Sistema in real-time PCR per la rilevazione rapida di patogeni invasivi
Creators: name: Ombres, Domenico
Publisher: name: Università degli Studi di Roma "Tor Vergata"
Year: 2009
Subjects: subject: rilevazione rapida; patogeni invasivi; real time PCR;
subject: rilevazione rapida
subject: patogeni invasivi
subject: real time PCR

description: In the last years a notable increase of the mortality is verified because of the infectious illnesses. The most alarming datum is the increase of the mortality for setticemia that from 1980 to 1992 has been of the 83% (1). The increase of the infections of the blood is undoubtedly tied up to the increase of the manoeuvres that facilitate the penetration and the colonization of the infecting agent in the circulatory stream. Often, there is a trend in increase of the antibiotic resistance, in fact a lot of batteriemies are sustained by multiresistenti germs that complicates the therapeutic approach. The batteriemies are divided in primary and secondary. The epidemiology and the eziologia of the BSIs is changed because of the increasing number of patients that need cares in the unity of intensive therapy and with the evolution of the medical cares more and more. If up to 1970 the patogenis of great isolation in the blood were the Gram - negative ¬, beginning from the eighties the Gram-positive ones have become the patogenis to great prevalence. Among the Gram-negative ones, those to great prevalence are: Escherichia coli and Pseudomonas aeruginosa. Bacteroides fragilis and Clostridium perfringenses are the most common anaerobis, while among the micetis the kind predominates is Candida strain. Among the gram-positive ones a log patogeno that in the next years you/he/she could represent a real world emergency it is the Staphilococcus aureus resistant (MRSA) meticillino. The Staphilococcus aureus is a bacterium gram + a lot of patogeno for the man, that provokes infections in any anatomical district In the resolution of the infections of the blood is fundamental a precocious therapeutic approach and contemplated. This last him deep without doubt on a timely and accurate diagnosis of laboratory. If the traditional crop of the bacterias remains the "standard gold" diagnostic in terms of accuracy and completeness of information that it furnishes, nevertheless fault in terms of rapidity. Purpose of this thesis has been that to introduce the experience matured in the application of new methodic molecular (REAL Time PCR) in the precocious diagnosis of the BSIs. If in fact the times of bacterial identification of the positive emocoltures wander around the 24-48 hours, the purpose of this thesis has been that to experiment a new system for the survey and identification of the bacterial Dna, present in the bottles of emocoltura positive the purpose of this thesis has been that to experiment a new system for the survey and identification of the bacterial Dna, present in the bottles of positive emocoltura, in more timely way. It has been considered a region of the ribosomial Dna between the gene 16S and the gene 23 Ss, the so-called region ISR or ITS. This is because its high variability in the nucleotidic sequence among the various kinds and its short dimension renders it the ideal target for the application of an experimental protocol of bacterial identification, based on the application of Real Time PCR technique. Our purpose has been to qualitatively discriminate the different bacterial strains, that more frequently cause BSIs in our Policlinico. A second aspect that has been considered is to appraise the prevalence of Stafilococcus aureus resistant (MRSA) metcillino in our policlinico., an emergent patogeno, that can create particular problems in the empirical therapy of the sepsis today based on the employment of glicopeptidi. For this germ it has also been valued the sensibility to the vancomicina (VRSA, VISA, h-VISA) and the presence of the leucocidina Panteon valentine (PVL). 300 positive emocoltures have been analyzed coming from patient of the PTV. In total there have been isolated 12 bacterial kinds. The prevailing kinds have been: 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%) and other germs (3%). The temperatures of melting of every bacterial kind were coincident or however contained in an interval considered important for the affiliation to the same kind (0,5 °Cs). For the evaluation of the meticillino resistance in Staphylococcus aureus 60 champions have been examined. Of these, around the 31% are results positive MRSA (12 strains). Of these MRSA positive champions 2 strains have shown some MICs (inhibiting least concentration) &gt; = 2, so that h-VISA to be considered h-VISA. VISA (MIC &gt; = 4) and VRSA (MIC &gt; = 8) have not been found. For these 2 champions it has been valued also the presence of the gene Van-A, possible inheritance acquired by the enterococchis, but the gene has not been found. Also the Leucocidina Panteon Valentine has been submitted to screening, but the presence of this protein has not been confirmed in the analized strains. Very probably this is due to the fact that the strains are predominantly hospital. However it confirms that in our hospital there has been no anybody exchange genico among the hospital and community strains of Staphilococcus aureus