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Abstract Background The modular approach to analysis of genetically modified organisms (GMOs) relies on the independence of the modules combined (i.e. DNA extraction and GM quantification). The validity of this assumption has to be proved on the basis of specific performance criteria. Results An experiment was conducted using, as a reference, the validated quantitative real-time polymerase chain reaction (PCR) module for detection of glyphosate-tolerant Roundup Ready® GM soybean (RRS). Different DNA extraction modules (CTAB, Wizard and Dellaporta), were used to extract DNA from different food/feed matrices (feed, biscuit and certified reference material [CRM 1%]) containing the target of the real-time PCR module used for validation. Purity and structural integrity (absence of inhibition) were used as basic criteria that a DNA extraction module must satisfy in order to provide suitable template DNA for quantitative real-time (RT) PCR-based GMO analysis. When performance criteria were applied (removal of non-compliant DNA extracts), the independence of GMO quantification from the extraction method and matrix was statistically proved, except in the case of Wizard applied to biscuit. A fuzzy logic-based procedure also confirmed the relatively poor performance of the Wizard/biscuit combination. Conclusions For RRS, this study recognises that modularity can be generally accepted, with the limitation of avoiding combining highly processed material (i.e. biscuit) with a magnetic-beads system (i.e. Wizard).

The aim of this study was to develop a sampling procedure to quantify genetically-modified Round-up Ready (GM RR) soybeans in a lot. Seven lots (35 kg each) fortified with three different percentages of GM RR soybeans (4 lots at 1%, 2 lots at 10%, and 1 lot at 5%) were prepared. Four sampling procedures were carried out on each lot. Different numbers and sizes of increment samples were collected from the lots tested and prepared by homogenising the sample with water. Separate analyses were carried out using quantitative Real Time PCR of split aliquots. The sampling procedures that used ten increment samples provided the best results, in terms of precision and accuracy. [ABSTRACT FROM AUTHOR]

Clostridium difficile (CD) diagnosis is very varied and under discussion. Different research groups disagree on the clinical significance of patients with negative direct toxin and positive polymerase chain reaction (PCR) or even more with direct toxin and glutamate dehydrogenase (GDH) both negatives, but CD detected by toxigenic culture (TC). The objective was to analyze the characteristics of patients with 3 different diagnostic criteria. We compared these 3 groups of patients: group 1: (GDH+/direct toxin+/PCR+), group 2: (GDH+/direct toxin-/PCR+) and group 3: (GDH-/direct toxin-/PCR not done/TC+). The proportion of patients with CD infection (CDI) (not colonization) for groups 1 to 3 was, respectively, 90.3%, 95.4%, and 59.1%. No differences between severity (40.8%, 38.5%, 27.3%), recurrence (20.3%, 24.1%, 7.6%), or related mortality (12.5%, 5.2%, 0%) were found within the 3 groups of patients. Laboratory clinical results should not be used as the only tool to differentiate CDI versus colonization or severity. We recommend that PCR or a second-look TC be performed on all patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: This study examines the impacts of a skin and soft tissue infection (SSTI) management program involving a rapid diagnostic algorithm (Gram stain plus real-time PCR, GeneXpert® MRSA/SA SSTI) performed directly on clinical samples plus antimicrobial stewardship (AMS) counseling of the responsible physician., Methods: Participants were 155 consecutive adult inpatients with SSTI and good quality clinical samples submitted to the microbiology laboratory from April 2016 to January 2017. Results of the rapid test and AMS recommendations were phoned through to the responsible physician. The comparison group was a historical cohort., Results: Most SSTI were surgical wound infections (41.3% vs 38.1% for the intervention and comparison groups respectively) followed by diabetic foot (14.2% and 18.1%), abscesses (13.5% both) and cellulitis (12.9% both). Isolated microorganisms were mostly Gram-negative bacilli (two-thirds), followed by Staphylococcus aureus (SA). The ratio methicillin-susceptible SA (MSSA) to methicillin-resistant SA (MRSA) was 4:1. Improvements in the intervention cohort were: DOT (22.0 vs. 24.3 days, p = 0.007), treatment duration per SSTI episode (14.1 vs. 15.0 days, p = 0.072), treatment cost (433.1 vs. 533.3 €, p = 0.039), length of stay (18.6 vs 20.7 days, p = 0.031), related mortality (1 vs. 4 patients, p = 0.022) and Clostridium difficile infection (CDI) (4 vs. 8 patients, p = 0.050). In 48 cases (31.4%) in the intervention group, advice was given to improve empiric antibiotic treatment., Conclusion: This type of program could help adjust antibiotic treatment when inappropriate, reducing antibiotic use and costs, length of stay, CDI and related mortality., (Copyright © 2018. Published by Elsevier B.V.)

