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A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample., (Copyright © 2021. Published by Elsevier Ltd.)

Among the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD 95 was 10 2 genome copies/g for HAV, HEV and norovirus GII and 10 3 genome copies/g for norovirus GI. The LOQ was 2.90 × 10 4 , 1.40 × 10 3 , 1.60 × 10 4 and 1.30 × 10 4 genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study investigated the obstacles in detecting enteric viruses from berry fruits, which are on the one hand often associated with outbreaks of viral enteric disease, and on the other hand recognized as a challenging food matrix for molecular detection of enteric viruses. According to the ISO 15216 protocol, for soft fruit samples, virus extraction is by elution with agitation followed by precipitation with polyethylene glycol/NaCl. As a result, first, the phenolic content in the berry eluate was found to be weakly correlated with the detection of coliphage MS2 spiked in the berry samples. Second and more importantly, it was observed that the gel-like pellets formed after precipitation could entrap considerable portions of viruses from being further purified and recovered for detection, suggesting that the low virus detection sensitivity from berries is largely due to the pectin content with complicated chemical structures in the berry fruits. Future research is needed to solve this problem in a targeted way.

Strawberry production and exports have been increasing in Spain in recent decades. However, little information is available about their microbiological quality. Due to the growing concern about the microbial safety of these fruits, the objective of this investigation was to study the microbiological quality and the prevalence of the main foodborne pathogens on strawberries sold in Spain. Fresh (n = 152) and frozen (n = 31) samples were obtained from marketplaces and fields in 2017 and 2018. The samples were assayed for total aerobic mesophilic microorganisms (TAM), moulds and yeasts (M&Y), total coliforms (TC), Escherichia coli, Salmonella spp., Listeria monocytogenes as well as Norovirus (NoV) GI and GII. The microbiological counts ranged from <1.70 (detection limit, dl) - 5.89 log 10 CFU/g (mean 3.78 log 10 CFU/g) for TAM; 2.10-5.86 log 10 CFU/g (mean 3.80 log 10 CFU/g) for M&Y; and <0.70 (dl) - 4.91 log 10 CFU/g (mean 2.15 log 10 CFU/g) for TC in fresh strawberries. In frozen strawberries, the counts were <1.70 (dl) - 3.66 log 10 CFU/g (mean 2.30 log 10 CFU/g) for TAM; <1.70 (dl) - 2.76 log 10 CFU/g (mean 1.82 log 10 CFU/g) for M&Y; and <0.70(dl) - 1.74 log 10 CFU/g (mean 0.77 log 10 CFU/g) for TC. All the samples in this study tested negative for Salmonella spp., L. monocytogenes. E. coli and NoV GI and GII genome. A global overview of all the data was executed using Principal Component Analysis (PCA), and the results showed that the scores and loadings according to principal components 1 (PC1) and 2 (PC2) accounted for 75.9% of the total variance, allowing a distinction between fresh and frozen samples. The presence of moulds was significantly higher in the supermarket samples whereas the presence of total coliforms was significantly higher in the field samples (p < 0.05). Although pathogenic microorganisms were not found, preventative measures and prerequisites in the strawberry production chain must be considered in order to avoid possible foodborne diseases related to the microbiological quality of the fruit., (Copyright © 2019. Published by Elsevier B.V.)

