Early warning and rapid public health response to prevent COVID-19 outbreaks in long-term care facilities (LTCF) by monitoring SARS-CoV-2 RNA in LTCF site-specific sewage samples and assessment of antibodies response in this population: prospective study protocol.

INTRODUCTION: The COVID-19 pandemic has an excessive impact on residents in long-term care facilities (LTCF), causing high morbidity and mortality. Early detection of presymptomatic and asymptomatic COVID-19 cases supports the timely implementation of effective outbreak control measures but repetitive screening of residents and staff incurs costs and discomfort. Administration of vaccines is key to controlling the pandemic but the robustness and longevity of the antibody response, correlation of neutralising antibodies with commercial antibody assays, and the efficacy of current vaccines for emerging COVID-19 variants require further study. We propose to monitor SARS-CoV-2 in site-specific sewage as an early warning system for COVID-19 in LTCF and to study the immune response of the staff and residents in LTCF to COVID-19 vaccines. METHODS AND ANALYSIS: The study includes two parts: (1) detection and quantification of SARS-CoV-2 in LTCF site-specific sewage samples using a molecular assay followed by notification of Public Health within 24 hours as an early warning system for appropriate outbreak investigation and control measures and cost-benefit analyses of the system and (2) testing for SARS-CoV-2 antibodies among staff and residents in LTCF at various time points before and after COVID-19 vaccination using commercial assays and neutralising antibody testing performed at a reference laboratory. ETHICS AND DISSEMINATION: Ethics approval was obtained from the University of Alberta Health Research Ethics Board with considerations to minimise risk and discomforts for the participants. Early recognition of a COVID-19 case in an LTCF might prevent further transmission in residents and staff. There was no direct benefit identified to the participants of the immunity study. Anticipated dissemination of information includes a summary report to the immunity study participants, sharing of study data with the scientific community through the Canadian COVID-19 Immunity Task Force, and prompt dissemination of study results in meeting abstracts and manuscripts in peer-reviewed journals.

Removal of Cryptosporidium surrogates in drinking water direct filtration.

Pilot-scale direct filtration challenge experiments were conducted to determine the impact of chemical pretreatment and filter design on the removal of Cryptosporidium surrogates dosed into the filter influent water at low temperatures (Average 0.5 °C). Copolymers-modified microspheres were identified as representative Cryptosporidium oocysts surrogates based on our previous findings and were used to evaluate the oocysts filtration removal at this pilot-scale study. The operational parameters examined included coagulant type (aluminum sulfate (alum) versus polyaluminium chloride (PACl)), filter aid polymer type (polyamine Magnafloc® LT-7981 versus poly(Dimethyl Diallyl Ammonium Chloride) (polyDADMAC) Magnafloc® LT-7995) and dose (0.5 versus 2.0 mg/L), and filter configuration (regular versus deep bed filters). The study found that higher Cryptosporidium surrogate removal was associated with higher polymer dose (2 mg/L) of polyDADMAC polymer and the deep bed filter configuration. The difference in surrogate removal between PACl and alum was no significant at cold temperature conditions tested. The deep bed filters were associated with higher surrogate removal, while exhibiting lower rates of flow reduction and longer filter run time. This work emphasizes the importance of optimizing chemical pretreatment and filter configuration for removing surrogates of Cryptosporidium oocysts in cold-water conditions in granular media water filtration processes. This pilot-scale study also demonstrated the exceed 2.5-log removal of Cryptosporidium surrogates (required from Guideline for Canadian Drinking Water Quality) can be achieved in the direct filtration during Edmonton cold-water condition when the pretreatment processes are optimized using 0.454 mg/L of alum as Al with addition of 0.5 mg/L poly DADMAC.

Prevalence, levels and seasonal variations of human enteric viruses in six major rivers in Alberta, Canada.

