1,721,088 research outputs found
Sistema in real-time PCR per la rilevazione rapida di patogeni invasivi
No full textNegli ultimi anni si è verificato un notevole incremento della mortalità a causa delle malattie infettive. Il dato più allarmante è l'aumento della mortalità per setticemia che dal 1980 al 1992 è stato dell' 83% (1).
L'aumento delle infezioni del sangue è legato indubbiamente all'aumento delle manovre che facilitano la penetrazione e la colonizzazione dell'agente infettante nel torrente circolatorio. Spesso, inoltrec’è un trend in aumento dell'antibiotico resistenza, infatti molte batteriemie sono sostenute da germi multiresistenti che complicano l'approccio terapeutico. Le batteriemie si suddividono in primarie e secondarie. L'epidemiologia e l'eziologia delle BSI è cambiata a causa del numero sempre più crescente di pazienti che necessitano di cure nella unità di terapia intensiva e con l'evoluzione delle cure mediche. Se fino al 1970 i patogeni di maggior isolamento nel sangue erano i Gram-¬negativi, a partire dagli anni ottanta i Gram-positivi sono diventati i patogeni a maggiore prevalenza. Tra i Gram-negativi, quelli a maggiore prevalenza sono: Escherichia coli e Pseudomonas aeruginosa. Bacteroides fragilis e Clostridium perfringens sono gli anaerobi più comuni, mentre tra i miceti predomina il genere Candida.
Tra i gram-positivi un ceppo patogeno che nei prossimi anni potrebbe rappresentare un vera e propria emergenza mondiale è lo Staphilococcus aureus meticillino resistente (MRSA). Lo Staphilococcus aureus è un batterio gram + molto patogeno per l’uomo, che provoca infezioni in qualsiasi distretto anatomico Nella risoluzione delle infezioni del sangue è fondamentale un approccio terapeutico precoce e mirato. Quest'ultimo si fonda senza dubbio su una diagnosi di laboratorio tempestiva e accurata. Se la coltura dei batteri tradizionale rimane il "gold standard" diagnostico in termini di accuratezza e di completezza di informazioni che fornisce, tuttavia pecca in termini di rapidità.
Scopo di questa tesi è stato quello di presentare l'esperienza maturata nell'applicazione di nuove metodiche molecolari (REAL TIME PCR) nella diagnosi precoce delle BSI. Se infatti i tempi di identificazione batterica delle emocolture positive si aggirano intorno alle 24-48 ore, lo scopo di questa tesi è stato quello di sperimentare un nuovo sistema per la rilevazione e identificazione del DNA batterico, presente nei flaconi di emocoltura positivi, in modo più tempestivo.
È stata presa in considerazione una regione del DNA ribosomiale compresa tra il gene 16S e il gene 23 S, la cosiddetta regione ISR o ITS. Questo perché la sua alta variabilità nella sequenza nucleotidica tra le varie specie e la sua ridotta dimensione ne fanno il target ideale per l'applicazione di un protocollo sperimentale di identificazione batterica, basata sull'applicazione in tempo reale con successiva curva di melting dell'amplificato.
Il nostro scopo è stato quello di discriminare qualitativamente i diversi ceppi batterici, che più frequentemente causano BSI nel nostro Policlinico.
Un secondo aspetto che è stato preso in considerazione è quello di valutare la prevalenza di Stafilococcus aureus metcillino resistente (MRSA) nel nostro policlinico., un patogeno emergente, che può creare particolari problemi nella terapia empirica delle sepsi ad oggi basata sull’impiego di glicopeptidi. Per questo germe è anche stata valutata la sensibilità alla vancomicina (VRSA,VISA, h-VISA) è la presenza della leucocidina Panteon valentie (PVL).
Sono stati analizzate 300 emocolture positive provenienti da pazienti del PTV. In totale sono state isolate 12 specie batteriche. Le specie prevalenti sono state: S.epidermidis (31%), S.aureus (20%), E.faecalis (14%), E.coli (14%), K.pneumoniae (9%), P.aeruginosa (4.40%), Efaecium (3%), A. baumannii (1.6 %) e altri germi (3%). Le temperature di melting di ciascuna specie batterica erano coincidenti o comunque contenute in un intervallo considerato importante per l'appartenenza alla stessa specie (0,5 °C).