X-ray is a non-thermal technology that has shown good efficacy in reducing pathogenic and spoilage bacteria, viruses and parasites. X-ray hygiene technology resulted in a high microbial loss in numerous food products, such as dairy products, ready-to-eat shrimp, oysters, fresh products, strawberries, shredded iceberg lettuce, and spinach leaves. Some X-ray studies on food safety have shown that X-ray is an effective technology and is also an appropriate alternative to the electron beam and gamma rays, and can be used in the food industry without side effects on human health. Besides, we reviewed the X-ray effect on the nutritional value of food. Therefore in this study, we aimed to review the available pros and cons of current studies regarding X-rays' effects on the food industry., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Detection of viruses on berries is a challenging task, often hampered by the presence of RT-qPCR inhibiting substances from berry juice. A direct extraction method for virus detection (murine norovirus and GA phage) on frozen raspberries was previously published. We expanded (different types of berries and viruses) and improved the method using MobiSpin S400 columns that filter nucleic acids based on size-exclusion chromatography. While no inhibition was detected in filtered RNA, unfiltered RNA needed from 1:2 to more than 1:8 dilution in order to remove inhibition. The modified method gave recoveries of bovine norovirus around 40.8 ± 4.5% (40.0 ± 7.0%), 48.0 ± 26.0% (50.5 ± 7.8%), 28.3 ± 2.6% (45.8 ± 6.6%) from frozen (fresh) raspberries, strawberries and blueberries, respectively. For the same samples, recoveries of hepatitis A virus were 34.0 ± 5.9% (34.0 ± 6.0%), 40.0 ± 13.3% (34.2 ± 10.5%) and 23.0 ± 6.8% (31.5 ± 7.9%). For adenovirus40 (DNA virus), recoveries were 21.2 ± 8.6%, 16.0 ± 3.2% and 5.7 ± 0.2% from fresh raspberries, strawberries and blueberries respectively and column filtration did not add any improved effect. The modified method is effective and timesaving for detection of viral RNA from both fresh and frozen berries. As an emerging detection and direct quantification method, droplet digital RT-PCR was compared to RT-qPCR and was much less influenced by inhibitors when detecting mengovirus in unfiltered RNA from berries. However, for low levels of pure RNA, RT-qPCR showed slightly higher sensitivity and more stable results., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Norovirus (NoV), a major food-borne virus, causes non-bacterial acute gastroenteritis in humans. Berries are generally harvested from low-growing bushes by hand and are minimally processed before being sold to consumers. Therefore, the consumption of berries has been linked to numerous food-borne gastroenteritis outbreaks caused by NoV in many countries. We performed a survey of NoV contamination in commercial fresh/frozen berry fruits collected from 2016 to 2017 in the Heilongjiang Province, the main berry-producing area in China, using a TaqMan-based real-time reverse transcription-PCR assay. Among 900 frozen and 900 fresh domestic retail berry samples, the prevalence of NoV was 9% (81/900) and 12.11% (109/900), including 35.80% (29/81) and 29.36% (32/109) of genotype GI alone, 54.32% (44/81) and 60.55% (66/109) of GII alone, and 9.88% (8/81) and 10.09% (11/109) of both GI and GII, respectively. No NoV was detected among the 677 frozen berry samples for export. Thus, the occurrence of NoV contamination was significantly higher in domestic berries than in exported berries and higher in fresh berries than in frozen berries. This study highlights the need for further risk surveillance for NoV contamination in berries produced in the Heilongjiang Province and recommends region-extended monitoring of retail berries for NoV., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Contamination of fresh vegetables and berries with human enteric viruses is a major cause of food poisoning. The aim of this study was to investigate the prevalence of norovirus GI, norovirus GII, hepatitis A virus (HAV), adenovirus, astrovirus, rotavirus, and male-specific coliphage systematically in fresh fruit and vegetables and associated agricultural environmental samples, including irrigation water, soil, and worker's gloves. Enteric viruses were detected by international standard methods (ISO/TS 15216), and male-specific coliphages were isolated using US EPA Method 1601. For the study, 773 samples were collected from June 2016 to April 2017, including Chinese cabbage ( n  = 244), cucumber ( n  = 98), lettuce ( n  = 73), strawberry ( n  = 120), soil ( n  = 191), irrigation water ( n  = 14), and gloves ( n  = 27). Two cucumber and two irrigation water samples were positive for norovirus GI, and one cucumber and two irrigation water samples were positive for norovirus GII. HAV was detected in one strawberry sample and one glove sample. The other tested foodborne viruses were not detected in any of the samples. Sixteen male-specific coliphages were isolated from Chinese cabbage, cucumber, lettuce, cherry tomato, soil, and irrigation water. The isolation of male-specific coliphage would be more practical to investigate the fecal contamination in produce rather than pathogenic viruses.

To acquire data on contamination with Norovirus in berry fruit and salad vegetables in the United Kingdom, one thousand one hundred and fifty two samples of fresh produce sold at retail in the UK were analysed for Norovirus. Of 568 samples of lettuce, 30 (5.3%) were Norovirus-positive. Most (24/30) lettuce samples which tested positive for Norovirus were grown in the UK and 19 of those 24 samples contained NoV GI. Seven/310 (2.3%) samples of fresh raspberries were Norovirus-positive. Most (6/7) of the positively-testing fresh raspberry samples were imported, but no predominance of a genogroup, or any seasonality, was observed. Ten/274 (3.6%) samples of frozen raspberries were Norovirus-positive. The country of origin of the positively-testing frozen raspberry samples was not identified in most (7/10) instances. The collected data add to the currently limited body of prevalence information on Norovirus in fresh produce, and indicate the need for implementation of effective food safety management of foodborne viruses., (Copyright © 2018. Published by Elsevier Ltd.)

