Egg and Milk Proteins as Hidden Allergens in Food: 5-Year (2010 to 2014) Results of Food Allergen Monitoring in Piedmont, Italy.

Cow's milk and egg allergies are two of the most common food allergies. Manufacturers of food products containing milk or eggs or their derivatives as an ingredient are required by European Union regulations to list their presence on the ingredient label. Under European Union legislation, member states are mandated to carry out food safety monitoring programs to verify compliance with food labeling requirements. Through the Regional Integrated Plan for Food Safety, the Piedmont (Italy) regional authority carries out an annual program to determine the presence of undeclared allergens in foods. In the 5-year period from 2010 to 2014, a total of 1,566 food samples were analyzed for the presence of hidden egg and milk proteins. The average positive percentage was 2.8% (3.6% egg and 2% milk proteins). Comparison between the allergen concentration and the published eliciting dose (ED) for egg proteins (0.03 mg) and for total milk proteins (0.1 mg) indicated a high risk of allergen exposure for sensitized consumers. The calculated exposure was up to 135× (for milk) the ED01 reported in the literature. Food manufacturers will need to improve their allergen control programs to reduce allergen exposure and risk.

Wound botulism after traumatic open fracture in Italy.

Seventeen days after a traumatic open fracture, a Clostridium botulinum wound infection was diagnosed, with self-limiting symptoms. This is the first report of wound botulism in Italy and the authors discuss the possible role of aerosolized contamination of the wound prior to hospital admission.

Consumers' Perception and Knowledge of Food Safety: Results of Questionnaires Accessible on IZSalimenTO Website.

The present survey was undertaken to investigate consumers' knowledge of the main foodborne agents and dietary regimen during pregnancy. Data were collected using monthly questionnaires available on IZSalimenTO website between March 2013 and January 2014. Hepatitis A virus questionnaire: 20 respondents (77%) recognized berries as foodstuff linked to the outbreak of hepatitis A. The majority correctly indicated as precautionary advice to boil berries before consumption. Botulism questionnaire: 29 respondents (62%) indicated pesto as food involved in botulism alert in July 2013. The risk of infant botulism in infant less than 1 year old due to honey consumption is known by 24 respondents (51%). Main foodborne disease questionnaire: the risk of infection by Salmonella after the consumption of foods made with raw eggs is known by the majority (94%; N=17) as well as the treatments to be applied in order to make fresh fish safe from parasites (76%). Pregnancy questionnaire: 20 respondents (74%) believed that washing vegetables and fruits with sodium bicarbonate or chlorate solution is able to inactivate Toxoplasma; only 4 (15%) reported both raw meat and vegetables washed with sodium bicarbonate as food at risk. Results indicate that all consumers should be trained on behaviour and dietary regimen to be adopted in pregnancy and in infant <1 year old. The website may be considered as a useful tool to assess consumers' knowledge: both the news section and the contents published may be a source of information and education for consumers on food safety.

Foodborne botulism associated with home-preserved turnip tops in Italy.

In Italy, foodborne botulism is a rare disease mainly due to home-preserved food. In the case reported here, clinical diagnosis was performed on the basis of clinical signs and referred consumption of home-preserved turnip tops in oil. Definitive diagnosis was performed by detection of botulinum toxin in sera and neuro-toxigenic organisms in stools and leftover food. This case report highlights the need of a high medical awareness, prompt clinical diagnosis, and synergic collaboration among the health authorities for a correct management of botulism as well as disease containment.

Evaluation of IFN-γ polymorphism+874 T/A in patients with recurrent tonsillitis by PCR real time mismatch amplification mutation assay (MAMA real time PCR).

Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP)+874∗T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donor's blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p<0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject (p=0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard.

Evaluation of Hygiene and Safety Criteria in the Production of a Traditional Piedmont Cheese.

Traditional products and related processes must be safe to protect consumers' health. The aim of this study was to evaluate microbiological criteria of a traditional Piedmont cheese, made by two different cheese producers (A and B). Three batches of each cheese were considered. The following seven samples of each batch were collected: raw milk, milk at 38°C, curd, cheese at 7, 30, 60, 90 days of ripening. During cheese making process, training activities dealing with food safety were conducted. Analyses regarding food safety and process hygiene criteria were set up according to the EC Regulation 2073/2005. Other microbiological and chemical-physical analyses [lactic streptococci, lactobacilli, pH and water activity (Aw)] were performed as well. Shiga-toxin Escherichia coli, aflatoxin M1 and antimicrobial substances were considered only for raw milk. All samples resulted negative for food safety criteria; Enterobacteriaceae, E.coli and coagulase-positive staphylococci (CPS) were high in the first phase of cheese production, however they decreased at the end of ripening. A high level of CPS in milk was found in producer A, likewise in some cheese samples a count of >5 Log CFU/g was reached; staphylococcal enterotoxins resulted negative. The pH and Aw values decreased during the cheese ripening period. The competition between lactic flora and potential pathogen microorganisms and decreasing of pH and Aw are considered positive factors in order to ensure safety of dairy products. Moreover, training activities play a crucial role to manage critical points and perform corrective action. Responsible application of good manufacturing practices are considered key factors to obtain a high hygienic level in dairy products.