The prevalence and seasonal variation of 7 viruses in 6 major rivers in Alberta were assessed using a combination of qPCR, cell culture and integrated cell culture with qPCR (ICC-qPCR). Water samples were collected monthly from rivers at different sites upstream and downstream of major urban centers. Seven viruses including rotavirus, adenovirus, astrovirus, norovirus, sapovirus, JC virus and enterovirus, were detected in at least one of the water samples at each site using qPCR. Rotavirus was most common with concentration ranging from 2.3 to 4.5 log10 genomic equivalent (GE) copies/L. Norovirus, sapovirus, astrovirus, adenoviruses and JC virus peaked during the winter (November to March). Viruses were most prevalent at the Bow River sampling site downstream of the City of Calgary, followed by the North Saskatchewan River site downstream of the City of Edmonton and the Red Deer River site downstream of the City of Red Deer. The detection rates and quantity of viruses had significant difference in the sampling sites between upstream and downstream of major urban centers (p < 0.001). 14% of the samples tested positive using viral culture indicating the presence of infectious viruses in river. Sequencing analysis identified human rotavirus in 75% of the samples collected from downstream versus 37% of the samples collected from upstream sites (p < 0.02). Multivariate binary regression showed that human activity in watersheds is a significant determinant of viruses in Alberta's Rivers. The discharge from wastewater treatment plants may be the possible sources of viral contamination. Seasonal coincidence of acute viral gastroenteritis outbreaks and monthly peak occurrence of enteric viruses in river water implies potential impact of waterborne viruses on human health.

Assessing UV Inactivation of Adenovirus 41 Using Integrated Cell Culture Real-Time qPCR/RT-qPCR.

Enteric adenoviruses are among most UV-resistant viruses in water. Cytopathic effects (CPE)-based cell culture TCID50 assay as a conventional virus assessment approach has major drawbacks for enteric adenovirus since it is selective on cell lines and takes longer time to show CPE. Integrated cell culture real-time quantitative PCR (ICC-qPCR) and reverse transcriptase (RT)-qPCR were applied in this study, in comparison with TCID50, to assess UV inactivation of adenovirus type 41 (Ad41) in water. Adenovirus type 41 was exposed to UV doses of 40, 80, 160, and 320 mJ/cm2 using a collimated beam apparatus. There was no significant difference of inactivation at conducted UV doses between measurements using TCID50 assay and ICC-RT-qPCR. Both assays fitted the Chick-Watson model at 95% confidence level. The inactivation measured by ICC-qPCR did not fit the Chick-Watson model. In summary, ICC-RT-qPCR is the most appropriate alternate to CPE-based assay for assessing UV inactivation of enteric adenoviruses.

Pre-analytical and analytical procedures for the detection of enteric viruses and enterovirus in water samples.

Practical pre-analytical and analytical procedures were developed and validated for detection of enteric viruses in three water matrices. Both RNA viruses (norovirus, coxsackievirus, echovirus, and rotavirus) and DNA virus (adenovirus 41) were included in the study. The NanoCeram 90mm laminated disc with electropositive filter and procedures of filtration, elution and flocculation were utilized to concentrate known amount of viruses in different water matrices. Real time quantitative PCR was used to evaluate the recovery of virus and cell culture to assess viral infectivity. There was no PCR inhibition using various concentrations and pH of beef extract eluting buffer. A good recovery of the viruses spiked in 10L of deionized water was achieved for serial dilutions of coxsackievirus (41-67%), echovirus (22-90%), norovirus (23-44%) and rotavirus (24-46%). Relatively lower recovery was observed for adenovirus 41 (24-35%). There was no significant difference in viral recovery from deionized, tap and river water samples. The infectivity of recovered adenovirus, coxsackievirus and echovirus was demonstrated using in vitro cell culture. The pre-analytical and analytic procedures attained consistent recovery of RNA and DNA viruses both as infectious viral particles and viral genome, provided effective removal of inhibitory substances, achieved reliable reproducibility, and were relatively inexpensive for monitoring viruses in water.

Comparison of the action spectra and relative DNA absorbance spectra of microorganisms: information important for the determination of germicidal fluence (UV dose) in an ultraviolet disinfection of water.

