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CLINICAL

MICROBIOLOGY
OUR VISION TO FUTURE
TECHNOLOGY
Dr.T.V.Rao MD

4/1/16

Dr.T.V.Rao MD

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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Dr.T.V.Rao MD

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
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Dr.T.V.Rao MD

Changing understanding on
Gram Negative Bacteria
Gram negative
bacteria by
measuring
molecular
masses of
proteins and
other bacterial
components
obtained from
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Dr.T.V.Rao MD

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-OfFlight (MALDI-TOF). Moreover, recent
4/1/16

Dr.T.V.Rao MD

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.
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Dr.T.V.Rao MD

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.
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Dr.T.V.Rao MD

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 2448 hours after
initial growth). Pathogens with
fastidious growth requirements and
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Dr.T.V.Rao MD

What are the Recent


advances in Blood culturing
and Identification
of
Rapid nucleic
acid amplification methods
such as real-time
PCR using melting curve
pathogens

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 .

4/1/16

Dr.T.V.Rao MD

Target oriented Assays


CHANGING FROM PHENOTYPIC TO
NEWER METHODS
These assays,
however, only
target specific
organisms;
require technical
expertise; and
specimens are
usually processed
in batches.
Turnaround times
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Dr.T.V.Rao MD

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A Rapid method to
Investigate Bacteremia
Brukers MALDI
and Septicaemias
Sepsityper
enables
identification of
gram-negative
bacteria, grampositive bacteria
and yeast from
positive blood

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Dr.T.V.Rao MD

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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.


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4/1/16

Dr.T.V.Rao MD

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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Dr.T.V.Rao MD

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A Rapid method to
Investigate Bacteremia
MALDI Sepsityper
and
Septicaemias
enables
faster
results, which
physicians can act
upon to manage
blood stream
infections, engage
in the fight
against
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Dr.T.V.Rao MD

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Dr.T.V.Rao MD

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Brukers MALDI
Every Minute Counts
to Life
Sepsityper
solution
provides a
rapid, highly
accurate
microbial
identification
directly from a
positive blood
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Dr.T.V.Rao MD

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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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Dr.T.V.Rao MD

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What are the


advantages of (MALDI-

The matrix assisted laser desorptionionization 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

TOF MS)

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Dr.T.V.Rao MD

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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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Dr.T.V.Rao MD

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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 . MALDITOF 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
4/1/16
Dr.T.V.Rao
MD blood culture bottle type,
20

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

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

4/1/16

Dr.T.V.Rao MD

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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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Dr.T.V.Rao MD

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MALDI-TOF MS
Overview
detection

ion detector
+ Vacuum

separation

tube

Uncharged
Drift region
Time of Flight
ring electrode

acceleration
+

ionization

desorption

Absorbs e
from LASER
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acceleration
zone

m
z

2eU
L

m: mass
z: charge
U: acceleration voltage
L: path length
t: time
e: elementary charge

matrix/analyte
Which
crystals
target

Multiple LASER shots

Dr.T.V.Rao MD

protein molecules?
Those that are easily desorbed,
Like ribosomal proteins
Courtesy of bioMrieux

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MALDI TOF Sample


Preparation

Step 1

Spot target slide


with direct colony
(can be up to 5
days old).

Step 2

Add matrix
solution*

NOTE:

Step 3

Load target
slides

Step 4

Create Spectra

Other sample types:


- sediment from positive blood cultures
- sediment from certain specimen (e.g. urines)

Air dry for 1-2


min.

Bacteria,
molds, yeasts,
mycobacteria

Target Slide
48 wells

Matrix
Solution: Dr.T.V.Rao
(0.5 lMD-cyano-4-hydroxycinnamic acid)
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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
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Dr.T.V.Rao MD

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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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Dr.T.V.Rao MD

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Benefits of Rapid
Positive Blood
Culture
Identifications
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Dr.T.V.Rao
MD

Direct Detection for Positive Blood


Culture
Bottles
By
MALDI
Purpose: Separate
human and
bacterial/yeast
ribosomal
proteins
Methods: Lysis/centrifugation or membrane filtration

Journal of Clinical Microbiology 51;805-809, 2013

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?

Bruker
Sepsityper
Kit
Journal of Clinical Microbiology 48;1584-1591, 2010

Definitive identifications as fast as current Gram stains!?


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Dr.T.V.Rao MD

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Should reality
be able to
Expectations Vs.
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
4/1/16

Dr.T.V.Rao MD

Positive effect on

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No Test Is Perfect
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
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Dr.T.V.Rao MD

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Dr.T.V.Rao
MD

No Test Is Perfect

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.
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Dr.T.V.Rao
MD

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
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Dr.T.V.Rao MD

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Limitations in
identification of
For staphylococci, the major goal is to differentiate S.
aureus
from coagulase negative staphylococci
and this
Staphylococcal
species
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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Dr.T.V.Rao MD

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


Laboratory
1. ReversalClinical
of sensitivity
by production of
carbapenemases
1. Modified Hodge test

2.Some -lactamases can be inhibited by


specific inhibitors
1. Clavulanic acid for some ESBLs
2. Boronic acid for KPC
3. Chelating agents for MBLs

3.Carbapenemase detection by MALDI4/1/16

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Dr.T.V.Rao
MD

Advantages with MALDITOF

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 Dr.T.V.Rao


be identified
prospectively rather than retospectively
4/1/16
MD

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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
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Dr.T.V.Rao MD

get better

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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

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Dr.T.V.Rao MD

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