The efficacy of EO waters on inactivating norovirus and hepatitis A virus in the presence of organic matter

The ability of acidic electrolyzed oxidizing water (AEO) and neutral electrolyzed oxidizing water (NEO) to inactivate the murine norovirus (MNV-1) surrogate for human norovirus and hepatitis A virus (HAV) in suspension and on stainless steel coupons in the presence of organic matter was investigated. Viruses containing tryptone (0.0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) were mixed with AEO and NEO for 1 min. In addition, stainless steel coupons containing MNV-1 with or without organic matter were treated with AEO or NEO for 3, 5, and 10 min. AEO was proven effective and generally killed more MNV-1 and HAV in suspension than NEO. Depending on the EO water generator, free chlorine concentrations are required to inactivate MNV-1 and HAV by 3-log PFU/mL or greater ranged from 30 mg/L to 40 mg/L after a 1 min contact time. The virucidal effect increased with increasing free chlorine concentration and decreased with increasing tryptone concentration in suspension. Both AEO and NEO at 70–100 mg/L of free chlorine concentration significantly reduced MNV-1 on coupons in the absence of organic matter. However, there was no significant difference between these two treatments in the presence of organic matter. In addition, the efficacy of these two EO waters on stainless steel coupons increased with the increasing treatment time. Results indicated that AEO and NEO can reduce MNV-1 and HAV in suspension. However, higher free chlorine concentrations and longer treatment times may be necessary to reduce viruses on contact surfaces or in the presence of organic matter.     

Estimated exposure to hepatitis E virus through consumption of swine liver and liver sausages

A quantitative risk assessment was undertaken following the Codex Alimentarius principles in order to predict the exposure of consumers to hepatitis E virus (HEV) through food consumption. Taking into account the tropism of HEV, fresh liver and liver sausages were regarded as having a higher risk of contamination. The model entailed a hypothetical food pathway and was based on worst case scenario where the intake of contaminated food derived from a 100% HEV-infected pig population was estimated. As no data on the prevalence of infectious HEV was available, the HEV-RNA prevalence in food matrices and the seroprevalence of HEV-specific antibodies in swine were assessed and adjusted for diagnostic misclassification and sampling uncertainty. Considering a HEV prevalence of 100% in pigs and excluding further cross-contamination events, a food portion consisting of 130 gr of liver or of 32.5 gr of sausage (containing 30% of liver) yielded an estimated exposure of 8047 and 210 RNA copies (median values), respectively. These findings take into account the effect of thermal treatment on the HEV-RNA concentration of food. Due to the lack of information concerning the correlation between HEV-RNA concentration and the amount of infectious virus as well as the dose-response relationship of HEV, the calculated RNA copies do not allow direct conclusions to be drawn on the risk of infection following ingestion of these food types. The true prevalence was estimated for Switzerland and Germany, leading to an overall prevalence of HEV-RNA in food of 6.2% (90% Highest Density Intervals (HDIs): 2.5%–11.2%). In comparison with fresh liver, liver sausages showed a higher prevalence, most likely due to the presence of more than one liver within the same sausage. The true prevalence of anti-HEV IgG ranged between 59.4% (HDIs 56.5%–62.4%) and 62.6% (HDIs 58.8%–64.3%) and between 7.6% (HDIs 3.3%–13.2%) and 30.5% (HDIs 23.2%–38.2%) in pigs and wild boars, respectively. The high prevalence of antibodies support the evidence that these animals can act as reservoirs for HEV and can contribute epidemiologically to the maintenance of the virus in the surroundings. This study is a preliminary investigation and highlights the major existing gaps needed to be filled in order to enable a refined HEV risk assessment that can drive future decisions for the implementation of food safety and of control measures.     

Contamination of mussels by hepatitis A virus: a public-health problem in southern Italy

The frequency of hepatitis A virus (HAV) contamination in mussels sold in south of Italy, where a high incidence of HAV infection both in resident people and in travelers is reported every year, was investigated during a three-year period.