Background: In patients with suspected ventilator-associated pneumonia, a rapid etiological diagnosis is crucial as incorrect or delayed treatment in the first few hours leads to a worse prognosis and a higher mortality rate. This study examines the efficacy of a rapid antibiogram on bronchial aspirates in patients with suspected ventilator-associated pneumonia (VAP)., Methods: The direct gradient diffusion susceptibility testing method (GDM) on respiratory samples was compared with a standard broth microdilution method (BMD) after quantitative cultures in patients with suspicion of VAP. Samples were preselected by Gram staining (for good quality microbiological samples with a predominant single bacterial morphotype). The antibiotics tested were ceftazidime, ceftobiprole, ceftolozane-tazobactam, meropenem, doripenem, and tedizolid., Results: Over a 16-month study period, 445 bronchial aspirate samples were selected from 1376 samples received at our laboratory from 672 adult patients. By direct plating on Mueller-Hinton agar, we recovered 504 (95.5%) of the 528 microorganisms identified by the standard semiquantitative method. Antimicrobial susceptibility testing by GDM was compared with the BMD method in 472 strains (216 Enterobacteriaceae , 138 P. aeruginosa and 118  S. aureus .) and 1652 individual microorganism-antimicrobial agent combinations. There was total agreement between both methods in 98% of combinations. The Kappa index between both techniques was excellent (over 80%). There was only one potential major error for P. aeruginosa susceptibility to ceftazidime., Conclusions: The six GDM strips directly placed on plated bronchial aspirates obtained from patients with a suspicion of VAP provided accurate and reliable susceptibility results within 24 h., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2019.)

Within the frame of the EU-funded MARLON project, background data were reviewed to explore the possibility of measuring health indicators during post-market monitoring for potential effects of feeds, particularly genetically modified (GM) feeds, on livestock animal health, if applicable. Four case studies (CSs) of potential health effects on livestock were framed and the current knowledge of a possible effect of GM feed was reviewed. Concerning allergenicity (CS-1), there are no case-reports of allergic reactions or immunotoxic effects resulting from GM feed consumption as compared with non-GM feed. The likelihood of horizontal gene transfer (HGT; CS-2) of GMO-related DNA to different species is not different from that for other DNA and is unlikely to raise health concerns. Concerning mycotoxins (CS-3), insect-resistant GM maize may reduce fumonisins contamination as a health benefit, yet other Fusarium toxins and aflatoxins show inconclusive results. For nutritionally altered crops (CS-4), the genetic modifications applied lead to compositional changes which require special considerations of their nutritional impacts. No health indicators were thus identified except for possible beneficial impacts of reduced mycotoxins and nutritional enhancement. More generally, veterinary health data should ideally be linked with animal exposure information so as to be able to establish cause-effect relationships., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This review explores the possibilities to determine livestock consumption of genetically modified (GM) feeds/ingredients including detection of genetically modified organism (GMO)-related DNA or proteins in animal samples, and the documentary system that is in place for GM feeds under EU legislation. The presence and level of GMO-related DNA and proteins can generally be readily measured in feeds, using established analytical methods such as polymerase chain reaction and immuno-assays, respectively. Various technical challenges remain, such as the simultaneous detection of multiple GMOs and the identification of unauthorized GMOs for which incomplete data on the inserted DNA may exist. Given that transfer of specific GMO-related DNA or protein from consumed feed to the animal had seldom been observed, this cannot serve as an indicator of the individual animal's prior exposure to GM feeds. To explore whether common practices, information exchange and the specific GM feed traceability system in the EU would allow to record GM feed consumption, the dairy chain in Catalonia, where GM maize is widely grown, was taken as an example. It was thus found that this system would neither enable determination of an animal's consumption of specific GM crops, nor would it allow for quantitation of the exposure., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Patients with cirrhosis are at high risk of Clostridium difficile infection (CDI). Rifaximin is commonly used in cirrhotic patients as prophylaxis for hepatic encephalopathy (HE). Several studies have demonstrated the efficacy of rifaximin in the treatment of CDI; however, resistance to rifaximin has also been reported. Few studies have assessed the risk of developing CDI in cirrhotic patients receiving rifaximin. Our objective was to assess the incidence and characteristics of CDI in patients with cirrhosis, especially in those who received rifaximin., Methods: We assessed the incidence and clinical characteristics of CDI in cirrhotic patients over a 6-year period in our hospital. Medical charts were retrospectively reviewed. Ribotyping and antimicrobial susceptibility testing of all strains against rifaximin were performed., Results: A total of 388 cirrhotic patients were included, of whom 127 patients had at least 1 episode of diarrhea in which a sample was sent to the laboratory. CDI was detected in 46 patients. Fourteen patients (30.4%) were receiving rifaximin as prophylaxis for HE. The main ribotypes detected were 001 (30.4%), followed by 014 (19.6%). Resistance to rifaximin was 34.1% overall, and 84.6% in patients who had received rifaximin. Multivariate analysis showed that rifamycin therapy and ribotype 001 were significant risk factors for having a rifaximin-resistant C. difficile strain., Conclusions: A high percentage of CDI cases were detected in cirrhotic patients receiving rifaximin, mostly owing to selection of rifaximin-resistant C. difficile strains. Clinicians should be aware of the risk of CDI in cirrhotic patients, even in those receiving rifaximin.

