Author Correction: Unrevealed genetic diversity of GII Norovirus in the swine population of North East Italy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rapid detection and quantification of plasmid-mediated colistin resistance genes (mcr-1 to mcr-5) by real-time PCR in bacterial and environmental samples.

AIM: The aim of the study was to validate a rapid method to detect and quantify colistin resistance genes (mcr-1 to mcr-5) by real-time polymerase chain reaction (RT-PCR) in diverse matrices. METHODS AND RESULTS: The detection limit of two newly designed SYBR Green real-time PCR assays for mcr-4 and mcr-5 and of previously published protocols for mcr-1 to mcr-3 was assessed using serial dilutions of reference strains. The assays could detect all five mcr genes with the lower limit of 102 copy numbers. Escherichia coli isolates (n = 1062) and environmental samples (n = 93) were tested for the presence of mcr genes. The assays enabled the detection of colistin resistance genes both in bacterial isolates and in complex environmental samples. CONCLUSIONS: This method represents a set of sensitive, rapid and effective assays for the screening of colistin resistance directly from the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Colistin is an antimicrobial commonly used in animals and has recently emerged as a last-resort treatment in humans. Plasmid-mediated mcr genes confer resistance to colistin and represent a major threat for public health since they can be easily disseminated through horizontal gene transfer. The rapid and sensitive detection of mcr genes is of utmost necessity.

Unrevealed genetic diversity of GII Norovirus in the swine population of North East Italy.

Noroviruses (NoVs) are one of the major causative agents of non-bacterial gastroenteritis in humans worldwide. NoVs, belonging to Caliciviridae, are classified into ten genogroups (G) and eight P-groups based on major capsid protein (VP1) and of the RNA-dependent-RNA-polymerase (RdRp), respectively. In swine, the main genogroup and P-group identified are GII and GII.P; which can infect humans too. To date, only one case of GIIP.11 have been identified in swine in Italy while the circulation of other P-types is currently unknown. In the present study, 225 swine faecal samples were collected from 74 swine herds in Veneto region through on-farm monitoring. NoV circulation was particularly high in older pigs. The phylogenetic analysis showed the co-circulation of NoVs belonging to two different P-types: GII.P11 and GII.P18, here described for the first time in Italy, presenting an extensive genetic diversity, never described before worldwide. Distinct NoV genetic subgroups and unique amino acid mutations were identified for each P-type for the first time. This study demonstrated the co-circulation of diverse swine NoVs subgroups in Italy, raising questions on the origin of such diversity and suggesting that continuous monitoring of swine NoVs is needed to track the emergence of potentially zoonotic viruses by recombination events.

N-glycosylation of infectious bronchitis virus M41 spike determines receptor specificity.

Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants. In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity.

Attenuated live infectious bronchitis virus QX vaccine disseminates slowly to target organs distant from the site of inoculation.

Infectious bronchitis (IB) is a highly contagious respiratory disease of poultry, caused by the avian coronavirus infectious bronchitis virus (IBV). Currently, one of the most relevant genotypes circulating worldwide is IBV-QX (GI-19), for which vaccines have been developed by passaging virulent QX strains in embryonated chicken eggs. Here we explored the attenuated phenotype of a commercially available QX live vaccine, IB Primo QX, in specific pathogens free broilers. At hatch, birds were inoculated with QX vaccine or its virulent progenitor IBV-D388, and postmortem swabs and tissues were collected each day up to eight days post infection to assess viral replication and morphological changes. In the trachea, viral RNA replication and protein expression were comparable in both groups. Both viruses induced morphologically comparable lesions in the trachea, albeit with a short delay in the vaccinated birds. In contrast, in the kidney, QX vaccine viral RNA was nearly absent, which coincided with the lack of any morphological changes in this organ. This was in contrast to high viral RNA titers and abundant lesions in the kidney after IBV D388 infection. Furthermore, QX vaccine showed reduced ability to reach and replicate in conjunctivae and intestines including cloaca, resulting in significantly lower titers and delayed protein expression, respectively. Nephropathogenic IBVs might reach the kidney also via an ascending route from the cloaca, based on our observation that viral RNA was detected in the cloaca one day before detection in the kidney. In the kidney distal tubular segments, collecting ducts and ureter were positive for viral antigen. Taken together, the attenuated phenotype of QX vaccine seems to rely on slower dissemination and lower replication in target tissues other than the site of inoculation.

