Athanassios Tsakris Medical School, University of Athens Greece

Transcription

1 Athanassios Tsakris Medical School, University of Athens Greece

2 Each hour of delay (over the first 6 hours) resulting in a 7.6% decrease in survival «In septic shock, time is life» Kumar A, Crit Care Med : Fraction of total patients survival fraction cumulative effective antimicrobial initiation 1 st ½ 2 nd ½ Time from hypotension onset (hours) >36

3 Days negative Only ~10% are positive 1. Poor specificity (false positive rates: 5 to 50% depending on the methods of collection) 2. Despite optimization of the technique only 15 25% of positive results can be anticipated 3. In up to 30% of the patients with fever clear results cannot be obtained at all (BC sensitivity for slow growing and fastidious organisms can be poor) Book M et al. Best Pract Res Clin Anaesthesiol. 2013;27:

4 Μοριακές μέθοδοι Type of molecular methods Hybridization Amplification Postamplfication detection strategies Non nucleic acid FISH PNA FISH Probe hybridization PCR Broad range PCR Pathogenspecific PCR PCR + Sequencing Pyrosequencing Hybridization Proteomics Spectroscopy Phage assays Microarrays Multiplex PCR MALTI TOF MS Venkatesh M et al. Expert Rev Anti Infect Ther. 2010;8: ; Mancini N et al. Clin Microbiol Rev 2010;23:

5 1. From pure culture rapid identification 2. From positive blood culture 3. Directly from whole blood Detection or not of antibiotic resistance genes

6 1. From pure culture rapid identification 2. From positive blood culture Mass spectrometry (MALTI TOF MS)

7 It is based on proteomic profiling of highly conserved proteins for the rapid (5 15 min) identification of bacteria and fungi Biological material (colony or blood culture concentrated) is placed on a plate that has a polymeric matrix Irradiated with a laser that vaporizes the sample, resulting in ionization of molecules, which are then aspirated into a vacuum flight tube and travel to a detector Protein spectra profile compared with database Clin Microbiol Infect 2010;16:1604

8 Identifying Microorganisms by Their Protein Fingerprint MALDI Biotyper VITEK MS Overall performance is comparable, and differences are more associated with software databases than with instrumentation.

9 Schieffer KM, et al. J Appl Microbiol. 2014;116: Jamal W, et al. Diagn Microbiol Infect Dis. 2013;76(4): Meex C, et al. J Med Microbiol. 2012;61(Pt 11): Martiny D et al. Eur J Clin Microbiol Infect Dis. 2012;31: MALDI Sepsityper product : Identification of microorganisms from positive blood culture bottles in < 30 min. Simple preparation protocols using just 1 ml sample material SE MALDI accurately identified 332 (80.8%) of 411 positive BC

10 Detection of microorganisms directly from whole blood LightCycler SeptiFast SepsiTest VYOO Multiplex PCR Detection limit: 3 30 CFU/mL for S. aureus 3 10 CFU/mL Time: 6 h 8 12 h 8 h Skvarc M Eur J Microbiol Immunol (Bp) Jun;3(2):97 104; Paulocci M Intern J Antimicrob Agents 2010;36S:S6

11 (1) extraction and purification of DNA from whole blood, (2) real time PCR amplification of the target DNA in three parallel reactions (Gram positive bacteria, Gram negative bacteria, fungi) and (3) detection by specific hybridization probes and automated identification of species within 6 h after the blood draw Detection and species differentiation of the 25 most relevant microorganisms covering around 90% of the most common pathogens causing BSI

12 SeptiFast panel: ~ 25 pathogens Detection and identification Gram negative bacteria Gram positive bacteria Fungi Escherichia coli Staphyloccus aureus Candida albicans Klebsiella (pneumoniae / oxytoca) CoNS (Coagulase negative Staphylococci) Candida tropicalis Serratia marcescens Streptococcus pneumoniae Candida parapsilosis Enterobacter (cloacae / aerogenes) Streptococcus spp. Candida krusei Proteus mirabilis Enterococcus faecium Candida glabrata Pseudomonas aeruginosa Enterococcus faecalis Asperigillus fumigatus Acinetobacter baumannii Stenotrophomonas maltophilia CoNS: S. epidermidis, S. haemolyticus, S. xylosus, S. hominis, S. cohnii, S. lugdunensis, S. saprophyticus, S. saprophyticus, S. capitis, S. pasteuri, S. warneri. Streptococci: S. pyogenes, S. agalactiae, S. mitis, S. mutans, S. oralis, S. anginosus, S. bovis, S. constellatus, S. cristatus, S. vestibularis., S. gordonii, S. intermedius, S. milleri, S. salivarius, S. sanguinis, S. thermophilus, S. parasanguinis.

13 Spacer Technology A multiplex PCR assay that uses post amplification melting point analysis to identify microorganisms 16S ITS (Spacer) 23S Generic Primers ( pan bacteria ) Amplification curve Group (e.g. Enterobacteriaceae) Specific Probes Tm depended upon fragment length, composition of sequence, and degree of homology between the hybridization probe and the target DNA Probe Melting Point(s) E. coli Enterobacter cloacae Cycles Tm (melting temperature )

14 Advantages of SeptiFast methodology Rapid diagnosis and implementation of targeted AB treatment positivity rate significantly higher than that of BC (x 2 times) less contaminations due to skin flora Diagnosis of invasive fungal infections detection and identification of 5 Candida spp. & A. fumigatus More sensitive than BC in patients already treated by AB LC SF is of high rule in value for early detection of septic patients. In a population with low pretest probability,lc SF test can still provide valuable information for ruling out bacteremia or fungemia. Chang S S et al., (2013) Multiplex PCR System for Rapid Detection of Pathogens in Patients with Presumed Sepsis A Systemic Review and Meta Analysis. PLoS ONE 8(5): e62323

15 High risk patients and patients with presumed sepsis Patients with febrile neutropenia Patients with onco hematological malignancies Neonates and children Transplants recipients Patients with serious burns Patients with suspected endocarditis Liesenfeld et al, Eur J Microbiol and Immunol 2014; Dark et al, Intensive Care Med 2015

16 Rapid innovative microbiological methods provide opportunities for antimicrobial stewardship programs to improve antimicrobial use and clinical and economic outcomes are considered game changers and represent a significant advancement in the management of infectious diseases