05/02/2023 Dr.T.V.Rao MD 1

MALDI-TOF IN

CLINICAL MICROBIOLOGY

OUR VISION TO FUTURE TECHNOLOGY

Dr.T.V.Rao MD

05/02/2023 Dr.T.V.Rao MD 2

Why we need Newer Technologies in Diagnostic Microbiology

■ Life is changing, Technology is changing and perception to diseases are changing

■ So we need better approaches to diagnose infectious diseases

■ Thoughts on how this technology will change the practice of Clinical Microbiology

■ Benefits of decreased time to detection of pathogens■ Directed patient care■ Antibiotic stewardship■Right drug, right dose, for the right duration

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Need for better Technologies in life threating Infections

■Blood cultures are the best approach to establish the etiology of bloodstream infections and infectious endocarditis. Moreover, rapid identification of etiological agent of such severe infections are pivotal to guide antimicrobial therapy. Thus, the impact of timely microbiology laboratory reporting is maximal at the notification of positive blood cultures . The matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows the identification at the species level in few minutes of both Gram positive

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Changing understanding on Gram Negative Bacteria ■Gram negative

bacteria by measuring molecular masses of proteins and other bacterial components obtained from whole bacterial extracts

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Diagnostics changed with mass spectrometry

■Since the early 1980s, mass spectrometry has emerged as a particularly powerful tool for analysis and characterization of proteins in research. Recently, bacteriologists have focused their attention on the use of mass spectrometry (MS) for bacterial identification, especially Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF). Moreover, recent publications have evaluated MALDI-TOF in microbiology laboratory for routine use.

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Trends of change with MALDI-TOF-MS

■MALDI-TOF-MS is a rapid, precise, and cost-effective method for identification of intact bacteria, compared to conventional phenotypic techniques or molecular biology. Furthermore, it allows identification of bacteria directly from clinical samples (blood cultures for example).

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Principles of MALDI-TOF ■Maldi is soft ionization technique used in

mass spectrometry, allowing the analysis of biomolecules such as DNA, proteins, peptides & sugar or polymers such as dendrimers and macromolecules • It is three steps method. I. The sample is mixed with a suitable matrix & applied to a metal plate. II. A pulsed laser irradiate a sample triggering desorption of matrix material. III. Ionization of analyte molecules

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What is happening in past with Phenotypic Methods ■Traditional phenotypic based

diagnostic methods for BSIs require the detection of bacterial growth in blood culture broths, followed by species identification and antimicrobial susceptibility testing (turnaround time 24–48 hours after initial growth). Pathogens with fastidious growth requirements and those difficult to identify by phenotypic methods require more time for identification

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What are the Recent advances in Blood culturing

and Identification of pathogens ■ Rapid nucleic acid amplification methods

such as real-time PCR using melting curve analysis, multiplex PCR, fluorescence in situ hybridization (FISH) and peptide nucleic acid-FISH (PNA-FISH) have been used to detect pathogens in blood cultures including Staphylococcus aureus, Enterococcus faecalis and Candida albicans .

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Target oriented AssaysCHANGING FROM PHENOTYPIC TO

NEWER METHODS ■These assays,

however, only target specific organisms; require technical expertise; and specimens are usually processed in batches. Turnaround times are up to 6 hours.

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A Rapid method to Investigate Bacteremia

and Septicaemias ■Bruker‘s MALDI Sepsityper enables identification of gram-negative bacteria, gram-positive bacteria and yeast from positive blood cultures within 30 minutes.

05/02/2023 Dr.T.V.Rao MD

MALDI-TOF MS: History■ Developed in 1980s by Karas and Hillenkamp■ Detection of large molecules using TOF by Tanaka and Yoshida■ Introduction of matrix compounds to analyze large molecules

■ First commercial instrument developed by Shimadzu■ First commercial database developed by Anagnostec (1998)■ Shimadzu scientist receives Nobel Prize in Chemistry

– Kiochi Tanaka (2002)

■ Technology in use in Europe for >10 years.12

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HISTORY OF MS • MALDI■ Term was coined in 1985 by

Franz Hillenkamp, Michael Karas • They found that amino acid alanine could be ionized easily if it was mixed with amino acid tryptophan & irradiated with pulsed 266nm laser. • Here, tryptophan absorbed the laser energy & helped to ionize the non absorbing alanine.

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A Rapid method to Investigate Bacteremia

and Septicaemias ■MALDI Sepsityper enables faster results, which physicians can act upon to manage blood stream infections, engage in the fight against resistance, and improve patient outcomes.