Viral enteropathogens are one of the leading causative agents of foodborne illnesses in both the United States and the European Union. While human noroviruses and hepatitis A virus cause the vast majority of outbreaks and illnesses, there are handful of human enteric viruses that contribute to sporadic outbreaks worldwide including astrovirus, sapovirus, rotavirus, enterovirus and Aichi virus. In addition, hepatitis E virus is increasingly being recognized as an emerging zoonotic threat within the food supply. This review aims to briefly describe the primary human enteric viruses of concern with respect to foodborne transmission. Next, we focus on the contamination and persistence of these viruses within three high-risk food commodities-leafy greens, soft red fruits and bivalve mollusks. As opposed to detailing the specific routes by which these foods can be contaminated with enteric viruses, we have chosen to focus on their persistence and specific interactions within the food itself. Therefore, the processes of attachment and internalization of the viruses in foods have been emphasized. Looking forward, the implications of these specific interactions of human enteric viruses with leafy greens, soft red fruits and bivalve mollusks are briefly considered within the context of future prevention and control strategies., (© 2018 The Society for Applied Microbiology.)

Objectives: Enteric viruses are responsible for foodborne and waterborne infections affecting a large number of people. Data on food and water viral contamination in the south of Italy (Sicily) are scarce and fragmentary. The aim of this study was to evaluate the presence of viral contamination in food, water samples, and surface swabs collected in Sicily METHODS: The survey was conducted on 108 shellfish, 23 water samples (seawater, pipe water, and torrent water), 52 vegetables, one peach and 17 berries, 11 gastronomic preparations containing fish products and/or raw vegetables, and 28 surface swabs. Hepatitis A virus (HAV), genogroup GI, GII, and GIV norovirus (NoV), enterovirus (EV), rotavirus (RoV), hepatitis E virus (HEV), adenovirus (AdV), and bocavirus (BoV) were detected by nested (RT) PCR, real-time PCR, and sequence analysis., Results: The most frequently detected viruses in shellfish were HAV (13%), NoV (18.5%), and EV (7.4%). Bocavirus was found in 3.7%, HEV in 0.9%, and AdV in 1.9% of the molluscs. Of the 23 water samples, 21.7% were positive for GII NoV and 4.3% for RoV and HEV genotype 3. Of the 70 vegetable samples, 2.9% were positive for NoV GI (GI.5 and GI.6), 2.9% for EV, and 1.4% for HEV. In the gastronomic preparations, only one EV (9%) was detected. No enteric viruses were detected in the berries, fruit, or swabs analyzed., Conclusions: Molecular surveillance of water and food samples clearly demonstrated that human pathogenic viruses are widely found in aquatic environments and on vegetables, and confirmed the role of vegetables and bivalve molluscs as the main reservoirs., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Human noroviruses (NoV) are recognized worldwide as important pathogens and the primary cause of foodborne disease outbreaks from contaminated food in the U.S. They are often transmitted by infected food handlers manipulating foods during preparation, such as fresh fruits and vegetables. This paper provides a study to model the transfer of NoV between food handlers and vegetables during salad preparation in school food services based on direct observation data. Three transfer pathways were modeled by considering different initial contamination sources (environment, handlers and contaminated produce). The probability of infection by NoV was also estimated based on the NoV levels at consumption obtained from each simulated transfer pathway. A scenario analysis ranging a wide concentration from 10 2 to 10 7 NoV infective particles was performed to represent different levels of NoV in the initial contamination sources. In addition, a sensitivity analysis was applied to identify the most important model inputs and determine the safest handling practices to be implemented in school food service operations. The pathway describing transfer from contaminated surfaces or handlers to foods indicated that initial levels of ≤10 4 NoV particles/fomite resulted in <0.5% cases per serving of NoV infection. When initial levels were higher, % cases of NoV infection was estimated to be ca. 3%. This rise in % cases of infection was linked to higher doses (5% serving with ≥15 NoV particles/serving) and prevalence levels at consumption (>0.2). In the pathway modeling cross contamination from contaminated vegetables to non-contaminated vegetables, all scenarios could lead to infected individuals, although number of cases of infection were lower (<1.3%), despite concentration levels were higher. On the contrary, for this pathway, prevalence was 2-fold lower than that observed in the pathways describing transfer from contaminated surfaces and hands. Based on the sensitivity analysis, NoV transfers to fresh produce may be minimized by improving hand washing, and therefore effective training programs need to be carried out specifically addressing hand washing. Moreover, the produce's washing step showed to be an effective control measure, depending on the desinfectant efficacy, by reducing % cases of NoV infection from 6 to 1%. The model in this study might be used, in the future, to evaluate the impact on the risk associated with NoV transmission of specific and effective training programs, aimed at food chain operators., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness, with common vehicles including bivalve molluscan shellfish, soft fruit and various vegetables. Outbreaks of viral illness due to contamination of the surfaces of foods, or food preparation surfaces by for example infected food handlers are also common. Virus analysis of food matrices can contribute towards risk management for these hazards and a two-part technical specification for determination of Hepatitis A virus and norovirus in food matrices (ISO/TS 15216:2013) was published jointly by the European Committee for Standardisation and the International Organization for Standardization in 2013. As part of the European Mandate No. M381 to validate 15 standards in the field of food microbiology, an international validation study involving 18 laboratories from 11 countries in Europe was conducted between 2012 and 2014. This study aimed to generate method characteristics including limit of detection, limit of quantification, repeatability and reproducibility for ISO 15216 - Part 1, the method for quantification, in seven food matrices. The organization and results of this study, including observations that led to improvements in the standard method are presented here. After its conclusion, the method characteristics generated were added to the revised international standard, ISO 15216-1:2017, published in March 2017., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Locally acquired hepatitis A infection is re-emerging in Australia owing to person-to-person outbreaks among men who have sex with men and imported frozen produce. This paper describes a multi-state foodborne outbreak in the first half of 2018. Enhanced human epidemiological investigation including a case-control study, as well as microbial surveillance and trace-back investigations concluded that the outbreak was caused by consumption of imported frozen pomegranate arils. A total of 30 cases of hepatitis A infection, genotype IB with identical sequences met the outbreak case definition, including 27 primary cases and three secondary cases. Twenty-five (83%) of the cases were hospitalised for their illness and there was one death. Imported frozen pomegranate arils from Egypt were strongly implicated as the source of infection through case interviews (19 of 26 primary cases) as well as from a case-control study (adjusted odds ratio 43.4, 95% confidence interval 4.2-448.8, P = 0.002). Hepatitis A virus (HAV) was subsequently detected by polymerase chain reaction in two food samples of the frozen pomegranate aril product. This outbreak was detected and responded to promptly owing to routine genetic characterisation of HAVs from all hepatitis A infections in Australia as part of a national hepatitis A enhanced surveillance project. This is now the third outbreak of hepatitis A in Australia from imported frozen fruits. A re-assessment of the risk of these types of imported foods is strongly recommended.

