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.

Fate of Human Noroviruses in Shellfish and Water Impacted by Frequent Sewage Pollution Events.

Knowledge of the fate of human noroviruses (NoV) in the marine environment is key to better controlling shellfish-related NoV gastroenteritis. We quantified NoV and Escherichia coli in sewage from storm tank discharges and treated effluent processed by a UV-disinfection plant following activated sludge treatment and studied the fate of these microorganisms in an oyster harvesting area impacted by frequent stormwater discharges and infrequent freshwater discharges. Oyster monitoring sites were positioned at intervals downstream from the wastewater treatment works (WwTW) outfall impacting the harvesting area. The decay rates of NoV in oysters as a function of the distance from the outfall were less rapid than those for E. coli that had concentrations of NoV of the same order of magnitude and were over 7 km away from the outfall. Levels of E. coli in oysters from more tidally influenced areas of the estuary were higher around high water than around low water, whereas tidal flows had no influence on NoV contamination in the oysters. The study provides comparative data on the contamination profiles and loadings of NoV and E. coli in a commercial oyster fishery impacted by a WwTW.

Methodological advances in the detection of biotoxins and pathogens affecting production and consumption of bivalve molluscs in a changing environment.

The production, harvesting and safe consumption of bivalve molluscs can be disrupted by biological hazards that can be divided into three categories: (1) biotoxins produced by naturally occurring phytoplankton that are bioaccumulated by bivalves during filter-feeding, (2) human pathogens also bioaccumulated by bivalves and (3) bivalve pathogens responsible for disease outbreaks. Environmental changes caused by human activities, such as climate change, can further aggravate these challenges. Early detection and accurate quantification of these hazards are key to implementing measures to mitigate their impact on production and safeguard consumers. This review summarises the methods currently used and the technological advances in the detection of biological hazards affecting bivalves, for the screening of known hazards and discovery of new ones.

Metabarcoding of Hepatitis E Virus Genotype 3 and Norovirus GII from Wastewater Samples in England Using Nanopore Sequencing.

Norovirus is one of the largest causes of gastroenteritis worldwide, and Hepatitis E virus (HEV) is an emerging pathogen that has become the most dominant cause of acute viral hepatitis in recent years. The presence of norovirus and HEV has been reported within wastewater in many countries previously. Here we used amplicon deep sequencing (metabarcoding) to identify norovirus and HEV strains in wastewater samples from England collected in 2019 and 2020. For HEV, we sequenced a fragment of the RNA-dependent RNA polymerase (RdRp) gene targeting genotype three strains. For norovirus, we sequenced the 5' portion of the major capsid protein gene (VP1) of genogroup II strains. Sequencing of the wastewater samples revealed eight different genotypes of norovirus GII (GII.2, GII.3, GII.4, GII.6, GII.7, GII.9, GII.13 and GII.17). Genotypes GII.3 and GII.4 were the most commonly found. The HEV metabarcoding assay was able to identify HEV genotype 3 strains in some samples with a very low viral concentration determined by RT-qPCR. Analysis showed that most HEV strains found in influent wastewater were typed as G3c and G3e and were likely to have originated from humans or swine. However, the small size of the HEV nested PCR amplicon could cause issues with typing, and so this method is more appropriate for samples with high CTs where methods targeting longer genomic regions are unlikely to be successful. This is the first report of HEV RNA in wastewater in England. This study demonstrates the utility of wastewater sequencing and the need for wider surveillance of norovirus and HEV within host species and environments.

Two-year systematic study to assess norovirus contamination in oysters from commercial harvesting areas in the United Kingdom.

