Improving the Quantification of Cyanotoxins Using a Mass Balance-Based Effective Concentration-Equivalent Concentration Approach.

Two commonly used methods for cyanotoxin analysis are enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Each method has its advantages and disadvantages, and discrepancies are commonly observed between the two methods due to various factors including the ELISA antibody cross-reacting to different cyanotoxin congeners. However, reliable cyanotoxin monitoring methods and accurate interpretation of results are needed for water utilities to guide recreational water planning and drinking water treatment operations. In this study, we explored an innovative "effective concentration-equivalent concentration" (EC-EQ) approach to improve the interpretation of ELISA results and the comparison to LC-MS/MS results. The precision of ELISA results was first improved by reporting the sample ECs and EQs derived from their ELISA dose curves. Concentrations of each cyanotoxin as measured by LC-MS/MS were then combined with their respective ELISA cross-reactivities to calculate their theoretical ELISA responses. Finally, instead of comparing the results from the two methods directly, the equivalent concentration based on one single reference cyanotoxin was used for reporting and comparison. This integrated mass balance-based approach provides a more reliable interpretation of results by considering the reactivity differences between toxins as well as their mixture effects. This approach has been successfully applied to microcystin (one main group of cyanotoxins) standard mixtures and cyanobacterial bloom samples to interpret and compare their ELISA and LC-MS/MS detection results. The study provides guidance to utilities on how to obtain more accurate cyanotoxin monitoring results and better understand the discrepancy between the two methods.

Reproducibility and sensitivity of 36 methods to quantify the SARS-CoV-2 genetic signal in raw wastewater: findings from an interlaboratory methods evaluation in the U.S.

In response to COVID-19, the international water community rapidly developed methods to quantify the SARS-CoV-2 genetic signal in untreated wastewater. Wastewater surveillance using such methods has the potential to complement clinical testing in assessing community health. This interlaboratory assessment evaluated the reproducibility and sensitivity of 36 standard operating procedures (SOPs), divided into eight method groups based on sample concentration approach and whether solids were removed. Two raw wastewater samples were collected in August 2020, amended with a matrix spike (betacoronavirus OC43), and distributed to 32 laboratories across the U.S. Replicate samples analyzed in accordance with the project's quality assurance plan showed high reproducibility across the 36 SOPs: 80% of the recovery-corrected results fell within a band of ±1.15 log10 genome copies per L with higher reproducibility observed within a single SOP (standard deviation of 0.13 log10). The inclusion of a solids removal step and the selection of a concentration method did not show a clear, systematic impact on the recovery-corrected results. Other methodological variations (e.g., pasteurization, primer set selection, and use of RT-qPCR or RT-dPCR platforms) generally resulted in small differences compared to other sources of variability. These findings suggest that a variety of methods are capable of producing reproducible results, though the same SOP or laboratory should be selected to track SARS-CoV-2 trends at a given facility. The methods showed a 7 log10 range of recovery efficiency and limit of detection highlighting the importance of recovery correction and the need to consider method sensitivity when selecting methods for wastewater surveillance.

Changes in Escherichia coli to Cryptosporidium ratios for various fecal pollution sources and drinking water intakes.

Assessing the presence of human pathogenic Cryptosporidium oocysts in surface water remains a significant water treatment and public health challenge. Most drinking water suppliers rely on fecal indicators, such as the well-established Escherichia coli (E. coli), to avoid costly Cryptosporidium assays. However, the use of E. coli has significant limitations in predicting the concentration, the removal and the transport of Cryptosporidium. This study presents a meta-analysis of E. coli to Cryptosporidium concentration paired ratios to compare their complex relationships in eight municipal wastewater sources, five agricultural fecal pollution sources and at 13 drinking water intakes (DWI) to a risk threshold based on US Environmental Protection Agency (USEPA) regulations. Ratios lower than the USEPA risk threshold suggested higher concentrations of oocysts in relation to E. coli concentrations, revealing an underestimed risk for Cryptosporidium based on E. coli measurements. In raw sewage (RS), high ratios proved E. coli (or fecal coliforms) concentrations were a conservative indicator of Cryptosporidium concentrations, which was also typically true for secondary treated wastewater (TWW). Removals of fecal indicator bacteria (FIB) and parasites were quantified in WWTPs and their differences are put forward as a plausible explanation of the sporadic ratio shift. Ratios measured from agricultural runoff surface water were typically lower than the USEPA risk threshold and within the range of risk misinterpretation. Indeed, heavy precipitation events in the agricultural watershed led to high oocyst concentrations but not to E. coli or enterococci concentrations. More importantly, ratios established in variously impacted DWI from 13 Canadian drinking water plants were found to be related to dominant fecal pollution sources, namely municipal sewage. In most cases, when DWIs were mainly influenced by municipal sewage, E. coli or fecal coliforms concentrations agreed with Cryptosporidium concentrations as estimated by the meta-analysis, but when DWIs were influenced by agricultural runoff or wildlife, there was a poor relationship. Average recovery values were available for 6 out of 22 Cryptosporidium concentration data sets and concomitant analysis demonstrated no changes in trends, with and without correction. Nevertheless, recovery assays performed along with every oocyst count would have enhanced the precision of this work. Based on our findings, the use of annual averages of E. coli concentrations as a surrogate for Cryptosporidium concentrations can result in an inaccurate estimate of the Cryptosporidium risk for agriculture impacted drinking water intakes or for intakes with more distant wastewater sources. Studies of upstream fecal pollution sources are recommended for drinking water suppliers to improve their interpretation of source water quality data.

