Dr. Sanghamitra Datta Consultant Dept. of clinical microbiology & Immunology Sir Ganga Ram Hospital

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1 Dr. Sanghamitra Datta Consultant Dept. of clinical microbiology & Immunology Sir Ganga Ram Hospital

2 Microbiology culture reports: most important information for patients management deciding factor in the outcome of critically ill patients. More than 70% of clinical decisions in ICU are based on laboratory results. Less than 25% of culture reports are used for guiding therapy. Reason C/S reports don t reach on time. Cunney et al. International Journal of Antimicrobial Agents 2000;14 :13 19

3 Crit Care Med, 34: , 2006.

4 Rapid Right Results generated and reported in a clinically useful timeframe State-of-the-art methodology to TAT Test results should have a high degree of analytical sensitivity, specificity and accuracy Relevant Reports should contain information of direct relevance to the clinical management of the patient

5 2 million people die each year due to infectious disease Need to integrate medicine and innovative technology in our health system RAPID ACCURATE COST EFFECTIVE RESULTS FOR IDENTIFICATION AND AST.

6 Shortens the turn around time Quicker results to clinician: affecting patient outcome less hands on time Improved reproducibility Accurate and precise measurement by eliminating measurement variation and human error Allows Large sample handling Electronic reports compatible with HIS are suitable for storing, archiving of data to understand the trend of the antibiotic resistance/sensitivity of a particular organism

7 Fails to recognise organisms not in data base Expensive: cost Some of instruments, infrastructure platforms have problems with inducible beta-lactamase mediated resistance in gramnegative bacilli, low level glycopeptide resistance in enterococci.

8 Turn Around Time!!!!

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10 Mainstream technologies still rely on Conventional agar dilution assays like E-test and disk diffusion Micro-Broth dilution assays: MIC test is miniaturised and standardised by use of small, plastic, disposable micro-dilution trays allow minimal volume the isolated growth and predetermined antibiotic concentrations. Commercially automated instruments

11 Gives the clinicians an option to choose the most optimum therapy for a particular infection Reveals the changing trends of local isolates Also a guide to choose empirical therapy in serious patients Allows judicious use in the face of the antibiotic resistance

12 Currently AST is accomplished using either classical manual methods OR Growth dependant automated systems such as biomerieux Vitek-2 Becton Dickinson s Phoenix Siemens Microscan Walkaway AST of all are BMD based: BMD has attained reference standard status - all AST methods are compared with it during development, verification, validation

13 MicroScan WalkAway Vitek-2 BD phoenix automated system Sensititre

14 Based on standard 96-well microdilution trays with automated sample handling robotics AST uses a photometer to detect bacterial turbidity in trays over hours

15 Uses a small AST cassette or card in 4564 well plate format Instrument is capable of handling AST cards Detects turbidity with bacterial growth over 410 hours to reveal AST results

16 Reads turbidity and colorimetric changes of up to 99 AST panels / cassettes Requires 6-16 hours starting from incubating pure cultures to obtain MIC for bacteria

17 Used standard 96 well microdilution panels (AST cassettes) Inoculated by SensititreAutoinoculator Capable of handling 64 panels/cassettes Bacterial growth in each panel is detected by fluorescent intensity monitored over hours

18 Streamlined Work Flow &Quantitative results Simplifies MIC determinations Disadvantages Still require isolated bacterial growth in pure culture Susceptibility testing are based on measuring bacterial growth and turbidity changes Lack the capability of analysing poly-microbial samples or heterogeneous response of bacterial populations to the antibiotics

19 Ist Generation 2nd Generation Vitek-2/PhoenixContinuous scanning & interpretation of B/C reaction: 8-10 hrs 3rd Generation API cards- automated interpretation of B/C reaction: hrs Gene sequencingmolecular fingerprinting: 46 hrs 4th Generation MALDITOF- Protein fingerprinting: < hour for detection

20 MALDI-TOF MS: Mass Spectrometry Method for rapid identification of organisms by protein finger printing Matrix Assisted Laser Desorption Ionization Time Of Flight

21 MALDITOF: ID in Minutes, not Hours Avg secs per Isolate! 21

22 Maussaoui et al., Clin. Micro. & Infec., 2010 MALDI-TOF MS: the quantum leap

23 Laser light causes the sample and matrix to volatilize. Matrix absorbs energyresults in desorption and ionization of protein molecules The ions formed are accelerated by a high voltage Flight of Ions: In the Long Tube they are separated according to their mass Ions detected at the end of the tube by mass-to-charge ratio (m/z). Creates a Mass Spectrogram Compared with a Library

24 protein mass signals (peaks) peaks: Mostly ribosomal proteins Ribosomal proteins are conserved within species Other peaks : mostly unknown proteins %Int m/z

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26 6 pm A 25 Y/ F presented at SGRH with c/o fever & Petechial rashes X 1day Relevant investigations Blood culture: Positive after 10 hours (4.00am) Grams Stain showing GNDC MALDI TOF: Neisseria meningitidis (16 hrs) 10.00am Conventional system: Day 1: S/c on blood agar plates Day2: Growth +ve, putting ID Day 3: Identification Patient shifted to isolation room in ICU Treated with I/V ceftriaxone All contacts given Rifampicin

27 Blood culture from 13 Y M (BacT alert 3D) Positive after 0.81days of incubation Gram positive bacilli Smear from growth: GPB Discarded as contaminants? Diptheroids MALDI-TOF MS identified it to be Listeria monocytogenes (immediately informed to clinician) Pt. a case of Meningitis Ampi + Genta

28 Several approaches are explored Documenting activity of antibiotic inactivating enzymes like beta-lactamases Changes in protein spectrum of an organism in the presence and absence of anti-microbial agent.