Subpopulations in aMPV vaccines are unlikely to be the only cause of reversion to virulence.

Avian metapneumovirus (aMPV) infects respiratory and reproductive tracts of domestic poultry, often involving secondary infections, and leads to serious economic losses in most parts of the world. While in general disease is effectively controlled by live vaccines, reversion to virulence of those vaccines has been demonstrated on several occasions. Consensus sequence mutations involved in the process have been identified in more than one instance. In one previous subtype A aMPV candidate vaccine study, small subpopulations were implicated. In the current study, the presence of subpopulations in a subtype B vaccine was investigated by deep sequencing. Of the 19 positions where vaccine (strain VCO3/50) and progenitor (strain VCO3/60616) consensus sequences differed, subpopulations were found to have sequence matching progenitor sequence in 4 positions. However none of these mutations occurred in a virulent revertant of that vaccine, thereby demonstrating that the majority progenitor virus population had not survived the attenuation process, hence was not obviously involved in any return to virulence. However within the vaccine, a single nucleotide variation was found which agreed with consensus sequence of a derived virulent revertant virus, hence this and other undetected, potentially virulent subpopulations, can be involved in reversion. Much deeper sequencing of progenitor, vaccine and revertant may clarify whether problematic virulent subpopulations are present and therefore whether these need to be routinely removed during aMPV vaccine preparation prior to registration and release.

A sensitive, reproducible, and economic real-time reverse transcription PCR detecting avian metapneumovirus subtypes A and B.

Use of real-time PCR is increasing in the diagnosis of infectious disease due to its sensitivity, specificity, and speed of detection. These characteristics make it particularly suited for the diagnosis of viral infections, like avian metapneumovirus (AMPV), for which effective control benefits from continuously updated knowledge of the epidemiological situation. Other real-time reverse transcription (RT)-PCRs have been published based on highly specific fluorescent dye-labeled probes, but they have high initial cost, complex validation, and a marked susceptibility to the genetic variability of their target sequence. With this in mind, we developed and validated a SYBR Green I-based quantitative RT-PCR for the detection of the two most prevalent AMPV subtypes (i.e., subtypes A and B). The assay demonstrated an analytical sensitivity comparable with that of a previously published real-time RT-PCR and the ability to detect RNA equivalent to approximately 0.5 infectious doses for both A and B subtypes. The high efficiency and linearity between viral titer and crossing point displayed for both subtypes make it suited for viral quantification. Optimization of reaction conditions and the implementation of melting curve analysis guaranteed the high specificity of the assay. The stable melting temperature difference between the two subtypes indicated the possibility of subtyping through melting temperature analysis. These characteristics make our assay a sensitive, specific, and rapid tool, enabling contemporaneous detection, quantification, and discrimination of AMPV subtype A and B.

[The general practitioner and clinical records: a survey of the knowledge of users of a software package]. / Medico di base e cartelle cliniche: indagine conoscitiva su utenti in possesso di un pacchetto software.

The FATMA project--subproject 6, Line of Research 2--aims at implementing a new computerized information network for general practitioners using the same kind of software and homogeneously located throughout Italy. Thus, patients' case form data can be collected for public health purposes. After a study of literature in order to check whether other countries had implemented similar projects and what they consisted of, a survey based on a computerized questionnaire was carried out on a group of g.p.s. who had already received a software for the management of their clinical and professional activities from Janssen Informedica. This survey aimed at judging some aspects of users in order to select potential monitors of the information network who would test a new software. The analysis regards both g.p.s' social and professional data and the use of a computer package with reference to the quality and quantity of the clinical data they input. 2,010 questionnaires were filled in all their parts and mailed back to Informedica. It came out that g.p.s. believe more and more they can improve the organization and management of their work by using a software equipped with routines and easy access procedures so that clinical data, diagnostic reports and treatment can be collected, placed on files and updated. Thus, a new software should be developed. While meeting g.p.s' needs, it must overcome the limits of packages now available concerning the possibility of carrying out epidemiological studies. The software can become a tool to collect clinical data of patients, diseases and diagnostic procedures through standard protocols directly from g.p.s records.

