Comparison of droplet digital PCR vs real-time PCR for Yersinia enterocolitica detection in vegetables.

Yersiniosis - the 4th most commonly reported zoonosis in the European Union - is caused by the consumption of food contaminated with the bacterium Yersinia enterocolitica. The number of human cases and contaminated food samples is probably underestimated since conventional molecular methods currently proposed for Yersinia enterocolitica detection proved to have several limitations. Critical issues associated with the detection of Yersinia enterocolitica in meat and/or meat product has already been investigated, whereas data on the possible limits of the molecular methods for Yersinia enterocolitica detection in vegetables are still lacking. According to ISO method (ISO 18867:2015), real-time polymerase chain reaction (rtPCR) should be adopted for Yersinia enterocolitica detection, even if it proved to be affected by some biases. Recently, Droplet Digital PCR (ddPCR) has been introduced as a useful tool to detect and quantify different pathogenic bacteria in complex food matrices. However, its potential application for Yersinia enterocolitica detection in vegetables has never been investigated before. In the present study two molecular platforms (rtPCR and ddPCR) were used to evaluate the pathogen's behaviour in experimentally contaminated leafy greens (Lactuca sativa L.) and to assess the rate of detection achievable after the incubation for eleven days at different temperatures. By comparing, noticeable differences emerged between the two technical approaches: only ddPCR allowed the detection of the pathogen in leafy greens when contaminated at low levels. Moreover, results of the present work highlighted the importance of length and temperature of incubation on the survival and/or the growth of Yersinia enterocolitica in vegetables: at 18 and 25 °C the concentration of the pathogen considerably decreases along incubation. Based on data, the use of rtPCR leads to an underestimation of the true prevalence of pathogenic Y. enterocolitica in vegetables, while temperature and time currently proposed for Y. enterocolitica (25 °C for 24 h), allow optimizing detection. To conclude, ddPCR may be undoubtedly proposed as a reliable alternative strategy for the quick detection of the pathogen in food samples.

An integrated approach to control Cystic Echinococcosis in southern Italy.

Cystic echinococcosis (CE) is a severe zoonosis, caused by the larval stage of the tapeworm Echinococcus granulosus. This helminth infection is of increasing public health and socio-economic concern due to the considerable morbidity rates that cause economic losses both in the public health sector and in the livestock industry. Control programmes against E. granulosus are considered long-term actions which require an integrated approach and high expenditure of time and financial resources. Since 2010, an integrated approach to control CE has been implemented in a highly endemic area of continental southern Italy (Campania region). Innovative procedures and tools have been developed and exploited during the control programme based on the following strategies: i) active and passive surveillance in livestock (using geospatial tools for georeferencing), ii) diagnosis in dogs (using the FLOTAC techniques and molecular analysis), iii) targeted treatment of farm dogs (using purpose-built confinement cages), iv) early diagnosis in livestock (by ultrasonography), v) surveillance in humans (through hospital discharge records analysis), vi) monitoring the food chain (analysing raw vegetables), vii) outreach activities to the general public (through dissemination material, e.g. brochures, gadgets, videos, virtual reality). Over eight years, the integrated approach and the new strategies developed have resulted in a noteworthy reduction of the parasite infection rates in livestock (e.g. up to 30 % in sheep). The results obtained so far highlight that using a one health multidisciplinary and multi-institution effort is of pivotal importance in preparing CE control programmes at regional level and could be extended to other endemic Mediterranean areas.

The potential role of trans-resveratrol/carboxymethylated (1.3/1.6)-ß-d-glucan minimizing symptoms and improve healing after functional endoscopic sinus surgery.