Mussels, collected from the markets of five big cities, were analysed by RT-nested-PCR to detect RNA-HAV and by an integrated method, cell culture-RT-PCR, to confirm the presence of infectious virus. On a total of 180 samples, 15.6% resulted contaminated by infectious HAV.

The high percentage of mussels, potentially dangerous for consumers, and the diffuse habit to consume them raw or slightly cooked, can contribute to maintain the endemic status of HAV infection in some areas of the south of Italy.     

Reported behavior,knowledge and awareness toward the potential for norovirus transmission by food handlers in Dutch catering companies and institutional settings in relation to the prevalence of norovirus

Norovirus (NoV) in ready to eat food has recently been defined as one of the virus-food commodity combinations with greatest public health concern. The role of food handlers therein has well been recognized. The aim of this study was to identify gaps in food handlers' education and to investigate possible associations between reported behavior, knowledge and awareness of NoV, and environmental presence of NoV. For this, face-to-face interviews were conducted using structured questionnaires in 1023 catering companies (i.e. restaurants mainly), 101 non-hospital health care centers, 52 hospital central kitchens and in 102 hospital in-patient units. In addition, three surface swabs were taken at each setting. Multivariate logistic regression was performed on data restricted to NoV high season months only, in which NoV was present in 21/374 (6%) catering companies and 37/233 (16%) institutional settings (p < 0.01). The two independent determinants of presence of NoV on environmental surfaces identified were being situated in an institutional setting and having an attitude to continue food handling while sick with vomiting complaints. Several gaps in education and training were identified, demonstrating that knowledge on NoV was low, although awareness of NoV was significantly higher among food handlers in institutional settings than in catering companies. This is the first time questionnaires and environmental testing have been combined in the same study to identify issues of improvement. Training on all important aspects of NoV according to the recently developed Codex Alimentarius guidelines to control viruses in food is strongly recommended.     

Detection of Norovirus and Feline Calicivirus in spiked molluscs subjected to heat treatments

Aim of the study was the evaluation of the effect of heat treatment in shellfish experimentally contaminated with human Norovirus (NoV). Feline Calicivirus (FCV), often used as surrogate for human NoV, was examined in parallel to test for virus infectivity after treatment. The experiments were performed subjecting suspensions and spiked mussels to heat treatment (60 °C and 80 °C) for various times. Analysis by rRT-PCR showed a limited reduction of NoV (less than 1 log RT-PCR units ml⁻¹) and FCV (0.3-1.2 log TCID₅₀ ml⁻¹) both in virus suspensions and in spiked mussels, with the exception of NoV suspensions treated at 80 °C (reduction of 3 log RT-PCR units ml⁻¹). Cell culture assay showed that the infectious FCV in suspensions decreased of 4 log after 3 min of treatment while a lower reduction (2 log) was obtained in spiked samples treated for 15 min. Data showed that mussel matrix plays a protective role against heat inactivation of viruses. Despite the efficacy of rRT-PCR for viral nucleic acid detection, its inability to provide information on virus infectivity, represents a limit to the evaluation of product safety. Caution should therefore be used in the evaluation of efficacy of heat treatments on virus-contaminated foods and when the judgment of food product safety is based exclusively on rRT-PCR results.     

A molecular survey of farmed and edible snails for the presence of human enteric viruses: Tracking of the possible environmental sources of microbial mollusc contamination

