmArray® Gastrointestinal Panel showed poor performance for the detection of norovirus, significantly underestimating its presence in wastewater and shellfish samples when compared with the singleplex norovirus GI and GII RT-qPCR assays. Therefore, improvement on detection efficiency for norovirus from environmental and food samples is necessary before using results from the FilmArray® Gastrointestinal Panel to assess associated public health risks.