Total and infectious Cryptosporidium oocyst and total Giardia cyst concentrations from distinct agricultural and urban contamination sources in Eastern Canada.

Cryptosporidium and Giardia (oo)cyst concentrations are frequently used for assessing drinking water safety. The widely used USEPA Method 1623 provides total counts of (oo)cysts, but may not be accurate for human health risk characterization, since it does not provide infectivity information. The total counts and infectious fraction of Cryptosporidium oocysts and the total counts of Giardia cysts were assessed in major fecal pollution sources. Fresh calf and cow feces, their manure, and the discharge point were sampled in a small rural sub-watershed (n = 20, 21, 10, 10). Median concentrations for total (oo)cysts were higher in calves (333 oocysts g(-1); 111 cysts g(-1)) than in cows (52 oocysts g(-1); 7 cysts g(-1)). Infectious oocysts were found in 17 (7%) of the samples and none were found in manure or at the discharge point. Urban sources were sampled in the influent and effluent (n = 19, 18) of two wastewater treatment plants. Peak concentrations were 533 oocysts L(-1) and 9,010 cysts L(-1) for influents and 89 oocysts L(-1) and 472 cysts L(-1) for effluents. Infectious oocyst fractions varied from below the detection limit to 7-22% in the effluent and influent respectively. These infectious fractions are significantly lower than those currently used for quantitative microbial risk assessment estimates.

Improved risk analysis by dual direct detection of total and infectious Cryptosporidium oocysts on cell culture in combination with immunofluorescence assay.

The inactivation of Cryptosporidium oocysts is a main driver in the selection of water treatment disinfection strategies, and microbial risk analysis provides a sound basis for optimizing water treatment processes. U.S. Environmental Protection Agency method 1622/23 provides an estimate of the total oocyst count; however, it cannot be used directly for risk assessment, as it does not determine the fraction of infectious oocysts. Improved assessment of the risk for designated sources or in treated water requires evaluation of the total number of oocysts and an estimate of their infectivity. We developed a dual direct detection method using differential immunofluorescent staining that allows detection of both oocysts and cell culture infection foci for each sample. Using Cryptosporidium parvum oocysts, various pH levels, proteases, and gastroenteric compounds and substrates were assessed to determine their abilities to enhance the number of infection foci. The results showed that the key trigger for oocyst stimulation was acidification. Addition of a low concentration of D-glucose (50 mM) to the infection media increased rates of infectivity, while a higher dose (300 mM) was inhibitory. The total number of oocysts in each sample was determined by counting the oocysts remaining on a cell monolayer and the oocysts recovered from cell monolayer washes during processing using a simple filtration technique. With the dual direct detection on cell culture with immunofluorescence assay method, it is now possible to determine the numbers of total and infectious oocysts for a given sample in a single analysis. Direct percentages of infectivity are then calculated, which allows more accurate assessments of risk.

Evaluation of swine-specific PCR assays used for fecal source tracking and analysis of molecular diversity of swine-specific "bacteroidales" populations.

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 "Bacteroidales" 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution.

Aged HCT-8 cell monolayers support Cryptosporidium parvum infection.

Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8- to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8- to 11-day-old and 11- to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.

Genotype diversity of Escherichia coli isolates in natural waters determined by PFGE and ERIC-PCR.

