RSEM-GSA 17 Role of Rapid Microbiology Assays in Sepsis Diagnosis

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1 RSEM-GSA 17 Role of Rapid Microbiology Assays in Sepsis Diagnosis Mohammed Omer Qutub, Ph.D., SM (ASCP), SM (NRM), D (ABMM), FCCM Senior Clinical Scientist in Clinical Microbiology and Molecular Infectious Diseases Section Head of Clinical Microbiology KFSHRCab

2 Outlines Overview of Different Diagnostic Methods Advantages & Disadvantages Rapid Molecular Testing State-of-the-Art Technology Its Clinical Values Role of the Microbiologist on use of Rapid Test Results

3 Key Terms Isolation (culture) Agar plate Liquid media Cell Line Tissue Culture Identification & taxonomy Family Genus Species Type Strain After culture Biochemical (physiological) tests Genetic tests Sequencing, Polymerase chain reaction (PCR) DNA-DNA homology Restriction enzymes (digests:rflp) Ribotyping Chemical - fatty acid/protein profiling (GLC) Immunological Direct detection (i.e. without culture) Staining (e.g. Gram stain) Antigen detection Serology (antibody detection) PCR

4 Stages

5 Stages

6 Advantages: Cost Limitation: In sufficient Accuracy e.g. B. cepacia complex (BCC) : ID as: Ralstonia sp. /Shigella spp./ Vs E coli Pseudomonas sp. Low Discrimination to the sp. Level TAT: Hours

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8 Why do we Need Rapid Diagnostics? Rapid Tailored Treatment Better Outcome Predicted Mortality in relation to time to antibiotics Reduce: Inadequate treatment Antibiotic resistance Adverse drug reaction Antimicrobial stewardship Conclusion: Initiation of Inappropriate Antimicrobial therapy Results in a Fivefold Reduction Survival in Human Septic Shock Ricard Ferrer et al., Critical Care Medicine. 2014: 42(8)

9 New Diagnostics Approach of 2000 PNA-FISH RealTime PCR

10 Faster Identification of Pathogens in Blood Cultures by FISH After Gram staining of growth-positive blood culture samples (i.e. BACTEC) Subculture on solid media>>> Provisional/Final ID Fluorescence in situ hydridisation (FISH) Peters et al. J Clin Microbiol 2006;44:

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12 Comparison of identification results and points in time: Identification of genus name: 91% of the micro-organisms of species name: 79% of the micro-organisms Time gain: (3.6 to 4.7 h) Vs 18 hours for bacteria 42 hours for yeasts Peters et al. J Clin Microbiol 2006;44:

13 Amplification Has Surprising Effects

14 Some of the Available Real-time PCR Instruments Roche LightCycler BioRad icycler ABI Prism 7700 Cephid SmartCycler

15 Melting of Hybridization Probes Molecular Beacons FRET Capillary Tube R TaqMan-Probe Q Fig. Courtesy of Roche

16 Testing Platform BACTERIA VIRUSES FUNGAL & PARASITES Group-A Streptococcus Group-B Streptococcus Slow Growing Bacteria Bartonella henselae Bordetella pertusis Agents of Community Aquired Pneumonia Chlamydophila pneumoniae Legionella pneumophila Mycoplasma pneumoniae Streptococcus pneumonia TB-Complex and OTTM Herpesviruses: HSV, VZV, EBV, CMV Enteovirus CNS Diseases BK and JC Poxvirus Respiratory Viruses: Influenza Parainfluenza SARAS-CoV Adenovirus RSV and others Viral Hepatitis: HBV, HDV, HCV, HBA, HEV HIV-1 and HIV-2 Aspergillus sp. Candida sp. Dimorphic Fungi Pneumocystis sp. Cryptococcus spp. Malaria Babesia sp. Trypanosoma sp. Leishmania sp. Toxoplasma sp. Giardia sp. Entamoeba sp. Cryptosporidium sp.

17 Provide the detection and the antibiotic Prescription within hours But Not a day Espy et al., JCM 2006;19:

18 Molecular Diagnosis of Multidrug-resistant Staph. aureus MDRSA MRSA VRSA meca gene vana gene

19 Conventional Versus Direct Molecular MRSA Diagnosis hrs Swab test Direct plating on agar Enrichment broth Subculture on blood agar suspicious : Slidex + Subcult BA + cefox disk diff hrs - 72 hrs cefoxitin R : MRSA screen I II DNA extract ( Real - time ) PCR + confirmation with conventional Pre - enrichment broth DNA extract test (Real- time ) PCR + Confirmation with conventional test Confirmation of positive results? + 24 (I) 48 ( II ) hrs - 4 (I) 24( II ) hrs Wertheim et al. J Clin Microbiol 2001;39:

20 Commercial MRSA Molecular Tests Confirmation in pure cultures sensitivity (%) specificity (%) MRSA Evigene (AdvanDx) Velogene geno id assay for MRSA (Alexon-Trend) Hyplex Staphyloresist (Alpha-Omega) Advantage versus protein detection in latex tests limited Direct test from clinical specimens sensitivity (%) specificity (%) Hyplex Staphyloresist (Alpha-Omega) Geno type MRSA-Direct (Hain) IDI MRSA (Gene Ohm Services) GenXper: Recently markted