Per la valutazione della meticillino resistenza nello Stafilococco aureo sono stati esaminati 60 campioni. Di questi sono risultati MRSA positivi circa il 31% (12 ceppi). Di questi campioni MRSA positivi 2 ceppi hanno mostrato delle MIC (concentrazione minima inibente) >=2, così da essere considerati h-VISA . Non sono stati trovati VISA (MIC>=4) e VRSA (MIC>=8). Per questi 2 campioni è stata valutata anche la presenza del gene Van-A, possibile eredità acquisita dagli enterococchi, ma il gene non è stato trovato.
Anche la Leucocidina Panteon Valentine è stata sottoposta a screening, ma non è stata confermata la presenza di questa proteina nei ceppi testati. Questo molto probabilmente è dovuto al fatto che i ceppi sono prevalentemente ospedalieri. Però conferma che nel nostro ospedale non c’è stato nessuno scambio genico tra i ceppi ospedalieri e comunitari di Staphilococcus aureus
From genomics to proteomics: beacon-based fluorescence in situ hybridization (bbFISH) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology in the clinical microbiology laboratory
No full textThe rapid and accurate identification of infectious agents and the early introduction of an
appropriate antimicrobial therapy is of crucial importance in the clinical practice, mainly in serious
infections such as blood stream infection (BSI), which may progress to sepsis /septic shock with
high morbidity and mortality rate. The delay in an appropriate antibacterial treatment has led to
increases non only in morbidity and mortality rates as well as in hospitalization time, in
nosocomial-acquired infections and make it challenging to implement the back-end approach of
the antimicrobial stewardship program, which has shown rewarding results in patient
management and the fight against the antimicrobial resistance. Moreover, the emergence of
resistant and multidrug- or even pandrug-resistant pathogens, especially the carbapenemaseproducing Enterobacteriacea (CPE) such as Klebsiella pneumoniae carbapenemase-producing (KPC) K. pneumoniae strains, is increasingly reported worldwide and is becoming an important issue in health care systems. Therefore, there is an urgent requirement to provide timely
information on infection status, which allow an optimal patient’s management and treatment.
Clinical microbiology has developed continuously and there has been a constant search for new
techniques and diagnostic methods with the aim to optimize the identification of pathogens, their
antimicrobial susceptibility profile and improve the patient’s management and treatment reducing
the social and economic impact on the health care system. Molecular methods seem to be an
appropriate choice, they are widely used in the diagnosis of BSIs, alongside to the conventional
culture-based methods. However, these techniques involve significantly increased cost and
technical complexity, both of which are likely to hamper their adoption in the laboratory routine in
the clinical setting.
In this paper are reported two novel technologies in clinical microbiology focused on the rapid
diagnosis of BSIs directly from clinical material in order to reduce the Turn Around Time (TAT) for
bacterial identification and improve patient’s management and treatment.
In the first part of this study has been evaluated the performance of a new molecular assay, the
beacon-based fluorescent in situ hybridization (bbFISH) in comparison to the conventional growthbased phenotypic identification of bacteria from positive blood culture vials in febrile patients. It has been also examined the bbFISH and the conventional identification assay’s total turnaround time (TAT) performance. Considering the good sensitivity and specificity of the hemoFISH® assays as well as the significant time saving in obtaining the final results (p-value 0.0001), the introduction of this assay could be reliable in the microbiology laboratories, supplementing traditional approaches, speeding up the diagnosis of bloodstream infections and identifying the majority of most important sepsis pathogens also in the case of polymicrobial infection. This assay has the potential to provide timely and cost effective information on infection status, thus allowing clinicians to make more informed decisions on appropriate antibiotic therapy at an earlier stage than is possible with culture-based approaches. hemoFISH® provides a same-day
identification of the majority of microorganisms thus the turnaround time is considerably lower
than microbiological culture.
