The complex association between drinking water consumption and endemic gastrointestinal illness as shown by Swedish cohort studies.

It is well known that municipal drinking water may be the cause of gastrointestinal illness (GII) outbreaks, but it is still unclear to what extent drinking water contributes to endemic GII. To explore this, we conducted a prospective cohort study among 6,955 adults in five municipalities in Sweden, collecting monthly GII episodes and mean daily cold drinking water consumption through SMS (Short Message Service). When the association between drinking water consumption and GII (all symptoms) and acute gastrointestinal illness (AGI, vomiting and/or three loose stools during a 24-h period) were assessed, there were indications that the association departed from linearity, following a unimodal shape. Among consumers in surface water areas, the highest risk of GII and AGI was generally seen among the average consumers, while the opposite was seen among groundwater consumers. The association however also seemed to be affected by neighbouring communities. The results of the study indicate that there is indeed an association between drinking water consumption and endemic GII, but the nature of this association is complex and likely affected by multiple factors, for example, water source type in the home and degree of exposure to drinking water from additional sources.

An international inter-laboratory study to compare digital PCR with ISO standardized qPCR assays for the detection of norovirus GI and GII in oyster tissue.

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

Hepatitis A Virus and Hepatitis E Virus as Food- and Waterborne Pathogens-Transmission Routes and Methods for Detection in Food.

Foodborne viruses are an important threat to food safety and public health. Globally, there are approximately 5 million cases of acute viral hepatitis due to hepatitis A virus (HAV) and hepatitis E virus (HEV) every year. HAV is responsible for numerous food-related viral outbreaks worldwide, while HEV is an emerging pathogen with a global health burden. The reported HEV cases in Europe have increased tenfold in the last 20 years due to its zoonotic transmission through the consumption of infected meat or meat products. HEV is considered the most common cause of acute viral hepatitis worldwide currently. This review focuses on the latest findings on the foodborne transmission routes of HAV and HEV and the methods for their detection in different food matrices.

European Union Reference Laboratories support the National food, feed and veterinary Reference Laboratories with rolling out whole genome sequencing in Europe.

The Inter European Union Reference Laboratories (EURLs) Working Group on Next Generation Sequencing (NGS) involves eight EURLs for microbiological food and feed hazards and has been working since 2017 to promote the adoption of NGS by the National Reference Laboratories (NRLs) in the European Union. This work illustrates the results of the first 5 years of activity. By working together, the EURLs involved have released guidance documents for assisting NRLs in all the steps of NGS, helping the transition from classical molecular methods towards whole genome sequencing while ensuring harmonization, with the final aim of improving preparedness in the use of NGS to characterize microbial hazards and trace the sources of infection.

rprimer: an R/bioconductor package for design of degenerate oligos for sequence variable viruses.

BACKGROUND: This paper presents a new R/Bioconductor package, rprimer, for design of degenerate oligos and PCR assays for sequence variable viruses. A multiple DNA sequence alignment is used as input data, while the outputs consist of comprehensive tables (data frames) and dashboard-like plots. The workflow can be run directly from the R console or through a graphical user interface (Shiny application). Here, rprimer is demonstrated and evaluated by using it to design two norovirus genogroup I (GI) assays: one RT-qPCR assay for quantitative detection and one RT­PCR assay for Sanger sequencing and polymerase-capsid based genotyping. RESULTS: The assays generated were evaluated using stool samples testing positive for norovirus GI. The RT-qPCR assay accurately amplified and quantified all samples and showed comparable performance to a widely-used standardised assay, while the RT-PCR assay resulted in successful sequencing and genotyping of all samples. Merits and limitations of the package were identified through comparison with three similar freely available software packages. Several features were comparable across the different tools, but important advantages of rprimer were its speed, flexibility in oligo design and capacity for visualisation. CONCLUSIONS: An R/Bioconductor package, rprimer, was developed and shown to be successful in designing primers and probes for quantitative detection and genotyping of a sequence-variable virus. The package provides an efficient, flexible and visual approach to degenerate oligo design, and can therefore assist in virus research and method development.

Comparison of Skimmed Milk and Lanthanum Flocculation for Concentration of Pathogenic Viruses in Water.