A new community-oriented network in Italy for the collection and processing of clinical and epidemiological information.

This study illustrates a project to implement a clinical information system based on a general practitioner's network covering the national territory. The project is important because there are no other such systems in operation in Italy. Moreover, as is well known, both the quality and the quantity of health information made available by the National Health Service are far from optimal. This community-oriented network enables end-users to share software for the management of outpatient clinics that is produced by Informedica Janssen and distributed throughout Italy to over 16,000 physicians. The features of potential participants in the network as well as the software they have adopted are described.

[Possibility of using B-mode echography in the early diagnosis of the carotid localization of atherosclerosis]. / Possibilità d'impiego dell'ecografia B-mode nell'accertamento precoce delle localizzazioni carotidee dell'aterosclerosi.

B-Mode ultrasonography is an important complementary tool in the early diagnosis of carotid pre-atheromatous vascular disease. The authors report their personal experience concerning 86 patients affected or not by neurological transitory or persistent symptoms, and point out two main semeiological remarks of endoluminal pathologic conditions. The findings obtained are related to the more recent physiopathological and pathological-anatomical data related the growth and the evolution of atheromatous lesions of the carotid district.

Biologic effects of ultrasound on liver cells of rat embryos.

Experiments on the biologic effects of ultrasound (US) were carried out on rat embryos exposed for 120 s to a US beam showing features similar to those already employed for diagnostic purposes. Electron microscopic investigation of the liver parenchyma of rat embryos confirmed results obtained from adult guinea pigs with the same duration of exposure, i.e. diffuse vacuolization of cytoplasma and varying degrees of mitochondrial damage.

Gold storage in the liver: appearance on CT scans.

The authors present one case in which high attenuation values in the liver parenchyma on computed tomographic scans were found in a patient who had been previously treated with colloidal gold for rheumatoid arthritis. Reticuloendothelial storage of gold was proved by biopsy.

[Quantitative evaluation of the phase image in radionuclide ventriculography ]. / Valutazione quantitativa dell'immagine di fase nella ventricolografia radionuclidica.

A functional image is a representation that enhances a particular characteristic of organ behaviour (feature enhancement) with a concomitant reduction or elimination of other informations. The functional image of phase (PI), obtained by Fourier analysis, may be interpreted to represent the onset of counts reduction or contraction in each region of the heart. Phase Spreading (PS), is a parameter derived from PI and represents ventricular temporal behaviour including some spatial information. Phase analysis and PS are very sensitive tools in the diagnosis and characterization of cardiac pathology. PS is a specific indicator of CAD, allows the introduction of normal ranges and is an example of quantitative evaluation of functional images.

[Experimental studies on possible biological effects of ultrasonic diagnosis: ultrastructural analysis of rat embryo]. / Ricerche sperimentali su ecodiagnostica e possibili effetti biologici: analisi ultrastrutturale su embrione di ratto.

Within the limits of a systematic research on the biological effects of the ultrasounds, the authors refer on their personal experience on rat's embrions treated, for 120 seconds, with a ultrasonic beam having the common characteristics used in diagnostic investigations. The ultra-structural studies on the hepatic parenchyma substantially confirm the previous results obtained in the adult cavy, exposed for the same period of time, i.e. a diffuse cytoplasmic vacuolization and an unstable mitochondrial damage. Various suggestions are advanced on the particular sensibility that the mitocondria show because of the ultrasonic action. The authors explain the limits of their experimentation in this specific sector of the scientific research.

[Initial cancer of the breast. Radiotherapy and radical surgical treatment]. / Il cancro iniziale della mammella. Radioterapia e terapia chirurgica radicale.

The controversy over the treatment of initial cancer of the breast is well known. An account is given of the factors that determine the prognosis, and the clinical and instrumental examinations on which staging is based. Lastly, radiotherapeutical approaches founded on precise classification dependent on systematic investigations of a radiological, radio-isotopic biochemical and immunological order, and, of course, on accurate information of surgical relevance, are presented.