A concise but explanatory presentation on the use, advantage and limitations of MALDI TOF in molecular diagnosis

1. MALDI-TOF as a diagnostic tool in
Microbiology (Advantages and
Limitations)
Dr. SuprakashDas
SeniorResident
Dept. of Microbiology

2. Introduction
 Rapid and accurate species identification of bacteria, fungi, and viruses is
a fundamental requirement in clinical and food microbiology and other
fields of microbiology diagnostics.
 Whereas virus recognition is usually achieved within hours by either
serological tests or genotyping approaches using various nucleic acid
detection systems, the conventional identification of bacteria and fungi
still mainly relies on methods that include laborious and time-consuming
initial cultivation and ensuing isolation of the microorganism.
 Though species identification of a pure culture is achievable within 24–
48 h with various (semi-)automated systems, additional isolation steps
are frequently necessary, which can extend the time until diagnosis by
days, e.g., if the potential pathogen must be separated from the
physiological background flora.
 Realistically species assignment of a putative pathogen from a non-sterile
specimen takes at least 2–3 days.

3. Introduction
 In many areas of patient care, elapsed time until diagnosis may
considerably reduce the therapeutic quality of care due to a lack of
information about the infecting pathogen.
 Therefore, a rapid species diagnosis is of high priority as a focused
therapy might be lifesaving for the patient.
 Molecular techniques which targets discriminatory genes offer
excellent species identification often missed by phenotypic methods
but they are limited by their high price per test, need of highly
expert persons and most of them lack vast database.
 Here, the power of matrix-assisted laser desorption/ionization
time-of- flight mass spectrometry (MALDI-TOF MS) is
demonstrated as a redevelopment that has evolved to revolutionize
the identification of prokaryotic and eukaryotic pathogens in
microbial laboratories during recent years.

4. History of MALDI-TOF
 Mass spectrometry (MS) technology has been used for
several decades in chemistry.
 In 1975, Anhalt and Fenselau suggested the use of this
tool for bacterial characterization, as they observed that
different and unique mass spectra were produced from
bacterial extracts of different genera and species.
 In the 1980s, desorption/ionization techniques (plasma
desorption, laser desorption, and fast atom
bombardment) that allow the generation of molecular
biomarker ions from microorganisms were developed,
opening the road for bacterial profiling.

5. History of MALDI-TOF
The first experiments were based on a bio-
molecule ionization processes that allowed the
generation of biomarker molecules of low
molecular masses, mostly bacterial lipids.
 Only in the late 1980s, Tanaka and
Fenn,[Noble-2002] thanks to the development of
soft ionization techniques (matrix-assisted laser
desorption/ionization [MALDI] and electrospray
ionization), made possible the analysis of large
biomolecules such as intact proteins.

6. History of MALDI-TOF
 For the first time, in 1996, MALDI-time of flight
(TOF) spectral fingerprints could be obtained from
whole bacterial cells by Holland et al., avoiding
pretreatment before the MS analysis.
 Nowadays, the matrix-assisted laser desorption
ionization time-of flight mass spectrometry (MALDI-
TOF MS) has emerged as a rapid, accurate, and
sensitive tool for microbial characterization and
identification of bacteria, fungi, and viruses, as
demonstrated by the exponential number of
publications about the issue.

7. Basic Terminology
 Adduct- Ion formed by the interaction of an ion with one or
more atoms or molecules to form an ion containing all the
constituent atoms of the precursor ion as well as the
additional atoms from the associated atoms or molecules.
 Analyte- Biomolecule or sample that is being analyzed.
 Chromophore- Functional group in a molecule that is
known to absorb light; this is necessary for the MALDI
matrix in order to absorb the energy of the laser beam.
 Desorption- The opposite of absorption; here a substance is
released from or through the surface rather than going into
it.
 TOF- Time taken by the ions to travel through the flight
tube when an electrostatic potential is applied at its ends

8. Basic Terminology
 Detector- The ions generated in a mass spectrometer after traveling
through the flight tube ultimately hit the analyzer, where they are
detected and converted into a digital output signal.
 Mass analyzer- Chamber having an electrostatic field; its purpose is
to separate the ions coming from the source depending on their
mass-to-charge ratio so that they can be detected by the detector.
 Matrix- Compound that is mixed with the sample that is being
analyzed; the matrix protects the sample molecules from being
destroyed by direct focus of the laser beams and facilitates the
sample’s vaporization and ionization.
 Sublimation- Passing from solid to gas without going through a
liquid phase.

9. Basic Principle of MALDI-TOF
 The genomic information within a microbial cell translates
into more than 2000 proteins, a substantial number of which
can be studied using proteomics.
 It is estimated that for genomes which contain less than
1000 genes, more than 50% of predicted proteome may be
identified from the genome.
 Similarly 30 and 10% predicted proteome may be identified
from genomes which carry 2500 and 4000 genes
respectively (Jungblut and Hecker, 2004).
 Thus, a microbial genome containing 600–7000 predicted
genes represents a medium-sized complex system where
application of proteomics may provide knowledge of a
substantial part of the microbe’s proteome.

