Option appraisal of matrix-assisted laser desorption ionisation time of flight systems for a diagnostic microbiology laboratory

Anish Pyne; Sanjay Bhattacharya

doi:10.4103/jacm.jacm_4_23

MINI REVIEW

Year : 2023 | Volume : 25 | Issue : 1 | Page : 8-15

Option appraisal of matrix-assisted laser desorption ionisation time of flight systems for a diagnostic microbiology laboratory

Anish Pyne¹, Sanjay Bhattacharya²
¹ Faculty of Biotechnology and Biosciences, School of Medical Science and Technology, Indian Institute of Technology, Kharagpur; Department of Microbiology, Tata Medical Center, Kolkata, West Bengal, India
² Department of Microbiology, Tata Medical Center, Kolkata, West Bengal, India

Date of Submission	09-Feb-2023
Date of Decision	02-May-2023
Date of Acceptance	08-May-2023
Date of Web Publication	1-Jun-2023

Correspondence Address:
Sanjay Bhattacharya
Department of Microbiology, Tata Medical Center, Kolkata - 700 160, West Bengal
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jacm.jacm_4_23

Abstract

A major challenge for a clinical diagnostic bacteriology/mycology laboratory is to identify the causal microorganism to the species level within a limited time frame. Conventional microbiological techniques such as culture, Gram stain, biochemical tests and molecular tests like 16S rRNA analysis can take days to yield the desired result. Matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) system serves as a potential solution as it can provide accurate identification results within minutes. While several MALDI-TOF MS instruments are in the market, choosing the right system and comparing all the relative pros and cons can be difficult. Our present study focuses on this aspect as we have performed an option appraisal between two leading MALDI-TOF MS instruments, namely the MBT Sirius IVD system (Bruker) and the VITEK-MS system (Biomerieux). We have discussed all the parts of MALDI-TOF systems in detail followed by a cost analysis categorising the total cost into capital and consumable cost which will provide clarity of the estimated budget. We have also discussed the principle involved in organism identification and have compared the two systems concerning technology, accuracy, database, optimal operating conditions, etc. This article aims to provide readers with a better understanding of MALDI-TOF systems and facilitate informed decision-making regarding the acquisition of such systems.

Keywords: Cost, matrix-assisted laser desorption ionisation-time of flight, MBT Sirius IVD, option appraisal, VITEK mass spectrometry

How to cite this article:
Pyne A, Bhattacharya S. Option appraisal of matrix-assisted laser desorption ionisation time of flight systems for a diagnostic microbiology laboratory. J Acad Clin Microbiol 2023;25:8-15

How to cite this URL:
Pyne A, Bhattacharya S. Option appraisal of matrix-assisted laser desorption ionisation time of flight systems for a diagnostic microbiology laboratory. J Acad Clin Microbiol [serial online] 2023 [cited 2023 Nov 30];25:8-15. Available from: https://www.jacmjournal.org/text.asp?2023/25/1/8/378070

Introduction

The identification of microorganisms in the clinical microbiological laboratory provides information about the possible aetiology of infection and facilitates the prevention/control/treatment of the infection. Application of phenotypic microbiological methods including culture, microscopy (Gram staining/Acid-Fast or Ziehl–Neelsen staining), biochemical tests (IMViC/catalase/oxidase, etc.), as well as genotypic methods including DNA sequencing, takes time and labour. This time requirement in traditional phenotypic and genotypic methods increases turn-around time (TAT). Identification of the organisms up to the species level is critical as it provides information about possible sources of infection, duration of antimicrobial therapy, infection control measures, intrinsic antimicrobial resistance and the possibility of acquired antimicrobial resistance. Matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) technology provides a solution for rapid and accurate organism identification. A study from the UK using MALDI-TOF observed that the first results were obtained after 5–10 min and analysis of a full 96-well target plate was completed in approximately 90 min. There were the savings of between GBP 1.79 and GBP 2.56 per isolate. The study showed 99.4% and 99.1% of organism identifications were in agreement between the MALDI Biotyper and conventional identification at the genus level, and 89.3% and 87.8% at the species level.^[1]

The pertinent questions related to the MALDI-TOF for a diagnostic or research laboratory are: (a) Are the acquisition and installation of a high capital system justified? (b) What are the advantages of the system over traditional microbiological methods? (c) What are the limitations of the system compared to conventional diagnostic techniques in microbiology? (d) Which one is more suitable among the different types of equipment available in the market from various manufacturers?