Snails have been grown or harvested for human consumption for centuries. Similarly to tissues or meat of other food animals, snail meat should be free from microbiological hazards including viruses. The aim of the study was to investigate the presence of human enteric viruses (hepatitis A and E virus, norovirus genogroup I, II and human adenovirus) in farmed and free-living snails intended for human consumption. In order to find the possible sources of snail contamination with viruses, the quality management system operating in the most widely-adopted snail farming system in Poland was reviewed. Additionally, the process hygiene criteria were checked through testing of snail meat and soil samples for bacterial contamination. Snails of genus Cornu and Helix were collected from two heliciculture farms and a purchase point. They were subjected to bacteriological (n = 180) and viral examination (n = 60). In the case of soil samples (n = 8) they were taken from different areas along the breeding cycle of the snails. For virus detection several real-time PCR protocols with an incorporated target-specific internal amplification control (IAC) were used. Determinations of the total aerobic bacteria count (TBC), number of Enterobacteriaceae (ENC) and enterococci (ECC) were performed using the international reference methods. Human enteric viruses were not detected in any of the tested snail samples. TBC for raw snail meat ranged from 5.1 up to 6.6 log cfu/g and ENC values did not exceed 4.8 log cfu/g. The ECC counts were below 2.0 log cfu/g. The overall microbiological status of snail meat indicated good hygiene and appropriate environmental conditions being maintained during snail breeding. TBC for soil samples was in the range of 6.0–7.7 log cfu/g indicating a low or moderate level of contamination. Only the soil samples from the plots used at the fattening stage were characterised by high ECC content increasing the risk of virus occurrence. In contrary to the sporadic finding of ECC, the ubiquitous presence of bacteria from the Enterobacteriaceae family in all types of soil samples was shown. A good microbial quality of snail meat resulted from the implemented GMP/GHP programmes and food safety management practices. This strategy appeared sufficient in reduction of microbiological risk and in prevention of virus contamination in snails. The study showed that the risk of foodborne virus infection due to consumption of snails and related products is considered negligible.     

Environmental testing for norovirus in various institutional settings using catering companies as sentinels for norovirus prevalence among the general population

Noroviruses (NoV) are among the most common causes of viral gastroenteritis (GE) worldwide and can be transmitted from person-to-person, via food or contaminated surfaces. The present study aimed to examine the prevalence of NoV RNA on surfaces in food preparation and sanitary areas in different health care settings and to compare the outcomes with the prevalence in nearby located catering companies, mainly restaurants, for general public, as sentinels.For this purpose, 1087 environmental swabs were taken for NoV analyses from surfaces in 241 institutional departments and 123 catering companies in The Netherlands without a recently reported outbreak of gastro-enteritis in high NoV season only. NoV RNA was detected in 15.1% of the 73 non-hospital health care institutions, 11.1% of the 54 hospital central kitchen departments, 14.9% of the 114 decentralized hospital kitchens (in-patient units) and 4.1% of the 123 nearby located catering companies. Twenty-five of the 49 positive environmental samples were genotyped by sequence analyses. In 7% of the investigated hospitals (4/58), NoV was detected in two or more departments. NoV prevalence was significantly lower in food preparation areas than in sanitary facilities (p < 0.05), but only in hospital central kitchen departments and non-hospital health care settings, and not in de-centralized hospital kitchens in in-patient units or in catering companies for the general public. This data suggests that there is a need for education on risks of NoV transmission by food handling of healthcare workers using in ward kitchen facilities.     

Effects of high hydrostatic pressure on the inactivation of norovirus and quality of cabbage Kimchi

In Korea, cabbage Kimchi is potentially regarded as a high-risk food with respect to enteric norovirus (NoV). This study investigated the effect of high hydrostatic pressure (HPP) treatment (100–500 MPa for 5 min at room temperature) on the inactivation of murine norovirus-1 (MNV-1, initial inoculum of 5–6 log₁₀ PFU/ml) as a human NoV surrogate in the Kimchi. The effects of HHP treatment on the microbiological, physicochemical properties and sensory qualities of cabbage Kimchi were also examined. The MNV-1 in the Kimchi after 100, 200, 300, and 400 MPa were 5.6 (0.1 log reduction), 5.5 (0.2 log reduction), 5.4 (0.3 log reduction), and 4.2 (1.5 log₁₀ PFU/ml), respectively. Complete inactivation of MNV-1 was determined in the cabbage Kimchi by 500 MPa. One-log reduction of total aerobic bacteria and lactic acid bacteria was only determined in the 500 MPa HHP treated Kimchi. The pH values and acidity (%) in all HPP treated Kimchi were 4.4–5.7 and 0.4–0.7%, respectively. The Hunter colors ‘a’ (redness) and ‘b’ (yellowness) and mechanical texture (hardness) in HHP Kimchi were not different between 0 MPa and HHP (100–500 MPa) treated Kimchi significantly (P > 0.05) different although the HHP treated Kimchi turned to very slight darkness. All sensory scores in HHP treated Kimchi were determined as > 5 point (like) in 7 point-hedonic scale. The current study suggest that 500 MPa for 5 min could potentially be used for direct application in commercial Kimchi processing and distribution for complete MNV-1 inactivation without causing any deleterious overall quality changes.     