Most library-dependent bacterial source tracking studies using Escherichia coli (E. coli) have focused on strain diversity of isolates obtained from known human and animal faecal sources for library development. In contrast, this study evaluated the genotype variation of E. coli isolated from natural surface water using pulsed field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) to better understand these naturally occurring populations. A total of 650 water samples were collected over a nine month period from eleven sampling stations from Lake Waco and Belton Lake in Central Texas. Of the 650 water samples collected, 412 were positive for E. coli, yielding a total of 631 E. coli isolates (1-12 isolates collected per sample). PFGE and ERIC-PCR patterns were successfully generated for 555 isolates and were compared using the curve-based Pearson's product-moment correlation coefficient. The 555 E. coli isolates represented 461 PFGE genotypes, with 84% (386/461) of the genotypes being represented by individual isolates. The remaining 75 genotypes were represented by 2-5 isolates each. Using ERIC-PCR, the 555 E. coli isolates represented 175 genotypes, with 63% (109/175) of the genotypes being represented by individual isolates. In contrast to the PFGE results, two ERIC-PCR genotypes represented 37% of the E. coli isolates, (83 and 124 isolates, respectively), and were found throughout the watersheds both spatially and temporally. Based on the PFGE genotype diversity of water isolates, there is little evidence that a small number of environmentally-adapted E. coli represent dominant populations in the studied waterbodies. However, with the lower discriminatory power technique ERIC-PCR, an opposing conclusion might have been drawn. These results emphasize the importance of considering the resolving power of the source tracking technique being used when assessing strain diversity and geographical stability.

Investigation of potential zooanthroponotic transmission of cryptosporidiosis and giardiasis through agricultural use of reclaimed wastewater.

A field study in the Juarez Valley of Mexico was performed to investigate the potential transmission of Cryptosporidium and Giardia to sheep livestock grazing on forage irrigated with reclaimed wastewater, and the potential for disease transmission back to humans. United States Environmental Protection Agency Method 1623 immunofluorescent assay (IFA) revealed high levels of pathogens in reclaimed wastewater, with 183 to >7000 Giardia cysts and 9 - 762 Cryptosporidium oocysts detected per litre. Infectious Cryptosporidium were detected in the reclaimed wastewater using the cell culture focus detection method (FDM). Polymerase chain reaction (PCR) analyses revealed reclaimed wastewater contained the C. parvum bovine (zoonotic) genotype, human-specific C. hominis subgenotypes, and G. lamblia (syn. G. duodenalis, G. intestinalis) Assemblage A genotypes (A2 and A3). Despite high levels of Cryptosporidium and Giardia in the reclaimed wastewater, these pathogens were rarely found on the forage plants, possibly due to environmental attenuation. Sheep fecal specimens were positive for only livestock-associated G. lamblia Assemblage E genotypes. Therefore, in this field study, there was no evidence of zooanthroponotic transmission of Cryptosporidium or Giardia.

Quantitative-PCR assessment of Cryptosporidium parvum cell culture infection.

A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.

Comparison of method 1623 and cell culture-PCR for detection of Cryptosporidium spp. in source waters.

Analysis of Cryptosporidium occurrence in six watersheds by method 1623 and the integrated cell culture-PCR (CC-PCR) technique provided an opportunity to evaluate these two methods. The average recovery efficiencies were 58.5% for the CC-PCR technique and 72% for method 1623, but the values were not significantly different (P = 0.06). Cryptosporidium oocysts were detected in 60 of 593 samples (10.1%) by method 1623. Infectious oocysts were detected in 22 of 560 samples (3.9%) by the CC-PCR technique. There was 87% agreement between the total numbers of samples positive as determined by method 1623 and CC-PCR for four of the sites. The other two sites had 16.3 and 24% correspondence between the methods. Infectious oocysts were detected in all of the watersheds. Overall, approximately 37% of the Cryptosporidium oocysts detected by the immunofluorescence method were viable and infectious. DNA sequence analysis of the Cryptosporidium parvum isolates detected by CC-PCR showed the presence of both the bovine and human genotypes. More than 90% of the C. parvum isolates were identified as having the bovine or bovine-like genotype. The estimates of the concentrations of infectious Cryptosporidium and the resulting daily and annual risks of infection compared well for the two methods. The results suggest that most surface water systems would require, on average, a 3-log reduction in source water Cryptosporidium levels to meet potable water goals.