An option appraisal enables the user to have informed decision-making by reviewing options and analysing the costs and benefits of the present options. Although initially, the MALDI-TOF mass spectrometry (MS) system was manufactured by Shimadzu Europa GmbH (Japan), several new manufacturers ventured into developing their own MALDI-TOF MS systems like Bruker Daltonics Inc. (Germany), BioMérieux (France), Charles River (US), ZyBio (UK), Thermo-Fisher (US), etc. However, there are two leading systems in Bruker and BioMérieux in terms of organism identification capability, high throughput, affordability, speed and quality which makes them the best options available in the market.^[2] This study presents the option appraisal between these two leading MALDI-TOF MS systems, i.e., Bruker's MBT Sirius IVD system and BioMérieux's VITEK MS system. The primary objective of this study is to help any diagnostic microbiology laboratory with information so that decision-making and system selection becomes easier.

Parts of the Matrix-assisted Laser Desorption Ionisation-time of Flight System

The instrumentation system and components of a MALDI-TOF can be categorised into mechanical, electrical and computerised parts [Figure 1] and [Figure 2].

Figure 1: Schematic representation of a MALDI-TOF. MALDI-TOF: Matrix-assisted laser desorption ionisation-time of flight

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Figure 2: Working principle for MALDI-TOF. MALDI-TOF: Matrix-assisted laser desorption ionisation-time of flight

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Sample plate

The sample plate includes a target plate, an organic matrix formed on one surface of the target plate and a Parylene (Poly-Para-Xylylene) thin film formed on the target plate on which the organic matrix is formed. The target plate is made up of stainless steel (Grade 316) where the samples can be vaporised and ionised after the sample is mixed with a suitable matrix material. The analytes are then analysed under various voltages to detect them based on their mass-to-charge ratios.

Matrix

The matrix performs two important functions; it absorbs photon energy from the laser beam and transfers it into excitation energy. The matrix serves as a solvent for the analyte so that the intermolecular forces are reduced and aggregation of the analyte molecules is held to a minimum. The matrix is composed of a mixture of silica gel, formic acid (FA) and α-cyano-4-hydroxycinnamic acid (HCCA). The matrix has to fulfil certain characteristics such as:

The matrix must possess a strong absorption at the emission wavelength of the laser typically, in the ultraviolet (UV) range at either 337 or 355 nm
The matrix ensures the ion formation of the analyte
The matrix should be stable under high vacuum for a long duration
A matrix should be able to isolate the generated ions and prevent the generation of analyte clusters like dimers.

Laser

The main function of the laser in MALDI-TOF is to transform the analyte molecules into the gas phase without fragmenting or decomposing them. This is done by striking the molecules after the sample has been prepared by solubilising it in the matrix. Lasers of both the UV and infrared (IR) wavelengths may be used. However, UV lasers are the preferred light sources in analytical MALDI. Among these, nitrogen lasers (337 nm) have been used as a laser source for MALDI-TOF, but more recently, frequency-tripled solid-state lasers (355 nm) have become more common due to their reliability and lifetime.

Variable voltage grid

The variable-voltage grid allows the application of grid voltage. This is defined as the percentage of accelerating voltage (18–20 kV) that is applied to the variable-voltage grid over the sample plate. In general, a higher grid voltage with a lower potential difference decreases ion fragmentation and vice-versa.