The analysis of food safety incidents in South Korea, 1998–2016

This study uses media reports to analyze food safety incidents in South Korea that occurred between January 1998 and March 2016. The total number of incidents during this period was 975, with an average of 51.3 per year and 4.3 per month. The top five types of food involved were fruits and vegetables, fish and fish products, meat and meat products, beverages, and confectionery. Of the 975 incidents, 406 (41.6%) were related to chemical hazards—such as food additives and pesticides—221 (22.7%) were due to biological hazards, and 199 (20.4%) were due to physical hazards. Through analysis, this study determines at what stage a breakdown in food safety is likely to occur: primary production is the most common stage with 615 (63.1%) incidents, followed by incidents during the handling and distribution stages, at 240 (24.6%) and 65 (6.7%), respectively. The results of this study can be used as data for risk analysis or food safety strategies.     

Viral disinfection of organic fresh produce comparing Polyphenon 60 from green tea with chlorine

Contamination of fresh produce with enteric viruses is a matter of concern because viruses may persist in food surfaces under normal storage conditions for long periods. Chlorinated compounds are the most common substances used to disinfect fresh produce but they present some disadvantages such as the formation of by-products. In this study, green tea extract (GTE), a natural compound from Camellia sinensis, was tested to disinfect fresh produce contaminated with 8 log₁₀ of human adenoviruses (HAdV) and 6 log₁₀ of infectious murine noroviruses (MNV), a surrogate of human norovirus. Chorine was also employed to compare the action of GTE with a common disinfectant. Results demonstrated that, although GTE was not as efficient as chlorine, it demonstrated good effectiveness to decrease the amount HAdV in fresh produce. Both disinfectants were more effective than just using water to reduce the load of infectious HAdV. It was also observed that both chlorine and GTE were more effective to remove HAdV than MNV, probably due to different interactions of the viruses with the plant tissues.     

Prevention and control of hazards in seafood

Seafood is high on the list of foods transmitting disease. However, the food safety issues are highly focussed and more than 80% of all seafood-borne outbreaks are related to biotoxins (ciguatoxin), scombrotoxin or the consumption of raw molluscan shellfish. The safety hazards in seafood production are listed and discussed. It is pointed out that there are serious safety concerns related to the consumption of raw fish and shellfish due to the presence of biological (bacteria, virus, parasites) and chemical (biotoxins) hazards. These hazards are present in the fish and shellfish pre-harvest and are difficult or impossible to control by applying presently available preventive measures. In contrast, the hazards related to contamination, recontamination or survival of biological hazards during processing are well-defined and can be controlled by applying Good Manufacturing Practice (GMP), Good Hygiene Practice (GHP) and a well designed HACCP-programme. Similarly, the means to prevent the growth of pathogenic microorganisms during distribution and storage of the final products are – with a few exceptions – available. Proper application of well-known preservative parameters including temperature is able to control growth of most pathogens. When this is not the always case, for example inhibition of Listeria monocytogenes in lightly preserved fish products, it is recommended to limit the stated shelf-life of these products to a period of no-growth for the pathogen of concern. There is a good agreement between the trends shown in disease statistics, the hazard analysis and the qualitative risk assessment of the various fish products. It is recommended that consumers should be informed of the risk of eating raw seafood – particularly molluscan shellfish and certain freshwater fish.     

