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84 The Open Virology Journal, 2013, 7, 84-90
1874-3579/13 2013 Bentham Open
Open Access
Application of MALDI-TOF Mass Spectrometry in Clinical Virology: A
Review
Fernando Cobo*
Section of Microbiology (Integrated Biotechnology Area), Hospital de Poniente. El Ejido, Almería, Spain
Abstract: MALDI-TOF mass spectrometry is a diagnostic tool of microbial identification and characterization based on
the detection of the mass of molecules. In the majority of clinical laboratories, this technology is currently being used
mainly for bacterial diagnosis, but several approaches in the field of virology have been investigated. The introduction of
this technology in clinical virology will improve the diagnosis of infections produced by viruses but also the discovery of
mutations and variants of these microorganisms as well as the detection of antiviral resistance. This review is focused on
the main current applications of MALDI-TOF MS techniques in clinical virology showing the state of the art with respect
to this exciting new technology.
Keywords: MALDI-TOF, mass spectrometry, viral diagnosis, genotyping.
INTRODUCTION
The matrix-assisted laser desorption ionization time-offlight
mass spectrometry (MALDI-TOF MS) is an analytical
method of microbial identification and characterization
based on the fast and precise assessment of the mass of
molecules in a variable range of 100 Da to 100 KDa.
The first description of the use of MS technology for
bacterial identification was in 1975 [1]. Furthermore,
MALDI-TOF MS was used by the mid-1990s for the
identification of bacteria in research settings [2, 3] but it took
a long time for the introduction of this technology in routine
microbiology. Then, in 2004, the first complete database for
bacterial identification was reported [4].
The possibility of MALDI-TOF to quickly characterize a
wide variety of microorganisms including bacteria, fungi and
viruses [5] increase it potential application in some areas of
microbiological diagnosis. Thus, this technology can be used
for rapid microbial identification at a relatively low cost and
it is an alternative for conventional laboratory diagnosis and
molecular identification systems. The major contribution of
this method is that has drastically improved the time to
identification of a positive sample.
The main use of this technology has been, until now, the
identification or typing of bacteria from a positive culture.
However, applications in the field of clinical virology have
been also performed and are opening up new opportunities,
although the introduction in the routine laboratory diagnosis
still remains a challenge.
In this review, MALDI-TOF technique is presented
including sample preparation as well as data management
*Address correspondence to this author at the Section of Microbiology
Section (Integrated Biotechnology Area), Hospital de Poniente. Ctra de
Almerimar S/N, 04700 El Ejido, Almería, Spain; Tel: +34 950 022 638;
Fax: +34 950 022 601; E-mail: fernando.cobo.sspa@juntadeandalucia.es
and quality control, mainly focused in viral research and
diagnosis.
MASS SPECTROMETRY PRINCIPLES
The mass spectrometer is composed of three main
components: an ion source to ionize and transfer sample
molecules ions into a gas phase, a mass analyser device that
separate molecules depending to their mass and finally a
detector to monitor all separated ions. There are several
ionization methods such as chemical ionization, electrospray
and MALDI. The choice of method basically depends to the
objective of the MS analysis and the nature of the sample,
although both MALDI and electrospray are techniques that
permit ionization and vaporization of a big quantity of
biomolecules [6].
In MS technique [7], samples are prepared by mixing
several biomolecules and embedded in a matrix that will
crystallize inside. The matrix is composed of small acid
molecules that have a strong optical absorption in the range
of the laser wavelength used, although the composition is
different according to the type of laser used and the
biomolecule to be analysed. The molecules are then both
desorbed and ionized by charge transfer by absorbing the
energy of a short laser pulse. The desorbed and ionized
molecules are first accelerated in an electrical field and
ejected through a metal flight tube and collide with a
detector at the end of the flight tube. This cycle is repeated at
a frequency of 50 or more hertz. Thus, biomolecules are
separated according to their mass, and create a mass
spectrum that is characterized by both the mass and the
intensity of the ions. Finally, the result is a spectral signature
(spike representation) that is then searched for in the
appropriate database for the identification of the
microorganism, comparing with values provides by the
manufacturer of the database (Fig. 1).