10. Basic Principle of MALDI-TOF
 Mass spectrometry is an analytical technique in which chemical
compounds are ionized into charged molecules and ratio of their
mass to charge (m/z) is measured.
 Though MS was discovered in the early 1900s, its scope was
limited to the chemical sciences.
 However, the development of electron spray ionization (ESI) and
matrix assisted laser desorption ionization (MALDI) in 1980s
increased the applicability of MS to large biological molecules like
proteins.
 In both ESI and MALDI, peptides are converted into ions by either
addition or loss of one or more than one protons. Both are based on
“soft ionization” methods where ion formation does not lead to a
significant loss of sample integrity.

11. Basic Principle of MALDI-TOF
 MALDI- TOF MS has certain advantages over ESI-
MS viz.
(i) MALDI- TOF MS produces singly charged ions, thus
interpretation of data is easy comparative to ESI-MS,
(ii) for analysis by ESI- MS, prior separation by
chromatography is required which is not needed for
MALDI-TOF MS analysis.
 Consequently, the high throughput and speed
associated with complete automation has made
MALDI-TOF mass spectrometer an obvious choice
for proteomics work on large-scale

12. MALDI – Principle and Methodology
 The sample for analysis by MALDI MS is prepared by
mixing or coating with solution of an energy-absorbent,
organic compound called matrix.
 When the matrix crystallizes on drying, the sample
entrapped within the matrix also co-crystallizes.
 The sample within the matrix is ionized in an automated
mode with a laser beam.
 Desorption and ionization with the laser beam generates
singly protonated ions from analytes in the sample.
 The protonated ions are then accelerated at a fixed
potential, where these separate from each other on the basis
of their mass-to- charge ratio (m/z).

13. MALDI – Principle and Methodology
 The charged analytes are then detected and measured using different
types of mass analyzers like quadrupole mass analyzers, ion trap
analyzers, time of flight (TOF) analyzers etc.
 For microbiological applications mainly TOF mass analyzers are
used. During MALDI-TOF analysis, the m/z ratio of an ion is measured
by determining the time required for it to travel the length of the
flight tube.
 A few TOF analyzers incorporate an ion mirror at the rear end of the
flight tube, which serves to reflect back ions through the flight tube to a
detector.
 Thus, the ion mirror not only increases the length of the flight tube, it
also corrects small differences in energy among ions (Yates, 1998).
 Based on the TOF information, a characteristic spectrum called peptide
mass fingerprint (PMF) is generated for analytes in the sample.

14. MALDI – Principle and Methodology
 Identification of microbes by MALDI-TOF MS is done by either
comparing the PMF of unknown organism with the PMFs contained in
the database, or by matching the masses of biomarkers of unknown
organism with the proteome database.
 In PMF matching, the MS spectrum of unknown microbial isolates is
compared with the MS spectra of known microbial isolates contained
in the database.
 For species level identification of microbes, a typical mass range m/z of
2–20 kDa is used, which represents mainly ribosomal proteins along with
a few housekeeping proteins.
 The characteristic pattern of highly abundant ribosomal proteins, which
represent about 60–70% of the dry weight of a microbial cell, in the mass
range of 2–20 kDa is used to identify a particular microorganism by
matching its PMF pattern with the PMFs of the ribosomal proteins
contained in an extensive open database.

15. MALDI – Principle and Methodology
 Although, the culture conditions might profoundly affect the
microbial physiology and protein expression profile (Welker, 2011)
they do not influence microbial identification by MALDI- TOF
MS.
 Valentine et al. (2005) cultured three bacterial species on four
different culture media and found that microbial MALDI-TOF MS
identification was independent of culture conditions.
 A number of organic compounds have been used as matrices for
MALDI-TOF MS but for microbiological applications,
 α-cyano-4-hydroxycinnamicacid(CHCA),
 2,5-dihydroxybenzoicacid(DHB), and
 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) have
been found to be the most useful.

16. MALDI – Principle and Methodology
 The matrix solution consists of water and a mixture of organic solvents
containing ethanol/methanol or acetonitrile and a strong acid like
trifluoro acetic acid (TFA), which dissolves the matrix.
 The solvents penetrate the cell wall of microorganisms and extract out
the intracellular proteins.
 When the solvent evaporates, ‘co-crystallization’ of protein molecules
and other cellular compounds entrapped within the matrix solution takes
place.
 Investigators have evaluated different sample preparation methods for
different groups of microorganisms.
 Some microbes might be identified directly by MS, called direct cell
profiling, while for some others whole cell lysates or crude cell
extracts are prepared.
 In direct cell profiling, a single colony of microorganism is picked and
spotted directly on to the sample plate and immediately overlaid with the

17. Role of the Matrix
 The energy pooling theory results in the following
mechanism, whereby the absorbed photon from the excited-
state matrix molecule (M*) is transferred to the second
excited matrix molecule, resulting in the formation of a
cationic matrix radical (M+), a nonradical matrix molecule
(M), and a free electron (e):
 MM→2hv
 M*M*→ M + + M + e-
 The second step of this two-part reaction involves a proton
transfer event from the excited matrix molecule to the
clinical sample (A), resulting in ionization of a molecule of
the clinical sample:

18. Role of the Matrix
 M* + A→ (M - H) + AH+
 Additional ions of the clinical specimen are formed by
secondary ion-molecule reactions between matrix-
matrix and matrix specimen interactions. These
reactions are thermodynamically favorable because the
proton affinity of MALDI matrices is typically lower
than that of peptides and proteins to be analyzed in
clinical material. This is modeled by the following
equations:
 M + + M→ MH+ + (M - H) and
 MH+ + A→ M + AH+

19. List of commonly used matrices in
UV-MALDI
PA- Picolinic Acid DHAP-2,6 dihydroxyacetophenone.
HPA- 3- hydroxy picolinic acid THAP- 2,4,6, trihydroxyacetophenone
SA- 3,5 dimethoxy-4-hydroxy cinnamic Acid
HABA- 2,4-Hydroxy phenylazo benzoic Acid
MBT- 2- mercaptobenzothiazole

20. Time of Flight Mass Analyzer
 The time of flight (TOF) analyzer is dependent upon the principle that
applying an electrostatic field (eV) to the ionized clinical material causes a
generated ion with a charge (z) to accelerate, imparting to it some amount
of kinetic energy (KE). The ions then move into a field-free drift region,
where the only force affecting ionic movement is the kinetic energy from
the acceleration step. The velocity (v) of the ionized molecule from a
clinical specimen can therefore be calculated by using the following
equation, where
 KE is kinetic energy,
 m is mass,
 v is velocity,
 z is the charge of the ion (1 for MALDI),
 eV is the voltage applied,
 D is the distance to the detector, and
 t is time:

21. Time of Flight Mass Analyzer
 In this context, D and eV are constant and t is measured,
allowing the m/z ratio to be determined. A simple
mathematical rearrangement results in the following
equation.
 KE= ½ mv2 = zeV, v= D/t, t= D.(Square root of m/2zeV).
 This equation demonstrates that drift time is directly
proportional to the m/z ratio. Larger ions will have a longer
drift time and smaller molecules will have a shorter drift
time, demonstrating separation of molecules based on mass.
This allows for separation of ions originating from clinical
material based on the m/z ratio.

22. Sample Preparation
 Gram negative bacteria like Neisseria spp., Yersinia
spp. and Vibrio spp. were identified by MALDI-TOF
MS using direct cell profiling.
 A ‘preparatory extraction’ of microbes with formic acid
(FA) reportedly increased the ability of MALDI-TOF
MS in identifying Gram-positive species.
 Due to the complex nature of their cell walls aerobic
actinomycetes, Nocardia and mycobacteria require
specialized processing procedures prior to MALDI-
TOF analysis.

23. Sample Preparation
Verroken et al. (2010) described a modified
procedure for identification of Nocardia spp. by
MALDI-TOF MS.
Bacteria were lysed in boiling water, followed by
ethanol precipitation of proteins.
 The precipitated proteins were dried, re-
suspended in 70% formic acid and acetonitrile
and analyzed by MALDI-TOF MS.
EI Khéchine et al. (2011) described a procedure
which combined inactivation and processing
methods.

24. Sample Preparation
Mycobacterial colonies collected in screw-cap
tubes containing water and 0.5% Tween-20 were
inactivated by heating at 95◦C for 1h.
Inactivated samples were centrifuged and
vortexed with glass beads to disrupt the
mycobacterial cell wall, the resultant pellet was
resuspended in formic acid, acetonitrile,and
centrifuged again.
Finally, the supernatant was deposited on to the
MALDI target plate and over laid with matrix.

25. Sample Preparation
 As in bacteria, various sample processing methods have
been investigated for identification of yeasts by MALDI-
TOF MS, out of which ‘preparatory extraction’ with
formic acid was reported to be most suitable.
 Investigators finally devised a protocol wherein fungi were
cultivated on Sabouraud gentamicin-chloramphenicol
agar for 72h at 27◦C, extracted with formic acid following
incubation in ethanol.
 Acetonitrile was added, the mixture was centrifuged and
supernatant was used for MALDI-TOF MS analysis.

26. Sample Preparation
Lau et al. (2013) reported a method based on
mechanical lysis for sample preparation of fungal
hyphae and spores.
 A small specimen from the mold isolate was
suspended in ethanol and
zirconia-silica beads, vortexed and centrifuged.
 The pellet was re suspended in 70% FA,
vortexed again, and centrifuged.
 The supernatant was used for subsequent analysis
by MALDI-TOF MS. e.g Penicillium spp.

29. Sample Preparation
 Suggestions for MALDI-TOF MS sample preparations for
use with different classes of microbes.
 Different groups of microorganisms vary fundamentally in
their cellular composition and architecture.
 These differences have been demonstrated to affect the
quality of spectra generated during MS experiments and,
thus, the accuracy of MALDI-TOF MS-derived
identifications.
 As such, investigators from a number of independent
studies have evaluated different methods for sample
preparation of different groups of microorganisms, ranging
directly from intact-cell to full-protein extraction- based
methodologies.