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Every Minute Counts to Life ■Bruker’s MALDI

Sepsityper solution provides a rapid, highly accurate microbial identification directly from a positive blood culture

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Features of MALDI-TOF MS

• Soft ionization - analyze intact biomolecules and synthetic polymers• Broad mass range - analyze a wide variety of biomolecules• Simple mixtures are okay• Relatively tolerant of buffers and salts• Fast data acquisition• Easy to use and maintain, no water or gas hook ups required• High sensitivity, superior mass resolution and accuracy

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What are the advantages of (MALDI-

TOF MS) ■The matrix assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of pathogens in blood culture broths, with results available within 2 hours. Although it does not provide antimicrobial susceptibility data (with the exception of methicillin resistant Staphylococcus aureus [MRSA]), it has good potential to guide empirical antimicrobial choice in the treatment of BSIs, yet there remain technical variables that may affect test performance

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APPLICATIONS Microbiology■ It is used for the identification

of microorganisms. • Species diagnosis by this procedure is much faster, more accurate & cheaper than other procedures based on biochemical tests. Forensic analysis Environmental analysis • Pesticides on foods • Soil and groundwater contamination

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What it means in Critical care

■ Identifying the etiologic pathogen, followed by antimicrobial susceptibility testing, is critical in the management of BSIs, as delays in effective antimicrobial therapy can adversely affect patient outcomes . MALDI-TOF MS has significant potential over phenotypic methods, as it is able to detect bacterial pathogens directly from blood culture broths reliably and quickly. However, the performance of MALDI-TOF MS is affected by blood culture bottle type, methodology in sample preparation prior to MS analysis, and the interpretive criteria employed

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(MALDI-TOF MS) is a Emerging Technology in Diagnostic Microbiology ■Matrix-assisted laser

desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of bacteria from blood culture broths

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MALDI-TOF Vs. Molecular Testing

■MALDI■ Rapid, efficient identification

from isolated colonies and liquids (MALDI-TOF/MS)

■ Molecular■ Direct detection from patient

specimens (Molecular)■ Direct detection of resistance

genes (MecA, VRE, CTX-M, KPC, NDM)

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MALDI-TOF MS Overview

desorption

ionization

acceleration

separation

detectionmz =

2eU

L²t²

m: massz: chargeU: acceleration voltageL: path lengtht: timee: elementary charge

ion detector

+

+ +

+

++

target

matrix/analytecrystals

accelerationzone

Time of Flight

ring electrode

Uncharged Drift regionVacuum

tube

Which protein molecules?Those that are easily desorbed,Like ribosomal proteins

Absorbs e from LASER

Multiple LASER shots Courtesy of bioMérieux

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Add matrix solution*

Air dry for 1-2 min.

MALDI TOF Sample Preparation

Create Spectra

Target Slide48 wells

Step 1 Step 2 Step 3 Step 4

Bacteria, molds, yeasts,mycobacteria

Spot target slide with direct colony

(can be up to 5 days old).

Load target slides

• Matrix Solution: (0.5 µl -cyano-4-hydroxycinnamic acid)

NOTE:Other sample types:- sediment from positive blood cultures- sediment from certain specimen (e.g. urines)

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Helps in the Rapid identification of Aetiological agents

■Helps identification of the etiological agents of life-threatening bloodstream infections. An alternative approach for rapid MALDI-TOF based bacterial identification starting from a short culture on agar might yield sufficient bacterial growth in 4 to 6 hours (data not shown). Given the importance of positive blood cultures, this delay may be clinically relevant as compared to the 30 to 45 minutes needed for the ammonium chloride erythrocytes-lysing procedure.

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Control of sample acceptability

■ Verification that appropriate sample(s) collected

■ Correct volume submitted■ Sample placed promptly in correct

transport media■ Optimal and timely transport

conditions■ Sample handled properly in

laboratory■ Shared samples■ Reflexed samples

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Benefits of Rapid Positive Blood

Culture Identifications

05/02/2023 Dr.T.V.Rao MD 28Definitive identifications as fast as current Gram stains!?

Direct Detection for Positive Blood Culture Bottles By MALDIPurpose: Separate human and bacterial/yeast ribosomal proteins

Methods: Lysis/centrifugation or membrane filtration

Journal of Clinical Microbiology 51;805-809, 2013

Journal of Clinical Microbiology 48;1584-1591, 2010

Issues:• Removal of human proteins

• Extraction protocol required• Bacterial concentration

• need~107/mL• Polymicrobial specimens

• Seen on Gram stain?• Charcoal• Antibiotic resistance genes• Yeasts?• Unique database, different

cutoffs?

BrukerSepsityperKit

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Expectations Vs. reality■ Should be able to

decrease the number of secondary identification systems required in Clinical Microbiology

■ Very good technology, but not perfect

■ Experienced technologists still needed

■ Better patient care through faster definitive results

■ Positive effect on workflow??