This study aimed to evaluate the elution-concentration methodology based on skimmed milk flocculation from three varieties of tomatoes (Solanum lycopersicum L. [globe], Solanum lycopersicum var. cerasiforme [cherry] and hybrid cocktail [grape tomato]) for further monitoring of field samples. Spiking experiments were performed to determine the success rate and efficiency recovery of human norovirus (NoV) genogroup II, norovirus murine-1 (MNV-1) used as sample process control virus and human adenovirus (HAdV). Mean values of 18.8%, 2.8% and 44.0% were observed for NoV GII, MNV-1 and HAdV, respectively with differences according to the types of tomatoes, with lower efficiency for cherry tomatoes. Analysis of 90 samples, obtained at commercial establishments in the metropolitan region of Rio de Janeiro State, revealed 4.5% positivity for HAdV. Bacterial analysis was also performed with no detection of Salmonella spp., L. monocytogenes and fecal coliforms. Data demonstrated that the skimmed milk flocculation method is suitable for recovering HAdV from tomatoes and highlights the need for considering investigation in order to improve food safety., (Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

High intensity ultrasound is becoming important and more widely used in the food industry for microorganisms decontamination. This sterilization technique has been evaluated to improve food safety and to replace common processing with chemical additive compounds. The efficiency of a horn-type power ultrasound treatment (300 W and 600 W, 28 kHz, 10-30 min) on Listeria monocytogenes, Bacillus cereus, Escherichia coli, Salmonella typhimurium bacteria suspensions and phytoviruses was examined in this study. The results of this study showed that ultrasonic treatment can be used to eliminate vegetative cells of gram-positive and gram-negative bacteria from 1.59 to 3.4 log in bacterial suspensions and some phytoviruses in fruits., (Copyright © 2018. Published by Elsevier Ltd.)

This study describes an outbreak investigation of 14 hepatitis A cases in the Netherlands. The hepatitis A virus (HAV) genotype IB sequences in cases were highly similar (459/460 nt). The origin of strains could be narrowed to Bulgaria based on information from EPIS-FWD. As an association with consumption of soft fruit was suspected, a case-control study was initiated using a questionnaire and a list of pictures of soft fruit available at the supermarket chain involved. Twelve out of 13 cases consumed a specific frozen raspberry/blueberry product shown on the list (OR 46.0, 95% CI 5.0-27). In multivariable regression analysis this product was the only risk factor (aOR 26.6, 95% CI 2.0-263). Laboratory analyses could not demonstrate HAV-RNA in batches that had been on the market in the incubation period of patients. Trace back of frozen fruit showed that raspberries had been traded by a producer in Bulgaria. After withdrawal of the product from the supermarket no new cases were reported. Use of advertisement pictures of consumed food was helpful in this investigation. Suspicion of the source was strengthened by data from molecular typing and food trace back activities, underlining the importance of good (inter)national cooperation between public health and food safety organisations.

