Generation of False-Positive SARS-CoV-2 Antigen Results with Testing Conditions outside Manufacturer Recommendations: A Scientific Approach to Pandemic Misinformation.

Antigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. However, misleading demonstrations of the Abbott Panbio coronavirus disease 2019 (COVID-19) Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside manufacturer recommendations. Using Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and Tween 20) as well as the impact of temperature (4°C, 20°C, and 45°C) and humidity (90%) on assay performance. Direct sample testing (without the kit buffer) resulted in false-positive signals resembling those obtained with SARS-CoV-2 positive controls tested under proper conditions. The likely explanation of these artifacts is nonspecific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside manufacturer claims. Our data support strict adherence to manufacturer instructions to avoid false-positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation. IMPORTANCE With the Panbio severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test being deployed in over 120 countries worldwide, understanding conditions required for its ideal performance is critical. Recently on social media, this kit was shown to generate false positives when manufacturer recommendations were not followed. While erroneous results from improper use of a test may not be surprising to some health care professionals, understanding why false positives occur can help reduce the propagation of misinformation and provide a scientific rebuttal for these aberrant findings. This study demonstrated that the kit buffer's pH, ionic strength, and buffering capacity were critical components to ensure proper kit function and avoid generation of false-positive results. Typically, false positives arise from cross-reacting or interfering substances; however, this study demonstrated a mechanism where false positives were generated under conditions favoring nonspecific interactions between the two antibodies designed for SARS-CoV-2 antigen detection. Following the manufacturer instructions is critical for accurate test results.

Gut Susceptibility to Viral Invasion: Contributing Roles of Diet, Microbiota and Enteric Nervous System to Mucosal Barrier Preservation.

The gastrointestinal lumen is a rich source of eukaryotic and prokaryotic viruses which, together with bacteria, fungi and other microorganisms comprise the gut microbiota. Pathogenic viruses inhabiting this niche have the potential to induce local as well as systemic complications; among them, the viral ability to disrupt the mucosal barrier is one mechanism associated with the promotion of diarrhea and tissue invasion. This review gathers recent evidence showing the contributing effects of diet, gut microbiota and the enteric nervous system to either support or impair the mucosal barrier in the context of viral attack.

Food Safety in Post-COVID-19 Pandemic: Challenges and Countermeasures.

Understanding food safety hazard risks is essential to avoid potential negative heath impacts in the food supply chain in a post-COVID-19 pandemic era. Development of strategies for virus direction in foods plays an important role in food safety and verification. Early warning, tracing, and detection should be implemented as an integrated system in order to mitigate thecoronavirus disease 2019 (COVID-19) outbreak, in which the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is critical as it not only concerns screening of populations but also monitoring of possible contaminated sources such as the food supply chain. In this review, we point out the consequences in different aspects of our daily life in the post-COVID-19 pandemic from the perspective of the food supply chain and the food industry. We summarize the possible transmission routes of COVID-19 in the food supply chain before exploring the development of corresponding detection tools of SARS-CoV-2. Accordingly, we compare different detection methods for the virus in foods, including different pretreatments of food matrices in the virus detection. Finally, the future perspectives are proposed.

Bacteriophages as antibiotic resistance genes carriers in agro-food systems.

Antibiotic resistance genes (ARGs) are a global health concern. Antibiotic resistance occurs naturally, but misuse of antibiotics in humans and animals is accelerating the process of antibiotic resistance emergency, which has been aggravated by exposure to molecules of antibiotics present in clinical and agricultural settings and the engagement of many countries in water reuse especially in Middle East and North Africa region. Bacteriophages have the potential to be significant actors in ARGs transmission through the transduction process. These viruses have been detected along with ARGs in non impacted habitats and in anthropogenic impacted environments like wastewater, reclaimed water and manure amended soil as well as minimally processed food and ready to eat vegetables. The ubiquity of bacteriophages and their persistence in the environment raises concern about their involvement in ARGs transmission among different biomes and the generation of pathogenic-resistant bacteria that pose a great threat to human health. The aim of this review is to give an overview of the potential role of bacteriophages in the dissemination and the transfer of ARGs to pathogens in food production and processing and the consequent contribution to antibiotic resistance transmission through faecal oral route carrying ARGs to our dishes.

