Development of a method for direct extraction of viral RNA from bivalve molluscs.

The detection of foodborne viruses in bivalve molluscs is a challenging procedure in relation to low virus concentration and to the presence of significant RT-PCR inhibitors. The aim of this study was the development of an efficient direct extraction method for foodborne viral RNA from bivalve molluscs. Using Mengovirus as a surrogate for foodborne viruses, five extraction methods based on RNA release by Trizol were compared on clams and oysters. A procedure consisting of Trizol, PureLink RNA Mini Kit, followed by Cetyltrimethylammonium bromide (CTAB) treatment and LiCl precipitation was found to provide RNA with the highest extraction efficiency and negligible inhibitory effect on real-time RT-PCR. This procedure was further compared to standard extraction method (ISO 15216) using clam, mussel and oyster samples spiked with Hepatitis A virus, Norovirus (NoV) GI and GII as well as bivalve samples naturally contaminated with NoV GI or GII. Results clearly demonstrated that the developed method provided, on average, a recovery 4·3 times higher than the standard reference protocol as well as good repeatability. SIGNIFICANCE AND IMPACT OF THE STUDY: A direct extraction procedure was developed to recover viral RNA from shellfish with improved efficiency in comparison to reference extraction method (ISO 15216). Without the need for specific equipment, this procedure offers an alternative for performing food safety controls and for risk assessment studies. Given the inclusion in this extraction method of several steps for the efficient removal of food components inhibiting PCR reaction, this approach could serve as a general scheme for the extraction of nucleic acids of other enteric viruses and/or from other food categories.

First Detection of Hepatitis E Virus in Shellfish and in Seawater from Production Areas in Southern Italy.

Shellfish samples (n = 384) from production areas, water samples from the same areas (n = 39) and from nearby sewage discharge points (n = 29) were analyzed for hepatitis E virus (HEV) by real-time and nested RT-PCR. Ten shellfish samples (2.6%) and five seawater samples (12.8%) tested positive for HEV; all characterized strains were G3 and showed high degree of sequence identity. An integrated surveillance in seafood and waters is relevant to reduce the risk of shellfish-associated illnesses.

Detection of Norovirus GII.17 Kawasaki 2014 in Shellfish, Marine Water and Underwater Sewage Discharges in Italy.

Norovirus (NoV) is a major cause of non-bacterial acute gastroenteritis worldwide, and the variants of genotype GII.4 are currently the predominant human strains. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been reported as the cause of gastroenteritis outbreaks in Asia, replacing the pandemic strain GII.4 Sydney 2012. The GII.17 Kawasaki 2014 variant has also been reported sporadically in patients with gastroenteritis outside of Asia, including Italy. In this study, 384 shellfish samples were subjected to screening for human NoVs using real-time PCR and 259 (67.4%) tested positive for Genogroup II (GII) NoV. Of these, 52 samples, selected as representative of different areas and sampling dates, were further amplified by conventional PCR targeting the capsid gene, using broad-range primers. Forty shellfish samples were characterized by amplicon sequencing as GII.4 (n = 29), GII.2 (n = 4), GII.6 (n = 2), GII.12 (n = 2), and GII.17 (n = 3). Sixty-eight water samples (39 seawater samples from the corresponding shellfish production areas and 29 water samples from nearby underwater sewage discharge points) were also tested using the above broad-range assay: eight NoV-positive samples were characterized as GII.1 (n = 3), GII.2 (n = 1), GII.4 (n = 2), and GII.6 (n = 2). Based on full genome sequences available in public databases, a novel RT-PCR nested assay specific for GII.17 NoVs was designed and used to re-test the characterized shellfish (40) and water (8) samples. In this second screening, the RNA of GII.17 NoV was identified in 17 additional shellfish samples and in one water sample. Upon phylogenetic analysis, these GII.17 NoV isolates were closely related to the novel GII.17 Kawasaki 2014. Interestingly, our findings chronologically matched the emergence of the Kawasaki 2014 variant in the Italian population (early 2015), as reported by hospital-based NoV surveillance. These results, showing GII.17 NoV strains to be widespread in shellfish samples collected in 2015 in Italy, provide indirect evidence that this strain has started circulating in the Italian population. Notably, using a specific assay, we were able to detect many more samples positive for GII.17 NoV, indicating that, in food and water matrices, broad-range assays for NoV may grossly underestimate the prevalence of some, less common, NoVs. The detection of the GII.17 strain Kawasaki 2014 in clinical, water and food samples in Italy highlights the need for more systematic surveillance for future disease control and prevention.