OBJECTIVE: This study aims to evaluate the effect of trans-resveratrol/carboxymethylated (1.3/1.6)-ß-d-glucan administered via nasal, after FESS, assessing nasal respiratory distress and nasal mucosa healing. PATIENTS AND METHODS: We enrolled 70 patients, from March 2019 to February 2020, with chronic nasal obstruction not responding to medical therapy and candidates to endoscopic nasal surgery. Patients were divided in two non-randomized groups: group A treated with trans-resveratrol/carboxymethylated (1.3/1.6)-ß-d-glucan administered via nasal, and group B treated with 0.9% nasal irrigation saline. Patients were clinically evaluated, in post-operative period, at 7 (T0), 15 (T1), and 30 days (T2) with fibroendoscopy. The CRS (chronic rhinosinusitis) questionnaire (Snot 20) was administrated at T0, T1, and T2. The findings were scored with respect to middle turbinate edema. In both Groups, the inferior turbinate's medial aspect was scraped using a sterile disposable Rhino-probe mucosal curette (Arlington Scientific, Inc., Springville, UT, USA) at T0, T1, and T2. RESULTS: Group A showed an improvement in Snot 20 in T1 and T2 both. The reduction of the mucosal edema and nasal secretion has been statistically significant in the Group A. A slight cell reduction was observed at T2 with respect to T1. This decreased pattern is more evident in nasal scraping from Group A. The appearance of epithelial cells at T2 of Group A is consistent with the reduction of inflammatory cells. CONCLUSIONS: We can assert that in Group A it appears less evident the presence of edema, nasal congestion and crusts, resulting in a quick recover.

Yersinia enterocolitica detection in pork products: Evaluation of isolation protocols.

Conventional methods for Yersinia enterocolitica detection in food samples are generally considered inadequate. Problems arise from the presence of the so-called "background flora", coupled to the low contamination level of the pathogen. Since, data on the microbial ecology occurring in competitive microflora are still lacking, MALDI TOF MS was used for strains 'identification after enrichment in PSB or ITC broths, and after plating on selective CIN medium at different incubation times. SYBR Green Real time PCR was used for the Y. enterocolitica strains' detection (4/O:3, 1A/O:5) in experimentally contaminated foods, as well as in naturally contaminated samples. A higher number of different bacterial genera (10 on CIN and 18 on PCA) was recorded after enrichment in PSB, whilst enrichment in ITC led to recovery of 6 and 10 genera on CIN and PCA, respectively. Yersiniaceae was the dominant family on the first day of incubation, but on the second day the percentage of isolation considerably decreased. By testing experimentally contaminated samples, substantial difficulties were encountered. The biotype 1A was always detected, whereas strain 4/O:3 proved to be poorly competitive. Based on the data, the enrichment media PSB and ITC, currently proposed for Y. enterocolitica detection, need to be improved to promote a successful pathogen's recovery.

Endogenous nerve growth factor stimulation: effects on auditory pathway neural cells in a mouse model.

OBJECTIVE: In the present study, we investigated whether high-pressure hypotonic saline solution (Hphss) affects the basal level of Nerve Growth Factor (NGF) and expression of receptors in the cochlea, bark earing, retina, and visual cortex. MATERIALS AND METHODS: For this study, we used three weeks old female Sprague Dawley (SD) rats (n = 12). Rats were housed in polypropylene cages and were kept under standard conditions (12 h light:12 h dark cycle) with free access to water and food (Purina chow food). A specific dispenser was employed to deliver sterile hypotonic saline at high pressure (pressing emission level (PEL): 7 g/s; emission time (ET): 0.5 s). Rats were divided into two groups: untreated (n = 6) and treated with Hphss (n = 6), three times per day, for 10 consecutive days. Treatment was performed in both nostrils with 50 µl of Hphss using a microsyringe equipped with a plastic tip. RESULTS: We observed a significant enhancement in the level of NGF in the cochlea and bark earing, but not in the retina and visual cortex. This is likely because the nasolacrimal duct pathway does not appear to have an effect on the retina, and the visual cortex appears to be too far from the cribriform plate to be reached by nasal NGF. CONCLUSIONS: This treatment can significantly protect and/or delay degeneration of cochlear auditory NGF-target cells. It is free from side effects and can be used in chronic diseases for as long as needed. It remains to be investigated whether the effects of short-term therapy are long-lasting, or if the treatment must be repeated.

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.

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.