21 Microarrays

22 Commercialized Arrays BD Max GeneXpert Verigene (Nanosphere) Luminex (TAG) BioFire-FilmArray IRIDICA (PELX) PyriSequance

23 FilmArray

24 Test for (20)-Respiratory Pathogens Panel: Viruses (17): Adneo, Corona(HKU1, NL 63, 229E, OC43), Metapneumovirus, Rhinovirus, ENT, Influenza (A, A/H1, A/H1-2009, A/H3), Innfluenza B, Para (1,2,3,4), RSV, Bacteria A typical pneumonia (3): Bordetella, Chlamy pne, Myco pne Test for 27 targets- BCID Panel: Gram Pos (8): Entero, List, Staph, Strep (A & B), pneumoniae Gram Neg (11): Acinto, Haemophilus, Neiss menin, Enter Yeast (5): Candida, albi, glabrata, krusei, para, tropicalis, Cryoto (gatti, neofor) Antibiotic Resistance (3): meca, vana/b, KPC Test for Herpes panel: CMV, EBV, HSV-1,2,6, VZV Test for (22)-GI Pathogens Panel FA Panels Bacteria (13): Camp, ClD, Sal, Shi, Plesio, Vibrio, Yers enter, 5-E coli Toxigenic strains Parasites (4): Cryptosp, Cyclo, Ent his, Giardia Viruses (5): Adeno (40/41), Astro, Noro, Rota, Sapovirus Test for (16)-CNS Pathogens Panel; Yeast : Crypto Bacteria (6): E cloi K1, H inf, Listeria, N menin, group-b Strep, Strep pne Viruses (8): HSV1&2, VZV, CMV, EBV, HHV-6, ENT, h-parechovirus

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26 Evaluation of FilmArray and Verigene Systems for Rapid Identification of Positive Blood Cultures By Bhatti. et al., ; JCM, Sep 2014, Vol-52 (9) 118 monomicro 82 +ve Vs 27 -ve Both ID 64 +ve correctly (100%)

27 Detection of Neisseria meningitidis from Negative Blood Cultures and Cerebrospinal Fluid with the FilmArray Blood Culture ID Panel By Pardo., et al., ; JCM, Sep 2014, Vol-52 (6) 5-month-old presented w fever, petechial rash, trunk, back, extremities Ceftriaxone 1h prior to 2xBC LP not done; Hemodynamic instability

28 TAT

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30 C albicans C parapsilosis C tropicalis C glabrata

31 IDWeek-2017

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33 Matrix-assisted Laser Desorption Ionization time of Flight Mass Spectrometry Base on species-specific spectra of peptides and protein masses by mass spectrometry Composed of three principal components : A specimen ionization chamber within which the laser-based vaporization of the specimen takes place a time of flight mass analyzer a particle detector MALDI-TOF MS

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35 Bacterial Identification by Mass Spectrometry ICCAC 2013

36 ICCAC 2013

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39 Enterobacteriaceae

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41 ICCAC: September, 2013

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44 Summary I Method No of Pathogens Panel Coverage (%) ID-Mono- Microbial (%) ID-Poly- Microbial (%) Verigene(GP/GN) 12/ Film Array Yeast Resistance Markers meca, vana, CTX-M, KPC, NDM, VIM,IMI, OXA meca, vana/b, KPC MALDI-TOF MS (Bruker/Vitek MS) > Low - Short-term culture & MALDI-TOF MS > Low - ICAAC-2015

45 Summary II Method FDA Approval TAT (min) Handson Time Prior to Test Invalid Test Results Verigene(GP/GN) Sept 2012/ Jan <5 min Gram stain 1-6% Film Array July <5 min Gram stain? 0-1.5% MALDI-TOF MS (Bruker/Vitek MS) +/ min Sample preparation? Short-term culture & MALDI-TOF MS <5 min Short culture? ICAAC-2015

46 Assessments of mrdts affects the mortality risk Time-to-therapy Los Reviewed 31 studies with 5920 patients: Laboratory methods varied among studies PCR and other microarray in 20/31 (64.5%) Followed by PNA-FISH in 6/31 (19.4%) MALDI-TOF in 4/31 (12.9%) ASP activities varied The presence of ASP facilitating mrdt represented the majority of the data (20/ %) The Clinical outcomes in BSI generally favored mrdt over conventional microbiology: Mortality risk (OR, 0.66; 95% CI ASP (OR, 0.64; 95% CI ) - ASP failed to decrease (0.75; ) Time to effective therapy by a weighted mean difference of hours Length of stay decreased by days CID, 2016

47 Role of the Microbiologist to Facilitate Clinician Acceptance and Use of Rapid Test Results The microbiologist must: Inform the clinicians of available rapid tests Create confidence and trust in the results reported Report results of rapid testing that correlate with the results of definite testing Convey rapid results to the clinician in a convenient manner Provide the evidence to validate the usefulness of the information provided by the rapid test result Condition the clinician to expect the results of rapid tests routinely (e.g. ESBL, MRSA, vana) Report results that coincide with the schedule of medical practice in the local setting

48 Rapid Conventional

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