Concentration of viruses in water is necessary for detection and quantification of the viruses present, in order to evaluate microbiological barriers in water treatment plants and detect pathogenic viruses during waterborne outbreaks, but there is currently no standardised procedure. In this study, we implemented a previously described fast and simple lanthanum-based protocol for concentration of norovirus genogroup I (GI), genogroup II (GII) and hepatitis A virus (HAV) in drinking and surface water. We compared the results with those of a widely used skimmed milk flocculation method, followed by nucleic acid extraction and RT-qPCR detection. Three seeding levels, with intended concentrations 5 × 103, 5 × 104 and 5 × 105 genome copies/10 L, were added to drinking water or surface water. All seed levels were detected with both flocculation methods. Samples extracted with skimmed milk flocculation had on average 1.82, 1.86 and 1.38 times higher measured concentration of norovirus GI, GII and HAV, respectively, than those extracted with lanthanum flocculation, across all seeding levels and water types tested. Mengovirus was used as a positive process control. Mengovirus recovery was higher for skimmed milk (40.7% in drinking water, 26.0% in surface water) than for lanthanum flocculation (24.4% in drinking water, 9.7% in surface water). Together, this indicates that skimmed milk flocculation provides higher viral recovery than lanthanum flocculation. However, lanthanum-based flocculation can be performed much faster than skimmed milk flocculation (1.5 h versus 16 h flocculation time) and thus could be a good alternative for rapid monitoring of viruses in water.

A new assay for quantitative detection of hepatitis A virus.

Hepatitis A virus (HAV) is mainly transmitted via contaminated food or water or through person-to-person contact. Here, we describe development and evaluation of a reverse transcription droplet digital PCR (RT-ddPCR) and reverse transcription real-time PCR (RT-qPCR) assay for detection of HAV in food and clinical specimens. The assay was evaluated by assessing limit of detection, precision, matrix effects, sensitivity and quantitative agreement. The 95 % limit of detection (LOD95 %) was 10 % higher for RT-ddPCR than for RT-qPCR. A Bayesian model was used to estimate precision on different target concentrations. From this, we found that RT-ddPCR had somewhat greater precision than RT-qPCR within runs and markedly greater precision between runs. By analysing serum from naturally infected persons and a naturally contaminated food sample, we found that the two methods agreed well in quantification and had comparable sensitivities. Tests with artificially contaminated food samples revealed that neither RT-ddPCR nor RT-qPCR was severely inhibited by presence of oysters, raspberries, blueberries or leafy-green vegetables. For this assay, we conclude that RT-qPCR should be considered if rapid, qualitative detection is the main interest and that RT-ddPCR should be considered if precise quantification is the main interest. The high precision of RT-ddPCR allows for detection of small changes in viral concentration over time, which has direct implications for both food control and clinical studies.

Micro and nano sized particles in leachates from agricultural soils: Phosphorus and sulfur speciation by X-ray micro-spectroscopy.

Colloids and nanoparticles leached from agricultural land are major carriers of potentially bioavailable nutrients with high mobility in the environment. Despite significant research efforts, accurate knowledge of macronutrients in colloids and nanoparticles is limited. We used multi-elemental synchrotron X-ray fluorescence (XRF) microscopy with multivariate spatial analysis and X-ray atomic absorption near-edge structure (XANES) spectroscopy at the P and S K-edges, to study the speciation of P and S in two fractions of leached particles, >0.45 and <0.45 µm respectively, collected from four tile-drained agricultural sites in Sweden. P K-edge XANES showed that organic P, followed by P adsorbed to surfaces of aluminum-bearing particles were the most common forms of leached P. Iron-bound P (Fe-P) forms were generally less abundant (0-30 % of the total P). S K-edge XANES showed that S was predominantly organic, and a relatively high abundance of reduced S species suggests that redox conditions were adverse to the persistence of P bound to Fe-bearing colloids in the leachates. Acid ammonium-oxalate extractions suggested that P associated with Al and Fe (Al-P and Fe-P) in most cases could be explained by the adsorption capacity of non-crystalline (oxalate-extractable) oxides of Al and Fe. These results improve our understanding of particulate P and S speciation in the vadose zone and helps in developing effective technologies for mitigating colloidal driven eutrophication of water bodies near agricultural land.

Endemic gastrointestinal illness and change in raw water source and drinking water production - A population-based prospective study.