In vitro CMV-infection model in fresh and glycerolized skin graft.

Viral infections, especially cytomegalovirus (CMV), are a cause of death in burned patients. Aim of this study was to perform an in vitro CMV-infection model comparing fresh and glycerol-treated fibroblasts and keratinocytes. Cells were plated in plates for the two conditions. Each plate was set up with CMV dilutions. Immunofluorescence and real time PCR assays were performed. The assays were negative in both fresh and glycerolized keratinocytes. For fibroblasts, CMV-DNA was positive in both conditions and immunofluorescence test only in fresh cells. Glycerol at 85% confirms its strong virucidal effect as reported also for other viruses.

Detection of Mimivirus in bronchoalveolar lavage of ventilated and nonventilated patients.

BACKGROUND/AIMS: We investigated the prevalence of Mimivirus in bronchoalveolar lavage (BAL) specimens from ventilated versus nonventilated patients. METHODS: The occurrence of Mimivirus DNA was evaluated by two previously developed real-time PCR assays in 69 BAL specimens: 30 from patients on mechanical ventilation for at least 48 h and 39 from nonventilated patients from different clinical settings, including lung transplant recipients. RESULTS: None of the BAL specimens from ventilated and nonventilated patients resulted positive for Mimivirus. CONCLUSION: This study, similarly to other studies that used molecular assays to detect Mimivirus, found no occurrence of the virus in the lower respiratory tract, thus being in contrast to serological investigations which reported a significant association between Mimivirus and the development of pneumonia. Gene polymorphism could explain these results or, alternatively, it could be hypothesized that Mimivirus does not represent a common cause of lower respiratory tract infection in either ventilated or nonventilated patients. Further studies on a larger population of patients from a different clinical setting evaluating both serology and DNA detection in lower respiratory tract specimens, including BAL and possibly tissue samples, could allow a better definition of the epidemiological and pathological role of Mimivirus in the development of pneumonia.

Development of a quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes.

Evidence demonstrating that human rhinovirus (HRV) disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of HRV infections and the increasing number of immunocompromised patients underline the need for rapid and accurate diagnosis of HRV infections. In this study, we developed the first quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes. We described a simple method to accurately quantify RNA target by computing the time to positivity (TTP) values for HRV RNA. Quantification capacity was assessed by plotting these TTP values against the starting number of target molecules. By using this simple method, we have significantly increased the diagnostic accuracy, precision, and trueness of real-time NASBA assay. Specificity of the method was verified in both in silico and experimental studies. Moreover, for assessment of clinical reactivity of the assay, NASBA has been validated on bronchoalveolar lavage (BAL) specimens. Our quantitative NASBA assay was found to be very specific, accurate, and precise with high repeatability and reproducibility.

Human cytomegalovirus glycoprotein B genotyping from bronchoalveolar lavage specimens.

The genes encoding glycoprotein complexes of human cytomegalovirus are often polymorphic; in particular, glycoprotein B (gB), which is essential for both in vivo and in vitro replication, is encoded by the highly polymorphic gene UL55. In this study, the distribution of gB genotypes was investigated in 44 bronchoalveolar lavage specimens from adult patients positive for human cytomegalovirus DNA by a multiplex nested fast PCR able to amplify 5 gB genotypes (gB1-gB5). The distribution of gB genotypes was as follows: 12 (27.3%) gB1, 11 (25%) gB2, 9 (20.4%) gB3, 4 (9.1%) gB4, 0 gB5, and 8 (18.2%) mixed genotypes. No difference in prevalence was found in relation to clinical features, including immunological status, non-transplant or transplant condition, and type of transplanted organ, or in follow-up specimens; while gB4 and gB3 were shown to be significantly more prevalent in patients with respiratory insufficiency, and gB4 and gB2 in those with pneumonia. The prevalence of gB genotypes in the lower respiratory tract was similar to that previously reported using other specimen types and patients, with gB1 found to be the most prevalent. The association of gB genotypes with specific clinical features should be further investigated.

Detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients.