Hepatitis A virus (HAV) is well known worldwide as a causative virus of acute hepatitis. In recent years, numerous cases of HAV infection caused by HAV-contaminated berries have occurred around the world. Because berries are often consumed without prior heating, reliable disinfection of the raw fruit is important in order to prevent HAV outbreaks. Previous studies have found that murine norovirus strain 1 (MNV-1) and human norovirus GII.4 were inactivated in heat-denatured lysozyme solution. In this study, we investigated whether or not heat-denatured lysozyme is effective in inactivating HAV and whether it could be an effective disinfectant for berries contaminated with HAV or MNV-1. We examined the inactivating effect of heat-denatured lysozyme on three strains of HAV and found that it reduced the infectivity of all three strains. We then immersed blueberries and mixed berries into solutions of HAV or MNV-1, and disinfected them by soaking them in 1% heat-denatured lysozyme for 1min. Consequently, the infectious HAV and MNV-1 contaminating the berries were decreased by >3.1 log units in all samples. Our results demonstrate that heat-denatured lysozyme effectively inactivates HAV and suggest that heat-denatured lysozyme may be an effective disinfectant for berry fruit, which is a potential source of HAV food poisoning., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Human norovirus (HuNoV) is a major foodborne virus causing gastroenteritis outbreaks in humans. Salad products can be vectors of transmission for foodborne viruses such as HuNoV when these products are contaminated naturally or through unsanitary food handling. Therefore, development of simple, reliable and sensitive techniques for the detection of HuNoV in salad products is needed to ensure food safety. The purpose of our study was to optimize a method for the detection of HuNoV in artificially contaminated salad products. To this end, 2 different kinds of salads (fruit salads and vegetable salads) were experimentally inoculated with HuNoV GI, HuNoV GII, and MS2 suspensions. The selected method was based on treatment with pectinase followed by Trizol-chloroform purification, and the recovery efficiencies were 6.07% to 26.52% for HuNoV GI and 5.54% to 37.36% for HuNoV GII. MS2 was used as the process control, and the recovery efficiencies for fruit salad and vegetable salad samples were 38.57% and 41.13%, respectively. The optimized method could be applied in diagnostic laboratories to identify NoV contamination in composite foods, such as salad products, should an event of foodborne outbreak occur., (© 2017 Institute of Food Technologists®.)