29 Detection of Carbapenem degradation products JOURNAL OF CLINICAL MICROBIOLOGY, 2011;49:

30 Documenting activity of antibiotic inactivating enzymes like beta-lactamases Carbepenemase activity of variety of GNBs using meropenem as substrate has been detected Organisms producing NDM-1 and IMP-1 hydrolyzed the drug in 1 hour and VIM, KPC, OXA hydrolyzed the drug much slowly The time to degradation correlated with the enzyme carried, not with the MICs of carbapenems 4 distinct peaks were recorded and stored in the data base

31 December 2014 Volume 52 Number 12 p MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) Bacteria are incubated in culture medium in the presence or absence of antibiotics After incubation, cells are lysed and spiked with an internal standard to facilitate the quantitative comparison of the acquired mass spectra. The peak intensities correlate to the amounts of bacterial peptides, which in turn correlate to bacterial growth. Evaluation of resistance status by comparing growth rates in bacteria with and without antibiotics

32 Changes in protein spectrum of an organism in the presence and absence of anti-microbial agent. MPCC: Minimal profile change concentration It is defined as a minimum concentration of drug at which a change in profile could be documented Researchers found a very high concordance between MPCC value and MIC values obtained by the CLSI broth based reference method.

33 December 2014 Volume 52 Number 12 p MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) Bacteria are incubated in culture medium in the presence or absence of antibiotics After incubation, cells are lysed and spiked with an internal standard to facilitate the quantitative comparison of the acquired mass spectra. The peak intensities correlate to the amounts of bacterial peptides, which in turn correlate to bacterial growth. Evaluation of resistance status by comparing growth rates in bacteria with and without antibiotics

34 All these methods require a pure isolated culture growth Novel approaches for MALDITOF in Clinical Microbiology: Direct ID in Samples : to make TAT even shorter.

35 A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. VITEK MS resulted in 94.0% correct organism identification to the species level. VITEK 2 resulted in 93.5 category agreement of antimicrobials tested, with 3.6% minor error, 1.7% major error, and 1.3% very major errors The average time to ID and AST was 11.4 hours by Combined approach compared to 56.3 hours by using conventional methods

36 Of 145 samples with 105 orgs/ ml, overall identification by by Vitek MS: 76.4% LF: 70 (98.6%), NLF: 3 (60.0%), GPC: 4 (44.4%), and Yeasts (20.0%), were correctly identified by Vitek MS.

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38 Wattal C, Oberoi JK. Microbial identification and automated antibiotic susceptibility testing directly from positive blood cultures using MALDI-TOF MS and VITEK 2. Eur J Clin Microbiol Infect Dis Jan;35(1): Wattal C, Oberoi JK, Goel N, Raveendran R, Khanna S. Matrix-assisted laser desorption ionization time of flight mass spectrometry ( MALDI-TOF MS) for rapid identification of micro-organisms in the routine clinical microbiology laboratory. Eur J Clin Microbiol Infect Dis 2017;36(5):807-12

39 Microorganis m LFM Standard Approach Time to ID & Time to ID Time to Time to ID AST (h) (h) AST (h) & AST (h) Time to ID (h) Time to AST (h) GNB GPC Total

40 Genexpert : GeneXpert MTB/RIF assay simultaneously detects DNA of Mycobacterium tuberculosis complex and resistance to rifampin (i.e. mutation of the rpob gene) in less than 2 hours Line probe assay ( MTB DR Plus) Detects INH and Rifampicin resistance :8-10 hours

41 27 pathogens Gram + Bacteria: Enterococcus spp. L. monocytogenes Staphylococcus S. aureus Streptococcus spp. S. agalactiae (Group B) S. pyogenes (Group A) S. pneumoniae Gram - Bacteria : A. baumannii Enterobacteriaceae Enterobacter cloacae Complex E. coli H. influenzae K. oxytoca K. pneumoniae N. meningitidis P. aeruginosa Proteus S. marcescens Fungi: C. albicans C. glabrata C. krusei C. parapsiolosis C. tropicalis Antibiotic Resistance: meca Van A/B KPC Sample : Positive Blood culture

42 Emerging technologies are actively pursued by commercial entities Promise rapid AST in a few hours Sample pretreatment may not be needed in some Applying them to slowly growing organisms require innovative approaches dealt in as future technologies

43 Imaging based AST Biochemical AST Non-imaging AST

44 MADM: Multiplexed automated digital microscopy Separates bacterial cells from clinical samples by gel filters Bacterial cells attached to the sensing surface by electrokinetic loading and FISH probes are used to identify them one hour Measures growth of bacterial masses in MH containing antibiotics compared to growth controls and growth curves made and reports in 5 hours Directly from a positive blood culture or BAL

45 Single cell morphological analysis Uses bright field microscopy to determine antibiotic induced morphological change in bacterial cells Captured images are processed using automated image processing algorithm.