Vacuum generator system

In a MALDI-TOF system, a low-pressure (high vacuum) environment is necessary for the generation of ions. In vacuum MALDI, ions are typically produced at 10 mTorr or less which is not the case in an AP-MALDI (atmospheric pressure-MALDI). The ions generated under the vacuum are then passed through the TOF tubes.

Flight tubes

In the flight tubes, the ions travel to the detector, following which the mass-to-charge ratio (m/z) of the sample ions can be calculated. The velocity of the ions traveling through the flight tubes is inversely proportional to the size of ions as the lighter ions travel faster whereas the heavier ions travel relatively slower and will hence require more time to reach the detector.

Time mass detectors

The ions are detected by the time mass detectors which analyse the m/z ratios of all the ions detected and produce a resultant spectrum that enables the user to analyse the molecular (e.g., protein) makeup of the sample. This spectrum is then compared against a large database of spectra. The identification can be done from a genus and species level. To analyse and compare the resultant spectra, the detector is attached to a computer which allows the user to perform the analysis in a user-friendly way.

The sample plate and TOF tubes constitute the mechanical part of the MALDI-TOF, whereas the lasers, variable voltage grid, and vacuum generator system constitute the electrical parts. These electrical parts have special electrical requirements that must be incorporated into the laboratory design for any laboratory that plans to install a MALDI-TOF system. The time and mass detectors also constitute the electrical parts as it allows the final analysis of the samples.^[3]

Principle of Matrix-assisted Laser Desorption Ionisation-time of Flight used in Bacterial Identification and Antimicrobial Susceptibility Testing

The following steps are performed inside the MALDI-TOF system for the generation of the spectra to be compared with the database:

The target slide is prepared and introduced to a high-vacuum environment, which is essential to acclimatise the sample to a high-vacuum environment. A high-vacuum environment is essential for sustaining the successful passage of the laser and a consistent electric field
A precise laser burst ionises the sample generating ions. Ionisation of the sample allows the generation of sample ions which is crucial for analysing the m/z ratio of these ions
A 'cloud' of proteins is released and accelerated by an electric charge
After passing through the ring electrode, the proteins' time of flight is recorded using the formula, where 't' is the time of flight required by the ionized peptides to cross the entire length of the flight tube, 'k' is the proportionality constant representing the factors related to the instrument settings and characteristics, 'm' is the mass of the ionized peptide and 'q' is the charge of the ionized peptide
Proteins are detected with the sensor to create a spectrum that represents the protein makeup of each sample. Each protein has a unique spectrum which is matched against an existing database containing spectra of over 1500 species, to identify the exact organism
A MALDI Biotyper score ≥2.0, indicates secure genus and probable species identification; and whereas a score ≥1.7 and <2.0 indicates probable genus identification.

MALDI-TOF Technology used in organism identification and antimicrobial susceptibility testing is operated based on the principle that organism proteins are analysed to generate unique graphical patterns which are then matched against a comprehensive database comprising clinically relevant organisms. The database is built using extensive analysis of a vast number of different strains, grown under various conditions and at different stages of growth, which allows distinctive identification of even closely related species and enables precise organism identification within minutes, reducing the TAT for general microbiological procedures. This can save time and cost as compared to conventional phenotypic and genotypic methods. The shorter TAT facilitates the early initiation of appropriate antimicrobial therapy.

A review from the University of Athens described the applications of MS in pathogen identification and especially in detecting biomarkers of antimicrobial resistance. The technology of MALDI-TOF for antimicrobial resistance detection has incorporated different methodologies such as: (I) the detection of differences of mass spectra of susceptible and resistant isolates of a given microorganism using the classical strain typing methodology; (II) the analysis of bacterial-induced hydrolysis of β-lactam antibiotics; (III) the detection of stable (non-radioactive) isotope-labelled amino acids and (IV) the analysis of bacterial growth in the presence and absence of antibiotics using an internal standard.^[4]

Comparative Analysis between MBT Sirius IVD and VITEK Mass Spectrometry Matrix-assisted Laser Desorption Ionisation-time of Flight Systems

MBT Sirius IVD system is an in vitro diagnostic MALDI-TOF mass spectrometer for clinical use in conjunction with IVD software, reference library, reagents and workflows for the accurate identification of biomolecules. The features offered include:

Accelerated data acquisition

Data generation is accelerated by minimising the number of laser shots needed to acquire meaningful spectra, hence also saving laser lifetime and shortening the TAT.