Development of a multiplex PCR method for testing six GM soybean events

Genetically modified (GM) soybean and derived products make up a large part of the biotech-derived food and feed market. As more GM soybean varieties have been approved for commercialization, labeling requirement by South Korea and other countries needs the technical testing methods. This paper reports the development of a multiplex PCR method for identifying six commercialized GM soybean events using the event-specific fragment. Event specific primers targeting Roundup Ready Soybean (RRS, GTS40-3-2), A2704-12, DP356043-5, MON89788, A5547-127, and DP305423-1 were designed, and a multiplex PCR assay consisting of six event-specific fragments and one endogenous lectin fragment was developed. The specificity of the event-specific PCR method was confirmed using 20 GM events of maize, soybean, cotton, and canola. The limit of detection (LOD) for each event in the multiplex PCR is approximately 0.05%. Intra-lab validation by two different operators confirmed the specificity and LOD of this multiplex PCR method. The method was used to test 30 soybean-derived foods from South Korean and US markets, and results revealed three varieties of GM soybean (RRS, A2704-12, and MON89788) in 19 of the 30 food samples tested. This work provides an efficient and cost-effective approach for event-specific analysis of six commercialized GM soybean varieties and related processed foods in Korea.     

Development and evaluation of a real-time PCR assay for detection of lobster,a crustacean shellfish allergen

Crustacean shellfish are a leading cause of food allergy in American adults, and the Food Allergen Labeling and Consumer Protection Act requires that different types of crustacean shellfish be distinguished from each other. In general ELISA assays are not capable of differentiating crustacean type, but PCR assays are. In this work, a real-time PCR assay for lobster, a crustacean shellfish allergen, was developed and evaluated. Food matrices were spiked with lobster meat at 0.1, 1, 10, 100, 1000, 10⁴, and 10⁵ parts per million (ppm). In addition to testing of several different food matrices, method performance was determined using conditions which have historically proven challenging for PCR analyses, specifically food matrices with low DNA content and acidic pH levels, as well as foods that were treated with combined high temperature and pressure. Real-time PCR standard curves were generated from spiked, treated foods and analyzed with respect to linear range and reaction efficiency. In most cases, the assay was linear over 6–8 orders of magnitude; lower limits of detection were 0.1–1 ppm and reaction efficiencies were within the preferred range of 100 ± 10%. A notable exception occurred in the case of heat treatment at acidic pH, which resulted in severe delay or complete loss of amplification signals.     

Recovery of structurally intact norovirus from food-contact surfaces

Human noroviruses (NoVs) are the most common cause of gastroenteritis and are responsible for at least 50% of all gastroenteritis outbreaks worldwide. NoV can be transmitted directly via person-to-person contact and contaminated fomites or indirectly via contaminated raw and ready-to-eat foods. However, there have not been enough studies that examine the detection and persistence of NoV on various food-contact surfaces, which may provide information regarding the transmission of NoV in public places, such as restaurants, hospitals, and nursing homes. In order to determine the persistence of NoV, the ability of NoV GII.4 to persist on six kinds of surfaces was investigated for up to 28 d post-inoculation, using an immuno-magnetic separation (IMS) procedure combined with quantitative real-time RT-PCR (qRT-PCR). NoV GII.4 was detected in all test samples, even after 672 h (28 d).     

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shellfish in Shanghai

Vibrio parahaemolyticus is a marine and estuarine bacterium that poses the greatest threat to human health worldwide. It has been the leading bacterial cause of seafood-borne illness. This study investigated the prevalence and drug resistance of V. parahaemolyticus isolated from retail shellfish in Shanghai. A total of 140 shellfish samples were collected from February 2014 to February 2015. The occurrence of V. parahaemolyticus in shellfish was 34.3%, which has increased compared to previous reports. In addition, discernible differences of total presumptive V. parahaemolyticus counts (TPVPC) were also observed in shellfish between market A and B. The results from PCR assays indicated that thermostable direct hemolysin (tdh) gene was positive in two isolates (2.1%), and the thermostable direct hemolysin-related hemolysin (trh) gene was not detected in all isolates. Antibiotic resistance profiles of those isolates were as follows: ampicillin (87.5%), cephazolin (31.3%), cephalothin (6.3%), amoxicillin/clavulanic acid (6.3%), piperacillin (6.3%), and amikacin (3.2%). Thirty-three out of 96 isolates were resistant to two or more antimicrobial agents. It is suggested that V. parahaemolyticus in retail shellfish could be a potential risk to consumers in Shanghai.     