The contamination of bivalve shellfish with norovirus from human fecal sources is recognized as an important human health risk. Standardized quantitative methods for the detection of norovirus in molluscan shellfish are now available, and viral standards are being considered in the European Union and internationally. This 2-year systematic study aimed to investigate the impact of the application of these methods to the monitoring of norovirus contamination in oyster production areas in the United Kingdom. Twenty-four monthly samples of oysters from 39 United Kingdom production areas, chosen to represent a range of potential contamination risk, were tested for norovirus genogroups I and II by using a quantitative real-time reverse transcription (RT)-PCR method. Norovirus was detected in 76.2% (643/844) of samples, with all sites returning at least one positive result. Both prevalences (presence or absence) and norovirus levels varied markedly between sites. However, overall, a marked winter seasonality of contamination by both prevalence and quantity was observed. Correlations were found between norovirus contamination and potential risk indicators, including harvesting area classifications, Escherichia coli scores, and environmental temperatures. A predictive risk score for norovirus contamination was developed by using a combination of these factors. In summary, this study, the largest of its type undertaken to date, provides a systematic analysis of norovirus contamination in commercial oyster production areas in the United Kingdom. The data should assist risk managers to develop control strategies to reduce the risk of human illness resulting from norovirus contamination of bivalve molluscs.

pilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of human health relevance.

The Gram-negative bacterium Vibrio vulnificus is a common inhabitant of estuarine environments. Globally, V. vulnificus is a significant foodborne pathogen capable of causing necrotizing wound infections and primary septicemia, and is a leading cause of seafood-related mortality. Unfortunately, molecular methods for the detection and enumeration of pathogenic V. vulnificus are hampered by the genetically diverse nature of this pathogen, the range of different biotypes capable of infecting humans and aquatic animals, and the fact that V. vulnificus contains pathogenic as well as non-pathogenic variants. Here we report an alternative approach utilizing the development of a real-time PCR assay for the detection of pathogenic V. vulnificus strains based on a polymorphism in pilF, a gene previously indicated to be associated with human pathogenicity. Compared to human serum reactivity, the real-time PCR assay successfully detected pathogenic strains in 46 out of 47 analysed V. vulnificus isolates (97.9%). The method is also rapid, sensitive, and more importantly can be reliably utilised on biotype 2 and 3 strains, unlike other current methods for V. vulnificus virulence differentiation.

An outbreak of norovirus infection linked to oyster consumption at a UK restaurant, February 2010.

BACKGROUND: We present the investigation of an outbreak of gastroenteritis at a UK restaurant incorporating both epidemiological and microbiological analysis. METHODS: Structured postal questionnaires were sent to 30 diners who ate at the restaurant during the outbreak period (5-7 February 2010). Stool specimens collected from staff and diners were submitted for bacterial culture and norovirus testing, and 15 Pacific oysters (Crassostrea gigas) from the batch served during the outbreak period were tested for norovirus. RESULTS: A strong association was observed between illness and oyster consumption (odds ratio undefined, confidence interval: 11.7 to infinity, P = 0.00001). Multiple different sequences of norovirus RNA were present in both stool and oyster specimens, typical of a shellfish origin. Several contemporaneous norovirus outbreaks throughout the UK were linked to oysters, particularly, though not exclusively, those sourced from Carlingford Lough in Ireland (as in this study), which were subsequently withdrawn from distribution. CONCLUSION: Despite the risk to human health, there is significant uncertainty surrounding the quantitative correlation between oyster norovirus levels and consumer illness. Continued research should help further our understanding of this crucial correlation and identify ways in which viral depuration of oysters can be enhanced.

The Foodborne Transmission of Hepatitis E Virus to Humans.

Globally, Hepatitis E virus (HEV) causes over 20 million cases worldwide. HEV is an emerging and endemic pathogen within economically developed countries, chiefly resulting from infections with genotype 3 (G3) HEV. G3 HEV is known to be a zoonotic pathogen, with a broad host range. The primary source of HEV within more economically developed countries is considered to be pigs, and consumption of pork products is a significant risk factor and known transmission route for the virus to humans. However, other foods have also been implicated in the transmission of HEV to humans. This review consolidates the information available regarding transmission of HEV and looks to identify gaps where further research is required to better understand how HEV is transmitted to humans through food.

Correction: Farkas et al. Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR. Methods Protoc. 2021, 4, 17.

There was an error in the original article [...].

Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR.