Smart-beam laser

MBT Sirius IVD system includes a smart-beam laser offering a 200 Hz repetition rate and a warranty of seven years or 500 million shots, whichever comes first.

High-capacity vacuum system

This system allows the accelerated generation of ions which brings down the target exchange time.

Integrated ion source cleaning

The ion source cleaning is performed within 15 min and without breaking the vacuum using the integrated IR-laser offering steady performance with low maintenance.

High resolution

The MBT Sirius IVD system offers a high mass spectrometric resolution across a wide mass range, tailored to distinctive microorganism identification.

VITEK MS system is an automated microbial identification system that provides identification results in minutes using the MALDI-TOF technology. The organism proteins are accurately analysed to generate unique fingerprint patterns that are matched against a database comprised of clinically relevant organisms. The features offered include:

Rapid identification

Accurate results: In a study from Canada, the Biotyper correctly identified 86.4% of the strains, while the VITEK MS correctly identified 92.3% of the strains^[5]
Optimized workflow-Its 4-slide capacity enables parallel preparation of samples by four different technologists, with placement on the instrument at the same time. With 48 sample spots per target slide, 192 isolates can be tested per run which enables the analysis of a large number of samples in a small period
Traceability of materials for quality assurance
Large Database covering 1316 species (1095 Bacteria, 221 Fungi) including 38 new species of Mycobacteria, 14 new species of Nocardia and 48 new moulds.

Disadvantages of the VITEK-mass Spectrometry System when Compared with the MBT Sirius IVD System

The VITEK MS system provides the user with a laser having a maximum pulse rate of 50 Hz which implies that the laser can fire 50 laser shots per second as compared with the MBT Sirius IVD system which provides the user with a laser having a maximum pulse rate of 200 Hz (200 laser shots per second). This ensures more accurate ionization of the analyte, and hence, a more accurate identification by the MBT Sirius system. This analysis can also be validated from a side-by-side comparison between these two performed by Lévesque et al.^[5]
The VITEK-MS weighs 330 Kg (floor standing model) excluding the data system as compared to the MBT Sirius IVD system which weighs 75 Kg (bench top model) and hence is much easier for the user to operate
The MBT Sirius IVD can also operate optimally at a wider temperature range (16°C–30°C) as compared to the VITEK-MS system which operates optimally at 18°C–26°C, which minimizes the comparative air-conditioning requirements for a lab working with an MBT Sirius IVD system as compared to a lab working with a VITEK MS system.

Advantages of the VITEK-mass Spectrometry System Compared to the MBT Sirius IVD System

The VITEK-MS 3.0 comes with a database that includes 38 new species of Mycobacterium, 14 new species of Nocardia, and 48 moulds, taking the total to about 1316 species enlisted, whereas in the MBT Sirius IVD system, there is a separate Mycobacteria module that needs to be incorporated with the mother instrument to manage Mycobacteria and Nocardia-related infections like tuberculosis, fungal infections and osteomyelitis caused by non-tuberculosis mycobacteria (NTM).
The MBT Sirius IVD system, costs about INR 21,063,000 including the installation, primary training, and warranty which is very high when compared to the VITEK MS system which costs around INR 18,000,000 including installation, primary staff training, warranty, and an Annual Maintenance Contract valid for four years.

Cost Analysis of a Matrix-assisted Laser Desorption Ionisation-time of Flight System

The total cost of the system is categorized into capital cost and consumable cost which can be defined as follows [Table 1], [Table 2], [Table 3], [Table 4].