On the other hand, electrospray ionization mass
spectrometry (ESI-MS) generally uses DNA amplicons that
Application of MALDI-TOF Mass Spectrometry in Clinical Virology The Open Virology Journal, 2013, Volume 7 85
are dissolved in a solvent and then injected in a conductive
capillary. Inside this capillary, high voltage is applied
resulting in the emission of aerosols of charged drops of the
sample. After this, the sample is sprayed through
compartments with diminishing pressure, resulting in the
formation of gas-phase multiple-charged analyte ions, which
are finally detected by the spectrometer. ESI-MS technology
permits high resolution analysing the basic composition of
amplicons [8].
Fig. (1). Principles of MALDI-TOF MS technique. The samples are first inserted and embedded in a matrix. A laser pulse is applied in the
matrix and the molecules are then both desorbed and ionized by charge transfer. The molecules are accelerated in an electrical field passing
through a flight tube and collide with a detector. Biomolecules are separated according to their mass. Finally, a mass spectrum is created
which is compared with a database for the identification of the virus.
Matrix
Laser
Flight tube
Detector
Acceleration
Specific spectrum
Detection
Time of flight
86 The Open Virology Journal, 2013, Volume 7 Fernando Cobo
There are also two available methods for typing using
this technology. The first method is the MALDI-RE that uses
the iSeq method [9, 10]. In this technique, RNA strands are
cleaved and the products of cleavage are analysed by mass
spectrometry, and their spectra are compared with the spectra
of the reference sequences. Initially, MALDI-RE was used
mainly for the detection of single-nucleotide polymorphisms,
but currently there are additional applications for this method
[11]. The second method for typing is based on the use of
ESI technology to analyse specific DNA amplicons (PCRESI-MS)
[12].
CURRENT CLINICAL VIROLOGY APPLICATIONS
Viral Diagnosis
Common laboratory techniques in viruses’ detection
include antibody analysis such as enzyme-linked
immunosorbent assay, molecular techniques like polymerase
chain reaction (PCR) and dot blot hybridization. However,
MALDI-TOF technology has been already used to
investigate a wide variety of viruses clinically relevant.
There are several approaches of this technology for
application in the field of virology.
A recent study performed a multiplex MALDI-TOF MS
method that successfully can detect human herpesviruses
(HHVs) from a wide variety of archival biological specimens
[13]. The concordance rate between the MALDI-TOF MS
technique and reference methods was high and the detection
limits of the MS methods were comparable to previous
reports of multiplex herpesvirus detection using an
oligonucleotide microarray and multiplex PCR techniques.
Low viral loads near the detection limit of the method and
weak cross-reactivity might contribute to the few discrepant
results with reference methods. Thus, this multiplex
approach can be very useful for large-scale epidemiological
research studies in which broad viruses detection is
necessary as well as may also become highly useful for
multiplex clinical diagnostic testing.
Other virus like influenza viruses remain a major health
problem for human beings. The recent cross-species
transmission of avian influenza viruses to humans and the
outbreak of the influenza H1N1 variant enhance the need for
a rapid and efficient diagnostic tools for these viruses. The
detection of influenza viruses is mainly performed using
PCR techniques (rRT-PCR) or antibody-based assays to
identify the nucleoproteins. Alternative methods have been
investigated for viral diagnosis, such as multiplexed flow
cytometry [14], microarrays [15] and mass spectrometry
[16]. MS has been considered as one of the best methods for
protein analysis because of its low detection limit and high
accuracy. It has been also demonstrated that the combined
use of antibody-magnetic nanoparticles and MALDI-TOF
MS is suitable for sensitive detection of influenza viruses
[17]. This method could also be used for the rapid screening
of virus subtypes, suggesting a promising application in
early and accurate diagnosis of influenza viruses.