ELIME assay vs Real-Time PCR and conventional culture method for an effective detection of Salmonella in fresh leafy green vegetables.

The detection of Salmonella according to EC regulation is still primarily based on traditional microbiological culture methods that may take several days to be completed. The purpose of this work is to demonstrate the applicability of an Enzyme-Linked-Immuno-Magnetic-Electrochemical (ELIME) assay, recently developed by our research group for the detection of salmonella in irrigation water, in fresh (raw and ready-to-eat) leafy green vegetables by comparison with Real-Time PCR (RTi-PCR) and ISO culture methods. Since vegetables represent a more complex matrix than irrigation water, preliminary experiments were carried out on two leafy green vegetables that resulted negative for salmonella by the ISO method. 25g of these samples were experimentally inoculated with 1-10 CFU of S. Napoli or S. Thompson and pre-enriched for 20h in two different broths. At this time aliquots were taken, concentrated at different levels by centrifugation, and analyzed by ELIME and RTi-PCR. Once selected the best culture medium for salmonella growth, and the optimal concentration factor suitable to reduce the sample matrix effect, enhancing the out-put signal, several raw and ready-to-eat leafy green vegetables were artificially inoculated and pre-enriched. Aliquots were then taken at different incubation times and analyzed with both techniques. Results obtained showed that 20 and 8h of pre-enrichment were required to allow the target salmonella (1-10 CFU/25g) to multiply until reaching a detectable concentration by ELIME and RTi-PCR assays, respectively. A confirmation with the ISO culture method was carried out. Based on the available literature, this is the first report of the application of an ELISA based method for the detection of Salmonella in vegetables.

Development and evaluation of an ELIME assay to reveal the presence of Salmonella in irrigation water: Comparison with Real-Time PCR and the Standard Culture Method.

A reliable, low-cost and easy-to-use ELIME (Enzyme-Linked-Immuno-Magnetic-Electrochemical) assay for detection of Salmonella enterica in irrigation water is presented. Magnetic beads (MBs), coupled to a strip of eight-magnetized screen-printed electrodes localized at the bottom of eight wells (8-well/SPE strip), effectively supported a sandwich immunological chain. Enzymatic by-product is quickly measured by chronoamperometry, using a portable instrument. With the goal of developing a method able to detect a wide range of Salmonella serotypes, including S. Napoli and S. Thompson strains responsible for various community alerts, different kinds of MBs, antibodies and blocking agents were tested. The final system employs MBs coated with a broad reactivity monoclonal antibody anti-salmonella and blocked with dry milk. For a simple and rapid assay these two steps were performed in a preliminary phase, while the two sequential incubations for the immuno-recognition events were merged in a single step of 1h. In parallel a Real-Time PCR (RTi-PCR) method, based on a specific locked nucleic acid (LNA) fluorescent probe and an internal amplification control (IAC), was carried out. The selectivity of the ELIME and RTi-PCR assays was proved by inclusivity and exclusivity tests performed analyzing different Salmonella serotypes and non-target microorganisms, most commonly isolated from environmental sources. Furthermore, both methods were applied to experimentally and not experimentally contaminated irrigation water samples. Results confirmed by the ISO culture method, demonstrated the effectiveness of ELIME and RTi-PCR assays to detect a low number of salmonella cells (1-10 CFU/L) reducing drastically the long analysis time usually required to reveal this pathogen.

Microbiological survey of raw and ready-to-eat leafy green vegetables marketed in Italy.

The presence of foodborne pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7, thermotolerant Campylobacter, Yersinia enterocolitica and norovirus) in fresh leafy (FL) and ready-to-eat (RTE) vegetable products, sampled at random on the Italian market, was investigated to evaluate the level of risk to consumers. Nine regional laboratories, representing 18 of the 20 regions of Italy and in which 97.7% of the country's population resides, were involved in this study. All laboratories used the same sampling procedures and analytical methods. The vegetable samples were screened using validated real-time PCR (RT-PCR) methods and standardized reference ISO culturing methods. The results show that 3.7% of 1372 fresh leafy vegetable products and 1.8% of 1160 "fresh-cut" or "ready-to-eat" (RTE) vegetable retailed in supermarkets or farm markets, were contaminated with one or more foodborne pathogens harmful to human health.