Disentangling host-parasite-pathogen interactions in a varroa-resistant honeybee population reveals virus tolerance as an independent, naturally adapted survival mechanism.

The ectoparasitic mite, Varroa destructor, is unarguably the leading cause of honeybee (Apis mellifera) mortality worldwide through its role as a vector for lethal viruses, in particular, strains of the Deformed wing virus (DWV) and Acute bee paralysis virus (ABPV) complexes. This multi-level system of host-parasite-pathogen interactions makes it difficult to investigate effects of either the mite or the virus on natural host survival. The aim of this study was to remove confounding effects of varroa to examine the role of virus susceptibility in the enhanced survival of a naturally adapted Swedish mite-resistant (MR) honeybee population, relative to mite-susceptible (MS) honeybees. Caged adult bees and laboratory reared larvae, from varroa-free colonies, were inoculated with DWV and ABPV in a series of feeding infection experiments, while control groups received virus-free food. Virus infections were monitored using RT-qPCR assays in individuals sampled over a time course. In both adults and larvae the DWV and ABPV infection dynamics were nearly identical between MR and MS groups, but MS adults suffered significantly higher mortality than MR adults. Results suggest virus tolerance, rather than reduced susceptibility or virus resistance, is an important component of the natural survival of this honeybee population.

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.

HEVnet: a One Health, collaborative, interdisciplinary network and sequence data repository for enhanced hepatitis E virus molecular typing, characterisation and epidemiological investigations.

Hepatitis E virus (HEV) is a common cause of acute hepatitis worldwide. In Europe, HEV is a zoonosis transmitted via contaminated pork meat or other pork food products. Genotype 3 is the most prevalent HEV type in the animal reservoir, as well as in humans. Despite an increased incidence of hepatitis E across Europe, much remains unknown about its spread, sources and transmission routes. A One Health approach is crucial to better understand the (molecular) epidemiology of HEV. HEVnet was established in April 2017 as a network and database for sharing sequences and accompanying metadata collected from human, animal, food and environmental sources. HEVnet members working in the public health, veterinary health, food, environmental and blood safety sectors have submitted 1,615 HEV sequences from nine countries as at January 2019. Most are from humans (89%), and sequences of animal (5%), food (6%) or environmental (0.3%) origin are rare. Metadata for human sequences capture mostly sex (93%), year of birth (92%) and sampling (100%); data on region of sampling (37%) and clinical information (hospitalisation 27%, symptoms 20% or mortality 8%) are limited. HEVnet aims to expand into a global network capable of performing cross-sectoral and supranational studies, with a joint repository of molecular and epidemiological data on HEV.

Role and regulation of the stress activated sigma factor sigma B (σB) in the saprophytic and host-associated life stages of Listeria monocytogenes.

The stress activated sigma factor sigma B (σB) plays a pivotal role in allowing the food-borne bacterial pathogen Listeria monocytogenes to modulate its transcriptional landscape in order to survive in a variety of harsh environments both outside and within the host. While we have a comparatively good understanding of the systems under the control of this sigma factor much less is known about how the activity of σB is controlled. In this review, we present a current model describing how this sigma factor is thought to be controlled including an overview of what is known about stress sensing and the early signal transduction events that trigger its activation. We discuss the known regulatory overlaps between σB and other protein and RNA regulators in the cell. Finally, we describe the role of σB in surviving both saprophytic and host-associated stresses. The complexity of the regulation of this sigma factor reflects the significant role that it plays in the persistence of this important pathogen in the natural environment, the food chain as well as within the host during the early stages of an infection. Understanding its regulation will be a critical step in helping to develop rational strategies to prevent its growth and survival in the food destined for human consumption and in the prevention of listeriosis.

Virus-Bacteria Interactions: Implications for Prevention and Control of Human Enteric Viruses from Environment to Host.