Breathing training on lower esophageal sphincter as a complementary treatment of gastroesophageal reflux disease (GERD): a systematic review.

OBJECTIVE: Gastroesophageal reflux disease (GERD) represents one of the most common gastrointestinal disorders, but is still a challenge to cure. Proton pump inhibitors (PPIs) are currently the GERD's standard treatment, although not successful in all patients; some concerns have been raised regarding their long term consumption. Recently, some studies showed the benefits of inspiratory muscle training in increasing the lower esophageal sphincter pressure in patients affected by GERD, thereby reducing their symptoms. MATERIALS AND METHODS: Relevant published studies were searched in Pubmed, Google Scholar, Ovid or Medical Subject Headings using the following keywords: "GERD" and physiotherapy", "GERD" and "exercise", "GERD" and "breathing", "GERD and "training". RESULTS: At the end of our selection process, four publications have been included for systematic review. All of them were prospective controlled studies, mainly based on the training of the diaphragm muscle. GERD symptoms, pH-manometry values and PPIs usage were assessed. CONCLUSIONS: Among the non-surgical, non-pharmacological treatment modalities, the breathing training on diaphragm could play an important role in selected patients to manage the symptoms of GERD.

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.

Salso-sulphide thermal water in the prevention of recurrent respiratory infections in children.

Recurrent respiratory infections (RRI) represent a social problem for both the pharmaco-economic impact and the burden on the family. Thermal water is popularly well accepted. However, there is no scientific evidence of its preventive activity on recurrent respiratory tract infections (RRI). Therefore, the purpose of this study was to evaluate the effects of Agnano thermal water nasal irrigation on RRI prevention in children.A total of 107 children (70 males, mean age 4.5 plus minus1.2 years) with RRI were enrolled in the study. At baseline, children were randomly assigned to the treatment with: A) inhaled crenotherapy with salso-sulphide water or B) isotonic saline (NaCl 0.9 percent). Inhaled therapy was performed using nasal washing by Rino-jet (ASEMA srl, Milan, Italy) b.i.d. for 12 days. Nasal washing lasted 2 minutes per nostril. Immediately before washing, children inhaled 1 l of water by stream inhalation per 2 minutes. Crenotherapy was capable of significantly reducing: the number of respiratory infections, nasal symptoms, neutrophil and bacteria count, turbinate and adenoidal hypertrophy, presence of biofilm, and blockage of ostiomeatal complex (OCM). In conclusion, this study provides the first evidence that Agnano crenotherapy may be capable of preventing RRI in children as it exerts some positive effects, such as reduction of nasal obstruction, OCM blockage, biofilm, and inflammatory events.

A novel synthetic peptide from a tomato defensin exhibits antibacterial activities against Helicobacter pylori.

Defensins are a class of cysteine-rich proteins, which exert broad spectrum antimicrobial activity. In this work, we used a bioinformatic approach to identify putative defensins in the tomato genome. Fifteen proteins had a mature peptide that includes the well-conserved tetradisulfide array. We selected a representative member of the tomato defensin family; we chemically synthesized its γ-motif and tested its antimicrobial activity. Here, we demonstrate that the synthetic peptide exhibits potent antibacterial activity against Gram-positive bacteria, such as Staphylococcus aureus A170, Staphylococcus epidermidis, and Listeria monocytogenes, and Gram-negative bacteria, including Salmonella enterica serovar Paratyphi, Escherichia coli, and Helicobacter pylori. In addition, the synthetic peptide shows minimal (<5%) hemolytic activity and absence of cytotoxic effects against THP-1 cells. Finally, SolyC exerts an anti-inflammatory activity in vitro, as it downregulates the level of the proinflammatory cytokines TNF-α and IFN-γ.

Occurrence of toxigenic Clostridium difficile in edible bivalve molluscs.