The occurrence of human rhinoviruses (HRV) and its relationship to clinical and histopathological findings were investigated in 127 bronchoalveolar lavage specimens from 36 lung transplant recipients by real-time RT-PCR. In addition, 286 samples from 235 other immunocompromised and immunocompetent patients were also studied. HRV was detected in 41.7% of lung transplant recipients vs 14.5% of other patients (p < 0.0001), and no differences in viral load were observed. Acute respiratory insufficiency was found in 15 cases, three of which were HRV positive (viral load, 6.3 x 10(6) RNA copies/ml in one patient with chronic graft dysfunction). A diagnosis of pneumonia was made in 10 out of 127 cases, two of which were HRV positive (viral load, 10(3)-10(4) in cases of co-infection). Acute rejection was diagnosed in 12 cases, three of which were HRV positive (viral load, 10(3) in two cases of co-infection and 10(5) in a single infection). HRV infection may involve the lower respiratory tract, particularly in the presence of an impaired pulmonary background, such as a transplanted lung. Clinical evaluation should take into account the viral load, with a load of >10(5) possibly being associated with clinical symptoms, although lower loads can be detected in both symptomatic and asymptomatic patients.

Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease and may contribute to the onset and development of Crohn's disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species.

Genotyping of polyomavirus BK by Real Time PCR for VP1 gene.

Polyomavirus BK latently persist in different sites, including the renourinary tract, and may reactivate causing nephropathy in renal transplant recipients or hemorrhagic cystitis in bone marrow recipients. Based on the sequence of the VP1 gene, four genotypes have been described, corresponding to the four serologically differentiated subtypes I-IV, with different prevalence and geographic distribution. In this study, the development and clinical validation of four different Real-Time PCR assays for the detection and discrimination of BKV genotypes as a substitute of DNA sequencing are described. 379 BK VP1 sequences, belonging to the main four genotypes, were aligned and "hot spots" of mutation specific for all the strains or isolates were identified. Specific primers and probes for the detection and discrimination of each genotype by four Real-Time PCR assays were designed and technically validated. Subsequently, the four Real-Time PCR assays were used to test 20 BK-positive urine specimens from renal transplant patients, and evidenced a prevalence of BK genotype I, as previously reported in Europe. Results were confirmed by sequencing. The availability of a rapid and simple genotyping method could be useful for the evaluation of BK genotypes prevalence and studies on the impact of the infecting genotype on viral biological behavior, pathogenic role, and immune evasion strategies.

Improvement of HRV quantification using cRNA-based standards for real time RT-PCR.

Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study, a cRNA-standard-based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us. Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intra- and inter-test variability, sensitivity, and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra- and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07 log was found, showing a significant underrate using plasmid standard-based quantification. The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.

Human cytomegalovirus load in fresh and glycerolized skin grafts.

This study evaluated the detection of Human Cytomegalovirus (HCMV)-DNA in donors' skin samples. HCMV-DNA was quantified in 100 skin specimens, including 50 fresh samples and as many corresponding glycerol-preserved specimens by a home-made Real Time PCR. HCMV-DNA was detected in 19/50 (38%) fresh specimens and 23/50 (46%) glycerol-preserved (p = n.s.). Nevertheless, the mere detection of HCMV-DNA does not imply the presence of infectious virions and therefore does not imply a risk of HCMV transmission, as treatment with glycerol is particularly efficacious in inactivating viral particles. Therefore, HCMV serology confirms its pivotal role in the setting of skin grafting.

Quantitative detection of the new polyomaviruses KI, WU and Merkel cell virus in transbronchial biopsies from lung transplant recipients.

BACKGROUND: Recently, three new polyomaviruses-KI, WU and Merkel cell (MCV)-have been discovered and their detection has been reported in different types of specimens, including respiratory samples, suggesting their shedding in the airways. In lung graft recipients, viral agents are associated with events that may limit the success of transplantation, including organ infection/disease and allograft rejection. AIMS: To evaluate the prevalence of KI, WU and MCV in transbronchial biopsies from lung transplant recipients and investigate the association with clinical and histopathological features. METHODS: The quantitation of new polyomaviruses DNA by real-time PCR and association with clinical and histopathological findings were evaluated in 66 transbronchial biopsies from lung transplant recipients. Results KI, WU and MCV were detected in 9.2%, 12.3% and 33.8% of specimens, respectively; with mean viral load ranging from 81 copies/10(4) cells for WU to 258 for MCV, thus not differing from that previously reported in native lungs. No significant association with clinical and histopathological findings (including acute respiratory insufficiency, interstitial and organising pneumonia, acute and chronic rejection) was found. CONCLUSIONS: Results showed a relatively high frequency of detection of the novel polyomaviruses in transbronchial biopsies from lung transplant recipients. It is likely that this accounted for the positive results found in some cases with different pathological background, although no significant association with a specific clinical and/or histopathological pattern was found.

