L8 _MALDI-TOF MS Bacterial ID_2025 (1)

The BTS is analyzed in parallel with samples in every run and consists of an E. coli extract spiked with two high-molecular-weight proteins, covering the entire mass range of proteins measured during identifications.

1. Purchase well-characterized organisms from American Type Culture Collection (ATCC).
2. Subculture from -80C freezer onto blood agar for purity check.
3. Subculture once more to another blood agar.
4. Perform MALDI-TOF MS ID using the direct transfer method.
5. Conduct testing quarterly.

1. Specimen registration
2. Put-up and cultivation
3. ID (Identification)
4. Drug ST (Susceptibility Testing)
5. Report

The average turnaround time for the entire workflow (ID + ST) is approximately 24 to 72 hours. This is a concern for patient management, especially in cases of sepsis and meningitis, where timely identification and treatment are critical.

Identifying 'difficult' pathogens poses several challenges:

1. Complicated biochemical tests are often required.
2. The identification process can be time-consuming.
3. In some cases, an accurate identification cannot be obtained at all.

The fundamental hypothesis is that the phenotypic differences among bacterial species are due to differences in their protein compositions. Each bacterial species has a unique protein fingerprint (protein spectrum) that allows for discrimination between closely related organisms.

Mass spectrometry is an ideal method for studying protein compositions based on the mass difference among protein molecules. It works by ionizing proteins to generate charged molecules or fragments, which are then accelerated in a magnetic or electrostatic field towards a detector. The flight time of the ionized proteins varies with their size, allowing for separation based on the mass-to-charge ratio (m/z ratio).

The primary method used is a laser beam that focuses on a small spot to accurately target the bacterial cell and remove electrons from protein molecules, sublimating the protein into the gas phase directly.

The problem was that bacterial proteins were degraded quickly by the high energy before being ionized, making the method infeasible.

The technique developed to solve the ionization problem is called Matrix-assisted Laser Desorption Ionization (MALDI).

The matrix serves two main purposes:

1. Absorption of Laser Energy: It absorbs high energy from the laser, protecting the bacterial proteins from direct ionization.

2. Indirect Ionization: It ionizes the bacterial proteins indirectly through energy transfer from excited matrix molecules.

The preparation process involves:

1. Mixing the matrix solution, which is a saturated solution of a low-mass organic compound, in excess with the sample.

2. Allowing the mixture to dry, resulting in the cocrystallization of the sample and matrix, forming a solid deposit of the sample embedded in the matrix.

MALDI-TOF MS is classified as a soft ionization technique.

MALDI-TOF MS operates on the principle of Matrix Assisted Laser Desorption Ionization, where a strong absorbance at the laser wavelength leads to the ionization of clinical specimens. The matrix must be soluble in compatible solvents to create an effective matrix-specimen mixture.

The common matrix used for bacterial identification in MALDI-TOF MS is α-cyano-4-hydroxycinnamic acid (HCCA), which is particularly suitable for large protein analysis.

The wavelength of the nitrogen laser used in MALDI-TOF MS is 337nm.

The irradiation time of the UV laser is kept short to avoid damage or fragmentation of proteins.

MALDI involves matrix molecules that absorb high energy from a laser beam, becoming ionized. This process excites the matrix molecules, which then ionize the protein molecules in the sample, leading to the vaporization of both the matrix and the sample.

Once ionized, proteins become positively charged and travel toward the negatively charged time-of-flight (TOF) tube.

Maintaining a vacuum condition in the TOF tube is essential as it allows molecules to be separated according to their velocities without interference from air molecules.

After entering the TOF tube, the ions move into a field-free drift region where they continue to travel towards the mass detector.

Protein molecules with a larger m/z ratio will have a longer drift time compared to those with a smaller m/z ratio.

Upon completion, a spectrum of proteins with different m/z ratios can be obtained.

The mass range of proteins captured for bacterial identification is 2,000 – 20,000 m/z.

Ribosomal proteins are significant because they are ubiquitously expressed throughout all phases of growth, making them more reproducible for analysis.

Signal intensity in MALDI-TOF MS is directly proportional to the quantities of the protein of a particular m/z.

The first step is to trace a small amount of bacterial mass from a single colony of an overnight culture.

After smearing, 1 microliter of matrix solution is overlaid on the bacterial smear.

The target plate should be left to dry in room air for 5 minutes.

The matrix solution assists in the ionization of the bacterial samples during the mass spectrometry process.

The color coding indicates the status of each spot, such as not occupied, prepared, measured, classified, or spectra present.

