D. De Medici

Inactivation of Hepatitis A virus in heat-treated mussels

L. CROCI, M. CICCOZZI, D. DE MEDICI, S. DI PASQUALE, A. FIORE, A. MELE and L. TOTI.1999.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 100u2003°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 105 50% tissue culture infectious dose ml−1, showed that, under our experimental conditions, the treatments at 60u2003°C for 30u2003min, 80u2003°C for 10u2003min and an immersion at 100u2003°C for 1u2003min were not sufficient to inactivate all the viruses; it was necessary to prolong the treatment at 100u2003°C for 2u2003min 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.

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.

Diagnostic PCR: Making internal amplification control mandatory

The explosive increase since the beginning of the 1990s in the number of publications reporting PCR-based methods for detection or molecular typing of foodborne pathogens has attracted the attention of end-user laboratories. However, the well-recognized difficulties in reproducing published tests because of variation in performance of PCR thermal cyclers (Schoder et al. 2003), in efficiency of different DNA polymerases, and in the presence of PCR inhibitors in the sample matrix, have hampered implementation in end-user laboratories. This particularly applies to laboratories with quality-assurance programmes. It is necessary to have PCR-based methods available as internationally recognized standards (Hoorfar and Cook 2002). Currently, lack of international standards often forces end-user laboratories to spend substantial resources on adaptation of the published tests. Although many commercial PCR kits are available, it is important that end-users and reference laboratories have access to open-formula, noncommercial and nonproprietary PCRs in which the information on target gene and reagents are fully available. The prerequisite for a PCR, published in the scientific literature, to be adopted as a standard is that it has to be nonproprietary, and to have been validated through multicentre collaborative trial according to the international criteria (Anon. 2001, 2002a; Hoorfar and Cook 2002). Multicentre trial validation of noncommercial PCRs for detection of zoonotic pathogens has been performed by a European validation and standardization project (FOODPCR: http://www.pcr.dk) involving 35 laboratories from 21 countries (Hoorfar 1999; Malorny et al. 2003). A major drawback of most published PCRs, surprisingly even to date, is that they do not contain an internal amplification control (IAC). An IAC is a nontarget DNA sequence present in the same sample reaction tube, which is co-amplified simultaneously with the target sequence. In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But, it could also mean that the reaction was inhibited, as a result of malfunction of thermal cycler, incorrect PCR mixture, poor polymerase activity and, not least, the presence of inhibitory substances in the sample matrix. Conversely, in a PCR with an IAC, a control signal will always be produced when there is no target sequence present. When neither IAC signal nor target signal is produced, the PCR reaction fails. Thus, when using a PCRbased method in routine analysis, an IAC, if the concentration adjusted correctly, will indicate false-negative results. It is the false-negative results that turn a risk into a threat for the population, whereas a false-positive result merely leads to a clarification of the presumptive results by re-testing the sample. The European Standardization Committee (CEN), in collaboration with International Standard Organization (ISO) has proposed a general guideline for PCR testing that requires the presence of IAC in the reaction mixture (Anon. 2002b). Therefore, only IAC-containing PCRs may undergo multicentre collaborative trials, which is a prerequisite for standardization. Scientific journals must provide the source of new PCRbased methods suitable for standardization. Therefore, we propose that henceforward the editorial boards of applied microbiology journals require inclusion of an IAC in diagnostic PCR reported in submitted manuscripts. This could be performed by providing a specific section devoted to PCR in their Instruction to Authors.

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.

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

Aims:u2002 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.

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.

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

Aims:u2002The objective of this study was to determine the presence of infectious hepatitis A virus (HAV) in molluscs naturally contaminated with viral HAV‐RNA.

Ongoing outbreak of hepatitis A in Italy: preliminary report as of 31 May 2013

Since January 2013, an unusual increase in hepatitis A cases has been detected in northern Italy. A total number of 352 cases were reported to the integrated surveillance system between January and the end of May 2013 and this represents a 70% increase compared to the same period of the previous year. The outbreak is ongoing and the public health authorities are continuing their investigations to establish the transmission vehicle and to control the outbreak.

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.

Diagnostic PCR: making internal amplification control mandatory.

The explosive increase since the beginning of the 1990s in the number of publications reporting PCR-based methods for detection or molecular typing of foodborne pathogens has attracted the attention of end-user laboratories. However, the well-recognized difficulties in reproducing published tests because of variation in performance of PCR thermal cyclers (Schoder et al. 2003), in efficiency of different DNA polymerases, and in the presence of PCR inhibitors in the sample matrix, have hampered implementation in end-user laboratories. This particularly applies to laboratories with quality-assurance programmes. It is necessary to have PCR-based methods available as internationally recognized standards (Hoorfar and Cook 2002). Currently, lack of international standards often forces end-user laboratories to spend substantial resources on adaptation of the published tests. Although many commercial PCR kits are available, it is important that endusers and reference laboratories have access to openformula, noncommercial and nonproprietary PCRs in which the information on target gene and reagents are fully available. The prerequisite for a PCR, published in the scientific literature, to be adopted as a standard is that it has to be nonproprietary, and to have been validated through multicentre collaborative trial according to the international criteria (Anon. 2001, 2002b; Hoorfar and Cook 2002). Multicentre trial validation of noncommercial PCRs for detection of zoonotic pathogens has been performed by a European validation and standardization project (FOOD-PCR: http://www.pcr.dk) involving 35 laboratories from 21 countries (Hoorfar 1999; Malorny et al. 2003). A major drawback of most published PCRs, surprisingly even to date, is that they do not contain an internal amplification control (IAC). An IAC is a nontarget DNA sequence present in the same sample reaction tube, which is co-amplified simultaneously with the target sequence. In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But, it could also mean that the reaction was inhibited, as a result of malfunction of thermal cycler, incorrect PCR mixture, poor polymerase activity and, not least, the presence of inhibitory substances in the sample matrix. Conversely, in a PCR with an IAC, a control signal will always be produced when there is no target sequence present. When neither IAC signal nor target signal is produced, the PCR reaction fails. Thus, when using a PCR-based method in routine analysis, an IAC, if the concentration adjusted correctly, will indicate false-negative results. It is the false-negative results that turn a risk into a threat for the population, whereas a false-positive result merely leads to a clarification of the presumptive results by re-testing the sample. The European Standardization Committee (CEN), in collaboration with International Standard Organization (ISO) has proposed a general guideline for PCR testing that requires the presence of IAC in the reaction mixture (Anon. 2002a). Therefore, only IAC-containing PCRs may undergo multicentre collaborative trials, which is a prerequisite for standardization. Scientific journals must provide the source of new PCRbased methods suitable for standardization. Therefore, we propose that henceforward the editorial boards of applied microbiology journals require inclusion of an IAC in diagnostic PCR reported in submitted manuscripts. This could be performed by providing a specific section devoted to PCR in their Instruction to Authors.