Enteroviruses are also a major cause of illness in children
causing gastroenteritis, hand, foot and mouth disease and
severe neurological complications such as aseptic
meningitis, encephalitis and acute neurogenic pulmonary
edema. Classical detection of enteric virus is mostly based
on conventional approaches such as indirect
immunofluorescence assay, neutralizing testing, and cell
culture and immunohistochemical detection. However, some
clinical samples remain undetectable because of the absence
of antibodies against viral proteins and cell culture and
neutralization tests are time-consuming and laborious.
Multiplex RT-PCR and real-time PCR have shown good
sensitivity and specificity [18]. For these viruses, the
MALDI-TOF MS technology has demonstrated high
capacity to diagnosis with high throughput and short
turnaround time. MALDI-TOF MS measures the intrinsic
physical properties of molecules directly. A PCR mass assay
combining multiplex PCR with MALDI-TOF MS
technology has shown simultaneous detection of eight
human enteric viruses such as hepatitis E virus,
coxsackievirus, poliovirus, astrovirus, norovirus, echovirus
and reovirus [19]. This method has a sensitivity ranging from
100 to 1000 copies/reaction and shows that this assay could
be used for the simultaneous detection of co-infections with
several viruses.
MALDI-TOF MS has been also used for the diagnosis of
hand, foot, and mouth disease that is caused by acute
enterovirus infections such as poliovirus, coxsackievirus A
and B and echovirus. These infections are clinically
indistinguishable but infection with echovirus 71 could be
complicated with aseptic meningitis, encephalitis and acute
flaccid paralysis. Recently, a study has developed a MALDITOF
MS technique for the rapid diagnosis of these viruses
[20]. The diagnosis of these pathogens can help to a more
comprehensive surveillance of enteroviruses to understand
the molecular epidemiology of enteroviruses detecting
multiple genotypes simultaneously. The main advantage of
this strategy is the identification of several genotypes at the
same time, opening up new possibilities for the diagnosis
and discovering pathological and epidemiological
characteristics.
On the other hand, infection with high-risk human
papillomaviruses (HPV) has been closely associated with
cervical cancer as well as other types of cancer such as anus,
vagina, penis and head and neck. The diagnosis of HPV
infection is based on several methods, but HPV DNA
detection is necessary for the following-up of the patients.
Currently, there are three HPV assays approved for clinical
use by the United States Food and Drug Administration.
Two non-PCR-based assays (e.g. Cervista, Hologic,
Marlborough, MA; Hybrid Capture 2, Qiagen, Valencia,
CA) and a PCR-based method such as the Cobas HPV assay.
The majority of these assays use GP5+/GP6+ consensus
primers, MY09/11 primers and their multiprimer derivates
PGMY09/11 primers and the SPF10 system. However, a
new method based on the amplification of a fragment of the
L1 gene region using GP5+/GP6+ consensus primers using
mass array technique based on the MALDI-TOF MS
technology has been recently developed to identify 14 highrisk
HPVs [21, 22]. This MS assay was found to be noncross
reactive with common low risk HPV genotypes
affecting the genital tract and more sensitive (General
sensitivity results ranging from 10 to 100 copies) than the
Hybrid Capture 2 and conventional PCR methods. An
advantage of this method is that it is possible to extend the
HPV genotype spectrum by adding new type-specific
primers. The use of this technique together with automated
Application of MALDI-TOF Mass Spectrometry in Clinical Virology The Open Virology Journal, 2013, Volume 7 87
DNA extraction and PCR pipetting procedures could
increase the viral detection which a decrease in the cost of
the overall assay.