The on plate extraction method is used for preparing samples from some Gram Positive organisms and Yeast for analysis in MALDI-TOF MS.

The key components include:

1. 70% Formic acid - used for sample extraction.
2. Matrix - essential for ionization of the sample during mass spectrometry.

The steps involved after sample preparation include:

1. Applying the sample to the MALDI target plate.
2. Inserting the target plate into the MALDI-TOF mass spectrometer.
3. Analyzing the results displayed on the computer screen, which includes a grid of wells and a table of identified organisms.

Once spectra are generated, they are compared to a database of defined reference spectra to lead to microbial identification.

A score value indicates the reliability of the identification, with higher scores suggesting more reliable identifications. For example, Clostridium perfringens has scores of 2.387 and 2.407, indicating reliable identification, while a score of 1.282 indicates 'no reliable identification.'

Comments provide additional context for the identification, such as the need for extraction to permit reliable species identification or that certain species are closely related and not definitively distinguishable at the moment.

The presence of closely related species, such as Escherichia coli and Shigella, complicates identification, as they may not be definitively distinguishable based on the spectra alone, necessitating further analysis or extraction for reliable identification.

The Acinetobacter baumannii /calcoaceticus complex indicates that multiple species may be present, and extraction must be performed to allow for reliable species identification, highlighting the complexity of microbial identification in this group.

A score value of 3.0 indicates fully identical protein spectra between the sample and the database.

The score value range of 2.000 to 2.299 indicates secure genus identification and probable species identification.

A score value of less than 1.700 indicates no identification can be made.

The score value range of 2.300 to 3.000 corresponds to highly probable species identification.

A score value between 1.700 and 1.999 indicates probable genus identification.

A high score value indicates a strong match between the analyte and the identified organism, suggesting reliable identification. Typically, a score above 2.0 is considered reliable, while scores below this may indicate uncertain identification.

The organism identified with the highest score value is Acinetobacter baumannii, with a score of 2.321 for analyte F4.

A score value of 1.994 for analyte A5 indicates that the identification is not reliable, as it falls below the threshold for reliable identification (typically 2.0).

For Clostridium perfringens, the score values are very close: the best match has a score of 2.387 and the second best match has a score of 2.357. This indicates a strong consistency in identification for this organism.

Analyte H1 has a score value of 1.813, indicating not reliable identification, while analyte H3 has a score of 2.084, which is considered a reliable identification. This shows a clear distinction in the reliability of the identifications between these two analytes.

A score value of 2.407 indicates a high confidence match for the sample, classified as '+++'.

The three consistency categories are:

1. Category A: Species consistency - best match is classified as 'green', with further matches being the same species or at least the same genus.
2. Category B: Genus consistency - best match is 'green' or 'yellow', with further matches having at least the same genus, but not fulfilling species consistency.
3. Category C: No consistency - neither species nor genus consistency is present.

Category A signifies species consistency, where the best match is classified as 'green', and further matches are of the same species or at least the same genus as the first match.

A match classified as '(-)' indicates a low confidence match, suggesting that the identified organism may not be closely related to the sample, and further investigation is needed.

Matched patterns are ranked based on their score values, with higher scores indicating better matches. The ranks range from '+++' for the best matches to '(-)' for the least confident matches.

A Score Value of 2.967 indicates a strong identification of Clostridium perfringens, suggesting high confidence in the match.

Organisms are ranked based on their Score Value, with higher scores indicating better matches to the analyte.

The matched pattern indicates the specific strain or variant of the organism that corresponds to the analyte, providing detailed identification beyond just the species level.

A Score Value of 1.288 signifies that the identification for analyte A5 is not reliable, indicating low confidence in the match.

A rank of (++) indicates a moderate quality match, suggesting that while the identification is likely correct, there may be some uncertainty compared to higher-ranked matches.

The best match has a score value >2.

All ranks with score value >2 (Green colour) must have the same species-level ID with the best match.

All ranks with score value 1.7-1.99 (Yellow colour) must have the same species-level OR Genus-level ID with the best match.

A Score Value of 2.576 indicates a high confidence in the identification of the organism, Arthrobacter sulfureus, as the best match for Sample D.

Organisms are ranked based on their Score Value, with higher scores indicating better matches to the sample analyzed. The rank also includes a quality classification, such as (+++) or (++).

The presence of a second best match with a lower Score Value suggests that while the first match is the best identification, there may be other closely related organisms, but with less confidence in their identification.

A Score Value of 1.332 indicates that the identification of the organism is not reliable, as it is below the threshold for confident identification in MALDI-TOF MS.

