Recreational, reclaimed and drinking source waters worldwide are under increasing anthropogenic pressure, and often contain waterborne enteric bacterial, protozoan, and viral pathogens originating from non-point source fecal contamination. Recently, the capsid integrity (ci)-qPCR, utilizing the azo-dyes propidium monoazide (PMA) or ethidium monoazide (EMA), has been shown to reduce false-positive signals under laboratory conditions as well as in food safety applications, thus improving the qPCR estimation of virions of public health significance. The compatibility of two widely used human adenovirus (HAdV) qPCR protocols was evaluated with the addition of a PMA/EMA pretreatment using a range of spiked and environmental samples. Stock suspensions of HAdV were inactivated using heat, UV, and chlorine before being quantified by cell culture, qPCR, and ci-qPCR. Apparent inactivation of virions was detected for heat and chlorine treated HAdV while there was no significant difference between ci-qPCR and qPCR protocols after disinfection by UV. In a follow-up comparative analysis under more complex matrix conditions, 51 surface and 24 wastewater samples pre/post UV treatment were assessed for enteric waterborne HAdV to evaluate the ability of ci-qPCR to reduce the number of false-positive results when compared to conventional qPCR and cell culture. Azo-dye pretreatment of non-UV inactivated samples was shown to improve the ability of molecular HAdV quantification by reducing signals from virions with an accessible genome, thereby increasing the relevance of qPCR results for public health purposes, particularly suited to resource-limited low and middle-income settings.
Faecal indicator bacteria (FIB) are commonly used as water quality indicators; implying faecal contamination and therefore the potential presence of pathogenic enteric bacteria, viruses, and protozoa. Hence in wastewater treatment, the most commonly used treatment process measures (surrogates) are total coliforms, faecal coliforms, Escherichia coli (E. coli), and enterococci. However, greywater potentially contains skin pathogens unrelated to faecal load, and E. coli and other FIB may grow within greywater unrelated to pathogens. Overall, FIB occurs at fluctuating and relatively low concentrations compared to other endogenous greywater bacteria affecting their ability as surrogates for pathogen reduction. Therefore, unlike municipal sewage, FIB provides a very limited and unreliable log-reduction surrogate measure for on-site greywater treatment systems. Based on our recent metagenomic study of laundry greywater, skin-associated bacteria such as Staphylococcus, Corynebacterium, and Propionibacterium spp. dominate and may result in more consistent treatment surrogates than traditional FIB. Here, we investigated various Staphylococcus spp. as potential surrogates to reliably assay over 4-log10 reduction by the final-stage UV disinfection step commonly used for on-site greywater reuse, and compare them to various FIB/phage surrogates. A collimated UV beam was used to determine the efficacy of UV inactivation (255, 265 and 285 nm) against E. coli, Enterococcus faecalis, E. faecium, E. casseliflavus, Staphylococcus aureus, and S. epidermidis. Staphylococcus spp. was estimated by combining the bi-linear dose-response curves for S. aureus and S. epidermidis and was shown to be less resistant to UV irradiation than the other surrogates examined. Hence, a relative inactivation credit is suggested; whereas, the doses required to achieve a 4 and 5-log10 reduction of Staphylococcus spp. (13.0 and 20.9 mJ cm−2, respectively) were used to determine the relative inactivation of the other microorganisms investigated. The doses required to achieve a 4 and 5-log10 reduction of Staphylococcus spp. resulted in a log10 reduction of 1.4 and 4.1 for E. coli, 0.8 and 2.8 for E. faecalis, 0.8 and 3.6 for E. casseliflavus and 0.8 and 1.2 for MS2 coliphage, respectively. Given the concentration difference of Staphylococcus spp. and FIB (3 to 5-log10 higher), we propose the use of Staphylococcus spp. as a novel endogenous performance surrogate to demonstrate greywater treatment performance given its relatively high and consistent concentration and therefore ability to demonstrate over 5-log10 reductions.
20Capsid-integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses 21 combining azo-dye pretreatment with qPCR, has been widely used in recent years; however, 22 variations in pretreatment conditions for various virus types can limit the efficacy of specific 23 protocols. By identifying and critically synthesizing forty-two recent peer-reviewed studies 24 employing capsid-integrity qPCR for viruses in the last decade (2009 to 2019) in the fields of food 25 safety and environmental virology, we aimed to establish recommendations for the detection of 26 infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided 27 the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of 28 attachment or genomic damage, are less well detected using this approach. Although optimizing 29 specific protocols for each virus is recommended, we identify a framework for general assay 30 conditions. These include concentrations of ethidium monoazide, propidium monoazide or its 31 derivates between 10 and 200 µM; incubation on ice or at room temperature (20 -25ºC) for 5 to 32 120 min; and dye activation using LED or high light (500 -800 Watts) exposure for periods 33 ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus 34 transmission in routine (water) monitoring settings and during viral outbreaks such as the current 35 COVID-19 pandemic or endemic diseases like dengue fever. 36 37 Graphical abstract 38 39 Keywords: (6) azo dye, EMA, PMA, microbial contamination, viability, water quality 40 41
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