Evaluating the impact of sample storage, handling, and technical ability on the decay and recovery of SARS-CoV-2 in wastewater

Wastewater based epidemiology (WBE) is useful for tracking and monitoring the level of disease prevalence in a community and has been used extensively to complement clinical testing during the current COVID-19 pandemic. Despite the numerous benefits, sources of variability in sample storage, handling, and processing methods can make WBE data difficult to generalize. We performed an experiment to determine sources of variability in WBE data including the impact of storage time, handling, and processing techniques on the concentration of SARS-CoV-2 in wastewater influent from three wastewater treatment plants (WWTP) in North Carolina over 19 days. The SARS-CoV-2 concentration in influent samples held at 4°C did not degrade significantly over the 19-day experiment. Heat pasteurization did not significantly impact the concentration of SARS-CoV-2 at two of the three WWTP but did reduce viral recovery at the WWTP with the smallest population size served. On each processing date, one filter from each sample was processed immediately while a replicate filter was frozen at -80°C. Once processed, filters previously frozen were found to contain slightly higher concentrations (<0.2 log copies/L) than their immediately processed counterparts, indicating freezing filters is a viable method for delayed quantification and may even improve recovery at WWTP with low viral concentrations. Investigation of factors contributing to variability during sample processing indicated that analyst experience level contributed significantly (p<0.001) to accepted droplet generation while extraction efficiency and reverse transcription efficiency contributed significantly (p<0.05) to day-to-day SARS-CoV-2 variability. This study provides valuable practical information for minimizing decay and/or loss of SARS CoV-2 in wastewater influent while adhering to safety procedures, promoting efficient laboratory workflows, and accounting for sources of variability

Lessons Learned from Implementing a Wet Laboratory Molecular Training Workshop for Beach Water Quality Monitoring

UA Open Access Publishing FundRapid molecular testing methods are poised to replace many of the conventional, culturebased tests currently used in fields such as water quality and food science. Rapid qPCR methods have the benefit of being faster than conventional methods and provide a means to more accurately protect public health. However, many scientists and technicians in water and food quality microbiology laboratories have limited experience using these molecular tests. To ensure that practitioners can use and implement qPCR techniques successfully, we developed a week long workshop to provide hands-on training and exposure to rapid molecular methods for water quality management. This workshop trained academic professors, government employees, private industry representatives, and graduate students in rapid qPCR methods for monitoring recreational water quality. Attendees were immersed in these new methods with hands-on laboratory sessions, lectures, and one-on-one training. Upon completion, the attendees gained sufficient knowledge and practice to teach and share these new molecular techniques with colleagues at their respective laboratories. Key findings from this workshop demonstrated: 1) participants with no prior experience could be effectively trained to conduct highly repeatable qPCR analysis in one week; 2) participants with different desirable outcomes required exposure to a range of different platforms and sample processing approaches; and 3) the collaborative interaction amongst newly trained practitioners, workshop leaders, and members of the water quality community helped foster a cohesive cohort of individuals which can advocate powerful cohort for proper implementation of molecular methods.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Lessons learned from implementing a wet laboratory molecular training workshop for beach water quality monitoring.

Rapid molecular testing methods are poised to replace many of the conventional, culture-based tests currently used in fields such as water quality and food science. Rapid qPCR methods have the benefit of being faster than conventional methods and provide a means to more accurately protect public health. However, many scientists and technicians in water and food quality microbiology laboratories have limited experience using these molecular tests. To ensure that practitioners can use and implement qPCR techniques successfully, we developed a week long workshop to provide hands-on training and exposure to rapid molecular methods for water quality management. This workshop trained academic professors, government employees, private industry representatives, and graduate students in rapid qPCR methods for monitoring recreational water quality. Attendees were immersed in these new methods with hands-on laboratory sessions, lectures, and one-on-one training. Upon completion, the attendees gained sufficient knowledge and practice to teach and share these new molecular techniques with colleagues at their respective laboratories. Key findings from this workshop demonstrated: 1) participants with no prior experience could be effectively trained to conduct highly repeatable qPCR analysis in one week; 2) participants with different desirable outcomes required exposure to a range of different platforms and sample processing approaches; and 3) the collaborative interaction amongst newly trained practitioners, workshop leaders, and members of the water quality community helped foster a cohesive cohort of individuals which can advocate powerful cohort for proper implementation of molecular methods

Comparison of Rapid Quantitative PCR-Based and Conventional Culture-Based Methods for Enumeration of Enterococcus spp. and Escherichia coli in Recreational Waters▿

