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

In April 2016, an outbreak of gastrointestinal illness (4,136 cases) occurred in Catalonia, Spain. We detected high levels of norovirus genotypes I and II in office water coolers associated with the outbreak. Infectious viral titer estimates were 33-49 genome copies/L for genotype I and 327-660 genome copies/L for genotype II.

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

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.

Standardized multiplex one-step qRT-PCR for hepatitis A virus, norovirus GI and GII quantification in bivalve mollusks and water.

A quadruplex Real-Time RT-PCR assay for the simultaneous quantitative detection of hepatitis A virus (HAV), norovirus (NoV) GI and GII, and mengovirus (used as process control for determination of the virus/nucleic acid extraction efficiency) has been developed. This multiplex assay has been comparatively evaluated with the individual monoplex assays and showed to be slightly less sensitive, with average ΔCq values of 0.90, 0.28 and 0.44 for HAV, NoV GI and NoV GII, respectively, in standard curves of viral RNA, or 0.32, 0.37 and 0.51 for the same viruses respectively, in naturally-contaminated samples. These ΔCq values were mostly negligible since it represented, in the worst case scenario, a loss of 0.43 log in genome copy numbers. The quadruplex assay shows similar theoretical detection limits than the monoplex assay for NoV GII, and 10 times higher for HAV and NoV GI. However, when naturally-contaminated food and water samples were tested, these theoretical detection thresholds were often exceeded and very low genome copy numbers (below the limit of detection) could be quantified. The quadruplex assay fulfills the requirements of the method developed by the European Committee on Standardization (CEN) for virus detection in selected foodstuffs with significant advantages in labor and reagent costs.