Propidium monoazide RTqPCR assays for the assessment of hepatitis A inactivation and for a better estimation of the health risk of contaminated waters

Abstract

The waterborne transmission of hepatitis A virus (HAV), the main cause of acute hepatitis, is well documented. Recently, two ISO proposals for sensitive determination of this pathogen by RTqPCR in water and food have been published (ISO/TS 15216-1 and ISO/TS 15216-2), and could enable the formulation of regulatory standards for viruses in the near future. However, since detected viral genomes do not always correlate with virus infectivity, molecular approaches need to be optimized to better predict infectivity of contaminated samples. Two methods involving the use of propidium monoazide (PMA), with or without Triton X-100, prior to RTqPCR amplification were optimized and adapted to infer the performance of infectious viral inactivation upon two different water treatments: free chlorine and high temperature. Significant correlations between the decrease of genome copies and infectivity were found for both inactivation procedures. The best procedure to infer chlorine inactivation was the PMA-RTqPCR assay, in which 1, 2 or 3-log genome copies reductions corresponded to reductions of infectious viruses of 2.61 ± 0.55, 3.76 ± 0.53 and 4.92 ± 0.76 logs, respectively. For heat-inactivated viruses, the best method was the PMA/Triton-RTqPCR assay, with a 1, 2 or 3-log genome reduction corresponding to reductions of infectious viruses of 2.15 ± 1.31, 2.99 ± 0.79 and 3.83 ± 0.70 logs, respectively. Finally, the level of damaged virions was evaluated in distinct types of water naturally contaminated with HAV. While most HAV genomes quantified in sewage corresponded to undamaged capsids, the analysis of a river water sample indicated that more than 98% of viruses were not infectious. Although the PMA/Triton-RTqPCR assay may still overestimate infectivity, it is more reliable than the RTqPCR alone and it seems to be a rapid and cost-effective method that can be applied on different types of water, and that it undeniably provides a more accurate measure of the health risk associated to contaminated waters.

Detection of Infectious Noroviruses from Wastewater and Seawater Using PEMAXTM Treatment Combined with RT-qPCR

“…Our results showed that we could not detect any infectious norovirus GI in seawater samples, possibly because of the low concentration of norovirus GI present in those samples. Similar results were reported in a previous study where 98% hepatitis A viruses present in river water were deemed non-infectious [33]. However, our results did indicate the low level of infectious norovirus GII present in one of the seawater samples.…”

Norovirus in Bottled Water Associated with Gastroenteritis Outbreak, Spain, 2016

“…Because molecular methods are unable to discern between infectious and noninfectious particles, we predicted the infectivity of norovirus in the concentrated samples by treating the samples with the nucleic acid intercalating dye PMA propidium monoazide; (50 µmol/L) and Triton X surfactant (0.5%) before RT-qPCR; this enabled us to distinguish between virions with intact and altered capsids ( 9 ). Following this approach, estimated infectious levels in the 2 positive samples were 49 and 327 genome copies/L for norovirus GI and 33 and 660 genome copies/L for norovirus GII (Table).…”

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

“…Photoactivatable intercalating dyes have begun to show promise in being able to selectively detect infectious HAV (Sanchez et al, 2012; Coudray-Meunier et al, 2015; Moreno et al, 2015; Fuster et al, 2016; Randazzo et al, 2018b) and human NoV (Parshionikar et al, 2010; Randazzo et al, 2016, 2018a; Jeong et al, 2017). Recently, Fraisse et al (2018) proposed PtCl 4 as a successful viability marker for human NoV.…”