Diagnosis of Mutations and Identification of Viral
Genotypes
Hepatitis B virus (HBV) and hepatitis C virus (HCV)
infections are still a major public health problem due to their
worldwide morbidity and mortality. For this reason,
screening for these infections is an important part of routine
laboratory activity. Serological and molecular markers are
key elements for the diagnosis, prognosis and monitoring of
treatment in HBV and HCV infections. Currently,
chemiluminiscence assays are widely being used for viral
diagnosis. Molecular techniques are routinely used for
detecting, quantifying viral genomes and analyzing their
sequence, in order to determine their genotype and detect
resistance to antiviral drugs. However, MassARRAY
systems based on nucleic acid analysis by MALDI-TOF MS
technology provides an alternative approach to HBV and
HCV genotyping [23], being accurate to identify all eight
HBV genotypes. MassARRAY system generates MS
patterns that are compared to sequences derived from known
HBV genotypes. The main advantages of this assay are its
rapidity, cost-effectively and versatility. The assay produces
automatic data reports and does not require any additional
data interpretation.
Moreover, this method can discriminate single-point
mutations and is capable of detecting wild-type and mutant
alleles [24, 25]. Variations in the HBV genome are
biologically and clinically important and might confer drug
resistance or affect virus replication capacity, resulting in
failure of antiviral therapy. Some MALDI-TOF MS
platforms are capable to detect 60 HBV variants with
accuracy and low detection limits [25]. The main
disadvantage of this method is that only HBV genotypes B
and C were tested.
Some characteristically mutations in HBV could cause
lamivudine resistance due to prolonged treatment with this
antiviral agent. Current methods for detecting such variants
(e.g. sequencing analysis, RFLP and hybridization-based
assays) are time-consuming and unsuitable for screening
large numbers of samples. For these reasons, it has been
developed a MALDI-TOF MS assay suitable for detecting
YMDD mutants [24]. This technique provides high rates of
accuracy for YMDD mutations and has advantages over
RFLP and direct DNA sequencing, including the ability to
more sensitively and specifically detects mixed populations
as well as being a simpler procedure without need for
cloning or multiple PCR steps producing earlier detection of
HBV mutations. Moreover, this platform is adaptable for the
detection of other mutations or polymorphisms. This method
will be very useful to evaluate the emergence of mutant
viruses and to monitoring of antiviral treatment of chronic
hepatitis B, correlating them to clinical features with regard
to response to drug therapy in these patients.
On the other hand, the identification of HCV genotypes
has become important in order to determine the clinical
course and the outcome of antiviral therapy. Many HCV
genotyping methods have been developed, including
genotype-specific PCR, RFLP, hybridization techniques and
heteroduplex motility assays. Currently, the method of
choice for this purpose is the nucleotide sequencing of a
subgenomic region, but all these methods are complex to
standardize, labor-intensive or non-useful for determining
multiple HCV genotypes. However, it is frequent
heterogeneity during the course of infection, producing
mixed-genotypes infections in some patients. Thus,
researchers have developed some MALDI-TOF MS-based
assays for HCV genotyping during last years [26-28]. For
genotyping purposes, MS provides accurate information on
the molecular mass of the analyte and the procedure can be
fully automated, as well as the direct analysis of molecules
with no labels to be introduced and no separation steps
followed by the image processing.
Other methods based on MALDI-TOF MS technology
for viral diagnosis purposes have been also recently
developed. A method based on MALDI-TOF MS technology
studied the possibility of genotyping JC virus using urine
samples in patients without clinical evidence of progressive
multifocal leukoencephalopathy [29]. MALDI-TOF showed
successfully genotype of these viruses and identifies
sequence variations in the target genome. Other study used
this technique in order to identify the possible mutations of
influenza A (H5) viruses for monitoring purposes [30].
Identification of Drug Resistance
Finally, the application of MALDI-TOF MS technique
could be useful for detecting drug resistance against some
antivirals. A recent study has developed a sensitive and rapid
method for the detection of ganciclovir resistance by PCR
based MALDI-TOF analysis [31].
Use of MALDI-TOF Techniques in Epidemiology
Other potential application of MALDI-TOF MS
technology is to provide critical epidemiological data about
viral infections. From an epidemiological point of view, one
of the main applications of these methods is determining
specific data of clinical isolates for viral diseases’ outbreaks.