A decade with nucleic acid-based microbiological methods in safety control of foods.

In the last decade, nucleic acid-based methods gradually started to replace or complement the culture-based methods and immunochemical assays in routine laboratories involved in food control. In particular, real-time polymerase chain reaction (PCR) was technically developed to the stage of good speed, sensitivity and reproducibility, at minimized risk of carry-over contamination. Basic advantages provided by nucleic acid-based methods are higher speed and added information, such as subspecies identification, information on the presence of genes important for virulence or antibiotic resistance. Nucleic acid-based methods are attractive also to detect important foodborne pathogens for which no classical counterparts are available, namely foodborne pathogenic viruses. This review briefly summarizes currently available or developing molecular technologies that may be candidates for involvement in microbiological molecular methods in the next decade. Potential of nonamplification as well as amplification methods is discussed, including fluorescent in situ hybridization, alternative PCR chemistries, alternative amplification technologies, digital PCR and nanotechnologies.

An innovative molecular detection tool for tracking and tracing Clostridium botulinum types A, B, E, F and other botulinum neurotoxin producing Clostridia based on the GeneDisc cycler.

Rapid and specific detection of botulinum neurotoxin (BoNT) producing Clostridia is a priority for public health authorities, in case of both natural and intentional botulism outbreaks. This study reports on the evaluation of a detection system based on the GeneDisc Cycler designed for simultaneously testing the bont/A, bont/B, bont/E and bont/F genes encoding for the botulinum neurotoxins types A, B, E and F. BoNT-producing Clostridia (n = 102) and non-BoNT-producing bacteria (n = 52) isolated from clinical, food and environmental samples were tested using this macro-array and results were compared to the reference lethality test on mice. The bont genes were correctly detected in all C. botulinum type A, B, E and F strains available, as well as in toxigenic C. baratii type F and toxigenic C. butyricum type E. No cross reactivity was observed with non human-toxigenic bacteria, C. botulinum types C, D and G. The identification of the bont genotype using the macro-array was correlated to toxino-typing of the BoNTs as determined by the mouse bioassay. An "evaluation trial" of the GeneDisc array performed blind in four European laboratories with 77 BoNT-producing Clostridia as well as 10 food and clinical samples showed that the developed macro-array is specific and reliable for identifying BoNT/A-, BoNT/B-, BoNT/E- and BoNT/F-producing clostridial strains and for screening naturally contaminated food and fecal samples. The test is robust, has a low detection limit (c.a. 5 to 50 genome copies in the PCR reaction microwell) and is promising for monitoring BoNT-producing Clostridia in different kinds of samples including food and clinical samples.

The evaluation of the microbial safety of fresh ready-to-eat vegetables produced by different technologies in Italy.

AIMS: The study was performed to evaluate the safety of whole and RTE vegetables and to investigate the effectiveness of different preventive strategies for the quality assurance of RTE vegetables collected from three Italian production systems. Producer 1, applied a strict system in compliance with GAP- GMP - HACCP, Producer 2 used chlorine disinfection at a second washing step, and Producer 3 using a physical microbial stabilization. METHODS: During the period 2005-2007, a total of 964 samples including whole vegetables and RTE salads, collected from three different producers in central Italy, were analysed to quantify the aerobic mesophilic count (AMC) and Escherichia coli, and for the presence of Salmonella spp, Listeria monocytogenes, E. coli O157:H7, hepatitis A virus and Norovirus (NoV). RESULTS: None of the whole vegetable samples were positive for L. monocytogenes, E. coli O157:H7, HAV and NoV; however, a low prevalence of Salmonella was found. No pathogens were detected with cultural methods in any of the RTE vegetables analysed, only two RTE samples were positive for L. monocytogenes with PCR, but were not confirmed by the cultural method. The median values of AMC in RTE vegetables measured 24 h after packaging were statistically different among the 3 producers (5·4 × 10(6), 1·5 × 10(7) and 3·7 × 10(7) CFU g(-1), respectively; P=0·011). The lowest level was detected in Producer 1. CONCLUSION: The products that were processed applying rigorously GAP, GMP and HACCP showed a better microbiological quality than those processed with chemical or physical stabilization. STUDY SIGNIFICANCE AND IMPACT: The results of the study evidenced the efficacy of GAP, GMP and HACCP in improving microbiological quality of whole and RTE vegetables.