The second part of the present work is instead dedicated to another novel proteomic assay, the
Matrix-Assisted Laser Desorption Ionization-Time of flight Mass Spectrometry (MALDI-TOF MS).
The application of this technology for the identification of pathogens in blood culture (BCs) has
been significantly improved the time required to obtain results with an excellent sensitivity and
specificity. Results were available on average after only a few minutes, representing a significant
difference in comparison with the time required for the results of conventional methods. Moreover the accuracy of this identification method seems to be considerable, with higher performance and cost effectiveness comparing with traditional methods. In addition, has been evaluated the applicability of this technology in order to predict susceptibility or resistance profiles against antibiotics, such as the carbapenems, on BCs derived directly from patients with Gram-negative bacteremia, in particular with carbapenemaseproducing Klebsiella pneumonia strains. The results of the present study have demonstrated that MALDI-TOF MS technology is a relevant tool also for the detection of antibiotic resistance. The enzymatic hydrolysis of the amide bond into the β-lactam ring of carbapenems, like ertapenem, by β-lactamases enzymes could be monitored by MALDI-TOF MS assay; hydrolyzed and nonhydrolyzed substances differ in their molecular weights and these differences can be detected by this assay within 1 to 3 hours with a great accuracy and high sensitivity. Compared to other alternative approaches like molecular genetics techniques this method is cost effective and easy to perform. The rapid detection of clinically important β-lactamases in routine diagnostic laboratories may be crucial for initial antibiotic therapy as well as for the prevention of the speed of β-lactamase-producing bacteria in health care setting. Combined with a proactive antimicrobial stewardship program of carbapenems this assay can improve patients outcomes. In order to provide even more timely information on infectious status, which allow an optimal patient’s management and treatment, always in the second part of this study has been also
demonstrated the reliability of a rapid antibiotic susceptibility test (AST) performed directly from
BCs flagged as positive to Gram-negative rods. The comparison of the antibiotic susceptibility data
obtained by the standard method and by the direct method has showed a concordance of 100%
while these data were obtained in a shorter time by the direct method than the traditional
culture-based susceptibility testing. Thus, this methodological approach has great potential to
become a routine method in any clinical microbiology laboratory while both this one method and
MALDI-TOF MS technology can likely modified the workflow of BCs providing a same-day final report available to clinicians and hospital epidemiologists
Microbiological Infections in Women with Cervical Cytological Reports of Atypical Squamous Cells of Undetermined Significance
No full text<it>Acinetobacter baumannii </it>in intensive care unit: A novel system to study clonal relationship among the isolates
Full text linkAbstract Background The nosocomial infections surveillance system must be strongly effective especially in highly critic areas, such as Intensive Care Units (ICU). These areas are frequently an epidemiological epicentre for transmission of multi-resistant pathogens, like Acinetobacter baumannii. As an epidemic outbreak occurs it is very important to confirm or exclude the genetic relationship among the isolates in a short time. There are several molecular typing systems used with this aim. The Repetitive sequence-based PCR (REP-PCR) has been recognized as an effective method and it was recently adapted to an automated format known as the DiversiLab system. Methods In the present study we have evaluated the combination of a newly introduced software package for the control of hospital infection (VIGI@ct) with the DiversiLab system. In order to evaluate the reliability of the DiversiLab its results were also compared with those obtained using f-AFLP. Results The combination of VIGI@ct and DiversiLab enabled an earlier identification of an A. baumannii epidemic cluster, through the confirmation of the genetic relationship among the isolates. This cluster regards 56 multi-drug-resistant A. baumannii isolates from several specimens collected from 13 different patients admitted to the ICU in a ten month period. The A. baumannii isolates were clonally related being their similarity included between 97 and 100%. The results of the DiversiLab were confirmed by f-AFLP analysis. Conclusion The early identification of the outbreak has led to the prompt application of operative procedures and precautions to avoid the spread of pathogen. To date, 6 months after the last A. baumannii isolate, no other related case has been identified.</p
COMBINATION THERAPY WITH THYMIC HORMONES AND CYTOKINES AFTER CHEMOTHERAPY IN CANCER-TREATMENT
No full text
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
- …