Human enteric viruses, specifically human norovirus (hNoV), are the most common cause of foodborne illness boasting a wide range of transmission routes. These include person to person, contact with contaminated fomites, as well as ingestion of contaminated water and food. Because of this, the control and prevention of enteric viruses in food and other relevant environments have been a research focus over the past few decades. Interestingly, viruses as well as many other pathogens are often studied in isolation even though it is known that microorganisms do not occur in isolation but rather as part of complex microbial communities-both external from the host and within the host. Therefore, the overall goal of this review is to present the current evidence on virus-microbe interactions as these relate to the infectivity as well as the control and prevention of epidemiologically relevant foodborne viruses (such as hNoV) within our food systems. Therefore, this review is divided into in vivo, in situ, and in vitro implications of virus-microbe interactions through discussion of studies investigating the complex relationships between human enteric viruses and microbial cohabitants, specifically hNoV and bacteria.

The qualified presumption of safety assessment and its role in EFSA risk evaluations: 15 years past.

Microorganisms are intentionally added at different stages of the food and feed chain (food or feed additive, novel food or plant protection product) and are subjected to regulation and safety assessment by the European Food Safety Authority. Safety evaluation is based on application dossiers for market authorisation to the European Commission. The qualified presumption of safety (QPS) concept was developed in 20031 to provide a harmonised generic safety pre-appraisal of the above microorganisms. Unambiguously defined biological taxonomic units (TUs) are assessed for their body of knowledge, their safety and their end use. Identified safety concerns for a certain TU can be, where reasonable in number and not universally present, reflected as 'qualifications.' Strains belonging to TUs having QPS status may benefit of a fast track evaluation. The lowest TU for which the QPS status is granted is the species level for bacteria and yeasts and the family for viruses. The QPS concept is also applicable to genetically modified microorganisms used for production purposes. Based on the current body of knowledge and/or the ambiguous taxonomic position, some TUs, such as filamentous fungi, bacteriophages, Enterococcus faecium, Escherichia coli, Streptomyces spp. and Oomycetes, are not considered liable for QPS status.

Hepatitis E infection in stem cell and solid organ transplantpatients: A cross-sectional study: The importance of HEV RNA screening in peri-transplant period.

BACKGROUND: Hepatitis E Virus (HEV) is a common cause of acute viral hepatitis worldwide. Typically associated with a self-limiting illness, infection may persist in immunosuppressed populations with significant morbidity and mortality. Based on clinical data published world-wide, UK blood safety guidance recommends the universal screening for HEV RNA of blood donors and donors of tissue, organs and stem cells. OBJECTIVES: This cross-sectional study aimed to determine the point prevalence of HEV viraemia and clinical course of viraemic patients in the peri-transplant period in solid organ transplant (SOT) and haematopoietic stem cell transplant (HSCT) recipients transplanted over a 3-year period (2013-2015). STUDY DESIGN: Nucleic acid extracts of whole blood from patients undergoing SOT or HSCT were tested by an in-house real-time reverse-transcriptase polymerase chain reaction assay for HEV RNA. Samples were tested at baseline (time of transplant), 30, 60 and 90 days post-transplant. RESULTS: 870 patients (259 HSCT, 262 liver and 349 kidney transplant) were included with 2554 samples meeting the inclusion criteria. No kidney transplant patients had HEV viraemia at time of testing. One HSCT and three liver transplant patients were found to be HEV RNA positive. Overall this represented 0.46% of the patients testing positive for HEV viraemia. CONCLUSIONS: Prevalence of HEV viraemia in SOT and HSCT patients in U.K. although higher than in the general population is low at baseline and remains low throughout the early post-transplant phase. Clearance of viraemia can be maintained despite ongoing immunosuppression. Prospective U.K. studies are necessary to inform screening policies in this population.

Foodborne viruses: Detection, risk assessment, and control options in food processing.

In a recent report by risk assessment experts on the identification of food safety priorities using the Delphi technique, foodborne viruses were recognized among the top rated food safety priorities and have become a greater concern to the food industry over the past few years. Food safety experts agreed that control measures for viruses throughout the food chain are required. However, much still needs to be understood with regard to the effectiveness of these controls and how to properly validate their performance, whether it is personal hygiene of food handlers or the effects of processing of at risk foods or the interpretation and action required on positive virus test result. This manuscript provides a description of foodborne viruses and their characteristics, their responses to stress and technologies developed for viral detection and control. In addition, the gaps in knowledge and understanding, and future perspectives on the application of viral detection and control strategies for the food industry, along with suggestions on how the food industry could implement effective control strategies for viruses in foods. The current state of the science on epidemiology, public health burden, risk assessment and management options for viruses in food processing environments will be highlighted in this review.