Wastewater-based epidemiology has become an important tool for the surveillance of SARS-CoV-2 outbreaks. However, the detection of viruses in sewage is challenging and to date there is no standard method available which has been validated for the sensitive detection of SARS-CoV-2. In this paper, we describe a simple concentration method based on polyethylene glycol (PEG) precipitation, followed by RNA extraction and a one-step quantitative reverse transcription PCR (qRT-PCR) for viral detection in wastewater. PEG-based concentration of viruses is a simple procedure which is not limited by the availability of expensive equipment and has reduced risk of disruption to consumable supply chains. The concentration and RNA extraction steps enable 900-1500× concentration of wastewater samples and sufficiently eliminates the majority of organic matter, which could inhibit the subsequent qRT-PCR assay. Due to the high variation in the physico-chemical properties of wastewater samples, we recommend the use of process control viruses to determine the efficiency of each step. This procedure enables the concentration and the extraction the DNA/RNA of different viruses and hence can be used for the surveillance of different viral targets for the comprehensive assessment of viral diseases in a community.

Comparison between quantitative real-time reverse transcription PCR results for norovirus in oysters and self-reported gastroenteric illness in restaurant customers.

Norovirus is the principal agent of bivalve shellfish-associated gastroenteric illness worldwide. Numerous studies using PCR have demonstrated norovirus contamination in a significant proportion of both oyster and other bivalve shellfish production areas and ready-to-eat products. By comparison, the number of epidemiologically confirmed shellfish-associated outbreaks is relatively low. This study attempts to compare norovirus RNA detection in Pacific oysters (Crassostrea gigas) by quantitative real-time reverse transcription PCR (RT-PCR) and human health risk. Self-reported customer complaints of illness in a restaurant setting (screened for credible norovirus symptoms) were compared with presence and levels of norovirus as determined by real-time RT-PCR for the batch of oysters consumed. No illness was reported for batches consistently negative for norovirus by real-time RT-PCR. However, norovirus was detected in some batches for which no illness was reported. Overall presence or absence of norovirus showed a significant association with illness complaints. In addition, the batch with the highest norovirus RNA levels also resulted in the highest rate of reported illness, suggesting a linkage between virus RNA levels and health risks. This study suggests that detection of high levels of norovirus RNA in oysters is indicative of a significantly elevated health risk. However, illness may not necessarily be reported after detection of norovirus RNA at low levels.

Application of mitochondrial DNA analysis for microbial source tracking purposes in shellfish harvesting waters.

We present a method for the reliable detection and source characterisation of faecal pollution in water and shellfish matrices, utilising real-time PCR analysis of mitochondrial DNA targets. In this study we designed real-time PCR (TaqMan) probes to target human, bovine, ovine and swine mtDNA. PCR amplification using species-specific TaqMan probes on faecal matter and mixed effluent slurries revealed no cross-reactions between species of interest and other vertebrate faecal matter. Performed as a single blind experiment we were able to correctly identify faecal material in 17/20 effluents (85% correct). mtDNA degrades relatively quickly in faecally-spiked water samples (approximately 2 weeks), a similar timeframe of environmental persistence to several bacterial faecal indictors, highlighting its applicability. The procedure described here is specific, rapid (<5 hours) and sensitive. These results confirm the suitability of using species-specific mtDNA as an indicator in source tracking studies in surface waters, shellfish harvesting areas and shellfish matrices.

Strategies to reduce norovirus (NoV) contamination from oysters under depuration conditions.

Depuration of oysters can effectively reduce levels of E. coli, however, may not be effective in safeguarding against viral contamination (EFSA, 2012). These trials assess the removal of Norovirus Genogroups I and II (NoV GI and GII) and F + RNA bacteriophage genogroup II (FRNAP-II) from oysters under depuration using molecular and viability assay methods. Our results show consistently better removal of NoV GII compared with Nov GI. We found approximately 46% removal of NoV GII at 18 °C after 2 days and 60% after 5 days compared with a maximum of 16% NoV GI removal. Twice the rate of NoV GII removal was achieved at 18 °C compared with 8 °C after 5 days. Results suggest better NoV removal when depuration water salinity is close to that prevailing in the harvesting area. Trials investigating algal feeding, light/dark and disturbance from pump vibration did not show any significant effect. We found that FRNAP-II was more readily removed than NoV. No significant difference was found between the rate of removal (as measured by RT-qPCR) and inactivation (as measured by bioassay) of FRNAP-II. This indicates that reduction in FRNAP-II may be primarily due to physical removal (or destruction) rather than in situ inactivation of the virus.

Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach.

Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family.

Development of a TaqMan qPCR assay for detection of Alexandrium spp and application to harmful algal bloom monitoring.

The Genus Alexandrium is a widespread dinoflagellate marine phytoplankton that is the primary causative organism causing Paralytic Shellfish Poisoning (PSP) intoxications in European waters. EU food safety directives specify that EU Member States must implement a routine monitoring programme to mitigate risks associated with bio-accumulation of biotoxins by bivalve shellfish, such as those produced by Alexandrium. This strategic drive comprises of both direct testing of bivalve flesh for the presence of regulated toxins and an early warning phytoplankton monitoring programme. In the UK the flesh testing moved away from animal bio-assays to analytical chemistry techniques, whereas phytoplankton monitoring methods have seen little technological advancement since implementation. Methods currently utilize light microscopy and manual enumeration of different algal species. These methods although proven are time consuming, reliant on highly trained staff, have high limits of detection (LOD) with low specificity, unable to reliably identify Alexandrium to species level. The implications of these limitations of the techniques mean that in the case of Alexandrium the LOD is also the action limit and as such it is easy to miss positive samples affecting the efficacy of any early warning strategy. This study outlines the development, preliminary method characterisation, validation and trial implementation of an alternative early warning technique, utilizing quantitative PCR to identify water samples containing Alexandrium cells. The approach outlined in this document, showed an improved correlation with flesh toxicity, improved sensitivity, improved throughput compared to traditional light microscopy methods and there was also good correlation with higher cell abundance samples when compared to the light microscopy results. The application of this approach to routine water samples was explored and was found to demonstrate potential as a corroborative method for use during flesh intoxication episodes. This study offers potential for future improvements in the accuracy and sensitivity of phytoplankton monitoring whilst ensuring continuity of public safety, providing cost savings and offering new research opportunities.

Assessment of the Applicability of Capsid-Integrity Assays for Detecting Infectious Norovirus Inactivated by Heat or UV Irradiation.

Human noroviruses are the leading cause of viral gastroenteritis. In the absence of a practical culture technique for routine analysis of infectious noroviruses, several methods have been developed to discriminate between infectious and non-infectious viruses by removing non-viable viruses prior to analysis by RT-qPCR. In this study, two such methods (RNase and porcine gastric mucin) which were designed to remove viruses with compromised capsids (and therefore assumed to be non-viable), were assessed for their ability to quantify viable F-specific RNA bacteriophage (FRNAP) and human norovirus following inactivation by UV-C or heat. It was found that while both methods could remove a proportion of non-viable viruses, a large proportion of non-viable virus remained to be detected by RT-qPCR, leading to overestimations of the viable population. A model was then developed to determine the proportion of RT-qPCR detectable RNA from non-viable viruses that must be removed by such methods to reduce overestimation to acceptable levels. In most cases, nearly all non-viable virus must be removed to reduce the log overestimation of viability to within levels that might be considered acceptable (e.g. below 0.5 log10). This model could be applied when developing alternative pre-treatment methods to determine how well they should perform to be comparable to established infectivity assays.

Determination of norovirus contamination in oysters from two commercial harvesting areas over an extended period, using semiquantitative real-time reverse transcription PCR.

