Comparison of PCR and MALDI-TOF MS for Bacterial Identification

This article compares the advantages and disadvantages of PCR and MALDI-TOF MS methods for bacterial identification. It discusses the sensitivity, workload, cost, and turnaround time of both methods. The article also highlights the limitations and cost-effectiveness of each method.

Journal Article Review

Introduction
Pathogen identification plays an integral role in ascertaining bacterial infection. It is also vital in
directing antimicrobial therapy. There has been constant development in clinical laboratories,
making the process cost-effective, rapid and reliable for bacterial identification. The edifice of
bacterial identification is culture-based methodologies, which need 24-hour isolation along with
an additional 24-48 hours for species identification.
Main body
While discussing about bacterial identification methods, molecular bacterial identification
methods like PCR and Matrix-assisted laser desorption ionization–time of flight mass
spectrometry (MALDI-TOF MS), are two of the mentionable methods. In order to compare both
the methods, advantages and disadvantages associated with each of the methods are needed to be
taken into consideration. On comparing both the methods, it can be stated that PCR is one of the
most sensitive methods for bacterial identification. A majority of PCR based identifications
available for present clinical use is dependent on amplification of preserved genes like RNA
polymerase (rpoB) (6), after detection of specific species genes (Cherkaoui et al., 2010). As
every PCR can be considered to have different reagent, issues with quality control becomes a
concern, as testing is disturbed with every additional gene target. When both the methods are
compared i.e. PCR and MALDI-TOF MS, it can be noticed that workload and cost that are
needed for multiplexing or microarray technology, at present prevents regular use on every
isolation. In terms of complications associated with each method, it can be noticed that PCR
based identification methods becomes more complex due to the procedure that are required to
collect the sample ready. Theoretically, PCR allows identifying slow-growing organisms and it
can be used for pathogenesis with the help of noncultivable organisms within the clinical
research. However, the reality of the situation is, majority of PCR based bacterial identification
that are performed within routine clinical laboratory, still requires nucleic acids that are gathered
from isolated colonies. There exists restriction with direct PCR of clinical samples, which are
restrained to detection of limited number of species and particular sample. It is further needed to
be extracted in such a manner wherein nucleic acid can be preserved, while eliminating PCR
inhibitors (Cherkaoui et al., 2010). The presence of these factors makes the use of PCR method
limited and restricted in nature which are the major disadvantages associated with PCR.
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MALDI-TOF MS is another mentionable molecular analytical tool, which is considered to be
useful for diagnostic analysis. When compared with PCR, it can be seen to have more preference
and use as a research tool for conducting protein analysis. When further analyzed and compared
with PCR in terms of applicability, it can be noticed to have been implemented in clinical
microbiology. From this perspective, it can be stated that MALDI-TOF MS has more
applicability (Cherkaoui et al., 2010). Further comparison with PCR or conventional phenotype
identification revels the fact that it has low requirement of sample volume, has the quality of
rapid turnaround time and cost effective in nature. Protein mass-to-charge (m/z) or peptide
values helps in developing mass spectral peaks. It indicates charge densities of components along
with molecular mass, which are present in a biological sample. It is with the help of these spectra
pathognomonic patterns can be generated, which is helpful in collecting unbiased identification
of specific species even the genotype of species. It is because of limited turnaround time and
collection of data that can be easily interpreted MALDI-TOF MS is more population in order to
identify protein in combination of average complexity, which is not the case for PCR as the
turnaround time is not rapid in its case, which is the major advantage associated with it
(Cherkaoui et al., 2010).
However, there is another aspect associated with MALDI-TOF MS that are needed to be taken
into consideration. A number of previous studies conducted on MALDI-TOF MS had stated that
the method has increased variability, limited reproducibility between and within laboratories. In
this context, the research conducted by (Cherkaoui et al., 2010), may be taken into consideration.
In the research results of MS system and phenotypic bacterial identifications in a periodic
manner. It reflected the fact that, MS system has excellent accuracy. For Bruker MS system, the
yield was higher that provided with higher confidence identification for 94.4% isolates. It was
further noticed that, MS provided with no wrong in identification of any enterococci, one of its
major strength. Another mentionable advantage associated with it is, it is with the development
of MALDI-TOF MS, more than three quarters of isolates can be identified with a single test in a
bacteriology laboratory to the level of species. Its effectiveness can be further established with
the help of the fact that this identification can be conducted in less than 5 minutes/sample.
Moreover, MALDI-TOF MS is cost effective can be established with the help of the fact the