The influence of food matrices on aptamer selection by SELEX (systematic evolution of ligands by exponential enrichment) targeting the norovirus P-Domain.

This study investigates the enrichment of aptamers targeting the norovirus protruding domain in the presence of foods often associated with norovirus outbreaks. The goal is to explore if and how the presence of food alters in vitro selection of aptamers and target binding of the enriched oligonucleotides. Our study demonstrates that the introduction of food to SELEX (systematic evolution of ligands by exponential enrichment) is either detrimental to enrichment of oligonucleotides with target-specific binding, or facilitates enrichment of non-target-specific oligonucleotides. Moreover, a relationship between target binding of enriched oligonucleotides in presence of food and their selection condition was not observed. Our findings also suggest that a pathogen specific aptamer with application in food does not need to be selected in presence of the particular food, but may require properties beyond high affinity and selectivity to be applied for pathogen extraction and detection in undiluted food matrices.

Rapid and label-free electrochemical DNA biosensor for detecting hepatitis A virus.

Diagnostic systems that can deliver highly specific and sensitive detection of hepatitis A virus (HAV) in food and water are of particular interest in many fields including food safety, biosecurity and control of outbreaks. Our aim was the development of an electrochemical method based on DNA hybridization to detect HAV. A ssDNA probe specific for HAV (capture probe) was designed and tested on DNAs from various viral and bacterial samples using Nested-Reverse Transcription Polymerase Chain Reaction (nRT-PCR). To develop the electrochemical device, a disposable gold electrode was functionalized with the specific capture probe and tested on complementary ssDNA and on HAV cDNA. The DNA hybridization on the electrode was measured through the monitoring of the oxidative peak potential of the indicator tripropylamine by cyclic voltammetry. To prevent non-specific binding the gold surface was treated with 3% BSA before detection. High resolution atomic force microscopy (AFM) confirmed the efficiency of electrode functionalization and on-electrode hybridization. The proposed device showed a limit of detection of 0.65pM for the complementary ssDNA and 6.94fg/µL for viral cDNA. For a comparison, nRT-PCR quantified the target HAV cDNA with a limit of detection of 6.4fg/µL. The DNA-sensor developed can be adapted to a portable format to be adopted as an easy-to- use and low cost method for screening HAV in contaminated food and water. In addition, it can be useful for rapid control of HAV infections as it takes only a few minutes to provide the results.

Recent knowledge on hepatitis E virus in Suidae reservoirs and transmission routes to human.

Hepatitis E virus (HEV) causes self-limiting acute hepatitis in humans that can eventually result in acute liver failures or progress to chronic infections. While in tropical and sub-tropical areas, HEV infections are associated with important waterborne epidemics, in Northern countries, HEV infections are autochthonous with a zoonotic origin. In the past decade, it has become clear that certain HEV genotypes are zoonotic and that swine, and more generally Suidae, are the main reservoir. Zoonotic transmissions of the virus may occur via direct contact with infected pigs, wild boars or consumption of contaminated meat. This review describes the current knowledge on domestic and wild Suidae as reservoirs of HEV and the evidence of the different routes of HEV transmission between these animals and humans.

Recent Trends in Salmonella Outbreaks and Emerging Technology for Biocontrol of Salmonella Using Phages in Foods: A Review.

Salmonella is one of the principal causes of foodborne outbreaks. As traditional control methods have shown less efficacy against emerging Salmonella serotypes or antimicrobialresistant Salmonella, new approaches have been attempted. The use of lytic phages for the biocontrol of Salmonella in the food industry has become an attractive method owing to the many advantages offered by the use of phages as biocontrol agents. Phages are natural alternatives to traditional antimicrobial agents; they have proven effective in the control of bacterial pathogens in the food industry, which has led to the development of different phage products. The treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases, and ultimately promotes safe environments for animal and plant food production, processing, and handling. After an extensive investigation of the current literature, this review focuses predominantly on the efficacy of phages for the successful control of Salmonella spp. in foods. This review also addresses the current knowledge on the pathogenic characteristics of Salmonella, the prevalence of emerging Salmonella outbreaks, the isolation and characterization of Salmonella-specific phages, the effectiveness of Salmonella-specific phages as biocontrol agents, and the prospective use of Salmonella-specific phages in the food industry.