There are indications that drinking water may contribute to endemic gastrointestinal illness (GII) even when the drinking water quality meets current standards, but the knowledge is limited. In this population-based prospective study, we assessed if changes in municipal drinking water production affected the GII incidence, by collecting self-reported GII episodes among the population in two municipalities during calendar time-specific inter-annual periods. About 2600 adults in central Sweden and 2600 adults (including 700 households with children aged 0-9 years) in Southwest Sweden, were followed during a baseline and a follow-up period in 2012-2016. Monthly reports of episodes and symptoms of GII were collected by SMS. The following drinking water related changes were assessed: Change 1 (adults); a municipality with a groundwater treatment, changed to a different groundwater source with UV treatment; Change 2 (adults); a municipality with a surface water treatment changed to a groundwater source with UV treatment; and Change 3a (adults) and 3b (children): a municipality with a surface water treatment changed to a new surface water source, having a treatment with a higher pathogen reduction. We observed no evidence that changes in raw water source and/or improved pathogen removal in the drinking water treatment affected the risk of GII among adults. Among children aged 0-9 years participating in Change 3b, we observed a 24% relative risk reduction in GII incidence. These results suggest that improved water treatment may reduce the disease burden of GII in children even in settings in which water treatment efficacy meets current quality standards.

Secretor Status is Associated with Susceptibility to Disease in a Large GII.6 Norovirus Foodborne Outbreak.

Norovirus is commonly associated with food and waterborne outbreaks. Genetic susceptibility to norovirus is largely dependent on presence of histo-blood group antigens (HBGA), specifically ABO, secretor, and Lewis phenotypes. The aim of the study was to determine the association between HBGAs to norovirus susceptibility during a large norovirus foodborne outbreak linked to genotype GII.6 in an office-based company in Stockholm, Sweden, 2015. A two-episode outbreak with symptoms of diarrhea and vomiting occurred in 2015. An online questionnaire was sent to all 1109 employees that had worked during the first outbreak episode. Food and water samples were collected from in-house restaurant and tested for bacterial and viral pathogens. In addition, fecal samples were collected from 8 employees that had diarrhea. To investigate genetic susceptibility during the outbreak, 98 saliva samples were analyzed for ABO, secretor, and Lewis phenotypes using ELISA. A total of 542 of 1109 (49%) employees reported gastrointestinal symptoms. All 8 fecal samples tested positive for GII norovirus, which was also detected in coleslaw collected from the in-house restaurant. Eating at the in-house restaurant was significantly associated with risk of symptom development. Nucleotide sequencing was successful for 5/8 fecal samples and all belonged to the GII.6 genotype. HBGA characterization showed a strong secretor association to norovirus-related symptoms (P = 0.014). No association between norovirus disease and ABO phenotypes was observed. The result of this study shows that non-secretors were significantly less likely to report symptoms in a large foodborne outbreak linked to the emerging GII.6 norovirus strain.

Optimisation and evaluation of an automated system for extraction of viral RNA from oysters.

The NucliSENS MiniMAG (Minimag) system from bioMérieux is widely used for extraction of viral RNA from oysters and is included as informative material in the ISO method for quantification of hepatitis A virus (HAV) and norovirus genogroups I and II (GI and GII) in food (ISO 15216-1:2017). However, the system is no longer on sale within the EU and alternative methods are therefore needed. We optimised and evaluated an automated benchtop system for extraction of viral RNA from oysters artificially contaminated with HAV, norovirus GI, norovirus GII and mengovirus, using the same reagents and a similar protocol as with the Minimag method. Using the automated system instead of Minimag increased measured viral concentration by on average 1.3 times, suggesting that the automated system extracts viral RNA more efficiently than Minimag. A drawback with the automated system was that it displayed higher variability in measured concentration for mengovirus. The median viral recovery was 17%, 37%, 44% and 41% for samples extracted with the automated system and 15%, 27%, 34% and 23% for samples extracted with Minimag for HAV, norovirus GI, norovirus GII and mengovirus, respectively. All samples displayed <75% inhibition in RT-qPCR when extracted with the automated system or Minimag. Together, these results suggest that the automated system can be a suitable alternative to Minimag in analysis of HAV, norovirus GI and norovirus GII in oysters. However, verification using naturally contaminated oysters is needed before it can be used for food safety control purposes.

Missing the Match Might Not Cost You the Game: Primer-Template Mismatches Studied in Different Hepatitis A Virus Variants.