30. Present Scenario
 The discovery of suitable matrices, usage of whole/intact
cells for recording PMF of microbes in the typical mass
range (m/z) of 2–20 kDa, followed by availability of
dedicated databases for microbial identification has made
MALDI-TOF MS a lucrative alternative for microbial
identification.
 The first MALDI-TOF MS system capable of microbial
identification, termed “MALDI Biotyper” was developed
by Bruker Daltonics.
 The MALDI Biotyper consisted of a basic MALDI-TOF
platform, operating and analysis softwares, an onsite
database and a simple method for extraction/preparation of
sample. This system also allowed for up gradation by
providing option of adding internal libraries of organisms.

31. Present Scenario
 Another MALDI platform for microbial identification
was introduced by Shimadzu in collaboration with
bioMérieux.
 Later, Andromas introduced a different kind of
database and software for routine bacteriological
identification which was compatible with both Bruker
and Shimadzu instruments.
 In the last 2 years, MALDI Biotyper CA System
(Bruker Daltonics Inc.) has been approved by the
US Food and Drug Administration (FDA) for
identification of cultured bacteria from human
specimens (in vitro diagnosis).

32. Present Scenario
Similarly, VITEKR MS (bioMerieux Inc.) was
approved by the USFDA for identification of
cultured bacteria and yeasts.
The list of microorganisms approved for
identification is unique to the individual system.
The organism databases are the key components
of commercial MALDI platforms. They are
continually increasing in size and are regularly
updated by the manufacturers with discovery of
new microbial species and annotations.

33. General schematic for the identification of bacteria and yeast by
MALDI-TOF MS using the intact-cell method. Bacterial or fungal growth is
isolated from plated culture media (or can be concentrated from broth
culture by centrifugation in specific cases) and applied directly onto the
MALDI test plate. Samples are then overlaid with matrix and dried. The
plate is subsequently loaded into the MALDI-TOF MS instrument and
analyzed by software associated with the respective system, allowing rapid
identification of the organism.

34. General schematic for MS analysis of ionized microbiological isolates and clinical material.
Once appropriately processed samples are added to the MALDI plate, overlaid with matrix,
and dried, the sample is bombarded by the laser. This bombardment results in the
sublimation and ionization of both the sample and matrix. These generated ions are
separated based on their mass-to-charge ratio via a TOF tube, and a spectral representation
of these ions is generated and analyzed by the MS software, generating an MS profile. This
profile is subsequently compared to a database of reference MS spectra and matched to
either identical or the most related spectra contained in the database, generating an
identification for bacteria or yeast contained within the sample.

40. Comparison of 2 FDA approved systems
Specification VITEK MS (Biomeriux) MALDI-Biotyper (Bruker)
Calibrator- Escherichia coli ATCC 8739 US IVD Bacterial Test Standard (BTS)
Matrix- -cyano-4-hydroxycinnamic
acid (VITEK MS-CHCA)
-cyano-4-hydroxycinnamic acid (US
IVD HCCA portioned);
must be reconstituted in accordance
with instructions provided by using
recommended solvent (standard solvent:
50 vol% ll acetonitrile, 47.5
vol% ll water, 2.5 vol% ll trifluoroacetic
acid)
Extraction- formic acid (VITEK MS-FA) (1) Deionized water
(2) Absolute ethanol (EtOH)
(3) Acetonitrile (ACN)
(4) Formic acid (70%)
(5) Microtube (Eppendorf), PCR clean
1.5 ml

41. Comparison of 2 FDA approved systems
Specification VITEK MS (Biomeriux) MALDI-Biotyper (Bruker)
Supplies-
Target
slides:
single-use, disposable slides consisting
of two acquisition
groups, each with 16 sample spots
(VITEK MS-DS target slides); each
group includes one calibration spot.
reusable, steel plates
consisting of spots for 48
test organisms
(US IVD 48 Spot Target);
there are also five cross-
joint positions
that should be used for the
BTS control.
Sterile inoculating loops (1 µl) Sterile inoculating loops (1
µL)
Precision micropipette (0.5 to 2.0 ll) Precision micropipette (0.5
to 2.0 ll)
Sterile pipette tips without filter to reduce
any protein contamination
Sterile pipette tips without
filter.

42. Comparison of 2 FDA approved systems
Specification VITEK MS (Biomeriux) MALDI-Biotyper (Bruker)
Equipment-
Instrument: VITEK MS, floor model with a class
1, 337-nm fixed focus, nitrogen laser
Microflex LT/SH mass
spectrometer; desktop model
with a class 1 337-nm fixed
focus, nitrogen laser.
Preparation
station:
VITEK MS Prep Station; location for
preparing target
slides, including a computer workstation
with barcode reader.
not applicable to MBT-CA
system.
Software: 1. VITEK MS Acquisition Station;
operates the MS to acquire spectral
data from each sample. The signal is
recorded as a spectrum of intensity
versus mass (in daltons [Da]).
1. MBT-CA System
Software Package, including
the MBT-CA System client
software, the MBT-CA
System Server, and the MBT-
CA System, DB Server
2. VITEK MS Analysis Server; manages
the VITEK MS workflow and
2. flexControl Software
Package,

43. Comparison of 2 FDA approved systems
Specification VITEK MS
(Biomeriux)
MALDI-Biotyper (Bruker)
Database VITEK MS
Knowledge Base;
current version
(V2.0) of the
reference database
contains 755
approved taxa
(645 bacteria and
100 fungi).
MALDI Biotyper for Clinical Applications
(MBT-CA); reference
database containing the identity of 210 species or
species groups,
covering 280 clinically relevant bacteria and yeast
species. The reference library was established
using type strains combined with 5 to 38 additional
clinical or culture collection strains per species.