The action spectra of Bacillus subtilis spores (ATCC6633) and Salmonella typhimurium LT2 were characterized using physical radiometry for irradiance measurements and a multiple target model to interpret the inactivation kinetics. The observed action spectrum of B. subtilis spores deviated significantly from the relative absorbance spectrum of the DNA purified from the spores, but matched quite well with the relative absorbance spectrum of decoated spores. The action spectrum of B. subtilis spores determined in this study was statistically different from those reported in previous studies. On the other hand, the action spectrum of S. typhimurium bacteria matched quite well with the relative absorbance spectrum of DNA extracted from vegetative cells, except in the region below 240nm. It is concluded that the common use of the relative DNA absorbance spectrum as a surrogate for the germicidal action spectrum can result in systematic errors when evaluating the performance of a polychromatic UV light reactors using bioassays. For example, if the weighted germicidal fluence (UV dose) calculated using the relative DNA absorbance spectrum as the germicidal weighting factor is found to be 40mJcm(-2) for a medium pressure lamp UV reactor, that calculated using the relative action spectrum of B. subtilis spores, as determined in this study, would be 66mJcm(-2).

Infectivity of Giardia lamblia cysts obtained from wastewater treated with ultraviolet light.

Several waterborne outbreaks of giardiasis have been linked to discharge of wastewater effluents into surface water. Little is known about the infectivity of Giardia lamblia cysts present in UV treated wastewater effluents. In this study, the infectivity of G. lamblia cysts, recovered from primary effluent and secondary effluent, both upstream and downstream of operating full-scale UV reactors at four wastewater treatment plants, was assessed using the Mongolian gerbil model. Infectivity of cysts obtained from the primary effluents was scored as either strong or moderate for induction of infection in gerbils at three out of four wastewater treatment plants. G. lamblia recovered from secondary effluent both upstream and downstream of the UV reactors caused weak infections in the gerbils. The probability of weak infections caused by inoculums of 50-1400 cysts per gerbil was, on the average, reduced by approximately 10% at the four wastewater UV installations with coliform reduction equivalent doses ranging from 6 to 18 mJ/cm2. The UV systems provided considerably less inactivation of the parasite than expected based on the UV dose response of Giardia reported in the literature.

The assessment of particle association and UV disinfection of wastewater using indigenous spore-forming bacteria.

Studies have shown that association between particles and coliform bacteria in wastewater influence the inactivation of these microorganisms by ultraviolet (UV) irradiation. This research investigated the potential use of indigenous aerobic spore-forming (ASF) bacteria for studying the particle - microorganism interaction and its effect on UV disinfection of protozoan pathogens, such as Giardia spp. and Cryptosporidium spp., present in effluents from full-scale municipal wastewater treatment plants. The effect of particle - ASF association was determined by homogenizing wastewater effluent samples before and after exposure to controlled UV doses delivered by a bench-scale collimated beam apparatus. Particle association between Bacillus subtilis spores added to wastewater and wastewater particles was also assessed. The results indicate that spores are not significantly associated with wastewater particulate matter and particle association does not significantly affect the inactivation of indigenous spores present in wastewater by UV radiation in this study.

Survival of Giardia lamblia trophozoites after exposure to UV light.

The ability of Giardia lamblia trophozoites to reproduce after exposure to different fluences of UV radiation was determined using an in vitro-cultured method. The rate of parasite reproduction following UV exposure was measured by direct enumeration of trophozoites cultured in Diamond's Trypticase Yeast extract-Iron (TYI)-S-33 medium. The results suggested that some G. lamblia trophozoites may survive or are reactivated following exposure to UV fluences up to 10 mJ cm(-2). In addition, trophozoites exposed to a UV fluence of 1 mJ cm(-2) were infectious to Mongolian gerbils. Evidence of survival or reactivation at UV fluences of 20 and 40 mJ cm(-2) was ambiguous and statistically inconclusive, while at 100 mJ cm(-2) there was no evidence of survival or reactivation. This finding may have implications for criteria used by the drinking water and wastewater treatment industry to ensure safe reduction of G. lamblia cysts by UV disinfection processes.

