Comparison of PCR and MALDI-TOF MS for Bacterial Identification
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Added on  2023/04/07
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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.
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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 Whilediscussingaboutbacterialidentificationmethods,molecularbacterialidentification methodslikePCRandMatrix-assistedlaserdesorptionionization–timeofflightmass 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 (Cherkaouiet 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 (Cherkaouiet 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. 2|P a g e
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.Fromthisperspective,itcanbestatedthatMALDI-TOFMShasmore applicability (Cherkaouiet 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 (Cherkaouiet 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 (Cherkaouiet 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(Cherkaouiet 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, 3|P a g e
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 caseofothercapitalequipment,themaintenancecostinthebudgetwouldbe10%of instrument’s price every year. That is not the case for MS. In order to organize instruments requiredforMS,thelabourcostissignificantlylesswhencomparedwithphenotype identification. Phenotype identification procedure is more labour intensive (Cherkaouiet 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(Cherkaouiet 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. 4|P a g e
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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 otheralternativestoMSlikenucleicacid-basedidentificationstrategies,nucleicacid amplification result in creating the concern for subsequent reactions that result in the creating concernofcontamination.Itneedsdifferentareaforsamplepreparation,analysisand 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 (Cherkaouiet 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. 5|P a g e
Reference Cherkaoui, A., Hibbs, J., Emonet, S., Tangomo, M., Girard, M., Francois, P. and Schrenzel, J., 2010.Comparisonoftwomatrix-assistedlaserdesorptionionization-timeofflightmass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level.Journal of clinical microbiology,48(4), pp.1169-1175. 6|P a g e