Real-time RT-PCR assay for the quantitation of polyomavirus BK VP1 mRNA levels in urine.

In renal transplant recipients, polyomavirus BK can reactivate resulting in graft nephropathy. Screening for BK virus replication may allow for earlier interventions with reduced allograft loss. The measurement of urinary cell BKV VP1 mRNA for identify viral replication levels at risk of developing nephropathy has been proposed. In this article, the development, optimization, and standardization of a Taqman real-time RT-PCR assay for the quantitation of BKV VP1 mRNA levels in urine is described. Subsequently, the method has been validated on urine specimens obtained from renal transplant recipients. The use of VP1 mRNA measurement as a marker for viral replication and a tool for noninvasive diagnosis of nephropathy should be regarded with great caution, given the potentially limited positive predictive value and the drawbacks associated with the complexity of the real-time RT-PCR assay requiring an expert well trained operator and the relatively poor cost-efficiency ratio.

Detection of human herpesvirus-7 DNA in bronchoalveolar lavage.

OBJECTIVES: Human herpesvirus-7 (HHV-7) is a highly seroprevalent virus that, following primary infection, establishes latency or persistence in some tissues, including lung. The aim of this study was to investigate the prevalence of HHV-7 in the lower respiratory tract of hospitalized adult patients. METHODS: The prevalence of HHV-7 DNA was determined by quantitative real-time PCR in 212 bronchoalveolar lavage (BAL) samples obtained from 153 patients. The molecular epidemiology and clinical role of HHV-7 were evaluated. RESULTS: HHV-7 DNA was positive in 44 of 212 specimens (20.7%), obtained from 40 of 153 patients (26.1%), in particular 22/68 (32.35%) and 18/86 (20.9%) in transplant and non-transplant patients, respectively (1 patient evaluated both before and after transplantation). No significant difference according to transplant condition or discharge diagnosis was found. Viral load was >100,000 genome equivalents/ml BAL in 6/22 (27.3%) transplant recipients and 4/18 (22.2%) non-transplant patients (p = n.s.). CONCLUSIONS: The evaluation of HHV-7 DNA in BAL may be useful to investigate its potential role in lower respiratory tract infection, alone or in association with other viral and/or non-viral pathogens and to distinguish latency from reactivation.

Non-organ-specific and anti-endothelial antibodies in relation to CMV infection and acute rejection in renal transplant recipients.

The presence of non-organ-specific (NOSA) and anti-endothelial antibodies (AECAs) and the onset of rejection in relation to cytomegalovirus (CMV) infection was investigated in 96 renal transplant recipients: 48 CMV pp65-antigenemia-negative (group 1) and 48 positive (group 2). The presence of autoantibodies (autoAbs) was evaluated before and following renal transplantation (first three months) by indirect immunofluorescence. Before transplantation, none of the patients was positive to AECAs, while eight (8.3%) were positive to NOSAs. Post-transplantation, AECA were found in none of patients from group 1 vs. 15/48 (31.2%) from group 2 (p<0.05); NOSAs were detected in 9/48 (18.8%) and 9/48 patients from group 1 and 2, respectively. An acute rejection was diagnosed in ten cases: six of interstitial type (antigenemia-, and AECA-negative; two NOSA-positive); four of vascular type (all of them NOSA-negative, 3/4 antigenemia-, and AECA-positive). CMV infection did not seem to be significantly associated with the appearance of NOSAs, while there was a significant correlation with the occurrence of AECAs. No significant correlation was found between acute rejection and the occurrence of NOSAs, while 75% of the cases of vascular rejection was associated to CMV infection and AECA-positivity, suggesting the pathogenic role of CMV-mediated endothelial damage.

Development of real time RT-PCR assays for detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes.

Rapid detection and subtyping of H5 and H7 subtypes influenza A viruses are important for disease control in poultry and potential transmission to humans. Currently, virus isolation and subsequent HA and NA subtyping constitute the standard for avian influenza viruses detection and subtype identification. These methods are highly accurate and sensitive but are also laborious and time-consuming. Reverse transcription PCR and real time reverse transcription PCR assays, suitable tests for rapid detection, have previously been used for the specific diagnosis of H5 and H7 viruses, however, at present, no primer and probe sets are available for the identification of all H5 and H7 strains. Herein, we have developed specific and sensitive real time reverse transcription PCR assays for the detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes and we have also compared these molecular assays with viral isolation in terms of sensitivity. Our results demonstrate that the real time reverse transcription PCR assays are more sensitive, specific, less expensive compared to viral isolation. In conclusion, molecular assays could represent an useful tool for rapid detection and screening of H5 and H7 isolates during influenza A virus outbreaks alternatively to viral isolation.