Mismatches between template sequences and reverse transcription (RT) or polymerase chain reaction (PCR) primers can lead to underestimation or false negative results during detection and quantification of sequence-diverse viruses. We performed an in silico inclusivity analysis of a widely used RT-PCR assay for detection of hepatitis A virus (HAV) in food, described in ISO 15216-1. One of the most common mismatches found was a single G (primer) to U (template) mismatch located at the terminal 3'-end of the reverse primer region. This mismatch was present in all genotype III sequences available in GenBank. Partial HAV genomes with common or potentially severe mismatches were produced by in vitro transcription and analysed using RT-ddPCR and RT-qPCR. When using standard conditions for RT-qPCR, the mismatch identified resulted in underestimation of the template concentration by a factor of 1.7-1.8 and an increase in 95% limit of detection from 8.6 to 19 copies/reaction. The effect of this mismatch was verified using full-length viral genomes. Here, the same mismatch resulted in underestimation of the template concentration by a factor of 2.8. For the partial genomes, the presence of additional mismatches resulted in underestimation of the template concentration by up to a factor of 232. Quantification by RT-ddPCR and RT-qPCR was equally affected during analysis of RNA templates with mismatches within the reverse primer region. However, on analysing DNA templates with the same mismatches, we found that ddPCR quantification was less affected by mismatches than qPCR due to the end-point detection technique.

Comparison between RT droplet digital PCR and RT real-time PCR for quantification of noroviruses in oysters.

Oysters are frequently associated with norovirus outbreaks, but the presence of norovirus RNA in oysters does not necessarily imply a health risk to humans. There is a close link between human illness and consumption of oysters with high levels of norovirus RNA, but oysters with low levels of norovirus RNA are more unlikely to be associated with illness. Reliable and precise quantification methods are therefore important for outbreak investigations and risk assessments. This study optimised and validated RT droplet digital PCR (RT-ddPCR) assays for quantification of norovirus genogroups I and II in artificially contaminated oysters, and compared them with the standard method, RT real-time PCR (RT-qPCR). The two methods had comparable 95% limits of detection, but RT-ddPCR generally showed greater precision in quantification. Differences between fluorometric measurements and quantification with RT-ddPCR were determined on in vitro transcribed RNA with targets for norovirus genogroups I and II. Quantification by RT-ddPCR was on average 100 times lower than the fluorometric value for norovirus GI and 15.8 times lower than the fluorometric value for norovirus GII. The large inter-assay difference observed highlights the need for monitoring the RT efficiency in RT-ddPCR, especially when results from different assays are compared. Overall, this study suggests that RT-ddPCR can be a suitable method for precise quantification of norovirus genogroups I and II in oysters.

Drinking water consumption patterns among adults-SMS as a novel tool for collection of repeated self-reported water consumption.

Studies have shown that the average drinking water consumption ranges between 0.075 and 3 L/day for adults with both national and regional differences. For exposure assessment of drinking water hazards, country-specific drinking water consumption data including sources of the consumed water may therefore be warranted. To estimate the amount and source of drinking water consumed among adults in Sweden, we collected self-reported estimates using both traditional methods (telephone interviews, web questionnaire) and a novel method (Short Message Service, SMS questionnaires) in a population from an average sized Swedish municipality. Monthly SMS questionnaires were sent out during one year to obtain longitudinal information as well. SMS showed to be a promising tool for collecting self-reported consumption, as most citizens could participate and the method showed high response rate. Data collected via the SMS questionnaire shows an average consumption of cold tap water of 4.9 glasses/24 h (one glass=200 ml), while the average estimates of cold tap water collected by the traditional methods range from 4.5 to 7.0 glasses/24 h. For statistical distributions, the mean daily consumption of cold tap water for the population was best fitted to a gamma distribution. About 70% of the cold tap water is consumed at home. Based on the results from the SMS study, we suggest using 1 l/day for the average adult population and 2.5 l/day for high consumers for risk assessment of cold tap water consumption. As 46% of the tap water consumed is heated, we suggest using 1.85 l/day for total tap water consumption.

Gastrointestinal illness linked to incidents in drinking water distribution networks in Sweden.

During recent years, knowledge gaps on drinking water-related gastrointestinal illness have been identified, especially for non-epidemic cases. Pathogen contamination of drinking water during distribution has been suggested to contribute to these cases, but the risk factors are not yet fully understood. During 2014-2015, we conducted an epidemiological study in five municipalities in Sweden, to assess whether incidents in the drinking water distribution system influence the risk of gastrointestinal illness. Telephone interviews were conducted in the affected areas and in reference areas 7-14 days after a reported incident. Symptoms of gastrointestinal illness occurring during the period were documented for each household member. The results showed a significantly elevated risk of vomiting and acute gastrointestinal illness (AGI) in the affected areas, compared to the reference areas (ORvom. = 2.0, 95% CI: 1.2-3.3; ORAGI = 1.9, 95% CI: 1.2-3.0). Certain conditions, or risk factors, during the incidents, such as sewage and drinking water pipelines at the same level in the trench, were associated with an elevated risk of AGI and vomiting. Safety measures taken during repair work, like flushing, were also associated with an elevated risk of AGI and vomiting. These results show that incidents in the drinking water distribution network contribute to endemic gastrointestinal illness, especially AGI and vomiting, and that external pathogen contamination of the drinking water is a likely cause of these cases of gastrointestinal illness. The results also indicate that safety measures used today may not be sufficient for eliminating the risk of gastrointestinal illness.