study conducted by (Cherkaoui et al., 2010) puts forward that the method has less marginal cost
when compared with PCR having greater or equal accuracy level. With the help of the research,
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it has been further stated that MS technology can bring major improvements in present clinical
laboratory in terms of its efficiency. It has further stated that, it is because of quality control
requirements, training, cost restrictions and rapid turnaround time associated with MS that makes
it appealing as compared to conventional identifications like PCR. This is one of the major
advantage of MS. In this regards it can be stated that instruments of MS are expensive. This
factor can be considered as major disadvantage associated with MS. However, the expense can
be compared to other usual bacteriology laboratory equipments like 16S sequencing devices and
automated blood culture, which is at par. When it is analyzed in an in depth manner, it can be
noticed that marginal cost associated with conventional identification strategies is higher, which
is significantly less in case of MS one of its major advantage. The reason behind this being, in
case of other capital equipment, the maintenance cost in the budget would be 10% of
instrument’s price every year. That is not the case for MS. In order to organize instruments
required for MS, the labour cost is significantly less when compared with phenotype
identification. Phenotype identification procedure is more labour intensive (Cherkaoui et al.,
2010).
Further comparison of MS with phenotype identification revels that phenotype identification
when involves the use of modern automated platform, it costs approximately US$10 for every
isolation. On the other hand, reagents involved in MS identification are less than $0.50. This
further establishes the advantage associated with it, i.e. cost-effectiveness. The devises required
for MS identification are usually commercially available, and simple in regards to its usage,
another mentionable advantage (Cherkaoui et al., 2010). The laboratory efficiency can also be
improved with the help of MS identification due to low margin cost and significant speed. It is
mentionable here, though it has been stated that MS provides with great accuracy in identifying
majority of isolates, every such isolate cannot be identified by MS, an aspect that can be
considered to be a disadvantage. Isolates that are not identified by MS are Gram positive
organisms. In spite of inaccuracy in case of Gram positive organisms in case of MS, the reagents
cost is so low in case of MS that conducting duplicate extraction on ever sample could be
cheaper when compared to present first-test strategy i.e. biochemical phenotyping. Even after
considering the flaw associated with MS, it can be stated that identification method associated
with MS is cost effective.
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While evaluating MS in terms of its advantages and disadvantages, its alternatives are also
needed to be taken into consideration. Nucleic acid-based identification strategy is one of the
molecular alternatives to MS. When analyzed in an elaborative manner, nucleic acid-based
identification strategies can be seen to have problematic limitations. To elaborate it further, in
order to conduct nuclear acid detection, enzymatic amplifications are used in which inhibitory
compounds that need particular purification and extraction strategies negatively affects it. On the
other hand, in case of MS there is no requirement of enzymatic step, another noticeable
advantage associated with its use. Hence, in this case, purification is not a concern. In case of
other alternatives to MS like nucleic acid-based identification strategies, nucleic acid
amplification result in creating the concern for subsequent reactions that result in the creating
concern of contamination. It needs different area for sample preparation, analysis and
amplification. In case of MS, no target amplification is required. As a result, of this, the real
estate demand for clinical laboratory in case of MS is limited, which can be considered as an
advantage (Cherkaoui et al., 2010). It has been further noticed that nucleic acid based method
has limit usefulness in microbial identification. It is limited to identification of limited individual
species in particular samples like Chlamydia in urine. In case of MALDI-TOF MS, the present
limitation is, it does not provide with sufficient data on antimicrobial susceptibility. In this case,
MS requires isolate for initial material.
Conclusion
Based on the above made discussion, it may be stated that, as MS provides with limited data on
antimicrobial susceptibility, there would be continued need of bacterial culture. However, there
is scope that with refinement of database, MS would provide with drastic identification of
antibiotic resistance characteristics. This it cannot be denied that with the help of MALDI-TOF
MS-based identification bacterial species can be identified in a faster and cheaper manner as
compared to conventional phenotypic identification methods.
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Reference
Cherkaoui, A., Hibbs, J., Emonet, S., Tangomo, M., Girard, M., Francois, P. and Schrenzel, J.,
2010. Comparison of two matrix-assisted laser desorption ionization-time of flight mass
spectrometry methods with conventional phenotypic identification for routine identification of
bacteria to the species level. Journal of clinical microbiology, 48(4), pp.1169-1175.
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