The quality ranking indicates the confidence level of the identification, with +++ representing high confidence, ++ indicating moderate confidence, and - suggesting low confidence in the identification of the organism.

In Category B, when the best match has a score value of 1.7-1.99 (Yellow colour), the following criteria must be met:

• All ranks with score value 1.7-1.99 must have the same species-level OR genus-level ID as the best match.

In Scenario 2 of Category B, when the best match has a score value greater than 2 (Green colour), the following conditions apply:

• One or more ranks with score value >2 may have different species-level IDs but must share the same genus-level ID as the best match.
• All ranks with score value 1.7-1.99 (Yellow colour) must have at least an identical genus-level ID with the best match.

The Score Value indicates the confidence level of the identification of an organism, with higher values representing more reliable identifications.

Category C indicates not reliable identification, suggesting that the results should be interpreted with caution and may not accurately represent the organism present.

The best match is the organism with the highest Score Value, while the second best match is the organism with the second highest Score Value, both providing potential identifications for the analyte.

A Score Value of 1.244 indicates a not reliable identification, suggesting that the identification of the organism is uncertain and should be verified with additional methods.

The organism with the highest Score Value for the best match is Bacteroides thetaiotaomicron with a Score Value of 2.051.

A Score Value of 2.253 indicates a strong match for the organism Alcaligenes eutrophus, suggesting high confidence in the identification based on the MALDI-TOF MS analysis.

Organisms are ranked based on their Score Value, with higher scores indicating better matches to the analyzed sample. The ranking also includes quality indicators such as (++) for the best match and (+) for secondary matches.

The second best match, Ralstonia eutropha with a Score Value of 1.994, provides an alternative identification option, which can be useful for confirming results or considering potential misidentifications.

A Type C classification indicates that the identification of the organism is less certain, often due to lower Score Values or ambiguous results, necessitating further validation or testing.

A score value of <1.7 indicates that the best match is not reliable for identification, falling into Category C.

A score value of 1.7-1.99 signifies that the best match has a different genus-level ID compared to all ranks with the same score range, which are highlighted in yellow.

A score value >2 indicates that the best match is reliable, but one or more ranks with this score may have a different genus-level ID compared to the best match, and all ranks with score values 1.7-1.99 also have different genus-level IDs.

Bacterial ID results with category A can generally be reported to the clinician, except for some species that share similar protein spectra, which require biochemical tests for confirmation.

For bacteria isolated from non-sterile sites (e.g., urine, stool), genus level identification is acceptable.

For bacteria isolated from sterile sites (e.g., blood, CSF, body fluids), species level (category A) reporting is necessary.

Confirmation methods include:

1. Re-measurement using MALDI-TOF MS with on-plate extraction (with FA)
2. Biochemical tests (manual or Vitek automatic system)
3. 16s rRNA sequencing

In category C, re-measurement with MALDI-TOF MS, biochemical test identification, or 16s rRNA sequencing must be performed to achieve species/genus level identification.

• Escherichia coli and Shigella species
• Streptococcus pneumoniae and Streptococcus mitis group
• Streptococcus pyogenes, Streptococcus canis, and Streptococcus dysgalactiae

MALDI-TOF MS cannot provide species-level identification for Salmonella spp.; serotyping is necessary for accurate identification.

MALDI-TOF MS is not useful for direct testing of clinical specimens, except for blood samples.

Identification is limited by the entries in the database; for example, the library used by HA hospital does not contain highly infectious agents such as Vibrio cholerae, Bacillus anthracis, Burkholderia pseudomallei/mallei, and Yersinia pestis.

Tiny or mucoid colonies may not be identified by MALDI-TOF MS, as they require extraction for proper identification.

Escherichia coli needs to be confirmed by:

1. Lactose fermentation
2. Indole production

Streptococcus pneumoniae can be differentiated by:

1. Optochin test susceptibility
2. Bile solubility test

Streptococcus pyogenes requires confirmation through:

1. Bacitracin test susceptibility
2. PYR test

For Salmonella spp., the next step is to proceed to serology for serotyping identification.

1. Vitek
2. 01, 0139 Serotyping
3. Salt tolerance

Burkholderia pseudomallei / Burkholderia mallei

1. Perform in BSC
2. Vitek

1. Perform in BSC
2. Motility test
3. Penicillin susceptibility
4. Haemolysis
5. Vitek

1. Perform in BSC

1. Check growth characteristics (Slow growing on BA /CHO and no growth on MAC)
2. Gram Stain
3. Oxidase
4. Serology typing
5. Vitek

To ensure that the spectra measurement is accurate and that all parameters and settings of the software are correct.