A metagenomic assessment of viral contamination on fresh parsley plants irrigated with fecally tainted river water.

Microbial food-borne diseases are still frequently reported despite the implementation of microbial quality legislation to improve food safety. Among all the microbial agents, viruses are the most important causative agents of food-borne outbreaks. The development and application of a new generation of sequencing techniques to test for viral contaminants in fresh produce is an unexplored field that allows for the study of the viral populations that might be transmitted by the fecal-oral route through the consumption of contaminated food. To advance this promising field, parsley was planted and grown under controlled conditions and irrigated using contaminated river water. Viruses polluting the irrigation water and the parsley leaves were studied by using metagenomics. To address possible contamination due to sample manipulation, library preparation, and other sources, parsley plants irrigated with nutritive solution were used as a negative control. In parallel, viruses present in the river water used for plant irrigation were analyzed using the same methodology. It was possible to assign viral taxons from 2.4 to 74.88% of the total reads sequenced depending on the sample. Most of the viral reads detected in the river water were related to the plant viral families Tymoviridae (66.13%) and Virgaviridae (14.45%) and the phage viral families Myoviridae (5.70%), Siphoviridae (5.06%), and Microviridae (2.89%). Less than 1% of the viral reads were related to viral families that infect humans, including members of the Adenoviridae, Reoviridae, Picornaviridae and Astroviridae families. On the surface of the parsley plants, most of the viral reads that were detected were assigned to the Dicistroviridae family (41.52%). Sequences related to important viral pathogens, such as the hepatitis E virus, several picornaviruses from species A and B as well as human sapoviruses and GIV noroviruses were detected. The high diversity of viral sequences found in the parsley plants suggests that irrigation on fecally-tainted food may have a role in the transmission of a wide diversity of viral families. This finding reinforces the idea that the best way to avoid food-borne viral diseases is to introduce good field irrigation and production practices. New strains have been identified that are related to the Picornaviridae and distantly related to the Hepeviridae family. However, the detection of a viral genome alone does not necessarily indicate there is a risk of infection or disease development. Thus, further investigation is crucial for correlating the detection of viral metagenomes in samples with the risk of infection. There is also an urgent need to develop new methods to improve the sensitivity of current Next Generation Sequencing (NGS) techniques in the food safety area.

Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples.

The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA- and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method.IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples.

Isolation and Characterization of Bacillus cereus Bacteriophages from Foods and Soil.

The aim of this study was to isolate and characterize Bacillus cereus bacteriophages of various origins. Twenty-seven bacteriophages against B. cereus were isolated from various Korean traditional fermented foods and soils. Plaque size, transmission electron microscopy, virulence profile, and in vitro lytic activity of bacteriophage isolates were examined. Transmission electron microscopy confirmed B. cereus bacteriophages belonging to the family Siphoviridae. Among B. cereus bacteriophages with broad host range, 18 isolates (66.7%) did not harbor any B. cereus virulence factors. Among them, bacteriophage strain CAU150036, CAU150038, CAU150058, CAU150064, CAU150065, and CAU150066 effectively inhibited B. cereus in vitro within 1 h. Therefore, they are considered potential candidates for controlling the contamination of B. cereus in food or other applications.

Human norovirus transmission and evolution in a changing world.

Norovirus infections are a major cause of gastroenteritis, and outbreaks occur frequently. Several factors are currently increasing the challenge posed by norovirus infections to global health, notably the increasing number of infections in immunocompromised individuals, who are more susceptible to disease, and the globalization of the food industry, which enables large norovirus outbreaks to occur on an international scale. Furthermore, the rapid rate of the genetic and antigenic evolution of circulating noroviruses complicates the development of vaccines and therapies that are required to counter these challenges. In this Review, we describe recent advances in the study of the transmission, pathogenesis and evolution of human noroviruses, and consider the ongoing risk of norovirus outbreaks, together with the future prospects for therapeutics, in a rapidly changing world.