Table 1: Capital cost for VITEK-mass spectrometry system (Biomerieux, 2022)

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Table 2: Capital cost for matrix-assisted laser desorption ionisation Biotyper Sirius in vitro diagnostics system (Bruker) (2021 quotation)

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Table 3: Consumable cost for VITEK-mass spectrometry system (Biomerieux)

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Table 4: Consumable cost for matrix-assisted laser desorption ionisation Biotyper Sirius in vitro diagnostics system (Bruker)

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Capital cost

These are the one-time expenditures incurred on the items at the time of purchasing on the acquisition, installation, or enhancement of significant fixed assets.

Consumable cost

These are the necessary recurrent operational expenses borne by the laboratory which include reagents, disposables, and other supplies, as well as maintenance contracts.

Maintenance cost

This is significant for MALDI-TOF as for comprehensive maintenance contract (CMC) companies may charge 5%–10% of the capital cost.

The consumables in the MALDI-TOF include a sample plate (disposable), matrix (containing silica gel, components needed for the preparation of matrix and sample plates), while the capital cost includes the other parts of the instruments including the flight tube and the electrical parts like lasers, variable voltage grid, vacuum generator system, and detectors.

The detailed cost analysis for the capital cost and consumables are given below to show a comparative analysis between the two types of MALDI-TOF systems available from Bruker and Biomerieux (BMX).

The cost of an in vitro MALDI-TOF system may be high owing to the database that each company has to offer along with the instrument. The number of organisms included in the database varies between about 4194 species (out of which 488 are clinically validated; 182 species of mycobacteria) in the Bruker systems and 1316 species in the BMX system.

Upgrade of the database may be done free of cost at least till a certain period inside the CMC after which the company may charge the user. The laser as well as the vacuum generator also adds up comprehensively to the total cost of the instrument, as a high-capacity vacuum system can minimize the target exchange time, hence minimizing the TAT.

Effect of Extraction on the Accuracy of Matrix-assisted Laser Desorption Ionization-time of Flight Systems

In a study from South Korea, it was reported that the accuracy of yeast identification was dependent on the method of sample preparation (extraction). Generally, in-tube acetonitrile (ACN) extraction was recommended before the analysis with MALDI Biotyper, but the direct on-plate FA extraction is simpler. The Biotyper correctly identified 8.7%, 30.4%, and 100% of 23 Cryptococcus neoformans isolates after performing initial FA extraction, additive FA, and FA/ACN extractions, respectively.^[6]

A study from the United States tested two simplified protein extraction protocols developed at the University of Washington (UW) and by BMX. Both extraction protocols included vortex with silica beads in the presence of ethanol. It was found that 94.9% of the isolates were correctly identified to the species level when extracted using the UW protocol and augmented Bruker database. The BMX protocol and Vitek MS system resulted in correct species-level identifications for 94.4% of these strains.^[7]

The MBT Sirius IVD system uses several protocols depending on the type of samples processed and generally uses an HCCA solution instead of a FA/ACN solution for sample preparation. One of the most famously used sample preparation protocols for processing clinical, peptide, or protein digests samples is known as the HCCA Dried Droplet Protocol, where the sample is mixed with the HCCA solution in a 1:1 ratio. 0.5 μL of this mixture is then deposited on the sample plate and allowed to dry before processing. The MBT Sirius IVD system resulted in 95% correct identification of these samples.