Comparison of different concentration methods for the detection of hepatitis A virus and calicivirus from bottled natural mineral waters.

Viral contamination of drinking water is frequently reported as the primary source of gastroenteritis or hepatitis outbreaks. The presence of viruses at low concentration levels in most environmental water poses major analytical problems when determining their concentration. To evaluate the efficiency of different recovery methods of viral RNA from bottled water, a comparison was made of 2 positively and 2 negatively charged membranes that were used for absorbing and releasing HAV virus particles during the filtration of viral spiked bottled water. All the 4 membranes, regardless of charge and pore size, had low level viral recovery. The results show that a considerable number of the virus particles passed through the pores of the membranes instead of being trapped by the electrostatic charges. Two different procedures were then compared using 1.5L polyethylene bottles spiked with 10-fold serial dilutions of HAV and FCV. The first procedure included an ultrafiltration-based method followed by MiniMag RNA extraction, and the second an ultracentrifugation-based method followed by RNA extraction using QIAamp viral RNA mini kit. The ultracentrifugation-based method resulted in a better recovery of HAV and FCV when compared to the ultrafiltration-based method.

Detection of hepatitis A virus in mussels from different sources marketed in Puglia region (South Italy).

Hepatitis A virus (HAV) infection is endemic in Puglia (South Italy). Epidemiological studies indicate that shellfish consumption, particularly mussels, is a major risk factor for HAV infection, since these products are eaten raw or slightly cooked. Nested reverse transcriptase-polymerase chain reaction (RT-PCR) has been shown to be a sensitive technique for the detection of HAV in mussels. The aim of the present study was to detect the presence of HAV in a large sample of mussels by nested RT-PCR and to confirm the presence of infectious viral particles in positive samples by cell culture infection and RT-PCR confirmation. Two hundred and ninety samples of mussels from different sources were collected between December 1999 and January 2000. One hundred samples were collected before being subjected to depuration, 90 after depuration, and 100 were sampled in different seafood markets. HAV-RNA was detected in 20 (20.0%) of non-depurated mussels, in 10 (11.1%) of depurated samples, and in 23 (23.0%) of samples collected in the shellfish markets, without any significant difference in the prevalence of positive samples by collection sources (chi2 = 4.79, p = 0.09). Of the 53 samples found positive by nested RT-PCR, 18 (34.0%) resulted positive by cell culture assay. No relationship between viral contamination and bacterial contamination was found (p = 0.41). This study confirms the usefulness of molecular techniques in detecting HAV in shellfish and, thus, for the screening of a large sample of naturally contaminated mussels. Improved shellfish depuration methods are needed to obtain virus-safe shellfish and reduce the risk for public human health.

Detecting the presence of infectious hepatitis A virus in molluscs positive to RT-nested-PCR.

AIMS: The objective of this study was to determine the presence of infectious hepatitis A virus (HAV) in molluscs naturally contaminated with viral HAV-RNA. METHODS AND RESULTS: One hundred and forty-two mollusc samples were analysed for the presence of viral HAV-RNA using RT-nested-PCR; positive samples were then analysed with an integrated method, cell-culture RT-PCR, to detect infectious virus. Viral HAV-RNA was detected in 34.5% of the samples while 12.7% of the total samples were positive for the presence of infectious virus. CONCLUSIONS: The results demonstrate the validity of the screening method (RT-nested-PCR) and the necessity of applying a method that is capable of detecting the presence of infectious HAV. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that in any case, to determine the safety for human consumption, the results of RT-nested-PCR must be confirmed with an integrated cell-culture PCR method.

Closed-circuit system for the depuration of mussels experimentally contaminated with hepatitis A virus.