44. Comparison of 2 FDA approved systems
VITEK MS (Biomeriux) MALDI-Biotyper (Bruker)
M
e
d
i
a
t
y
p
e
s
:
a. Columbia blood agar with 5% sheep
blood
b. TSA with 5% sheep blood
c. TSA
d. Chocolate polyvitex agar
e. Campylosel agar
f. MAC (use of this medium from some
suppliers may show less optimal
performance)
g. Modified Sabouraud dextrose agar
(glucose: 20 g/liter)
h. chromID CPS
a. Columbia blood agar with 5% sheep
blood (Gram-negative aerobic bacteria)
b. TSA with 5% sheep blood (Gram-
negative aerobic bacteria, Gram-positive
aerobic bacteria, yeast)
c. Chocolate agar (Gram-negative aerobic
bacteria and Gram-positive aerobic
bacteria)
d. MAC (Gram-negative aerobic bacteria)
e. Columbia CNA agar with 5% sheep
blood (Gram-positive aerobic bacteria)
f. Brucella agar with 5% horse blood
(Gram-negative and Gram-positive
anaerobic bacteria)
g. CDC anaerobe agar with 5% sheep
blood (Gram-negative and Gram-positive
anaerobic bacteria)

45. Comparison of 2 FDA approved systems
VITEK MS
(Biomeriux)
MALDI-Biotyper (Bruker)
Age
of
cultu
re:
For both bacteria
and yeast
protocols,
isolates should
be obtained from
cultures after 24
to 72 h of
incubation
under
appropriate
growth
conditions.
In general, bacteria and yeast isolates should be obtained
from cultures after 18 to 48 h of incubation, with continued
stability up to
12 additional hours at room temperature (maximum 60 h).
The following
organisms, however, require specific considerations:
a. Bordetella: incubation on BG agar should not be longer
than 24 h (+12
h storage at RT)
b. Campylobacter: incubation can be prolonged to 72 h
(+12h storage at
RT)
c. Streptococcus pneumoniae: incubation should not be
longer than 24 hours
(+12 h storage at RT) due to possible autolysis of organism.

46. Comparison of 2 FDA approved systems
Speci
ficati
on
VITEK MS (Biomeriux) MALDI-Biotyper (Bruker)
Ident
ificat
ion
Resu
lts.
(1) Single identification: When only one
significant organism or organism
group is identified, a single identification is
displayed with a confidence value of 60 to
99.9.
(2) Low discrimination identification:
When more than one significant
organism or organism group is retained, but
not more than four, low discrimination
identifications for each organism are
displayed. The
sum of the confidence values equals 100.
(3) Nonidentified: When more than four
organisms or groups are identified,
the list of possible organisms is displayed but
the sum of confidence
values is less than 100.
(4) Nonidentified, U (unclaimed
identification): No match is found.
Using the FDA-approved algorithm,
an organism identification is
reported with high confidence if the
log(score) is greater than or equal to
2.000. An organism identification is
reported with low confidence if the
log(score) is between 1.700 and
2.000. No identification is given if
the log(score) is less than 1.700.
(1) In general practice, a score of
greater than or equal to 2.000 allows
for species-level identification,
whereas a score of 1.700 to 1.999
allows for genus-level
identification. Again, a log(score)
of less than 1.700 indicates no
identification.

47. MALDI-TOF in Clinical Bacteriology
 A number of researchers have shown that MALDI-TOF MS
can be used for early identification of bacteria in blood
cultures, urinary tract infections (UTIs), cerebrospinal
fluids, respiratory tract infections, stool samples etc.
 Many studies have shown that MALDI-TOF MS equal or
even surpassed the conventional diagnostic methods in
speed and accuracy in detecting blood stream infections.
 A few studies suggested that additional pre-treatment of
body fluids by ammonium chloride, formic acid or short-
term incubation on solid medium further improved the
diagnostic potential of MALDI-TOF MS.

48. MALDI-TOF in Clinical Bacteriology
 When conventional methods for identification of urinary tract
pathogens for diagnosis of UTIs were compared with MALDI-
TOF MS based identification systems, it was found that MALDI-
TOF MS required minimal processing time and identified bacteria
from urine samples in the presence of even more than two uro-
pathogens.
 Diagnosis of infectious diarrhea in laboratory is usually done by
culture and identification of bacteria in the stool samples. This is a
costly and time consuming process requiring 3–5 days for
detection and identification of enteric bacterial pathogens.
 He et al. (2010) found that the entire procedure for identification
by MALDI- TOF MS, from smear preparation to reporting of the
final result was completed within 30 min, thus shortening the
turnaround time of the test by 2–3 days.