Investigation of the experience of foodborne illness and estimation of the incidence of foodborne disease in South Korea

The national incidence of foodborne illness reported by public health surveillance systems is lower than the actual incidence due to underdiagnosis and underreporting. Therefore, it is necessary to estimate the actual cases of foodborne illness at a national level. This study used a simple method of polling and estimation that overcomes the problem of lack of data. A national survey of the experience, diagnosis with and/or treatment for, and/or hospitalization for foodborne illness among a representative sample in South Korea was conducted. The survey results were then used to estimate the actual incidence of foodborne disease using a simulation method that expressed the estimated values as 95% confidence intervals (CI) to address uncertainty. Among the 1439 respondents for whom valid data were collected, 19.18% had experienced, 3.13% had been diagnosed with and/or treated for, and 0.28% had been hospitalized for foodborne disease. Using these results, it was estimated that 9.59 (95% CI: 8.57–10.61) million individuals experience, 1.56 (95% CI: 1.11–2.01) million are diagnosed with and/or treated for, and 140,000 (95% CI: 30,000–280,000) are hospitalized for foodborne diseases annually in South Korea. The estimated number of patients hospitalized for foodborne disease is approximately 20 times higher than that of the number of reported cases in Ministry of Korean Food and Drug Safety, indicating that a large number of cases are unreported. Consideration of these results will be useful for the prevention and control of foodborne disease in South Korea and allow for international comparison of the incidence of foodborne illness.     

Detection of sixteen genetically modified maize events in processed foods using four event-specific pentaplex PCR systems

To efficiently identify genetically modified (GM) maize events in foods and feeds, we report here the development of four individual pentaplex PCR analysis systems for event-specific identification of sixteen GM maize events approved in South Korea. In addition to the maize endogenous reference gene, zSSIIb, the four pentaplex PCR assays target group 1 containing TC6275, MON810, T25, and NK603; group 2 with TC1507, MON863, GA21, and DAS-59122-7; group 3 with MIR604, Bt11, Bt176, and MON89034; group 4 with event 3272, LY038, MIR162, and MON88017. These amplicons were designed to be smaller than 200 bp to make the testing system suitable for analyzing processed foods/feeds derived from these 16 GM maize events. After optimizing the reaction condition, the limits of detection of these four assays were approximately 0.25% for all of the 16 GM maize events. This multiplex PCR method for sixteen GM maize events was validated by three operators, and the data confirmed the reliability of the developed assays. Furthermore, 74 food samples containing maize ingredients from the USA, China, Japan, and South Korea were analyzed, and we observed 18 food products containing one or more GM maize events. These results suggest that the developed multiplex system is applicable for use in specific testing of sixteen GM maize events in foods and feeds in South Korean market.     

Comparison between thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar Vibrio for isolating Vibrio parahaemolyticus

Considering its widespread distribution in marine environments, its fast replication times and low infectious doses and the rapid spread of its strains in recent years, intensive and continuous monitoring of potentially pathogenic Vibrio parahaemolyticus is strongly recommended in order to assess the human health risk arising from shellfish consumption. The lack of epidemiological data points to the need to develop specific methods for detecting V. parahaemolyticus. In this note, the authors compare two plating media currently available for isolating V. parahaemolyticus in shellfish. Both approaches involve pre-enrichment of V. parahaemolyticus. One uses thiosulphate-citrate-bile salt sucrose (TCBS) as the isolation medium, while the other uses a chromogenic medium (CHROMagar Vibrio). Next, biochemical identification of isolates was performed with API 20E, followed by PCR assay aimed at the toxR gene to confirm the cultural and biochemical identification. Comparison of the two methods highlighted that CHROMagar Vibrio is more accurate and specific than TCBS. The analysis of data from 160 shellfish samples showed an accuracy and specificity of just 51% and 71% for TCBS compared with 88% and 95% for CAV.