Measuring sporadic gastrointestinal illness associated with drinking water - an overview of methodologies.

There is an increasing awareness that drinking water contributes to sporadic gastrointestinal illness (GI) in high income countries of the northern hemisphere. A literature search was conducted in order to review: (1) methods used for investigating the effects of public drinking water on GI; (2) evidence of possible dose-response relationship between sporadic GI and drinking water consumption; and (3) association between sporadic GI and factors affecting drinking water quality. Seventy-four articles were selected, key findings and information gaps were identified. In-home intervention studies have only been conducted in areas using surface water sources and intervention studies in communities supplied by ground water are therefore needed. Community-wide intervention studies may constitute a cost-effective alternative to in-home intervention studies. Proxy data that correlate with GI in the community can be used for detecting changes in the incidence of GI. Proxy data can, however, not be used for measuring the prevalence of illness. Local conditions affecting water safety may vary greatly, making direct comparisons between studies difficult unless sufficient knowledge about these conditions is acquired. Drinking water in high-income countries contributes to endemic levels of GI and there are public health benefits for further improvements of drinking water safety.

Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing.

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C q shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.

Efficacy of water treatment processes and endemic gastrointestinal illness - A multi-city study in Sweden.

Outbreaks of acute gastrointestinal illnesses (AGI) have been linked to insufficient drinking water treatment on numerous occasions in the industrialized world, but it is largely unknown to what extent public drinking water influences the endemic level of AGI. This paper aimed to examine endemic AGI and the relationship with pathogen elimination efficacy in public drinking water treatment processes. For this reason, time series data of all telephone calls to the Swedish National Healthcare Guide between November 2007 and February 2014 from twenty Swedish cities were obtained. Calls concerning vomiting, diarrhea or abdominal pain (AGI calls) were separated from other concerns (non-AGI calls). Information on which type of microbial barriers each drinking water treatment plant in these cities have been used were obtained, together with the barriers' theoretical pathogen log reduction efficacy. The total log reduction in the drinking water plants varied between 0.0 and 6.1 units for viruses, 0.0-14.6 units for bacteria and 0.0-7.3 units regarding protozoans. To achieve one general efficacy parameter for each plant, a weighted mean value of the log reductions (WLR) was calculated, with the weights based on how commonly these pathogen groups cause AGI. The WLR in the plants varied between 0.0 and 6.4 units. The effect of different pathogen elimination efficacy on levels of AGI calls relative non-AGI calls was evaluated in regression models, controlling for long term trends, population size, age distribution, and climatological area. Populations receiving drinking water produced with higher total log reduction was associated with a lower relative number of AGI calls. In overall, AGI calls decreased by 4% (OR = 0.96, CI: 0.96-0.97) for each unit increase in the WLR. The findings apply to both groundwater and surface water study sites, but are particularly evident among surface water sites during seasons when viruses are the main cause of AGI. This study proposes that the endemic level of gastroenteritis can indeed be reduced with more advanced treatment processes at many municipal drinking water treatment plants.

Polymorphisms and variants in the prion protein sequence of European moose (Alces alces), reindeer (Rangifer tarandus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in Scandinavia.

The prion protein (PrP) sequence of European moose, reindeer, roe deer and fallow deer in Scandinavia has high homology to the PrP sequence of North American cervids. Variants in the European moose PrP sequence were found at amino acid position 109 as K or Q. The 109Q variant is unique in the PrP sequence of vertebrates. During the 1980s a wasting syndrome in Swedish moose, Moose Wasting Syndrome (MWS), was described. SNP analysis demonstrated a difference in the observed genotype proportions of the heterozygous Q/K and homozygous Q/Q variants in the MWS animals compared with the healthy animals. In MWS moose the allele frequencies for 109K and 109Q were 0.73 and 0.27, respectively, and for healthy animals 0.69 and 0.31. Both alleles were seen as heterozygotes and homozygotes. In reindeer, PrP sequence variation was demonstrated at codon 176 as D or N and codon 225 as S or Y. The PrP sequences in roe deer and fallow deer were identical with published GenBank sequences.