Apostolos Vantarakis

Distribution of Human Virus Contamination in Shellfish from Different Growing Areas in Greece, Spain, Sweden, and the United Kingdom

ABSTRACT Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.

Virus hazards from food, water and other contaminated environments

Abstract Numerous viruses of human or animal origin can spread in the environment and infect people via water and food, mostly through ingestion and occasionally through skin contact. These viruses are released into the environment by various routes including water run‐offs and aerosols. Furthermore, zoonotic viruses may infect humans exposed to contaminated surface waters. Foodstuffs of animal origin can be contaminated, and their consumption may cause human infection if the viruses are not inactivated during food processing. Molecular epidemiology and surveillance of environmental samples are necessary to elucidate the public health hazards associated with exposure to environmental viruses. Whereas monitoring of viral nucleic acids by PCR methods is relatively straightforward and well documented, detection of infectious virus particles is technically more demanding and not always possible (e.g. human norovirus or hepatitis E virus). The human pathogenic viruses that are most relevant in this context are nonenveloped and belong to the families of the C aliciviridae, A denoviridae, H epeviridae, P icornaviridae and R eoviridae. Sampling methods and strategies, first‐choice detection methods and evaluation criteria are reviewed.

Evaluation of Potential Indicators of Viral Contamination in Shellfish and Their Applicability to Diverse Geographical Areas

ABSTRACT The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.

Ultraviolet light and ultrasound as non-thermal treatments for the inactivation of microorganisms in fresh ready-to-eat foods.

The effects of two non thermal disinfection processes, Ultraviolet light (UV 254 nm) and Ultrasound (US) on the inactivation of bacteria and color in two freshly cut produces (lettuce and strawberry) were investigated. The main scope of this work was to study the efficacy of UV and US on the decontamination of inoculated lettuce and strawberries with a cocktail of four bacteria, Escherichia coli, Listeria innocua, Salmonella Enteritidis and Staphylococcus aureus. Treatment of lettuce with UV reduced significantly the population of E. coli, L. innocua, S. Enteritidis and S. aureus by 1.75, 1.27, 1.39 and 1.21 log CFU/g, respectively. Furthermore, more than a 2-log CFU/g reduction of E. coli and S. Enteritidis was achieved with US. In strawberries, UV treatment reduced bacteria only by 1-1.4 log CFU/g. The maximum reductions of microorganisms, observed in strawberries after treatment with US, were 3.04, 2.41, 5.52 and 6.12 log CFU/g for E. coli, S. aureus, S. Enteritidis and L. innocua, respectively. Treatment with UV and US, for time periods (up to 45 min) did not significantly (p>0.05) change the color of lettuce or strawberry. Treatment with UV and US reduced the numbers of selected inoculated bacteria on lettuce and strawberries, which could be good alternatives to other traditional and commonly used technologies such as chlorine and hydrogen peroxide solutions for fresh produce industry. These results suggest that UV and US might be promising, non-thermal and environmental friendly disinfection technologies for freshly cut produce.

Cytogenetic study in cultured human lymphocytes treated with three commonly used preservatives.

Potassium sorbate, sodium benzoate and potassium nitrate have been tested for their genotoxic, cytostatic and cytotoxic potential in human peripheral blood cells in vitro. Potassium nitrate has shown no activity in the test system. When potassium sorbate and sodium benzoate were used at concentrations of 2.0, 0.2 and 0.02 mM no cytostatic activity was detected. However, concentrations of 4 and 8 mM have shown a weak cytostaticity. Additionally, a genotoxic activity using the SCE methodology has been observed at 8 mM of sodium benzoate and at 4 and 8 mM of potassium sorbate. No cytotoxic activity has been induced by the three preservatives. Data demonstrate that the preservatives at low concentrations can be considered as non genotoxic under conditions tested.

Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels

Multiplex PCR amplification of invA and virA genes was developed enabling simultaneous detection in mussels of Salmonella spp. and Shigella spp., respectively. Simultaneous amplification of products of 215 and 275 bp was obtained either by using mixtures of individual strains of Sh. dysenteriae and Salm. typhimurium or spiked contaminated mussels with both bacteria. In the case of the mussels, 10–100 cells of Salmonella spp. and Shigella per millilitre of homogenate were detected by the multiplex PCR following a pre‐enrichment step to increase sensitivity and to ensure that detection was based on the presence of cultivable bacteria. Also, the sensitivity and specificity of this method was evaluated. Multiplex PCR amplification was shown to be an effective, sensitive and rapid method for the simultaneous detection of pathogens in mussels.