In Italy, the consumption of raw or slightly cooked mussels represents the most important risk factor for the transmission of hepatitis A virus (HAV). Although there exist effective methods for the bacterial depuration of contaminated mussels, these methods are poorly effective on enteric viruses. The objective of the present study was to evaluate the effectiveness of a closed-circuit depuration system that uses both ozone and UV light for disinfecting water and that allows salinity and temperature, important parameters for the metabolism of mussels (Mytilus galloprovincialis), to be maintained at constant levels. The results showed that this depuration method decreased the viral load (from 1.72 log 50% tissue culture infective dose [TCID50] ml(-1) to <1 log TCID50 ml(-1) within 24 h and from 3.82 log TCID50 ml(-1) to <1 log TCID50 ml(-1) within 48 h). However, in both cases, after 120 h of depuration, a residual amount of virus capable of replicating in cells was detected. These results show that depuration, even if performed with advanced systems, may not guarantee the absence of virus.

Determination of enteroviruses, hepatitis A virus, bacteriophages and Escherichia coli in Adriatic Sea mussels.

The aim of the present study was to evaluate the incidence of enteric viruses in mussels and to verify the possibility of using phages as indirect indicators of mussel viral contamination. Mussels (36 samples) collected from three different areas of the Adriatic Sea were analysed to determine the following parameters: Escherichia coli, somatic coliphage (T6 phage), F-Plus (MS2 phage), B40-8 (phage of Bacteroides fragilis), enteroviruses and hepatitis A virus. Most of the results of the bacteriological analysis (most probable number (MPN) ml-1) were in accordance with the bacteriological limits established by European law, with the exception of seven samples. The bacteriophage analyses were always negative for F-Plus and B40-8, with the exception of a few samples, whereas the somatic coliphages were generally between 0 and 20 MPN g-1, with the exception of two samples (110 MPN g-1). The virological analysis showed five samples positive for the presence of enteroviruses and 13 for the presence of hepatitis A virus (in three samples both viruses were present). Most of these samples presented acceptable bacteriological parameters and the bacteriophages were absent or their value was generally very low. The results show that the detection of E. coli and phages does not seem to be a good indicator of viral contamination.

Detection of hepatitis A virus in shellfish by nested reverse transcription-PCR.

A method for the detection of HAV in shellfish, based on the use of guanidinium isothiocyanate-containing solution for RNA extraction and purification steps, followed by nested PCR, is hereby proposed. Tests were carried out on mollusc samples spiked with HAV strain FG. Results showed that in samples subjected only to one round of PCR it was possible to detect HAV at concentrations of 10(3)-10(4) TCID50/10 g of mollusc. The use of the nested PCR renders the system more sensitive and specific enabling the identification of HAV concentrations as low as 1 TCID50/10 g of mollusc. Furthermore thus method, in addition to allowing the avoidance of confirming tests, such as hybridization, proved to be inexpensive and simple to perform.

Inactivation of hepatitis A virus in heat-treated mussels.

Hepatitis A is a widespread infectious disease world-wide. In Italy, shellfish consumption was shown to be a major risk factor for hepatitis A infection, especially when these products are eaten raw or slightly cooked. The aim of the present study was to evaluate Hepatitis A virus (HAV) resistance in experimentally contaminated mussels treated at different temperatures (60, 80 and 100 degrees C) for various times. The presence of HAV was evaluated by cell culture infection and reverse transcriptase-polymerase chain reaction confirmation. The experiments, carried out on HAV suspension and contaminated mussel homogenate both containing about 10(5) 50% tissue culture infectious dose ml-1, showed that, under our experimental conditions, the treatments at 60 degrees C for 30 min, 80 degrees C for 10 min and an immersion at 100 degrees C for 1 min were not sufficient to inactivate all the viruses; it was necessary to prolong the treatment at 100 degrees C for 2 min to completely inactivate the virus. Thus it is advisable to eat only cooked shellfish, paying particular attention to the times and temperatures used in the cooking process, since evidence suggests that the shellfish body may protect the virus from the heat effect.

Application of reverse transcriptase-nested-PCR for detection of poliovirus in mussels.

In order to identify polioviruses in molluscs, we hereby propose a method based on precipitation with PEG 6000 followed by the use of a commercial kit (RNAfast II-Molecular System-San Diego) for the extraction and purification of viral RNA. The RT-PCR phase is followed by a second amplification using nested primers to increase the sensitivity and specificity of the method. Tests were carried out on mollusc samples spiked with Poliovirus 1. Results showed that in samples subjected only to one round of PCR it was possible to detect Poliovirus concentrations as small as 10(3)TCID50/ml. The use of nested-PCR makes the system more sensitive and specific enabling the identification of Poliovirus concentrations as small as 1 TCID50/ml.