Correlation of SARS-CoV-2 RNA in wastewater with COVID-19 disease burden in sewersheds.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease (COVID-19), is shed in feces and the viral ribonucleic acid (RNA) is detectable in wastewater. A nine-week wastewater epidemiology study of ten wastewater facilities, serving 39% of the state of Utah or 1.26 M individuals was conducted in April and May of 2020. COVID-19 cases were tabulated from within each sewershed boundary. RNA from SARS-CoV-2 was detectable in 61% of 126 wastewater samples. Urban sewersheds serving >100,000 individuals and tourist communities had higher detection frequencies. An outbreak of COVID-19 across two communities positively correlated with an increase in wastewater SARS-CoV-2 RNA, while a decline in COVID-19 cases preceded a decline in RNA. SARS-CoV-2 RNA followed a first order decay rate in wastewater, while 90% of the RNA was present in the liquid phase of the influent. Infiltration and inflow, virus decay and sewershed characteristics should be considered during correlation analysis of SAR-CoV-2 with COVID-19 cases. These results provide evidence of the utility of wastewater epidemiology to assist in public health responses to COVID-19.

Template length, concentration and guanidine and cytosine content influence on multiple displacement amplification efficiency.

Detection of low abundance human health pathogens in environmental samples is a challenge for water monitoring. This limitation can be overcome by the introduction of multiple displacement amplification (MDA) where a minute amount of genetic material can be amplified using a phi-29 DNA polymerase. However, the genetic makeup and the concentration of the polynucleotides might influence the amplification process due to inherent assay bias. Herein, a series of experiments were designed to demonstrate the effect of genome length, guanidine and cytosine content, and template concentration on the efficiency of MDA. Quantitative polymerase chain reaction (qPCR) was performed to quantify pre- and post-MDA concentrations of selected genes. Linear regression between pre- and post-MDA log gene copies L-1 of both environmental and lab-grown samples showed a positive correlation (F = 77.59, P < 0.001, R2 = 0.7, slope = 1.01). Correlation between relative polynucleotide increase after MDA and target organism length and gene target guanidine and cytosine (G + C) content (F = 4.3, P = 0.02) shows that lower G + C and higher genome length is favored in the MDA process. The MDA process was shown to favor a longer genome over a shorter genome (1.19 and 1.04 change in log gene copy L-1, respectively) and a lower G + C content over a higher G + C content (1.11 and 0.61 change in log gene copy L-1, respectively). There was no MDA bias observed when polynucleotides had the same G + C and genome length but different initial concentrations. This study highlights the need for increased caution when interpreting relative abundance of organisms amplified by MDA such as in next generation sequencing.

Informing water distribution line rehabilitation through quantitative microbial risk assessment.

Poor urban water quality has been linked to diminished source water quality, poorly functioning water treatment systems and infiltration into distribution lines after treatment resulting in microbiological contamination. With limited funding to rehabilitate distribution lines, developing nations need tools to identify the areas of greatest concern to human health so as to target cost effective remediation approaches. Herein, a case study of Hyderabad, Pakistan was used to demonstrate the efficacy of combining quantitative microbial risk assessment (QMRA) for multiple pathogens with spatial distribution system modeling to identify areas for pipe rehabilitation. Abundance of Escherichia coli, Enterococcus (enterococci), Salmonella spp., Shigella spp., Giardia intestinalis, Vibrio cholera, norovirus GI and adenovirus 40/41, were determined in 85 locations including the source water, treatment plant effluent and the city distribution lines. Bayesian statistics and Monte Carlo simulations were used in the QMRA to account for left-censored microbial abundance distributions. Bacterial and viral abundances in the distribution system samples decreased as follows: 9400 ± 19,800 norovirus gene copies/100 mL (average ± standard deviation, 100% of samples positive); 340 ± 2200 enterococci CFU/100 mL (94%), 71 ± 97 Shigella sp. CFU/100 mL (97%), 60 ± 360 E. coli CFU/100 mL (89%), 35 ± 79 adenovirus gene copies/100 mL (100%), and 21 ± 46 Salmonella sp. CFU/100 mL (76%). The QMRA revealed unacceptable probabilities of illness (>1 in 10,000 illness level) from the four exposure routes considered (drinking water, or only showering, tooth brushing, and rinsing vegetables consumed raw). Disease severity indices based on the QMRA combined with mapping the distribution system revealed areas for targeted rehabilitation. The combined intensive sampling, risk assessment and mapping can be used in low- and middle-income countries to target distribution system rehabilitation efforts and improve health outcomes.

Probabilistic risk assessment of water distribution system in Hyderabad, Pakistan reveals unacceptable health hazards and areas for rehabilitation.