Comparison of levels of inactivation of two isolates of Giardia lamblia cysts by UV light.

The effects of 254-nm UV irradiation on two human isolates (WB and H3) of Giardia lamblia cysts were assessed using a collimated beam protocol and a Mongolian gerbil model. The levels of infection of cysts in the gerbils were assessed based on the presence of cysts in feces and the presence and activity of trophozoites in the small intestine of inoculated gerbils. The results suggest that there were differences in the infectivities of the WB and H3 isolates, as well as in susceptibilities of the parasites to UV light. Without UV exposure, gerbils were more readily infected by isolate H3 cysts. After UV exposure of the cysts, however, the gerbils were more susceptible to isolate WB cysts.

Synergistic inactivation of Cryptosporidium parvum using ozone followed by monochloramine in two natural waters.

The effect of sequential exposure to ozone followed by monochloramine on inactivation of Cryptosporidium parvum oocysts suspended in untreated natural surface water from two different sources was studied in bench-scale batch reactors. Animal infectivity using neonatal CD-1 mice was used to measure oocyst inactivation. A statistically significant synergistic effect on oocyst inactivation was measured in both natural water samples studied. The magnitude of the effect measured in the natural water with lower turbidity, colour, and organic carbon concentration was comparable to that previously reported for oocysts suspended in buffered de-ionized water but was reduced considerably in the natural water with higher turbidity, colour and organic carbon concentration. Synergy increased with initial pH and with the degree of ozone pre-treatment but was independent of temperature. For water treatment plants with adequate disinfectant contact times, ozone followed by monochloramine may be a practical means of achieving additional C. parvum inactivation, however, the influence of water quality characteristics should be considered.

Synergistic inactivation of Cryptosporidium parvum using ozone followed by free chlorine in natural water.

The synergistic effect of sequential exposure to ozone followed by free chlorine on inactivation of Cryptosporidium parvum oocysts suspended in natural waters was studied in bench-scale batch reactors. Animal infectivity using neonatal CD-1 mice was used to measure oocyst inactivation. The synergistic effect measured in two alkaline (pH 8.1) natural waters was statistically significant but was considerably smaller than previously reported in buffered de-ionized water at pH 6.0. Temperature, ozone primary treatment level, and water type did not have measurable impacts on the synergistic effect. Efforts to increase the synergistic effect by reducing the pH from 8 to 6 by acid addition were unsuccessful. In the two low alkalinity (pH 6.0) natural waters tested, the measured synergistic effect was greater than in the alkaline waters, but was still less than that measured previously in buffered de-ionized water. It was concluded that the synergistic effect reduction in the natural waters tested was due in part to alkalinity and in part to other unidentified water quality characteristics. Sequential treatment with ozone followed by free chlorine may only be a feasible strategy for achieving synergistic C. parvum inactivation credit for water treatment facilities with natural waters having a low pH (near 6.0).

Effect of turbulent gas-liquid contact in a static mixer on Cryptosporidium parvum oocyst inactivation by ozone.

Static mixers may be used to dissolve gaseous ozone in water treatment facilities in order to provide protection against the waterborne parasite Cryptosporidium parvum. The objective of this study was to determine the effect of a brief exposure to turbulent gas-liquid mixing conditions in a static mixer on inactivation of C. parvum oocysts by ozone. Inactivation measured in an ozone contacting apparatus that employed a static mixer for ozone dissolution was compared to predictions based on a previously published kinetic model of C. parvum inactivation by dissolved ozone in gently stirred batch reactors. Although initial contact in the static mixer had no immediate effect on the oocysts, a 20% increase in the rate of inactivation during subsequent contact with dissolved ozone was observed. Increasing the degree of turbulence within the static mixer did not yield additional inactivation. Use of static mixers for dissolution of ozone in drinking water treatment systems may provide limited enhancement of C. parvum inactivation by dissolved ozone.