46 Imaging growth of a population of bacterial cells in a fluid sample with antibiotics in microfluidic chambers over a period of time Recorded images are processed using imaging algorithms Reports - 5 hours

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48 Novel imaging approaches of bacterial morphokinetic changes applied to AST using smart phones and other high resolution cameras are being studied.

49 BacterioScan ( Fisher Scientific) Detects forward laser light scattering (FLLS) analyses the angular variation in the intensity of scattered light and determines the number size of bacterial cells suspended in antibiotic solution Can perform AST directly on clinical samples with minimal sample preparation Unable to differentiate spp. In polymicrobial flora AST in 3-5 hours

50 Technology measures the mass of bacterial cells upon passage through microfluidic channels inside of the micro-cantilevers Micro-channel resonator sensors permit quantitation of bacteria & measures mass changes of individual cells to assess antibiotic activity Advantages : ability to perform sensitive mass and morphology measurements on single cell and promise AST in 3 hours Can be used as a point of care test Yet to be commercialized

51 They measure the molecular and biochemical signatures e.g. changes in 16SRNA, DNA and ATP of growing bacterial cells Biosensor based AST assay is being developed by Genefluidics measures bacterial growth via quantification of 16srRNA molecules which are specific for each bacterial spp. in presence of antibiotics After DNA probes hybridise specifically 16SrRNA molecules electrochemical signals quantify it. Directly done from samples

52 Smarticles Technology in development by Roche introduces recombinant bacteriophages with DNA probes Binding of probes with bacteria leads to luciferase expression Light intensity quantifies the bacteria and perform rapid AST Promises Point of care test While promising, these emerging approaches require further studies and evaluations

53 AFM Cantilever: Atomic force Microscopy cantilevers PIT: Plasmonic Imaging and Tracking Flow Cytometry IMC- Isothermal microcalorimetry

54 AFM consists of a cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. The cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of nanometers. The

55 Bacterial cells attached to microcantilevers and deflection of the cantilever associated with micro-motions is detected as a signature of bacterial metabolism in antibiotic of diff. concentrations. AST will be given in two hours

56 Used to track 3D motions of single bacterial cell associated with metabolic viability Rapid AST Built on inverted optical microscope, where light from a luminescence diode is directed onto sensor chip which is a gold coated glass film with immobilized bacterial cells Image contrast fluctuations are due to bacterial cell movement due to metabolism

57 Measures changes in morphology, cellular numbers,viability. Dye is used to stain viable cells which flow through a channel into a reader zone Light scatterring is used to measure counts and viabilities in presence of antibiotics Rapid AST in 3-4 hours

58 Not widely used Presence of auto-fluorescence Staining inefficiency of dyes Inability to differentiate cellular damage caused by bacteriostatic nd cidal drugs Lack of clinical databases for validation

59 Novel technique: Measures cumulative heat and generates heat curves of growing bacterial cells Heat curves of growing bacteria are similar to growth curve measured by standard turbidity instruments

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61 IMC produces results within 3 hours using patient s urine sample Lower detection limit is 104cfu/m Have been effectively studied in E.coli, S.aureus. M.tuberculosis. However further studies and novel purification is required for samples like blood and sputum l

62 Solid media cultures Time of AST Direct on pt. Real MIC sample Agar Dilution Assay hours No No Disk Diffusion hours No No E-Test hours No Yes Broth dilution Assay 12-24hrs NO yes Microscan WalkAway No yes Vitek1/Vitek hours No yes BD Phoenix 9-15 hrs No yes Sensititre No yes Liquid Media Cultures

63 Imaging Principle Time of AST Direct on pt. sample Real MIC MADM Quantify growth rate 3-5 hours Yes (urine & Blood) yes SCMA Image of morphology change 3-4 hours Urine yes ocelloscope Measure of bacterial cells 1-4 hours Urine yes Bacterioscan Measures Bacterial numbers and sizes 3-10 hours urine yes MC resonator Count bacterial cells and morphology changes on single cells after antibiotic action >3hours no yes Genefluidics Count of 16sRNA as proxy to bacterial growth 4 hours urine yes Smarticles Bacteriophages that express luciferase on growing cells Non-Imaging

64 Technology Principle Time for AST Direct from sample MIC AFM cantilever Measures fluctuations originating from bacterial cells denoting bacterial metabolism <2 hours no Yes PIT Measures nanometric motion of bacteria <2 hours yes no Flow cytometry Count viable bacteria using dye 2-3 hours no yes IMC Heat signature of 3-14 hours growing cells yes yes

65 In near future we anticipate that the Emerging and Future innovative technologies will lead to the next wave of more powerful AST tools for rapid clinical diagnosis Will enable a one hour AST within the time span of an OPD visit Rapid real time AST will potentially slow the evolution of antibiotic resistance and improve antibiotic stewardship

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