49. MALDI-TOF in Clinical Bacteriology
 Bacterial meningitis is a neurological emergency.
MALDI-TOF MS has been used for direct detection of
bacteria causing meningitis in cerebrospinal fluids
(Segawa et al., 2014).
 It has also been used for rapid identification of
atypical, Gram-negative environmental organisms
and respiratory tract pathogens which chronically
infect patients with cystic fibrosis .
 Guembe et al. (2014) reported that MALDI-TOF MS
can perform better than conventional culture methods
in diagnosis of catheter- related bloodstream
infections.

50. MS Identification of Bacteria Directly from Patient
Specimens
 While MALDI-TOF MS has been extensively evaluated as a
universal platform for the proteomic analysis and identification of
bacteria and yeasts from culture media,
 The technology is also being exploited to analyze patient
specimens directly, completely bypassing the need for culture
by detecting the presence or absence of pathogens in the clinical
specimen proper.
 This type of direct analysis proved impossible before the advent of
molecular analysis.
 The ability of both molecular and proteomic approaches to
identify targets in these types of samples can also be enhanced by
preliminary processing of these samples, removing some of the
elements (proteins, nucleic acids, and cellular debris, etc.) that can
inhibit analysis.

53. MS Identification of Bacteria Directly from
Patient Specimens
 Current position of MALDI-TOF MS in the workflow of the clinical
microbiology laboratory, including the current options for analysis
of bacteria directly from patient specimens.
 The MALDI-TOF MS instrument fits easily into the clinical
microbiology workflow, occupying the position once held by
instruments for
 automated phenotypic-based identifications (blue arrows).
 Evaluated mechanisms for the processing of samples directly from
patient specimens are included (hatched red arrows),
 as are options for the use of traditional (green arrows)
 and MALDI-TOF MS (hatched green arrows) mechanisms.
 Finally, results are imported into the laboratory information system
from the MADLI-TOF MS instrument or other instruments and
reported to physicians and pharmacists as indicated.

54. Food- and Water-Borne Bacteria
The genus Aeromonas which is indigenous to
surface waters is currently composed of 17
species, of which seven can cause severe water-
borne outbreaks.
Donohue et al. (2007) used the m/z signature of
known strains of Aeromonas to assign species to
unknown environmental isolates.
 Their results showed that MALDI-TOF MS
rapidly and accurately classified unknown species
of the genus Aeromonas, which was suitable for
environmental monitoring.

55. Food- and Water-Borne Bacteria
 MALDI-TOF MS has also been applied successfully in food
microbiology for various purposes like,
I. Identification and classification of lactic acid bacteria in
fermented food
II. Detection of bacteria involved in spoilage of milk and pork
III. Identification of bacteria isolated from milk of dairy cows
IV. Identification of bacteria present in probiotics and yogurt
V. Identification of pathogenic bacteria contaminating powdered
infant formula-food
VI. Characterization of biogenic amine-producing bacteria
responsible for food poisoning and
VII. Identification of causative agents of seafood-borne bacterial
gastroenteritis.

56. Detection and Identification of Agents of
Biological Warfare
 Fast and reliable identification of microbes which pose
threats as agents of bioterrorism is required, not only to
combat biological-warfare attacks , but also to prevent
natural outbreaks caused by these organisms.
 Conventionally, organisms which pose severe threats as
agents of bioterrorism have been identified by phenotypic,
genotypic, and immunological identification systems which
are slow, cumbersome and pose significant risk to the
laboratory personnel.
 Recently various researchers reported MALDI-TOF MS as
a simple, rapid and reliable approach to identify highly
pathogenic organisms like Brucella spp., Coxiella burnetti,
Bacillus anthracis, Francisella tularensis, and Y. pestis.

57. Detection and Identification of Agents of
Biological Warfare
 Further work is being carried out to develop safe and
MS- compatible protocols for inactivation of vegetative
cells and spores of highly pathogenic organisms (TFA
& Ethanol), which can be integrated into a routine
microbiological laboratory.
 MALDI-TOF MS has also been shown to be useful for
detection of protein toxins, such as
staphylococcal enterotoxin B,
botulinum neurotoxins,
Clostridium perfringens epsilontoxin,
shiga toxin etc. which can be used as potential agents
for bioterrorism when delivered via an aerosol route.

58. Detection and Identification of Agents of
Biological Warfare
Alam et al. (2012) developed a simple method of
sample processing for identification of protein
toxins by MALDI-TOF/TOF MS method.
Nebulizer was used to generate aerosols which
were collected using a cyclone collector.
 Tandem MS data with information from peptide
sequences was used for detecting toxins that
originated from organisms of any geographical
location.

59. Bacteria Identified in various studies using
MALDI-MS
Bacteria Genus Species or Group Evaluated
Gram-positive organisms Staphylococcus Coagulase-negative staphylococci
S. aureus
Coagulase positive, non-S. aureus
Micrococcus Micrococcus spp.
Streptococcus Beta-hemolytic species
Group A streptococci
Group B streptococci
Streptococcus pneumoniae
Viridans group streptococci
Nutritionally variant streptococci
Enterococcus Enterococcus spp.
Lactococcus Lactococcus spp.
Bacillus Bacillus spp.