Clostridium difficile is an anaerobic bacterium commonly considered to be responsible for antibiotic-associated gastrointestinal diseases, ranging from diarrhea of varying severity to pseudomembranous colitis. The aim of this study was to assess the occurrence of C. difficile in marine edible bivalve molluscs, which, as filter feeding organisms, are able to accumulate particles suspended in water, including microorganisms. Samples of Mytilus galloprovincialis, Tapes philippinarum, and Venus verrucosa were collected from mussel farms and fishmongers in the province of Naples (Southern Italy). C. difficile was found in 49% of the 53 samples investigated. Sixteen isolates were grouped in 12 known different PCR ribotypes (001, 002, 003, 010, 012, 014/020, 018, 045, 070, 078, 106, and 126), whereas 10 additional isolates were grouped in 8 new PCR riboprofiles. Two toxinotypes (0 and V) were found. Fifty eight percent of the isolates were toxigenic. These findings indicate that toxigenic C. difficile strains can be isolated in bivalve molluscs. Marine filter feeding organisms, therefore, may be considered as reservoir of toxigenic strains of C. difficile. The ingestion of raw or poorly cooked contaminated seafood and the high temperature resistance of the spore-forming C. difficile could represent an important source of exposure and pose human health concern.

Detection of Coxiella burnetii in buffaloes aborted fetuses by IS111 DNA amplification: a preliminary report.

The aim of this study was to evaluate by PCR analyses the presence of Coxiella burnetii infection in fetuses of water buffalo (Bubalus bubalis) in the Campania region (Southern Italy). Samples were collected only from aborted fetuses and C. burnetii presence was evaluated by one-tube nested PCR amplification of the IS111 repetitive element. Of the 164 fetuses examined 14 (17.5%) were positive after DNA amplification, showing that C. burnetii occurs in this population of water buffaloes. However, more extensive prevalence studies need to be carried out to define the role of buffaloes as reservoirs for this pathogen and also the role of C. burnetii as an abortive agent in this animal.

Safety and effectiveness of combining carotid artery stenting with cardiac surgery: preliminary results of a single-center experience.

AIM: Optimal strategy (staged or combined) for the treatment of patients with concurrent severe carotid and cardiac disease is still controversial. Moreover, carotid artery stenting (CAS), has become a valid alternative to carotid endarterectomy (CEA) and has been proposed for the treatment of cardiac patients. The authors report the preliminary results of a new therapeutic strategy consisting in combined CAS and cardiac surgery. METHODS: An initial series of 22 patients underwent combined CAS and cardiac surgery in the same operating room and under general anesthesia. All filter-protected CAS procedures were performed under only heparin and aspirin. A cervical approach (3-cm cervicotomy) was used in patients with documented vessel tortuosity or severe aorto-iliac occlusive arteriopathy. In all the other cases a femoral access was used. A double antiplatelet regimen was initiated in the early postoperative period, once major bleedings were excluded. RESULTS: Among the 22 patients who underwent this combined procedure, no deaths, no myocardial infarctions and one controlateral stroke (overall complication rate: 4.5%) were observed. This stroke was observed after transcervical CAS, coronary artery bypass and mitral valve replacement. No major postoperative bleedings nor stent thrombosis were observed. CONCLUSIONS: Combined carotid stenting and cardiac surgery, performed in the same operating room under only heparin and aspirin, seems a safe and effective strategy for the treatment of patients with concomitant carotid and cardiac disease.

The role of cattle in the epidemiology of Echinococcus granulosus in an endemic area of southern Italy.

An epidemiological and molecular survey was conducted to investigate the role of cattle in the transmission chain of cystic echinococcosis (CE) in the Campania region of southern Italy. Out of a total of 434 cattle examined for CE, 45 (10.4%) were found infected. A total of 363 cysts were collected from the infected animals: 239 in the liver and 124 in the lungs. The cysts were either sterile (42.7%) or calcified/caseous (57.3%); no fertile cysts were found. Most of the cysts had sizes <3 cm (77.1%) and were unilocular (78.8%). The results of the linear regression model did not show any significant correlation between the age of infected cattle and the number of cysts. The sequencing of the mitochondrial cytochrome C oxidase subunit 1 (CO1) gene of 40 hydatid cysts produced sequences of 419 bp for each sample analyzed. Alignment of the obtained sequences with those present in GenBank showed 100% identity with the common sheep G1 (n=21 cysts), the Tasmanian sheep G2 (n=2 cysts), and the buffalo G3 (n=17 cysts) strains, which constitute the species Echinococcus granulosus sensu stricto. The findings reported in the present study show that CE is widespread in cattle bred in the Campania region of southern Italy. However, the absence of fertile cysts and of the cattle strain (G5, E. ortleppi) suggests that cattle would not have any role in the persistence of this important zoonosis but rather a role as indicators of CE infection in this endemic area.

