Comparison of three new molecular methods for identification of bacteria from positive blood cultures

Sepsa je kljub napredkom v protimikrobni terapiji in podpornemu zdravljenju povezana z visoko obolevnostjo in umrljivostjo. Zgodnja identifikacija povzročiteljev sepse in njihove občutljivosti za antibiotike je tako ključnega pomena. V magistrski nalogi smo primerjali tri nove molekularne metode za identifikacijo bakterij iz pozitivnih hemokultur. Primerjali smo saponinsko metodo in komplet reagentov Sepsityper (Bruker), ki sta različna postopka priprave vzorca za pregled z MALDI-TOF sistemom masne sprektrometrije, ter vgnezden mnogokratni PCR FilmArray BCID (BioFire Diagnostics, ZDA). Metode smo primerjali s standardnimi rutinskimi metodami kultivacije in identifikacije ter določanja občutljivosti za antibiotike. Metode smo uporabili na 50 pozitivnih hemokulturah (42 monomikrobnih in 8 polimikrobnih) z 61 izolati. Med metodami smo primerjali pravilnost identifikacij in trajanje. Največ identifikacij (95,2 %), vendar včasih le do višjih taksonomskih nivojev, smo pri monomikrobnih vzorcih dobili z metodo FilmArray. Največ identifikacij do vrste (88,1 %) smo dobili s kompletom reagentov Sepsityper, s saponinsko metodo pa 66,7 %. Pri vseh treh metodah so bile najboljše identifikacije pri po Gramu negativnih bakterijah. Pri identifikaciji bakterij iz polimikrobnih vzorcev se je s 73,7 % najbolje obnesel FilmArray, ki je edini sposoben hkratne identifikacije več mikroorganizmov. Z metodo FilmArray smo sočasno dobili tudi podatke o prisotnosti determinant za odpornost proti antibiotikom mecA, vanA/B in blaKPC. Saponinska metoda je bila s povprečnim časom 14,4 minut hitrejša od kompleta reagentov Sepsityper (41,9 min) in FilmArraya (68,6 min).Sepsis is, despite the advances in antimicrobial therapy and supportive treatment, associated with high morbidity and mortality. Early identification of the causative agents of sepsis and their susceptibility to antibiotics is therefore crucial. In the master\u27s thesis, we compared three new molecular methods for identifying bacteria directly from positive blood cultures. We compared saponin method and the Sepsityper kit (Bruker), which are two methods of preparing the sample for analysis with MALDI-TOF mass spectrometry and nested multiplex PCR FilmArray (BioFire Diagnostics, USA). All three methods were compared with standard routine methods of cultivation, identification and susceptibility testing for antibiotics. We tested 50 positive blood cultures (42 monomicrobial and 8 polymicrobial) with 61 isolates. We compared the methods according to the correctness of the identification and duration. FilmArray method gave the highest number of identifications in monomicrobial samples (95,2 %) but sometimes only to the higher taxonomic levels. The majority (88,1 %) of identifications to the species level in monomicrobial samples were obtained with Sepsityper kit and 66,7 % with saponin method. All methods gave best results in Gram-negative bacteria. When identifying bacteria from the polymicrobial samples FilmArray was the most succesful (73,7 %), being the only one capable of simultaneously identifying several microorganisms. With the FilmArray method, we also received data on the presence of determinants for resistance to antibiotics mecA, vanA/B and blaKPC. Saponin method was the fastest, with the average time of 14,4 minutes, compared to Sepsityper (41,9 min) and FilmArray (68,6 min)