Raw fruits may harbour many pathogens of public health concern including enteric viruses, which are the leading cause of foodborne outbreaks. Recently, consumption of soft berries has been associated with increasing reports of norovirus and hepatitis A virus outbreaks in Europe. Due to their low infectious doses and low concentrations in food samples, an efficient and sensitive analytical method is required for virus detection. In this study we explored two different ways to improve the reference method for the detection of enteric viruses in soft fruits (ISO/TS 15216-1; 15216-2): an additional purification step after RNA extraction; and the detection of enteric viral genome by an absolute quantification method (microfluidic digital RT-PCR). Both of these approaches led to an improvement of enteric virus detection in soft berries by greatly lowering PCR inhibition, raising viral extraction efficiencies and enabling validation of controls using pure RNA extracts. The PCR inhibitor removal step can be easily included in the routine method. Absolute quantification by digital RT-PCR may be a relevant alternative method to standardize quantification of enteric viruses in foodstuffs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human norovirus (HuNoV) has been an increasing concern of foodborne illness related to fresh and frozen berries. In this study, high hydrostatic pressure (HHP) inactivation of HuNoV on fresh strawberries, blueberries, and raspberries and in their purees was investigated. Porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs) and real-time reverse transcriptional polymerase chain reaction (RT-qPCR) were utilized for infectious HuNoV discrimination and quantification. Strawberry puree inoculated with HuNoV genogroup I.1 (GI.1) strain was HHP-treated at 450, 500 and 550 MPa for 2 min each at initial sample temperatures of 0, 4 and 20 °C. HuNoV GI.1 strain became more sensitive to HHP treatment as the temperature decreased from 20 to 0 °C. HuNoV GI.1 or genogroup II.4 (GII.4) strains were inoculated into three types of berries and their purees and treated at pressure levels from 250 to 650 MPa for 2 min at initial sample temperature of 0 °C. For the purees, the HHP condition needed to achieve >2.9 log reduction of HuNoV GI.1 strain and >4.0 log reduction of HuNoV GII.4 strain was found to be ≥ 550 MPa for 2 min at 0 °C. HHP treatment showed better inactivation effect of HuNoV on blueberries than on strawberry quarters and raspberries. HuNoV GI.1 strain was more resistant to HHP treatment than HuNoV GII.4 strain under different temperatures and environment. The physical properties and sensory qualities of HHP-treated and untreated blueberries and the three types of berry purees were evaluated. Color, pH and viscosity of blueberries and three berry purees showed no or slight changes after HHP treatment. Sensory evaluation demonstrated that HHP treatment of 550 MPa for 2 min at 0 °C did not significantly reduced the sensory qualities of three berry purees. The results demonstrated that the HHP treatment of 550 MPa for 2 min at 0 °C could be a potential nonthermal intervention for HuNoV in berry purees without adversely affecting their sensory qualities and physical properties., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Berries, in particular raspberries, have been associated with multiple recalls due to norovirus contamination and were linked to a number of norovirus (NoV) outbreaks. In the present study a total of 130 samples of frozen raspberries were collected from 26 batches in four different raspberry processing companies. In two companies the samples consisted of bulk frozen raspberries serving as raw material for the production of raspberry puree (an intermediate food product in a business to business setting). In two other companies, the samples consisted of bulk individually quick frozen (IQF) raspberries serving as raw material for the production of frozen fruit mixes (as a final food product for consumer). Enumeration of Escherichia coli and coliforms was performed as well as real-time reverse transcription PCR (RT-qPCR) detection of GI and GII NoV (in 2 × 10 g). In addition, in cases where positive NoV GI or GII RT-qPCR signals were obtained, an attempt to sequence the amplicons was undertaken. Six out of 70 samples taken from the 14 batches of frozen raspberries serving raspberry puree production provided a NoV RT-qPCR signal confirmed by sequencing. Four of these six positive samples clustered in one batch whereas the other two positive samples clustered in another batch from the same company. All six positive samples showed NoV RT-qPCR signals above the limit of quantification of the RT-qPCR assay. These two positive batches of frozen raspberries can be classified as being of insufficient sanitary quality. The mean NoV level in 20 g of these raspberry samples was 4.3 log genomic copies NoV GI/20 g. The concern for public health is uncertain as NoV RT-qPCR detection is unable to discriminate between infectious and non-infectious virus particles. For the IQF raspberries, one batch out of 12 tested NoV positive, but only 1 out of the 5 samples analyzed in this batch showed a positive RT-qPCR GI NoV signal confirmed by sequencing. The RT-qPCR signal was below the limit of quantification of the assay used (<3.7 log genomic copies/20g). It was shown that the applied protocol for sequencing of the amplicon to confirm the specificity of the RT-qPCR signal was successful for GI NoV amplicons but often failed and provided an inconclusive result for GII NoV amplicons., (Copyright © 2014 Elsevier B.V. All rights reserved.)

According to the results of complex microbiological examination of samples of vegetables, fruits and grapes there was established significant contamination of them with opportunistic bacteria, antigens of intestinal viruses and cysts of intestinal Protozoa, that confirms the epidemiological role of these products as factors in transmission of acute intestinal infections. There was revealed ribonucleic acid of enteric viruses in experimentally infected pulp from the surface of tomatoes and apples, that indicates to the possibility of penetration of these pathogens into the fruits and vegetables through intact (having no visible damages) surface.

Multiple outbreaks of human norovirus (hNoV) have been associated with fresh produce, such as soft berries and lettuce. Even though food handlers are considered an important source for the introduction of hNoV into food chains, their contribution to public health risks associated with hNoV remains unknown. To assess to which extent food handlers contribute to the introduction and spread of hNoV in fresh produce chains quantitative virus transfer data are needed. We estimated transfer proportions of hNoV GI.4, GII.4, murine norovirus (MNV-1), a culturable surrogate of hNoV, and human adenovirus (hAdV-2), a human pathogen proposed as an indicator for human faecal pollution, between gloved fingertips and raspberries, strawberries, and lettuce, by quantitative RT-PCR and cell culture if applicable. Virus transfer proportions were corrected for virus-matrix specific recoveries, and variability and uncertainty of the parameters were estimated. Virus transfer from gloves to soft berries was generally lower as compared to lettuce, with mean transfer proportions ranging between 0.1 to 2.3% and 9 to 10% for infectious MNV-1 and hAdV-2, respectively. Transfer from produce to glove was mostly greater than transfer from glove to produce, adding to the likelihood of virus transfer due to cross contamination from contaminated produce via food handlers. HNoV GI.4 and hNoV GII.4 showed no significant difference between their mean transfer proportions. Using the estimated transfer proportions, we studied the impact of low and high transfer proportions on the public health risk, based on a scenario in which a food handler picked raspberries with contaminated fingertips. Given the made assumptions, we could show that for a pathogen as infectious as hNoV, low transfer proportions may pose a greater public health risk than high transfer proportions, due to a greater viral spread. We demonstrated the potential of food handlers in spreading hNoV in food chains, showing that prevention of virus contamination on food handlers' hands is crucial for food safety. Nevertheless, complete prevention of virus contamination on fresh produce cannot be achieved in reality, and reliable and effective intervention measures are consequently required. We estimated that, especially for low transfer proportions, a robust one log10-unit reduction of infectious hNoV on contaminated produce, and on food handlers' hands, could lower the public health risk substantially. Using the obtained data in quantitative risk assessment will aid in elucidating the contribution of food handlers in hNoV transmission., (© 2013 Elsevier B.V. All rights reserved.)