Because of their complexity, some viral typing techniques
require specialized test methods and additional diagnostic
devices. For this reason, the typing is not routinely
performed in every laboratory, so these samples are being
sent out of the hospital and this fact could delay the
investigation of these outbreaks and hospital-associated
infections. The introduction of MALDI-TOF MS technology
in the laboratories can facilitate the performing of tests
leading to improve the information to the infection control
staff. The introduction of MS techniques into the clinical
laboratory can provide accurate and rapid epidemiological
data and it could have a critical role in hospital infection
control and surveillance of viral outbreaks.
Table 1 summarizes the main applications of MALDITOF
MS to clinical virology.
ADVANTAGES OF MALDI-TOF MS TECHNOLOGY
AND COST-EFFECTIVENESS
With regard to viral diagnosis, there are few studies
comparing MALDI-TOF MS technology with other
conventional based methods (e.g. viral, culture, nucleic acid
based techniques). MALDI-TOF method successfully detects
viruses in a wide variety of biological specimens, and the
88 The Open Virology Journal, 2013, Volume 7 Fernando Cobo
concordance rate between MALDI-TOF and these
techniques is high. PCR based identification strategies have
some limitations such as much longer turnaround time than
MS, reagent and labour costs and some technical problems.
On the other hand, MALDI-TOF technology can also be
used to identify viruses in co-infected samples. These
methods are capable of simultaneous detection of multiple
pathogens in a single assay and can then prevent
misdiagnosis and delays in treatment without increasing
costs or adding a new step to the process.
Table 1. Main Applications of MALDI-TOF Techniques to
Clinical Virology
Applications
Identification of viruses from clinical samples
Diagnosis of mutations for viruses
Screening of viral subtypes
Identification of antiviral resistance
Epidemiology of viral infections
With regard to the technical problems, PCR methods
could have inhibitory particles and issues of contamination;
thus, separate areas for sample preparation, amplification and
analysis are needed. Furthermore, nucleic acid based
techniques are expensive and time-consuming and appear in
most cases less convenient than MS for routine laboratory
identifications. For MALDI-TOF there must be significant
progress made in closing the upfront of PCR processing to
include DNA extraction and the PCR process itself.
MALDI-TOF MS methods also allow large-scale
research studies not only for fresh samples but also on
archival samples from several biological specimens.
The main advantages and disadvantages of MALDI-TOF
MS technology are shown in Table 2.
Table 2. Advantages and Disadvantages of MALDI-TOF MS
Technology for Viral Analysis Compared to Other
Methods
Advantages Disadvantages
Characterization of a wide
variety of viruses
Reliable and rapid identification
Accurate and sensitive method
High throughput
Easy sample preparation
Cost-effective
Improve the patient management
Automation possible
Identification limited by database
For sequencing of genetic markers:
Time-consuming
High cost
Different genetic markers
The most remarkable differences between MALDI-TOF
method and other conventional techniques are the estimated
time and costs required for sample identification. There are
no studies of cost-effectiveness in viruses, but it is estimated
that the cost of bacterial identification represent only 17-32%
of the costs of conventional identification methods [32].
Other studies have shown that the cost of reagents required
for phenotypic and nucleic acid identification is higher than
for MS techniques [33]. The expensive cost of MALDI-TOF
devices is comparable with other laboratory equipment.
Moreover, MALDI-TOF is a rapid technique if compared
with other methods, so this technology improves the working
time (e.g. pre-analytical procedure) as well as the turnaround
time (e.g. analytical proceduce). The time needed for
microorganism identification is < 15 minutes for MS
method, whereas conventional techniques need 5-48 hours.
One of the main weaknesses of MALDI-TOF is that the
identification is limited by database. The mass profile is used
as a mass spectrum to compare with those of wellcharacterized
organisms in a database. The spectrum
typically includes specific peaks, so that with a
comprehensive library of spectra, the identification might be
performed by using bioinformatics.
Currently, there are a wide number of references in the
database from the available commercial platforms for
MALDI-TOF techniques. These mass spectra can establish
the comparison and further identification of microorganisms,
but until now these spectra are limited. The database will
expand due to the collaboration between commercial
companies and the hospitals of several countries, so the
available databases should be improved in the near future. It
will be very important the comparison of data from different
companies’ instruments, but this fact is currently not
feasible.
DATA MANAGEMENT AND QUALITY CONTROL
Sample preparation by MALDI-TOF MS remains
laborious today, although some robots are being developed
in order to improve the deposit of the sample and the matrix
on the target. Moreover, some aspects of MALDI-TOF MS
technology are being also investigated; for example, the laser
pulse frequency, disposable target slides and the mass
spectrometers as well as the software used for the
communication between laboratory equipment and
laboratory information systems.
Data management primarily depends on the interpretation
of crude data. Currently, some methods for data
interpretation have been investigated [34]. An important fact
is the integration of information communication systems to
physicians that requires hospital or laboratory information
systems (HIS, LIS).
Reference databases require updating if new information
is obtained. The addition of new isolates or mutations could
improve this tool and the final diagnosis of the diseases. A
robust system of information for regional prevalence of
infectious diseases, emerging and re-emerging infectious
pathogens, the discovery of new mutations and local or
pandemic changes in epidemiology is necessary.
The MALDI-TOF MS system is increasingly used in
clinical diagnostic laboratory for viral identification,
mutation analysis, genotyping and antiviral resistance
studies. However, there are still some problems arising
during sample extraction affecting to the extraction protocol
and to the adequate reagent conservation for the assay. These
problems could be avoided by the introduction of an
adequate quality control program. The extraction step should
Application of MALDI-TOF Mass Spectrometry in Clinical Virology The Open Virology Journal, 2013, Volume 7 89
be daily checked by training technicians as well as the
introduction of routinely internal quality controls.
Other aspect of this technique that must be submitted to
an adequate quality control is the deposit of the sample on
the microplate and the cleaning of this microplate. It is
necessary implement a control about these issues in order to
the correct identification of the samples.
Finally, it is a good practice for these devices the
adequate maintenance of the MALDI-TOF based on a global
quality control program. In this sense, the calibration control
is a tool useful to recalibrate the spectrometer device and to
identify technical problems. Appropriate maintenance of
these devices is essential to carry out an adequate and
accurate viral identification.
It is essential both to continuously improve the quality of
the database and to introduce a quality control program for
the MS device. The implementation of these programs can
help to improve the quality of the overall procedure, so it
will be the basis to improve the usefulness and the accuracy
of MALDI-TOF.
CONCLUDING REMARKS
MALDI-TOF MS is a relatively recent innovation for the
identification of microorganisms and is being used in many
clinical microbiology laboratories. The main application of
this technology is, at the moment, the identification of
bacteria to species level in less than a minute, although this
method is about to become the standard of identification of
microorganisms in most laboratories.
MALDI-TOF MS technology is also being introduced in
virology assays. The main applications in this field are the
identification of viruses from clinical samples, the discovery
of mutations for these viruses, the rapid screening of virus
subtypes, its use in molecular epidemiology and the
detection of resistance to several antiviral agents.
Future applications of MALDI-TOF MS in clinical
virology will be developed in order to provide more accurate
identification on clinical specimens and will include the
detection of resistance protein markers and specific virulence
for several viruses. However, the introduction of this
technology routinely for virology diagnosis would enable a
change in working practices. Moreover, this fact will require
a complete integration into laboratory workflow and several
technical improvements such as to detection sensitivity. The
continuous updating of databases for microorganisms will be
also an important issue, as well as the possibility for the
diagnosis directly from clinical samples.
MS methods can be used currently for viral diagnosis but
refinement of technology will be an important approach for
the introduction of MALDI-TOF in routine viral diagnosis.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
ACKNOWLEDGEMENTS
Declared none.
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Received: May 3, 2013 Revised: August 1, 2013 Accepted: August 2, 2013
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