Microbiological challenges in critical care

Transcription

1 12/13/2014

2 Microbiological challenges in critical care Limited time, narrow window to diagnose, treat, intervene Organ failure tissue penetration of antibiotics, drug metabolism / elimination (altered Pk & Pd) High antimicrobial usage cost, AMR, CDAD (C. difficile associated diarrhoea) Lack of isolation facilities leading to greater infection control risks

3 Strategies for Infection Management in Critical Care ICU rounds: microbiologists with intensivists on infection management Prompt communication of microbiology results Change, stop, optimize / augment antimicrobial Rx Infection prevention and control Microbiology result interpretation / feedback to ICU team regarding AMR, audit and policy implementation Rational utilization of diagnostic, therapeutic and infection prevention / control resources

4 Sepsis: SIRS vs sepsis Systemic response to infection, characterized by hemodynamic, metabolic and inflammatory derangement, presenting as SIRS (mild) or progress to severe sepsis and septic shock (ACCP/ SCCM Consensus Conference Committee 1992) SIRS: by heart rate, respiratory rate, temperature, WBC count Sepsis: SIRS + high clinical suspicion of / microbiologically confirmed infection

5 Current status Confirmation of sepsis ID of triggering pathogens to guide Rx Conventional approach Culture, mostly blood biochemical ID susceptibility Lacks sensitivity Slow Promotes prolonged pre-emptive and empirical broad-spectrum antibiotics abuse escalates AMR 25-40% of culture +ve cases retrospectively didn t get proper antibiotic

6 Blood culture Central role in detection of bacteremia, differentiate SIRS from sepsis Enable antibiotic Rx to be rationalized by pathogen ID & AMST Provide clinical gold standard in D of bloodstream infections Can establish diagnosis in difficult, deep-seated infections Surveillance data- important epidemiological tool for empirical Rx

7 Current status Confirmation of sepsis ID of triggering pathogens to guide Rx Conventional approach Culture, mostly blood biochemical ID susceptibility Lacks sensitivity Slow Promotes prolonged pre-emptive and empirical broad-spectrum antibiotics abuse escalates AMR 25-40% of culture +ve cases retrospectively didn t get proper antibiotic

8 Conventional time scale

9 Current status Confirmation of sepsis ID of triggering pathogens to guide Rx Conventional approach Culture, mostly blood biochemical ID susceptibility Lacks sensitivity Slow Promotes prolonged pre-emptive and empirical broad-spectrum antibiotics abuse escalates AMR 25-40% of culture +ve cases retrospectively didn t get proper antibiotic

10 Need for growth independent: Detection Identification Susceptibility

11 Nucleic acid tests (NAT) NAT revolutionized diagnosis of infectious agent PCR RT-PCR Multiplexing Advantages Results in few hours Not affected by viability, prior antimicrobial Rx Higher sensitivity

12 Multiplex technologies for D from + blood cultures Prove-it TM Sepsis StripArray (Mobidiag, Finland) PCR + microarray ID from incubated blood cultures DNA extraction broad-range PCR biotin-amplicon of topoisomerase genes, gyrb & pare hybridization to microarray at bottom of well streptavidin-peroxidase 60 bacteria, 13 fungi, meca, vana/b, in 3.5 h Sn 95 %, Sp 99 %, 100 % accuracy for MRSA With fungi added, Sn 99 %, Sp 99 % (Aittakorpi et al, JCM 2012)

13 Prove-it TM

14 Multiplex technologies for D from + blood cultures Prove-it TM Sepsis StripArray (Mobidiag, Finland) PCR + microarray ID from incubated blood cultures DNA extraction broad-range PCR biotin-amplicon of topoisomerase genes, gyrb & pare hybridization to microarray at bottom of well streptavidin-peroxidase 60 bacteria, 13 fungi, meca, vana/b, in 3.5 h Sn 95 %, Sp 99 %, 100 % accuracy for MRSA With fungi added, Sn 99 %, Sp 99 % (Aittakorpi et al, JCM 2012)

15 Multiplex technologies for D from + blood cultures Verigene Gram positive blood culture NAT (BC-GP, Nanosphere Technology, US) Bact DNA extracted by magnetic beads hybridized to cdna on glass slide bifunctional oligo binds to target + gold nanoparticle 13 Gm + bacteria, meca, vana/b Hands-on <5 m, TAT 2.5 h, FDA approved 88 % agreement, Sn 93 %, 94 % MRSA, 98 % cefox R Gm negative 8 genera, 5 spp, CTX-M, 5 carbapenemases 8 yeasts

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17 Multiplex technologies for D from + blood cultures FilmArray (BioFire Diagnostics, US) DNA purified by silica magnetic beads amplified by stage 1 PCR 2 nd nested PCR with specific primers dsdna fluorescent dye 24 organisms (Gm+, Gm-, Candida, meca, vana/b, KPC) 2 m hands-on, TAT 1 h Sn 91 %

18 Clinical implications for Rx Progressive improvements in diagnosis of sepsis All require blood culture as starting point Escalation / de-escalation faster Critical Rx window / Golden Hour missed due to dependence on blood culture Limited targets can never have Sn 100 %