Recreational water quality is currently monitored using culture-based methods that require 18 to 96 h for results. Quantitative PCR (QPCR) methods that can be completed in less than 2 h have been developed, but they could yield different results than the conventional methods. We present two studies in which samples were processed simultaneously for Enterococcus spp. and Escherichia coli using two culture-based methods (EPA method 1600 and Enterolert/Colilert-18) and QPCR. The proprietary QPCR assays targeted the 23S rRNA (Enterococcus spp.) and uidA (E. coli) genes and were conducted using lyophilized beads containing all reagents. In the first study, the QPCR method developers processed 54 blind samples that were inoculated with sewage or pure cultures or were ambient beach samples. The second study involved 163 samples processed by water quality personnel. The correlation between results of QPCR and EPA 1600 during the first study (r2) was 0.69 for Enterococcus spp., which was less than that observed between the culture-based methods (r2, 0.87). During the second study, the correlations were similar. No false positives occurred in either study when QPCR-based assays were used with blank samples. Levels of reproducibility measured through coefficients of variation were similar for results by Enterococcus QPCR and culture-based methods during both studies but were higher for E. coli QPCR results in the first study. Regarding the concentration at which beach management decisions are issued in the State of California, the agreement between results of Enterococcus QPCR and EPA method 1600 was 88%, compared to 94% agreement between EPA method 1600 and Enterolert. The beach management decision agreement between E. coli QPCR and Colilert-18 was 94%. The samples showing disagreement suggested an underestimation bias for QPCR

Trends in total Vibrio spp. and Vibrio vulnificus concentrations in the eutrophic Neuse River Estuary, North Carolina, during storm events

Vibrio spp. are ubiquitous members of aquatic microbial food webs that can be pathogenic to humans and a range of other organisms. Previously published predictive models for Vibrio spp. concentrations in estuarine and coastal waters, based only on salinity and temperature, are 70 to 75% accurate during 'normal' conditions (e.g. not during storms or drought). We have conducted a preliminary comparison of the output from this type of model to the natural concentrations of both total Vibrio spp. and the potentially pathogenic Vibrio vulnificus when measured during tropical storms. Water samples were collected in situ from a deployed platform in the Neuse River Estuary (NRE), North Carolina, USA, during 2 storm events: Hurricane Ophelia and Tropical Storm Ernesto. Total Vibrio spp. concentrations were measured using culture-based methods and V vulnificus levels were determined using a newly developed, rapid quantitative polymerase chain reaction (QPCR) assay. Results were analyzed in relation to environmental parameters and to concentrations of the fecal indicator bacteria Escherichia coli (EC) and Enterococcus spp. (ENT). Total concentrations of Vibrio spp. in the NRE were often orders of magnitude higher than those predicted by a previously published model. These large deviations from model predictions may indicate contributions from storm forcing (e.g. resuspension, surges) that are missing from the calm weather observations used to build these models

Participant written evaluation responses focused on the workshop environment.

<p>Q5: The workshop was well organized; Q6: The atmosphere of the workshop was professional; Q7: The lodging arrangements were clean and appropriate for this venue; Q8: The food selection was appropriate, on time, and enjoyable; Q9: Transportation during the workshop was on time, comfortable, and drivers were courteous.</p

Comparison of Transcription-Mediated Amplification and Growth-Based Methods for the Quantitation of Enterococcus Bacteria in Environmental Waters▿

An assay based on transcription-mediated amplification (TMA) technology was used to quantitate Enterococcus fecal indicator bacteria in environmental water samples. The results generated by this and two growth-based methods relative to the 104 most-probable-number or CFU-per-100-ml threshold show that the three methods are in good qualitative agreement when tested against a range of water samples taken from different locations. The results demonstrate sensitive and rapid detection (approximately 4 h from sample collection to result) and quantitation of Enterococcus bacteria compared to the results with the growth-based methods

Multitiered Approach Using Quantitative PCR To Track Sources of Fecal Pollution Affecting Santa Monica Bay, California

The ubiquity of fecal indicator bacteria such as Escherichia coli and Enterococcus spp. in urban environments makes tracking of fecal contamination extremely challenging. A multitiered approach was used to assess sources of fecal pollution in Ballona Creek, an urban watershed that drains to the Santa Monica Bay (SMB) near Los Angeles, Calif. A mass-based design at six main-stem sites and four major tributaries over a 6-h period was used (i) to assess the flux of Enterococcus spp. and E. coli by using culture-based methods (tier 1); (ii) to assess levels of Enterococcus spp. by using quantitative PCR and to detect and/or quantify additional markers of human fecal contamination, including a human-specific Bacteroides sp. marker and enterovirus, using quantitative reverse transcriptase PCR (tier 2); and (iii) to assess the specific types of enterovirus genomes found via sequence analysis (tier 3). Sources of fecal indicator bacteria were ubiquitous, and concentrations were high, throughout Ballona Creek, with no single tributary dominating fecal inputs. The flux of Enterococcus spp. and E. coli averaged 10(9) to 10(10) cells h(−1) and was as high at the head of the watershed as at the mouth prior to discharge into the SMB. In addition, a signal for the human-specific Bacteroides marker was consistently detected: 86% of the samples taken over the extent during the study period tested positive. Enteroviruses were quantifiable in 14 of 36 samples (39%), with the highest concentrations at the site furthest upstream (Cochran). These results indicated the power of using multiple approaches to assess and quantify fecal contamination in freshwater conduits to high-use, high-priority recreational swimming areas

Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution▿ †

Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 × 106 copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters