Marta Hernández

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.

Hepatitis E Virus in Pork Production Chain in Czech Republic, Italy, and Spain, 2010

Processing does not substantially abate endogenous virus.

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples.

We developed a novel filtration-based method that can eliminate dead or severely damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p<0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R(2)>0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.

Prevalence and transmission of hepatitis E virus in domestic swine populations in different European countries

BackgroundHepatitis E virus (HEV) genotype 3 and 4 can cause liver disease in human and has its main reservoir in pigs. HEV investigations in pigs worldwide have been performed but there is still a lack of information on the infection dynamics in pig populations.FindingsThe HEV transmission dynamics in commercial pig farms in six different European countries was studied. The data collected show prevalence in weaners ranging from 8% to 30%. The average HEV prevalence in growers was between 20% and 44%. The fatteners prevalence ranged between 8% and 73%. Sows prevalence was similar in all countries. Boar faeces were tested for HEV only in Spain and Czech Republic, and the prevalence was 4.3% and 3.5% respectively. The collected data sets were analyzed using a recently developed model to estimate the transmission dynamics of HEV in the different countries confirming that HEV is endemic in pig farms.ConclusionsThis study has been performed using similar detection methods (real time RT-PCR) for all samples and the same model (SIR model) to analyse the data. Furthermore, it describes HEV prevalence and within-herd transmission dynamics in European Countries (EU): Czech Republic, Italy, Portugal, Spain, The Netherlands and United Kingdom, confirming that HEV is circulating in pig farms from weaners to fatteners and that the reproductive number mathematical defined as R0 is in the same range for all countries studied.

Occurrence of Human Enteric Viruses in Commercial Mussels at Retail Level in Three European Countries

In this study, the prevalence of different enteric viruses in commercial mussels was evaluated at the retail level in three European countries (Finland, Greece and Spain). A total of 153 mussel samples from different origins were analysed for human norovirus (NoV) genogroups I and II, hepatitis A virus (HAV) and hepatitis E virus (HEV). Human adenovirus (HAdV) was also tested as an indicator of human faecal contamination. A full set of controls (such as sample process control, internal amplification controls, and positive and negative controls) were implemented during the process. The use of a sample process control allowed us to calculate the efficiencies of extraction, which ranged from 79 to 0.5xa0%, with an average value of 10xa0%. Samples were positive in 41xa0% of cases, with HAdV being the most prevalent virus detected (36xa0%), but no significant correlation was found between the presence of HAdV and human NoV, HAV and HEV. The prevalences of human norovirus genogroup II, HEV and human NoV genogroup I were 16, 3 and 0.7xa0%, respectively, and HAV was not detected. The estimated number of PCR detectable units varied between 24 and 1.4xa0×xa0103xa0g−1 of digestive tract. Interestingly, there appeared to be a significant association between the type of mussel species (M. galloprovincialis) and the positive result of samples, although a complete overlap between country and species examined required this finding to be confirmed including samples of both species from all possible countries of origin.

European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.

Multicenter Collaborative Trial Evaluation of a Method for Detection of Human Adenoviruses in Berry Fruit

The qualitative performance characteristics of a qPCR-based method to detect human adenoviruses in raspberries were determined through a collaborative trial involving 11 European laboratories. The method incorporated a sample process control (murine norovirus) and an internal amplification control. Trial sensitivity or correct identification of 25-g raspberry samples artificially contaminated with between 5u2009×u2009102 and 5u2009×u2009104xa0PFU was 98.5%; the accordance and concordance were 97.0%. The positive predictive value was 94.2%. The trial specificity or percentage correct identification of non-artificially contaminated samples was 69.7%; the accordance was 80.0% and the concordance was 61.7%. The negative predictive value was 100%. Application of a method for the detection of human adenoviruses in food samples could be useful for routine monitoring for food safety management. It would help to determine if a route of contamination exists from human source to food supply chain which pathogenic viruses such as norovirus and hepatitis A virus could follow.

Survival kinetics of Listeria monocytogenes on raw sheep milk cured cheese under different storage temperatures

Raw sheep milk cured cheese produced in the Castilla y Leon region (Spain) constitutes a traditional semi-hard aromatic cheese typically aged for three to six months. This product is catalogued as ready-to-eat since it is not submitted to any further treatment before consumption. Thus, foodborne pathogens such as Listeria monocytogenes can represent a health concern for susceptible consumers. This study was aimed at evaluating the survival of L. monocytogenes on raw sheep milk cured cheese under different storage temperatures. Log-linear+shoulder and Weibull type models were fitted to data observed in order to estimate kinetic parameters. The Arrhenius relationship was further used to predict the impact of temperature on L. monocytogenes behavior during storage at 4, 12 and 22°C. Additionally, growth of lactic acid bacteria (LAB) as a representative group of the indigenous microbiota was evaluated. Results obtained indicated that the time to eradication (time when absence of L. monocytogenes in the analyzed samples was observed) was 114, 104, and 77 days for cheese samples stored at 4, 12 and 22°C, respectively. The LAB population showed an increase at 12 and 22°C during storage. However, an increase of 1 log CFU/g was observed during the first 2 weeks irrespectively of the storage temperature. The log-linear+shoulder model indicated a good fit to observed data. Likewise, the Arrhenius relationship explained sufficiently the dependency of temperature on L. monocytogenes behavior. This study demonstrated that cheese storage at ambient temperatures could lead to the preservation of its quality properties as well as its safety against L. monocytogenes.

Foods confiscated from non-EU flights as a neglected route of potential methicillin-resistant Staphylococcus aureus transmission.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in food-producing animals has provoked a great concern in the presence of MRSA in associated foodstuff. In this study, we have assessed for the first time the presence of MRSA in food confiscated from non-EU flights. We performed a search for MRSA among 195 food samples confiscated from passengers on flights from twenty-one non-EU countries in 2012 and 2013. One hundred and seventeen meat samples of diverse animal origin (including antelope, beef, chicken, duck, guinea pig, pork, rodents, and turkey), 75 dairy products (74 cheeses and 1 butter) and 3 eggs were analyzed. All S. aureus were studied by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. MRSA isolates were further characterized by multilocus sequence typing (MLST), SCCmec typing, and tested for the presence of Panton-Valentine leukocidin (PVL) virulence factors. Overall, 66 food samples were positive for S. aureus (33.9%). Six S. aureus strains were MRSA (9.1%), all of them in flights from Bolivia (and 5 from the same passenger). Among methicillin-sensitive S. aureus (MSSA) (60 out of 66S. aureus strains), 44.1% were resistant to penicillin, 10.2% to tetracycline, 8.5% were resistant to aminoglycosides (amikacin and tobramycin) and 3.4% exhibited the M phenotype. MRSA isolates were sensitive to all non-β-lactam antibiotics tested. SmaI-PFGE analysis provided 40 genotypes among the S. aureus isolates (three genotypes among the six MRSA). Five MRSA isolates belonged to ST8 and harboured SCCmec type IVc as well as PVL genes. One isolate belonged to ST1649, harboured SCCmec type IVc and tested negative for the presence of the PVL genes. In conclusion, in this study, we report for the first time the presence of CA-MRSA in food confiscated from non-EU flights: ST8/ST1649-MRSA-IV. These results confirm the illegal entrance of food as a neglected route of transmission as well as the dissemination of successful CA-MRSA lineages among countries via illegal foods. As a result, illegally imported food could play a role in the prevalence and evolution of MRSA clones in the community.