60. Bacteria Identified in various studies
using MALDI-MS
Bacteria Genus Species or Group Evaluated
Gram-positive
organisms
Listeria Listeria spp.
Corynebacterium Corynebacterium spp.
Arcanobacterium/Trueperell
a
Trueperella spp./A.
haemolyticum
Nocardia/myc-
obacteria
Nocardia Nocardia spp.
Mycobacterium Mycobacterium spp.
Gram-negative
bacteria
Enterobacteriacea
e
Salmonella Salmonella spp.
Escherichia/Shigella E. coli/Shigella spp.
Cronobacter Cronobacter spp.

61. Bacteria Identified in various studies
using MALDI-MS
Bacteria Genus Species or Group Evaluated
Enterobacteriacea
e
Enterobacter Enterobacter cloacae complex
Pantoea Pantoea spp.
Plesiomonas P. shigelloides
Klebsiella/Raoultella K. oxytoca/Raoultella spp.
Yersinia Yersinia spp.
Y. enterocolitica
Y. pestis/Y. pseudotuberculosis
Nonfermenting
rods
Acinetobacter Acinetobacter
Burkholderia B. cepacia complex
B. mallei/B. pseudomallei
Pseudomonas Pseudomonas spp.

62. Bacteria Identified in various studies
using MALDI-MS
Bacteria Genus Species or Group Evaluated
Nonfermenting rods Stenotrophomonas Stenotrophomonas maltophilia
Fastidious organisms Brucella Brucella spp.
Bartonella Bartonella spp.
Francisella Francisella spp.
Haemophilus Haemophilus spp.
Vibrio Vibrio spp.
Aeromonas Aeromonas spp.
Campylobacter Campylobacter spp.
Helicobacter Helicobacter spp.
Neisseria Neisseria gonorrhoeae/N.
meningitidis
Moraxella Moraxella catarrhalis
Legionella Legionella spp.

63. Bacteria Identified in various studies
using MALDI-MS
Bacteria Genus Species or Group Evaluated
Anaerobic
bacteria
Propionibacterium P. acnes
Bacteroides Bacteroides spp.
Clostridium Clostridium spp.
Clostridium difficile

64. Bacteria in which MALDI-TOF
MS was used for identification
and strain typing.

65. Detection of Antibiotic Resistance in Bacteria
 MALDI-TOF MS has been shown to generate PMFs capable
of discriminating lineages of methicillin-resistant S. aureus
strains
 MALDI-TOF MS has been shown to be of great use in
identifying vancomycin-resistant enterococci.
 The production of β-lactamases is detected by MALDI-TOF
MS employing a‘mass spectrometric β-lactamase (MSBL)
assay.
 The MSBL assay has been applied for detection of resistance
to β-lactam antibiotics like penicillin, ampicillin, piperacillin,
cetazidime, cefotaxime, ertapenem, meropenem, and
imipenem.

66. Detection of Antibiotic Resistance in
Bacteria
Using MSBL assay researchers have successfully
detected β-lactamase producing organisms like
 Escherichia coli,
 Klebsiella pneumoniae ,
 Pseudomonas aeruginosa,
 Acinetobacter baumanni,
 Citrobacter freundii,
 Enterobacter cloaceae,
 Salmonalla spp. etc.

67. Detection of Antibiotic Resistance in Bacteria
 Recently, Johansson et al. (2014) developed a MALDI-TOF MS
method for detection and verification of carbapenemase
production in anaerobic bacterium, Bacteroides fragilis as early
as2.5h .
 Hart et al. (2015) suggested that instead of using intact bacterial
cells for MS, the periplasmic compartment should be extracted
(since β-lactamases are located in the periplasm); in-solution
digested with trypsin, separated by nano-LC before MALDI-TOF
MS analysis.
 Using this approach they reported the peptide sequence of
biomarkers for several classes of β-lactamases like CTX-M-
1group extended spectrum β-lactamase, TEM β-lactamase, VIM
a metallo-β-lactamase and CMY-2, an ampC β-lactamase.

68. Bacterial Strain Typing and Taxonomy
Proteomics represents the functional aspect of
genomics and can be used as a taxonomic tool.
Gel-based whole cell protein profiling may be as
cumbersome and time consuming as any other
genomic technique.
 On the other hand MALDI-TOF MS intact cell or
whole cell PMF based typing is a rapid and
sensitive method for bacterial identification.
In many cases it has shown resolution and
reproducibility which is better than gel- based
protein or DNA fingerprinting techniques.

69. MALDI-TOF MS in Clinical Virology
 The use of MALDI-TOF MS in virology has advanced less as it has in
bacteriology or mycology.
 This might be a consequence of the relatively low protein content of
viruses (Kliem andSauer,2012), higher molecular weight of viral
proteins(>20,000 Da)and a probable carry over of debris of the cell
substrate in which viruses are cultured in vitro.
 Many researchers have proved the utility of MALDI-TOF MS for diagnosis
of various infectious viruses in clinical samples like
 influenza viruses
 enteroviruses
 human papilloma viruses (HPVs)
 herpes virus
 hepatitis virus etc.
 Interestingly in most of the studies, the viral genetic material was amplified
by PCR and the amplicons were analyzed/identified by MALDI.

70. MALDI-TOF MS in Clinical Virology
 Yi et al. (2011) reported the use of a PCR-based MS
method for detection of high-risk HPVs, a prime cause
of human cervical cancer.
 Piao et al. (2012) combined the multiplex PCR with
MALDI-TOF MS and developed a PCR-Mass assay
which simultaneously detected eight distinct viruses
associated with enteric infections in humans.
 Calderaro et al. (2014) reported that MALDI- TOF
MS was an effective, rapid and inexpensive tool which
identified various poliovirus serotypes from different
clinical samples.

71. Viral Genotyping, Sub-typing, and
Epidemiological Studies
 Apart from viral identification, MALDI-TOF MS has also been
used in virology for genotyping of JC polyomaviruses, hepatitis
B and hepatitis C viruses and for detection of mutations in
hepatitis B viruses.
 Many researchers have demonstrated the application of MALDI-
TOF MS for screening of influenza virus subtypes and for tracking
epidemiology of influenza viruses.
 Downard (2013) described a method for detection of strains of
influenza viruses using whole virus protein digests. This
‘proteotyping approach’ was successful in typing, subtyping, and
tracing the lineage of human influenza viruses.
 MALDI-TOF MS has proved efficacy in detecting drug resistance
to ganciclovir in cytomegaloviruses which frequently infect
transplant recipients

72. MALDI-TOF MS in Clinical Mycology
 Conventional methods for identification of fungi are based on
morphological, biochemical, and/or immunological properties which
might span 2–5 days, or more, and often require combining several
phenotypic methods for conclusive interpretations.
 The molecular methods based on analysis of genes encoding 18S
rRNA and the internal transcribed spacer regions 1 and/or 2 (ITS
1/2) are tedious and time consuming.
 Fungal identification by MALDI-TOF MS in the medical mycology
laboratory has moved at a slower pace than bacterial identification,
owing to their inherent biological complexity which makes their
study as a whole difficult.

73. MALDI-TOF MS in Clinical Mycology
 In order to obtain reproducible PMF results, parameters
like culture media, quantity/type of colony material and
incubation time, need to be carefully standardized.
Also, the fungal cells might require additional treatment
with trifluoroacetic acid, formic acid, or acetonitrile
along with beating with beads to disrupt strong cell
walls.
 Among fungi, highly reproducible PMF spectra have
been reported for the ascomycetous and
basidiomycetous yeasts including organisms like
Candida, Cryptococcus, and Pichia.

74. Fungus Identified using MALDI-TOF in
various studies
Fungi Genus Species
Yeasts Candida Candida spp.
Cryptococcus Cryptococcus spp.
Filamentous fungi/molds Aspergillus Aspergillus spp.
Fusarium Fusarium spp.
Dermatophytes
Pseudallescheria-
Scedosporium
Pseudallescheria-
Scedosporium
Penicillium Penicillium spp.

75. Limitations of MALDI-TOF
identification.
 The sensitivity of MALDI-TOF MS analysis for identification of microorganisms
largely depends on the quality of the reference database used
Difficult to identify microorganisms using MALDI-TOF MS-
 Enterobacteriaceae-
 E. coli/Shigella: As with many genotypic and phenotypic identification systems,
E. coli and Shigella species cannot be differentiated by using MALDI-TOF MS
because of their high degree of similarity at the genomic level.
 Salmonella: Per FDA approval of the VITEK MS system, confirmatory testing is
recommended before a final identification of Salmonella is made using MALDI-
TOF MS.
 Enterobacter cloacae complex: Resolution to the species-level cannot always be
accomplished by using MALDI-TOF MS analysis for strains within the E.
cloacae complex. Combination of MALDI-TOF MS analysis and other molecular
approaches, such as use of polymerase chain reaction (PCR) detection of specific
gene targets, may be required for reliable identification of species within this
complex.

76. Limitations of MALDI-TOF
identification.
 Nonfermenting Gram-negative bacteria-
 Neisseria: Per FDA approval of the VITEK MS system,
confirmatory testing is recommended before a final identification
of Neisseria gonorrhoeae is made using MALDI-TOF MS.
 Burkholderia cepacia complex: MALDI-TOF MS analysis can
reliably identify isolates within the B. cepacia complex to the
genus-level but may misidentify or fail to identify isolates at the
species-level.
 Gram-Positive Bacteria-
 Streptococcus mitis group: MALDI-TOF MS does not reliably
distinguish between members of the S. mitis group. Therefore,
additional testing, such as Optochin sensitivity and bile solubility,
may be required to differentiate Streptococcus pneumoniae from
other members of the group.

77. References
 Matrix-Assisted Laser Desorption Ionization–Time of
Flight Mass Spectrometry: a Fundamental Shift in the
Routine Practice of Clinical Microbiology. Clinical
Microbiology Reviews p. 547–603July 2013 Volume 26
Number 3.
 Singhal N, Kumar M, Kanaujia PK and Virdi JS (2015)
MALDI-TOF mass spectrometry: an emerging technology
for microbial identification and diagnosis. Front. Microbiol.
6:791. doi: 10.3389/fmicb.2015.00791 .
 Clinical Microbiology Procedures Handbook, 4th Ed.
 Advanced Techniques in Diagnostic Microbiology. 2nd Ed.

78. THANK YOU