Effect of Matrix-assisted Laser Desorption Ionization-time of Flight Database and Threshold Level on Accuracy Scores

A standard MALDI-TOF system shows high sensitivity and specificity in terms of identifying common bacteria like Escherichia coli and Staphylococcus aureus which can be attributed to their widely informative databases, lack of erroneous identification, and high accuracy.^[8] In terms of mycobacterial identification, a standard MALDI-TOF MS system provides an estimated accuracy of over 95% which varies with the databases incorporated in each of these systems. This is exactly why the VITEK-MS system has an edge over the MBT Sirius IVD system in terms of Mycobacterial identification.^[9] In a study from the UW, only 79.3% of the Mycobacteria strains were identified to the species level by the nonaugmented Bruker database, although the use of a lower identification score threshold (≥1.7) increased the identification rate to 93.9%.^[6]

In the case of fungal identification, the VITEK-MS system uses a solid media cultivation method against a 'Knowledge 3.2' database which is different from the liquid cultivation method used by the MBT Sirius IVD system against a FilFungal Database. Unfortunately, no direct comparison is available between these two systems.^[10] However, in terms of comparing both the systems and their respective databases from growth on solid media, it was concluded that the VITEK MS system performed significantly better than the MBT Sirius IVD which can again be attributed to its newly updated and extensive database as mentioned above.^[11],[12]

The VITEK-MS system also holds an edge over the MBT Sirius IVD system in terms of identification of uncommon bacteria like Nocardia which can once again be attributed to its newly updated database.

Limitations of a standard matrix-assisted laser desorption ionization-time of flight system

A standard MALDI-TOF system also has a significant advantage over traditional organism identification methods in terms of direct detection from clinical samples. This can be attributed to a study conducted by Tang et al.^[13] which showed reduced TAT, better reproducibility, higher accuracy, and reliability of the MALDI-TOF systems when compared with the traditional organism identification methods in terms of identification of organisms directly from clinical samples like urine. Tang et al. also talks about certain limitations that these systems encounter while processing clinical samples, which mainly deal with the purity and concentration of the samples.^[13] Similar observations can also be seen with other clinical samples like blood and cerebrospinal fluid. Despite the above-mentioned limitations, a standard MALDI-TOF system can provide a significant reduction in the TAT when compared to conventional methods in identifying organisms from clinical samples.

Like every standard technology, MALDI-TOF MS systems has also several limitations in terms of organism identification. This can be attributed to a steep decline in the estimated specificity and accuracy in identifying inherently similar organisms. This also owes to a limited number of spectra available in the database which associates a MALDI-TOF system with an increasing number of misidentifications and very poor discriminations between organisms in the same genus.^[14] The lack of efficient organism identification in MALDI-TOF systems can also be witnessed in processing complex environmental or clinical samples containing thousands of diverse microorganisms. A metagenome analysis might be a far better solution in terms of the microbiome diversity analysis. MALDI-TOF systems also struggle in providing highly accurate results in virus and parasite identification so far, which can also be attributed to one of its limitations.

Conclusion

A modern-day diagnostic microbiology laboratory needs to have a MALDI-TOF MS for organism identification within minutes which would result in the quick diagnosis of several bacterial and fungal infectious diseases by reducing the TAT and facilitating definitive antimicrobial treatment. Although a standard MALDI-TOF MS system can be used for several other research purposes as well like analysing large non-volatile or thermally unstable molecules, characterising these molecules based on their mass, mass-analysis of peptide mixtures and protein identification, it will take some time for them to be used diagnostic tool in any clinical set-up for all such applications.^[15] Comparing the MBT Sirius IVD and the VITEK-MS system on several facets such as technology, efficiency, accuracy, cost, etc., it can be concluded that both these devices have certain disadvantages amidst several merits. Hence, a laboratory needs to understand its requirements before deciding to buy any one of these two instruments.

Before a purchase decision considerations should be given to the epidemiology of infection in the area where the laboratory is located, the average sample load, the budget available, the remit of the lab (diagnostic or reference lab) and alternative technology in place (genotypic and phenotypic). The MALDI-TOF for the immediate and foreseeable future is likely to be an add-on system where conventional diagnostic methods such as microscopy, culture, polymerase chain reaction, sequencing and antimicrobial susceptibility have to continue. This additional investment needs to be justified based on the sample load for microbial identification (bacteria, mycobacteria and fungus).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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