EVOLUTIE BINNEN HET MICROBIOLOGISCH LAB: STREVEN NAAR EEN HOGERE KLINISCHE IMPACT

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1 Pentalfa 24 maart 2016 EVOLUTIE BINNEN HET MICROBIOLOGISCH LAB: STREVEN NAAR EEN HOGERE KLINISCHE IMPACT Katrien Lagrou, Laboratoriumgeneeskunde, UZ Leuven

2 Evolutions in the clinical microbiology lab IMPLEMENTATION OF Matrix-assisted laser desorption/ionization (MALDI)- Time of flight (TOF) Mass Spectrometry Molecular methods/antigen detection assays Liquid microbiology/automation of specimen inoculation/ Total laboratory automation

3 MALDI-TOF MS: PRINCIPLE

4 MALDI-TOF MS: WORKFLOW Comparison with reference database Similarity with main spectrum profiles (MSP) in the database is expressed. Figures adapted from A. Wieser, et al. Appl Microbiol Biotechnol. 2011

5 MALDI-TOF MS Results in < 2 min Identification rates for routine clinical bacterial isolates range from 90-95% compared to 16S rrna Excellent tool for the identification of yeasts (filamentous fungi) Two technical limitations: o o minimum of 1.5 x10 5 CFU required inability to identify mixed (polymicrobial) cultures

6 Application of MALDI-TOF MS for detection of antibiotic resistance Approach Applications Remarks Detection of β-lactamases Resistance test with stable isotope-labeled amino acids Quantitative MALDI-TOF MS Strain typing Detection of carbapenemases, β-lactam resistance in enterobacteriaceae, Detection of MRSA; ciprofloxacin, meropenem and tobramycin resistance in P. aeruginosa Meropenem resistance in Klebsiella spp. Detection of vancomycinresistant enterococci, MRSA, carbapenem resistant Bacteroides fragilis Needs special skills for interpretation of spectra or special software; incubation phase with antibiotic Incubation in medium with the normal or heavy amino acid Spiking with an internal standard, peak intensities correlate to the amounts of bacterial peptides and thus bacterial growth May require specific sample preparation, only feasible for the detection of resistance mechanisms that are clearly related to distinct clonal groups J. Hrabak et al., CMR 2013, 26: ; I Wybo et al., JCM 2011, 49: ; M DeMarco et BA Ford, Clin Lab Med 2013, 33: ; C. Lange et al., JCM 2014, 52: ; J. S. Jung et al., Eur J Clin Microbiol Infect Dis 2014,

7 Molecular methods Clinical virology: shift from culture to molecular tests, loss of expertise of viral culture is a threat Still many in house developed methods Fully automated random access instruments are becoming available Different approaches: o Real time PCR for a single target o Test panels/multiplex PCRs Belgian reimbursement rules restrict the implementation of molecular tests

8 Early diagnosis of bloodstream infections G. Dubourg and D. Raoult. Expert Rev Mol Diagn 2016, 16:

9 Impact of rapid ID (and resistance gene testing) of positive blood cultures Prospective randomized controlled trial evaluating outcomes associated with rapid multiplex PCR (rmpcr, FilmArray Blood Culture ID Panel, BioFire Diagnostics biomérieux) for detection of 19 bacteria, 5 Candida species and 3 resistance genes Mayo Clinic, Rochester, Aug March 2014 All groups: MALDI-TOF ID and PBP2a test for S. aureus, baseline institutional stewardship interventions Primary outcome: duration of antimicrobials in the 4 days after enrollment Secondary outcomes: time to antimicrobial de-escalation or escalation, length of stay, mortality and cost R. Banerjee et al., CID 2015, 61:

10 R. Banerjee et al., CID 2015, 61:

11 Impact of rapid ID (and resistance gene testing) of positive blood cultures: results control n=207 rmpcr n=198 rmpcr + AS (24/7) n=212 Piperacillin-tazobactam use 56h 44h 45h p= 0.01 Narrow-spectrum β-lactam use 42h 71h 85h p= 0.04 Treatment of contaminants 25% 11% 8% p= No difference in mortality, length of stay, total costs AS: antimicrobial stewardship R. Banerjee et al., CID 2015, 61:

12 Subset of patients with organisms represented on the rmpcr panel (n=481) (19% not in panel) R. Banerjee et al., CID 2015, 61:

13 Molecular testing for respiratory pathogens in CAP Patients: o From 2 tertiary care hospitals in Edinburg o Sept 2012-Feb 2014 o Clinical and radiological evidence for CAP Samples: lower respiratory tract specimen obtained within 48h of admission Methods: o Multiplex real-time PCR: combination of multiplex assays for 26 pathogens (viruses and bacteria) o Quantification of bacterial load for S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus, E. coli, K. pneumoniae, P. aeruginosa and A. baumannii (in house real-time PCR, CFUs/mL) NJ Gadsby et al., CID 2016, 62:

14 Results 323 specimens (96% sputa and 4% endotracheal aspirate) from 323 patients +/- double pathogen detection (87% versus 39%) by PCR compared to culture Specimens from 8.4% of patients grew bacteria that were not included in PCR assays 84.8% of patients received AB during the 72 hours before the specimen was obtained (significantly associated with culture-negative result) NJ Gadsby et al., CID 2016, 62:

15 NJ Gadsby et al., CID 2016, 62:

16 NJ Gadsby et al., CID 2016, 62:

17 Next generation sequencing-based metagenomics: the future? Advantages of molecular tests and culture-based methods: host and pathogen-derived nucleic acids are detected and sequenced without priori knowledge of expected pathogens Enables genotyping, assessment of molecular markers for drug resistance and molecular epidemiologic studies But implementation in diagnostic laboratories remains to be demonstrated: complexities of lab workflow, speed of sequence analysis and expertise for analysis and interpretation are concerns EH Graf et al. JCM 1016, Epub ahead of print

18 Unbiased Detection of Respiratory Viruses Using RNA-seq-Based Metagenomics RNA-seq analysis of viral RNA extracted from pediatric nasopharyngeal swabs Comparison with multiplex PCRbased test (GenMark esensor Respiratory Virus Panel, 14 viruses) + in house qpcr Detected 86% of respiratory viruses detected by panel (4/6 undetected were negative by qpcr) 16% extra positive samples *Undetected by RVP EH Graf et al. JCM 2016, Epub ahead of print

19 Culture remains essential! For study of virulence, antibiotic susceptibility and genomic sequence in order to facilitate the understanding and treatment of infectious diseases Rebirth of culture prompted by microbiologists specializing in intracellular bacteria and microbiome researchers

20 Specific strategies for fastidious bacteria Intracellular bacterial culture Coculture with amoeba Cell culture Axenic culture: sterile media containing no living organisms except the one being cultivated Optimization of transport time and the use of antioxidants will likely further increase the culture of anaerobic bacteria

21 Increasing incidence of multidrug resistant organisms challenges microbiology laboratories

22 OXA48 and KPC immunochromatographic tests 100% sensitivity and specificity for the detection of OXA-48 and KPC enzymes in 15 min Glupczynski Y et al., JAC 2016, epub ahead of print

23 Conversion of samples to a liquid-phase More efficient release of microorganisms when put in liquid In combination with liquid nonnutritive transport medium rather than a gel

24 Laboratory automation Two main manufacturers: BD Kiestra and Copan Advantages: reduction of repetitive low-value-added tasks (reduction of manipulation of plates up to 90%), increase reproducibility A. Croxatto et al., CMI 2016, 22:

25 BD-Kiestra TM TLA

26 Copan WASPTM and WASPLabTM

27 Impact of total laboratory automation? Difficult to assess: Reduction of of 2.4 FTE/14.5 FTE: 16.5% reduction estimation according to Croxatto et al. Extension of opening hours to 24/7 service is likely one of the major changes needed to obtain maximal efficiency and thus a positive impact on patient management and hospital costs Minimum sample volume required to consider automation? Rapid development of new technologies may affect the impact of lab automation Remain to be investigated and demonstrated in peer-reviewed publications A. Croxatto et al., CMI 2016, 22:

28 And what about the diagnosis of fungal infections?

29 Which tests can the lab offer to you for the diagnosis of fungal infection? Conventional Tests Antigen/Serological /DNA Histopathology Direct Microscopy Culture Antigen detection Galactomannan (GM) 1,3 β-d-glucan (BDG) Lateral Flow Assays (LFA) Antibody detection PCR Panfungal Panfungal Mass Spectrometry (MALDI-TOF) Susceptibility testing Invasive aspergillosis Cryptococcosis Panfungal Panfungal (not Pneumocystis) Chronic pulmonary aspergillosis Panfungal

30 Novel tests for diagnosis of IA in patients with underlying respiratory disease 286 BAL from 221patients with underlying respiratory diseases (without hematological malignancy or SOT) 14% proven/probable IPA 02/ /2014, Graz, Austria Prattes J et al. Am J Resp Crit Care Med 2014; 190:

31 The Aspergillus lateral flow device: a point-of-care test Murine MAb JF5: IgG 3 immunoglobulin Recognizes an extracellular, constitutive, glycoprotein antigen Antigen is secreted during active growth of hyphae and is not produced by dead or quiescent spores Displays superior specificity to rat MAb EB-A2 (Bio-Rad Platelia GM-EIA) Used to develop a rapid, user-friendly, diagnostic test for detection of IA 1 Cryptococcus neoformans 2 Candida albicans 3 Fusarium solani 4 Rhizopus oryzae 5 Aspergillus fumigatus Thornton CR. Clin Vaccine Immunol Jul;15(7):

32 Localisation of JF5 antigen Expert Review of Clinical Immunology (2014) When commercially available? Current Fungal Infection Reports (2013) 7:

33 Cryptococcus antigen detection Antigen: the major capsular polysaccharide glucuronoxylomannan (shed during infection) Detection in serum/csf Latex-agglutination/Lateral flow assay Excellent sensitivity and specificity One of the most useful serologic tests in mycology

34 Conclusions Bacterial culture remains an essential test in clinical microbiology MALDI-TOF MS has revolutionized clinical microbiology Increasing use of molecular based diagnostics but lack of RIZIV reimbursement is a hurdle Full automation in now also entering the clinical microbiology laboratory Effect of new evolutions on patient management and outcome: few data available.