19 Multiplex technologies for direct D from blood SeptiFast (Roche Diagnostics, Germany) Most widely used FRET probes targeting ITS 16S 23S (bact), 18S 5.8S (fungi) 25 pathogens (Gm +, Gm -, A. fumigatus, Candida spp) TAT 4.5 h Real-time quantitative results correlates with severity Lacks R markers Not automated, needs technically demanding hands-on time

20 Multiplex technologies for direct D from blood SepsiTest (Molzym, Germany) Multiplex PCR-based, universal 16S rrna gene electrophoresis sequencing Detects >345 pathogens Hands-on 2.5 h, TAT 4 h (10 h with sequencing) Sn 87 %, Sp 86 %

21 Multiplex technologies for direct D from blood VYOO (SIRS Lab, Germany) DNA enrichment by affinity resin amplified for 33 bacteria, 7 fungi, 5 R genes meca, vana/b, blashv, blactx- M) gel electrophoresis Sn 60 %, Sp 75 %, TAT 24 h

22 Clinical implications Growth independent, all produce valuable molecular info fast Minimally confounded by antimicrobial Rx All require substantial technical expertise Two require preprocessing to remove human genomic DNA VYOO, SepsiTest require electrophoresis, not routine procedure, and sequencing, demands time None approved in US Lack of RCT to prove- mol diagn info can directly improve patient care Cost FilmArray : 3 m hands-on, 1 h TAT, best potential

23 Biomarker Characteristic that can be objectively measured and evaluated as an indicator of: normal biology pathological process pharmacological response to therapeutic intervention (NIH) Readily obtainable from tissue / body fluids Results available in a short period

24 Biomarkers Classification on clinical application: Diagnostic / screening health vs disease Prognostic likely outcome if untreated Monitoring evaluate progress, response to intervention Surrogate end-point outcome Stratification

25 Biomarkers Classification on clinical application: Diagnostic / screening health vs disease Prognostic likely outcome if untreated Monitoring evaluate progress, response to intervention Surrogate end-point outcome Stratification

26 Sepsis biomarkers are metabolites, chemicals, proteins or genes associated with: Biomarkers oxygenation state of tissues / organs oxidative injury by reactive species inflammatory & immune response alteration of coagulation or complement system endothelial dysfunction or tissue necrosis

27 Markers of acute inflammation ESR, CRP (APR, cytokines hepatocytes CRP) Nonspecific; inflammation, not infection CRP useful in severity and progression of disease, cannot differentiate SIRS vs sepsis Normal <10 mg/l, in 4-6h, several 100x in h, in 48h IL-6 (pro-inflam cytokine), LPS-binding protein (LBP) Good predictive & prognostic value but non-specific (trauma, surgery, stroke) LBP better than IL-6 & PCT in critically ill but needs validation in bigger RCTs

28 Biomarkers Markers of organ damage fms-like tyrosine kinase 1, E-selectin, ICAM-1, VCAM-1, plasminogen activator inhibitor 1 associated with disease severity, organ dysfunction and mortality

29 Procalcitonin Potential marker of infection 116 aa precursor of calcitonin, secreted by thyroid in health, in infection cytokines + bacterial products secreted by all tissues / cell types 1000x increase Distinguishes SIRS from bacterial (& fungal) sepsis Attenuated by viral-induced g-ifn in bacterial infections, in fungal infections rapidly after Rx Good diagnostic efficacy not affected by steroid / antiinflammatory Rx Studied as monitoring biomarker for antibiotic stewardship

30 Biomarkers Markers of impaired metabolism SvO 2, ScvO 2, serum lactate Biomarkers of tissue hypoxia & AnO 2 metabolism Markers of innate immune response CD64 (high-affinity Ig Fcg receptor I) Expressed constitutively on monocytes but rarely on PMN In infection, CD64 expression on PMN Pooled Sn 79%, Sp 91% One study Sn 95%, Sp 89% TREM-1 (triggering receptor expressed on myeloid cells 1)

31 MALDI-TOF Reduces ID time from ON to minutes Allows species ID directly from flagged blood culture bottles in 30 m Facilitates isolation of fastidious bacteria Potential for targeted AMR detection emerging

32 Direct species ID from blood culture by MALDI-TOF 1147 blood cultures flagged +ve subjected to direct MALDI-TOF Species identified in 558 (48.6 %) E. coli A. baumannii K. pneumoniae CoNS 47 P. aeruginosa Salmonella S. aureus Others C. jejuni 2 C. coli 2 G. adiacens 1 A. defectiva 2 P. acnes 2 Act. odontolyticus 1 Peptoniphilus harei 1 H. cinaedi 1 H. influenzae 2 H. parainfluenzae 2 Facilitated growth of fastidious organisms

33 Carbapenemase detection by MALDI-TOF Drug solution Imipenem in MiliQ 0.5mg/ml Matrix used HCCA Machine Microflex Program - ClinProtMix (to include lower range of m/z) Readings/sample at 0,1,2,3,6,8 hr incubation

34 Carbapenemase detection by MALDI TOF-MS Figure 5. MALDI TOF-MS analysis of imipenem hydrolysis. Blue graph Drug solution (DS) peaks at 489m/z and 104m/z. AUC 1988a.u and 157a.u, respectively. Black graph Matrix only(mo) peaks at 326m/z and 189.9m/z. Green graph Negative control (NC) ATCC A. baumannii. Red graph Test isolate (F2) at 3hr incubation. AUC for 489 and 104 was 20a.u and 84.9a.u, respectively. Ratio<0.5

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