Poor water quality exacerbates multidimensional poverty in developing nations. Often centralized treatment facilities generate acceptable water quality, but the water is contaminated during distribution. Methods to assess sources of contamination in water distribution systems are lacking. A case study of two methods, human risk assessment linked to water distribution system sampling was conducted in Hyderabad, Pakistan to determine areas requiring infrastructure rehabilitation. Water samples from source water (i.e., the Indus River), treatment plant effluent and from taps in the water distribution system were analyzed by atomic adsorption spectroscopy for metals and metalloids (As, Cd, Cr, Hg, and Pb) and water quality parameters (dissolved and suspended solids, pH, conductivity, and total organic carbon). Source water exceeded acceptable drinking water levels for As, Cd, total Cr, and Pb, while the treatment plant effluent concentrations were acceptable. Concentrations of all metals and metalloids, except Hg, increased in the water distribution system post-treatment, exceeding safe drinking limits in at least one location, suggesting contamination of the water during distribution. A deterministic and a probabilistic risk assessment were conducted to evaluate two scenarios: (1) unrestricted use of piped water for all household purposes, including as drinking water and (2) restricted use of the water for purposes other than drinking in the household, including only dermal and inhalation exposure pathways. The water was deemed unsafe for unrestricted use as the sole source of drinking water by both risk assessment methods. Yet when an alternative source of drinking water was assumed and the piped water was used only for bathing and dish washing, the probabilistic risk assessment revealed acceptable health risks to the population, while the overly conservative deterministic risk assessment suggested unacceptable risks. The combined methods of water sampling, risk assessment and correlation analysis suggested areas for rehabilitation of the water distribution system in Hyderabad, Pakistan and these methods can be adopted in other developing nations to target limited funds for infrastructure rehabilitation.

Roofing material and irrigation frequency influence microbial risk from consuming homegrown lettuce irrigated with harvested rainwater.

Rooftop harvested rainwater has become an alternative, potable, and non-potable water source used around the world. In the United States, rooftop harvested rainwater is most commonly used for irrigation. Rooftop harvested rainwater may contain contaminants from bird or animal feces that may present a risk to water users. Different roofing materials may influence the survival of fecal bacteria on the rooftop prior to runoff during rainfall. In this study, three pathogen groups (E. coli, enterococci and Salmonella enterica) in rooftop runoff from three, replicated roof types (asphalt shingle, synthetic slate, and wood shake) were quantified in multiple rain events. Matched roofs were selected from locations with differing amounts of tree cover. Enterococci were the most frequently detected bacteria from all roof types. Wood shake and asphalt shingle roofing materials had the poorest microbial water quality. Rainwater runoff from two of the six buildings failed to meet United States Food and Drug Administration microbial standards for irrigation water. A quantitative microbial risk assessment indicated that the annual probability of illness from consuming lettuce irrigated with rooftop harvested rainwater varied by roofing material, irrigation water withholding period, and exposure frequency. Consuming lettuce immediately after irrigation with rooftop rainwater presented the highest human health risk based on the probability of illness from E. coli and enterococci exposure. Withholding irrigation by 1 day prior to harvest decreased the annual probability of illness from E. coli by 2 log, but had a minimal effect on the risk from enterococci.

Elucidating Waterborne Pathogen Presence and Aiding Source Apportionment in an Impaired Stream.

Fecal indicator bacteria (FIB) are the basis for water quality regulations and are considered proxies for waterborne pathogens when conducting human health risk assessments. The direct detection of pathogens in water and simultaneous identification of the source of fecal contamination are possible with microarrays, circumventing the drawbacks to FIB approaches. A multigene target microarray was used to assess the prevalence of waterborne pathogens in a fecally impaired mixed-use watershed. The results indicate that fecal coliforms have improved substantially in the watershed since its listing as a 303(d) impaired stream in 2002 and are now near United States recreational water criterion standards. However, waterborne pathogens are still prevalent in the watershed, as viruses (bocavirus, hepatitis E and A viruses, norovirus, and enterovirus G), bacteria (Campylobacter spp., Clostridium spp., enterohemorrhagic and enterotoxigenic Escherichia coli, uropathogenic E. coli, Enterococcus faecalis, Helicobacter spp., Salmonella spp., and Vibrio spp.), and eukaryotes (Acanthamoeba spp., Entamoeba histolytica, and Naegleria fowleri) were detected. A comparison of the stream microbial ecology with that of sewage, cattle, and swine fecal samples revealed that human sources of fecal contamination dominate in the watershed. The methodology presented is applicable to a wide range of impaired streams for the identification of human health risk due to waterborne pathogens and for the identification of areas for remediation efforts.IMPORTANCE The direct detection of waterborne pathogens in water overcomes many of the limitations of the fecal indicator paradigm. Furthermore, the identification of the source of fecal impairment aids in identifying areas for remediation efforts. Multitarget gene microarrays are shown to simultaneously identify waterborne pathogens and aid in determining the sources of impairment, enabling further focused investigations. This study shows the use of this methodology in a historically impaired watershed in which total maximum daily load reductions have been successfully implemented to reduce risk. The results suggest that while the fecal indicators have been reduced more than 96% and are nearing recreational water criterion levels, pathogens are still detectable in the watershed. Microbial source tracking results show that additional remediation efforts are needed to reduce the impact of human sewage in the watershed.