Multidrug-resistant (MDR) Klebsiella pneumoniae is one of the most important causes of nosocomial infections worldwide. After the spread of strains resistant to beta-lactams at the end of the previous century, the diffusion of isolates resistant to carbapenems and colistin is now reducing treatment options and the containment of infections. Carbapenem-resistant K. pneumoniae strains have spread rapidly among Italian hospitals, with four subclades of pandemic clonal group 258 (CG258). Here we show that a single Italian hospital has been invaded by three of these subclades within 27 months, thus replicating on a small scale the "Italian scenario." We identified a single clone responsible for an epidemic outbreak involving seven patients, and we reconstructed its star-like pattern of diffusion within the intensive care unit. This epidemiological picture was obtained through phylogenomic analysis of 16 carbapenem-resistant K. pneumoniae isolates collected in the hospital during a 27-month period, which were added to a database of 319 genomes representing the available global diversity of K. pneumoniae strains. Phenotypic and molecular assays did not reveal virulence or resistance determinants specific for the outbreak isolates. Other factors, rather than selective advantages, might have caused the outbreak. Finally, analyses allowed us to identify a major subclade of CG258 composed of strains bearing the yersiniabactin virulence factor. Our work demonstrates how the use of combined phenotypic, molecular, and whole-genome sequencing techniques can help to identify quickly and to characterize accurately the spread of MDR pathogens., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The principles for the safety assessment of genetically modified (GM) organisms (GMOs) are harmonised worldwide to a large extent. There are, however, still differences between the European GMO regulations and the GMO regulations as they have been formulated in other parts of the world. One of these differences relates to the so-called 'stacked GM events', that is, GMOs, plants so far, where new traits are combined by conventional crossing of different GM plants. This paper advocates rethinking the current food/feed safety assessment of stacked GM events in Europe based on an analysis of different aspects that currently form the rationale for the safety assessment of stacked GM events., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the present study the evolution of mechanical strength and the leaching behavior of major and trace elements from activated incinerator bottom ash/Portland cement mixtures were investigated. Chemical and mechanical activation were applied with the purpose of improving the reactivity of bottom ash in cement blends. Chemical activation made use of NaOH, KOH, CaCl(2) or CaSO(4), which were selected for the experimental campaign on the basis of the results from previous studies. The results indicated that CaCl(2) exhibited by far the best effects on the evolution of the hydration process in the mixtures; a positive effect on mechanical strength was also observed when CaSO(4) was used as the activator, while the gain in strength produced by KOH and NaOH was irrelevant. Geochemical modeling of the leaching solutions provided information on the mineral phases responsible for the release of major elements from the hardened materials and also indicated the important role played by surface sorption onto amorphous Fe and Al minerals in dictating the leaching of Pb. The leaching of the other trace metal cations investigated (Cu, Ni and Zn) could not be explained by any pure mineral included in the thermodynamic database used, suggesting they were present in the materials in the form of complex minerals or phase assemblages for which no consistent thermodynamic data are presently available in the literature., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Ochratoxin A is a mycotoxin frequently found as a contaminant both in food and in animal feed. It can reach humans through the food chain and can then be excreted in biological fluids, one of which is human milk; it can therefore be transmitted from mother to child during breast-feeding. This fact prompted us to carry out the present study, aimed at the determination of ochratoxin A in human milk in Italy, as done elsewhere. Fifty samples of human milk were collected randomly over one year and analysed by a high-performance liquid chromatography method. Nine samples were found to contain levels in the range of 1.7-6.6 ng/ml.

A study was undertaken to evaluate the total aflatoxin content in naturally contaminated eggs. Two pools of eggs from laying hens were collected after 2 and 7 days of treatment with aflatoxin B1 (AFB1). An HPLC method has been developed for the determination of AFB1, aflatoxin M1 (AFM1), aflatoxin B2a (AFB2a) and aflatoxicol (Ro) both in free form and after release from water-soluble conjugates. The bound form was cleaned up after acid hydrolysis of the aqueous phase. Eggs collected after 2 days of treatment revealed residues of AFB1, AFB2a and Ro in the organic phase, but none in the aqueous portion. After 7 days of treatment both AFB1 and its hydroxy derivative were found in the organic phase but the aqueous portion showed only hydroxylated metabolites accounting for 35% of the total aflatoxin content.

36 samples representative of the 1981 Italian durum wheat crop were separated by an industrial Italian milling process and the products obtained analyzed by AAS for their Pb, Cd, Cr, Cu, Zn content. The results obtained were: Pb ranging from 0.04 to 0.80 ppm, Cd from 0.02 to 1.39 ppm, Cr from 0.05 to 1.60 ppm, Cu from 1.8 to 39.7 ppm and Zn from 8.8 to 117.6 ppm. Although Cr content was relatively homogeneous, Pb, Cd, Cu and Zn distribution in the various mill products proved to be rather inhomogeneous. Maximum contamination for Pb, Cd and Cr, was appreciably lower than in previous studies.