Professional Documents
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MICROBIOLOGY
OUR VISION TO FUTURE
TECHNOLOGY
Dr.T.V.Rao MD
4/1/16
Dr.T.V.Rao MD
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
4/1/16
Dr.T.V.Rao MD
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.
4/1/16
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
Dr.T.V.Rao MD
4/1/16
Dr.T.V.Rao MD
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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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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TOF MS)
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Dr.T.V.Rao MD
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APPLICATIONS Microbiology
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
(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
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Dr.T.V.Rao MD
21
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
Dr.T.V.Rao MD
protein molecules?
Those that are easily desorbed,
Like ribosomal proteins
Courtesy of bioMrieux
23
Step 1
Step 2
Add matrix
solution*
NOTE:
Step 3
Load target
slides
Step 4
Create Spectra
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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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
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
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
29
No Test Is Perfect
E. coli Vs. Shigella
Very closely related and cannot be differentiated
Molecular methods
Dr.T.V.Rao MD
4/1/16
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Dr.T.V.Rao
MD
No Test Is Perfect
32
Dr.T.V.Rao
MD
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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35
Dr.T.V.Rao
MD
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
Reduced Hands-on-Time
36
get better
37
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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doctortvrao@gmail.com
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Dr.T.V.Rao MD
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