The human health risk associated with the consumption of molluscan shellfish grown in sewage-contaminated waters is well established. Noroviruses, which cause gastroenteritis, are the principal agents of shellfish-related illness. Fecal-indicator quality standards based on Escherichia coli are well established in Europe and elsewhere. However, norovirus outbreaks after consumption of shellfish meeting these standards still occur, and the need to improve consumer health protection is well recognized. Alternative approaches proposed include direct monitoring of viral pathogens and the use of alternative indicator organisms capable of providing a better indication of virus risk. This study applies a recently developed TaqMan PCR assay to assess norovirus contamination in shellfish. Comparison was made with E. coli as the existing sanitary standard and a male-specific RNA bacteriophage as a possible alternative. Two commercial pacific oyster (Crassostrea gigas) harvesting areas were monitored over a 31-month period. The results show peaks of norovirus contamination in both areas during winter months, with average levels approximately 17 times higher in oysters sampled October to March than during the remainder of the year, consistent with epidemiological data for the United Kingdom showing oyster-associated illness is confined to winter months. While there was no apparent association with E. coli, an association between levels of norovirus contamination and the male-specific RNA bacteriophage was noted, with average norovirus levels over 40 times higher in samples with male-specific RNA bacteriophage counts of >1,000 PFU/100 g than in samples with <100 PFU/100 g. Overall, these results suggest that norovirus monitoring in shellfish production areas could be an effective strategy for reduction of virus risk.

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.

A model for estimating pathogen variability in shellfish and predicting minimum depuration times.

Norovirus is a major cause of viral gastroenteritis, with shellfish consumption being identified as one potential norovirus entry point into the human population. Minimising shellfish norovirus levels is therefore important for both the consumer's protection and the shellfish industry's reputation. One method used to reduce microbiological risks in shellfish is depuration; however, this process also presents additional costs to industry. Providing a mechanism to estimate norovirus levels during depuration would therefore be useful to stakeholders. This paper presents a mathematical model of the depuration process and its impact on norovirus levels found in shellfish. Two fundamental stages of norovirus depuration are considered: (i) the initial distribution of norovirus loads within a shellfish population and (ii) the way in which the initial norovirus loads evolve during depuration. Realistic assumptions are made about the dynamics of norovirus during depuration, and mathematical descriptions of both stages are derived and combined into a single model. Parameters to describe the depuration effect and norovirus load values are derived from existing norovirus data obtained from U.K. harvest sites. However, obtaining population estimates of norovirus variability is time-consuming and expensive; this model addresses the issue by assuming a 'worst case scenario' for variability of pathogens, which is independent of mean pathogen levels. The model is then used to predict minimum depuration times required to achieve norovirus levels which fall within possible risk management levels, as well as predictions of minimum depuration times for other water-borne pathogens found in shellfish. Times for Escherichia coli predicted by the model all fall within the minimum 42 hours required for class B harvest sites, whereas minimum depuration times for norovirus and FRNA+ bacteriophage are substantially longer. Thus this study provides relevant information and tools to assist norovirus risk managers with future control strategies.

Use of Mytilus edulis biosentinels to investigate spatial patterns of norovirus and faecal indicator organism contamination around coastal sewage discharges.

Bivalve shellfish have the capacity to accumulate norovirus (NoV) from waters contaminated with human sewage. Consequently, shellfish represent a major vector for NoV entry into the human food chain, leading to gastrointestinal illness. Identification of areas suitable for the safe cultivation of shellfish requires an understanding of NoV behaviour upon discharge of municipal-derived sewage into coastal waters. This study exploited the potential of edible mussels (Mytilus edulis) to accumulate NoV and employed the ISO method for quantification of NoV within mussel digestive tissues. To evaluate the spatial spread of NoV from an offshore sewage discharge pipe, mesh cages of mussels were suspended from moorings deployed in a 9 km2 grid array around the outfall. Caged mussels were retrieved after 30 days and NoV (GI and GII), total coliforms and E. coli enumerated. The experimentally-derived levels of NoV GI and GII in mussels were similar with total NoV levels ranging from 7 × 101 to 1.6 × 104 genome copies g-1 shellfish digestive gland (ΣGI + GII). NoV spread from the outfall showed a distinct plume which matched very closely to predictions from the tidally-driven effluent dispersal model MIKE21. A contrasting spatial pattern was observed for coliforms (range 1.7 × 102 to 2.1 × 104 CFU 100 g-1 shellfish tissue) and E. coli (range 0-1.2 × 103 CFU 100 g-1 shellfish tissue). These data demonstrate that hydrodynamic models may help inform effective exclusion zones for bivalve harvesting, whilst coliform/E. coli concentrations do not accurately reflect viral dispersal in marine waters and contamination of shellfish by sewage-derived viral pathogens.