05/02/2023 Dr.T.V.Rao MD

■E. coli Vs. Shigella – Very closely related and cannot be differentiated– Molecular methods

■Streptococcus pneumoniae Vs. Streptococcus mitis group– Very closely related, new databases can give a

definitive ID– Differentiate by Bile solubility or optichin disk

■Bordetella pertussis Vs. Bordetella bronchioseptica – Very closely related and cannot be differentiated– Rarely cultured

No Test Is Perfect

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• Stenotrophomonas maltophilia Vs. Pseudomonas hibiscola, Ps. gentculata, Ps. betelli■ Very closely related and cannot be differentiated■ Biochemical ID required

• The Acinetobacter baumanii-calcoaceticus complex (A. baumanii, A. calcoaceticus, A. genospecies 3, A. genospecies : ■ Species differentiation can be difficult.

– A. baumanii and A. calcoaceticus can be differentiated, there are several members of the “Genospecies 3” clustering with A. baumanii or A. calcoacteticus, this can lead to “A. genospecies 3” ID result where biochemistry will identify A. baumanii or A. calcoaceticus

No Test Is Perfect

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Limitations with Identification of Streptococcal species

■The lower yield of valid MALDI-TOF MS results with streptococci and staphylococci might be due: (i) to the close relatedness of the different species of streptococci belonging to the S. mitis group (i.e. S. pneumoniae, S. mitis, S. sanguinis, S. oralis, …), (ii) to some relatedness of different coagulase negative staphylococci, (iii) to the cell wall composition of Gram positive bacteria conferring increased resistance to lysis, and (iv) partially to the possible presence of some residual blood proteins

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Limitations in identification of

Staphylococcal species For staphylococci, the major goal is to differentiate S. aureus from coagulase negative staphylococci and this may be accurately done on blood 108 culture bacterial pellets using the MALDI-TOF MS. In the routine practice, the difficulty in identifying S. pneumoniae from other species of the S. mitis group is much more clinically 1relevant and represents a current limitation of the MALDI-TOF MS. The presence of a capsule may also partially explain the low identification rate of S. pneumoniae, H. influenza and K. pneumoniae. Improved extraction protocols specifically designed for encapsulated bacteria are thus warranted.

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1. Reversal of sensitivity by production of carbapenemases1. Modified Hodge test

2.Some β-lactamases can be inhibited by specific inhibitors1. Clavulanic acid for some ESBLs2. Boronic acid for KPC3. Chelating agents for MBLs

3.Carbapenemase detection by MALDI-TOF1. Directly measure changes in M/S by hydrolysis

and, in some cases, decarboxylation of the antibiotic

Future Uses and on going work on - Detection of Carbapenemases in the

Clinical Laboratory

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Advantages with MALDI-TOF ■ Rapid identification ~ 1min per isolate

■ Consolidation of identification testing onto a single platform– Current Phenotypic methods

■ Gram stain, Vitek 2, Microscan, numerous API methods, disks on media, growth characteristics, selective media, chromogenic media, biochemical tests, serologic tests, enzymatic reactions

– Genotypic methods■ amplified nucleic acid methods, nucleic acid sequencing

■ Reduced cost per test– Cost will be <$1.50 per determination

■ Reduced Hands-on-Time■ Tech setup time 2-3 minutes

■ Flexibility - each bench get their own target slide■ High throughput – 192 isolates/run (4 hours)■ Outbreak strain typing is possible, eventually. May need different matrix

– Local strains can be included in a user defined database– Outbreaks can be identified prospectively rather than retospectively

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Next big change in Clinical Microbiology■ MALDI-TOF/MS is faster, better, ■ Better than current full identification methods

– Modify to fit your laboratory. ■ Use in conjunction with rapid methods■ RUO Vs. IVD databases

– Amount of validation required??

– Same identification expertise on all shifts– Retrospective outbreak evaluations– Identification directly from positive blood

culture bottles and other body fluids– Identification not dependent on interaction

with biochemicals■ Limited reference spectra in database for some

genera and species– Identifications will get better

■ Can be automated

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References ■ Identification of Bacteria in Blood Culture Broths

Using Matrix-Assisted Laser Desorption-Ionization Sepsityper™ and Time of Flight Mass SpectrometryJen Kok,1,2,* Lee C. Thomas,1 Thomas Olma,1 Sharon C. A. Chen,1 and Jonathan R. Iredell1, PLoS One. 2011; 6(8): e23285. NCBI resource

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■Program Created by Dr.T.V.Rao MD for Benefit of Medical and Technical

Professionals on newer and emerging technologies in Diagnostic

Microbiology with resources from world wide web

■ doctortvrao@gmail.com