Loop-Mediated Isothermal Amplification (LAMP) for the Detection of Salmonella in Food

Salmonella infection represents a considerable global burden, with significant health and economic impacts. Salmonellosis is most often attributed to the consumption of contaminated foods such as poultry, beef, pork, eggs, milk, seafood, nut products, and fresh produce. Increased public awareness related to food-borne contamination resulted in greater efforts to develop more sensitive, rapid, and inexpensive methods of pathogens detection. Loop-mediated isothermal amplification (LAMP) constitutes a promising solution for rapid diagnosis of food-borne pathogens and is increasingly been applied for the specific diagnosis of different pathogens, Salmonella included. We have reviewed the application of LAMP for the specific detection of Salmonella in food matrices, compared with conventional culture techniques, and in terms of applicability, food matrices, type of assays, target genes, assay temperature, time and equipment, specificity, sensitivity, and robustness. The pros and cons of Salmonella LAMP assays are presented. The potential of LAMP for the development of new on-site diagnostics for the food and agricultural industries and its use as a routine Salmonella screening tool are discussed. Salmonella-specific LAMP assays are expected to provide a very robust, innovative, and powerful molecular diagnostic method for food safety testing services and public health authorities.

Quantitative farm-to-fork risk assessment model for norovirus and hepatitis A virus in European leafy green vegetable and berry fruit supply chains

Fresh produce that is contaminated with viruses may lead to infection and viral gastroenteritis or hepatitis when consumed raw. It is thus important to reduce virus numbers on these foods. Prevention of virus contamination in fresh produce production and processing may be more effective than treatment, as sufficient virus removal or inactivation by post-harvest treatment requires high doses that may adversely affect food quality. To date knowledge of the contribution of various potential contamination routes is lacking. A risk assessment model was developed for human norovirus, hepatitis A virus and human adenovirus in raspberry and salad vegetable supply chains to quantify contributions of potential contamination sources to the contamination of produce at retail. These models were used to estimate public health risks. Model parameterization was based on monitoring data from European supply chains and literature data. No human pathogenic viruses were found in the soft fruit supply chains; human adenovirus (hAdV) was detected, which was additionally monitored as an indicator of fecal pollution to assess the contribution of potential contamination points. Estimated risks per serving of lettuce based on the models were 3×10(-4) (6×10(-6)-5×10(-3)) for NoV infection and 3×10(-8) (7×10(-10)-3×10(-6)) for hepatitis A jaundice. The contribution to virus contamination of hand-contact was larger as compared with the contribution of irrigation, the conveyor belt or the water used for produce rinsing. In conclusion, viral contamination in the lettuce and soft fruit supply chains occurred and estimated health risks were generally low. Nevertheless, the 97.5% upper limit for the estimated NoV contamination of lettuce suggested that infection risks up to 50% per serving might occur. Our study suggests that attention to full compliance for hand hygiene will improve fresh produce safety related to virus risks most as compared to the other examined sources, given the monitoring results. This effect will be further aided by compliance with other hygiene and water quality regulations in production and processing facilities.

DETECTION OF ENTEROVIRUSES, ADENOVIRUSES AND HEPATITIS A VIRUSES IN RAW SEWAGE AND TREATED EFFLUENTS BY NESTED-PCR

To determine the good working condition of the biological treatment plant of the University Hospital of Patras, Greece, twenty-four raw sewage samples and twenty-four effluent samples were analyzed for the presence of enteroviruses, adenoviruses and Hepatitis A virus (HAV), during the period of March 1995 to March 1996. We used a nested-PRC approach, to increase the sensitivity of the detection. Enteroviruses and adenoviruses were detected in twelve samples (50%) and fourteen samples (58.3%) of raw sewage, respectively. HAV was not detected in any of the raw sewage samples. The more frequent isolation of adenoviruses in raw sewage, indicates their stability as virological indicators of the pollution of the environment. In addition, a seasonal distribution of the detection of enteroviruses and adenoviruses was observed. The absence of HAV is in agreement with the lack of HAV infections in the hospital during that period of time. In contrast with the raw sewage, we were unable to detect the presence of enteroviruses, adenoviruses in samples collected after the biological treatment plant. This fact indicates the effective treatment of sewage by the local biological purification plant.

Environmental Mycobacteria in Drinking Water Before and After Replacement of the Water Distribution Network

Environmental mycobacteria (M. chelonae (8), M.flavescens (6), M. gordonae (6), M. fortuitum (5), M. kansasii (4), M. phlei (2) and M. terrae (2)) were isolated from21.3% of drinking water samples before replacement of thedistribution network of Patras. After replacement of the networkonly 1.8% of the samples contained environmental mycobacteria(M. chelonae (2)). The identification of environmentalmycobacteria was performed by Restriction EnzymeAnalysis-Polymerase Chain Reaction. Our results showed no statistically significant correlationbetween the presence of mycobacteria and the bacteriologicalfecal indicators (p >0.05). Moreover, we determined thatconcentrations of free residual chlorine equal to or greaterthan 0.5 mg L-1 in the water supply network were needed toeliminate environmental mycobacteria.We conclude that the presence of a biofilm in the old Patrasdrinking water network significantly increased the number ofenvironmental mycobacteria in the drinking water. This problemwas resorted after the replacement of the network pipes.