Human noroviruses (hNoV) have been detected on soft fruits. Especially raspberries have been found to be associated with outbreaks of gastroenteritis suggesting persistence of hNoV on these fruits. Therefore, the persistence of hNoV GII.4 and GI.4, murine norovirus (MNV-1, a culturable surrogate for hNoV), and human adenovirus (hAdV, an indicator for human fecal contamination), on raspberries, strawberries and in phosphate buffered saline (PBS) at 4°C, 10°C and 21°C, mimicking commonly applied storage conditions was studied by molecular and cell culture techniques. Monophasic, biphasic and Weibull models were fitted to virus counts with maximum likelihood estimation. The tested viruses were persistent (≤0.5 log(10)-unit reduction in viral titer) under all studied conditions in PBS, at 4°C and 10°C on raspberries, and at 4°C on strawberries. The difference in viral persistence on raspberries and strawberries was most pronounced at 21°C. Here, infectious MNV-1 and hAdV particles decayed rapidly on strawberries with TFL-values (time for the first log(10)-unit reduction) of only 1day (95% CI of 0.6-1 and 0.8-1days, respectively). On raspberries, however, the TFL-value of infectious MNV-1 was found to likely exceed the shelf life of the berries with 3days (95% CI of 2.8-3.1days); hAdV remained infectious with only 0.3 log(10)-unit reduction (95% CI of 0.2-0.4) in viral titer. For hNoV GI, a TFL-value of 2days (95% CI 1-4days) was determined based on the targeted genome fragment, whereas the TFL-value of hNoV GII exceeded the shelf life of strawberries at 21°C. The greater viral persistence on raspberries as compared to strawberries, especially at 21°C, may at least in part explain why raspberries are more frequently associated with hNoV outbreaks than strawberries. Moreover, our results show that due to the high persistence of the virus already low contamination levels of the highly infectious hNoV may be associated with an infection risk of humans after consumption of raspberries. The estimated decay parameters and uncertainties of this study serve as important input requirements in the quantitative assessment of public health risks from the consumption of soft fruits., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A total of 91 fig and 185 date samples were analyzed by reverse transcription (RT) real-time PCR for the presence of hepatitis A virus (HAV) RNA. Two batches of dates tested positive, and the HAV RNA detected was genotyped as IA. These findings warrant further development of methods applicable to food which is consumed untreated and is exported from countries in which HAV is endemic.

In recent years the importance of prepared salads as potential vehicles of gastrointestinal infection has been highlighted by several large outbreaks both nationally and across international boundaries. Between 1992 and 2006, 2274 foodborne general outbreaks of infectious intestinal disease were reported in England and Wales, of which 4% were associated with the consumption of prepared salads. In total, 3434 people were affected, with 66 hospitalizations and one death reported. The attribution of prepared salad types and pathogens among prepared salad associated outbreaks are presented and discussed. Findings from UK studies on salad vegetables, fruit and mixed salads from 1995 to 2007 (21 247 samples) indicate that most bacteria of concern with regard to human health are relatively rare in these products (98.6% of satisfactory quality); however, outbreaks of salmonellosis were uncovered associated with bagged salad leaves and fresh herbs during two such studies. Although it is known that fresh salad vegetables, herbs or fruit may become contaminated from environmental sources, only in recent years has the association of foods of nonanimal origin, such as salad vegetables, with foodborne illness become evident and recurrent, demonstrating that major health problems can arise from consumption of contaminated prepared salads if hygiene practices breakdown.

Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform.

An outbreak of acute gastroenteritis occurred in Hamburg among guests of a canteen in August 2005. A total of 241 persons were found ill. In stool samples of 16 of them Norovirus Genogroup I was identified. Neither bacterial nor viral pathogens could be detected in food samples. Among 162 guests enrolled in a cohort study, 69 (42.6 %) met the case definition. A desert dish made from blackberries and yoghurt showed a significant association with the disease (RR 6.9; 95% CI 3.36-14.16). In addition, a protective effect of the decision for the alternative desert dish ice cream was observed (RR 0.9; 95% CI 0.08-0.45). The desert, which was prepared in the canteen kitchen from yoghurt and frozen blackberries, was the most likely source of the outbreak. Frozen berries should be included in the HACCP concepts for canteen kitchens.