Molecular update on cystic echinococcosis in cattle and water buffaloes of southern Italy.

Cystic echinococcosis (CE)--caused by the larval stage (hydatid cyst) of the cestode Echinococcus granulosus--is one of the most widespread zoonoses of veterinary and medical importance. Molecular techniques have allowed the identification of 10 different genotypes (G1-G10) of the parasite. The present paper is an update regarding the E. granulosus genotypes infecting water buffaloes and cattle bred in the Campania region of southern Italy. The molecular study was performed on 30 hydatid cysts (11 from water buffaloes and 19 from cattle). Two different mitochondrial DNA genes, namely the cytochrome c oxidase subunits 1 and the 12S ribosomal DNA (12S rDNA) were used as genetic markers. Three different genotypes of E. granulosus were unequivocally identified, i.e. the G1 (common sheep), G2 (Tasmanian sheep) and G3 (buffalo) genotypes, as well as some G1 and G2 variants. It should be noted that the present study demonstrated for the first time: (i) the presence of the G2 genotype in water buffaloes from a Mediterranean area; and (ii) the fact that the analysed portion of the 12S rDNA gene can not discriminate between the G2 and G3 genotypes of E. granulosus. The finding of the G1, G2 and G3 genotypes in large ruminants from southern Italy is of epidemiological relevance and immediate public health importance because of their recognized infectivity in humans.

ENA screen chemiluminescence immunoassay: a random access method in autoimmunology.

Recently, autoimmunity, due to an increase in examination requests, has become an independent area of laboratory research, which needs management optimization in terms of quality, time, and flexibility. Therefore, we have evaluated the screening of extractable nuclear antigens (ENA) antibodies both with a chemiluminescence immunoassay and the enzyme-linked immunosorbent assay (ELISA) method, which was used in our laboratory, as a reference kit. The most important difference between these two methods is the possibility of processing serum samples with a random access system, which is different from batch methods.

Characterization of the 28S and the second internal transcribed spacer of ribosomal DNA of Dicrocoelium dendriticum and Dicrocoelium hospes.

Isolates of Dicrocoelium dendriticum (n=150) from sheep and cattle bred in southern Italy and isolates (n=5) of D. hospes from a Bos indicus from Senegal were characterized genetically. The 28S region and the second internal transcribed spacer (ITS-2) plus flanking 5.8S and 28S sequences (ITS-2+) of ribosomal DNA (rDNA) were amplified by polymerase chain reaction and sequenced from individual flukes. Regarding the 28S rDNA, sequences of 568 and 581 bp were obtained for D. dendriticum and D. hospes, respectively. No intraspecific variation was observed between the 28S rDNA of all the D. dendriticum specimens studied and the D. dendriticum 28S rDNA sequence present in GenBank. However, intraspecific variation was observed in the 28S rDNA of the D. hospes specimens compared to the sequence present in GenBank. Regarding the ITS2+ rDNA, sequences of 402 and 428 bp were obtained for D. dendriticum and D. hospes, respectively; both sequences were deposited in GenBank. Variations intra- and interpopulation were observed for D. dendriticum, whereas 100% identity was observed in all the ITS2+ sequences of D. hospes. With respect to the interspecific variations, the ITS-2+ of D. dendriticum and D. hospes differed in 33 positions. The findings of the present study showed an ITS2+ sequence variability (8.2-8.5%) between D. dendriticum and D. hospes, thus demonstrating the utility of this sequence to discriminate the two species.