Although there is a large body of evidence incriminating foods as vehicles in the transmission of norovirus, little is known about virus survival in foods and on surfaces. Feline calicivirus was used as a surrogate for norovirus to investigate its survival in representative foods of plant and animal origin and on metal surfaces. Known concentrations of feline calicivirus in a natural fecal suspension were deposited onto lettuce, strawberries, ham, or stainless steel and incubated for 7 days at refrigeration or room temperatures. Virus was recovered at 1-day intervals, and the titers of the virus were determined by plaque assay. Infectious virus was recoverable until day 7 from lettuce, ham, and stainless steel. Statistically higher titers of feline calicivirus (P < 0.05) were recovered from ham under all conditions than from lettuce, strawberries, or stainless steel. These data provide valuable information for epidemiological and monitoring purposes as well as for the development of food processing practices and appropriate strategies to inactivate norovirus and control its transmission via foods and surfaces.

Between 1992 and 2000, 1,518 foodborne general outbreaks of infectious intestinal disease (IID) were reported to the Public Health Laboratory Service (PHLS) Communicable Disease Surveillance Centre (CDSC), of which 83 (5.5%) were associated with the consumption of salad vegetables or fruit (SVF). The pathogens most frequently reported were salmonellas (41.0%) and Norwalk-like virus (NLV) (15.7%). In total 3,438 people were affected; 69 were admitted to hospital and one person died. Most outbreaks were linked to commercial catering premises (67.5%). Three community outbreaks, of Salmonella enterica serovar Typhimurium Definitive Phage Type (DT) 104, S. Typhimurium DT 204b and Shigella sonnei infection, were found to be associated with lettuce contaminated at source, and these accounted for 501 (14.6%) cases. The latter two outbreaks were international, involving several European countries. This demonstrates how contamination of SVF during production/processing can result in major, geographically widespread, outbreaks of infection with serious public health consequences.

A series of studies were performed, using poliovirus, to ascertain the potential for enteric pathogenic viruses to survive on various foodstuffs. The extraction protocols, which could be performed in just a few hours, were developed for use with quantities of food that would normally constitute a portion for consumption. The protocols were based on elution of viruses from food surfaces, followed by differential centrifugation to remove food debris and concentrate viruses. The studies were mostly performed using fresh produce stored at refrigeration temperature for 2 weeks or so, which was considered to represent the maximum time elapsing between purchase and consumption. Each food sample was inoculated with a viral suspension, and samples were analyzed immediately and at intervals throughout the experiment. Statistical analyses were performed on the results, and the decimal reduction times (D-values), or number of days after which the initial virus numbers had declined by 90%, were calculated. In summary, the resulting D-values were as follows: lettuce, 11.6 days; green onion, no decline; white cabbage, 14.2 days; fresh raspberries, no decline; and frozen strawberries, 8.4 days. The results showed that enteric viruses may persist on fresh fruit and vegetables for several days under conditions commonly used for storage in households. Therefore, if contamination has occurred before purchase, there will always be a risk of infection from consumption of the food.

Poliovirus kept on the cut surfaces of fully ripe papaya cubes placed in an ice box showed a sharp and significant reduction in the recovery of infectious virus about 15 minutes after exposure. Thereafter, a very gradual decrease ensued and infectious residual virus was detected up to the end of the 6-hour exposure period. Papaya cubes washed or kept overnight before virus inoculation, and from less ripe fruits produced a similar survival pattern. A very small proportion of the inoculum was recovered from the mashed content of the inoculated papaya cubes thus suggesting that most of the non-recovered virus particles were inactivated. The results suggest that the importance of poliovirus-contaminated cut papayas as a transmission vehicle for the virus is greatly reduced by the rapid decline in the infectivity of a large proportion of the virus soon after contamination. Nevertheless, the potential to transmit remains as a small residual pool of infectious poliovirus is able to survive for a relatively long period.

The ability of two strains of Salmonella enteritidis PT4 to cross-contaminate from inoculated egg droplets on surfaces onto melon or beef (sterile or non-sterile) was investigated. When the foods were placed on these surfaces where egg droplets were still wet, cross-contamination occurred within 1 s onto every piece of food. It took at least 1 min for all the food pieces to be contaminated when egg droplets had been allowed to dry. Both strains were capable of rapid growth on melon and beef (sterile or non-sterile) at 20 degrees C, but growth rates on beef appeared to be slowed by pre-exposure to either 4 or -18 degrees C.