Clinical Microbial Identification by MALDI-TOF Mass Spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) is a powerful analytical mass spectrometry technique that has generated numerous diagnostic and clinical applications, especially for the identification of microorganisms for medical diagnosis. MALDI-TOF measures the mass of molecules from a sample that has been embedded in a matrix by using a laser to ablate and desorb the molecules with minimal fragmentation.¹ The sample’s molecules are ionized in the resultant hot plume of ablated gases and are funneled into a TOF mass spectrometer that records the ions’ mass-to-charge (m/z) ratio. This is achieved by measuring the time the ions take to traverse a known length under acceleration by an electric field of known strength. The resultant mass spectrum is produced from the pattern (i.e., position and relative intensity) of detected m/z peaks, generating a distinct profile for a particular sample. The uniqueness of mass spectra can be leveraged for identification purposes when a comparison reference spectrum is available.

Applications abound for MALDI-TOF

MALDI-TOF is extremely versatile and can analyze a broad range of samples, which has led to numerous applications in basic research and clinical diagnostics. It can be utilized to identify proteins and glycoproteins,^2,3 biomolecule complexes,⁴ lipidomics profiles,⁵ small molecules and natural products,^6-8 cancer and disease biomarkers,^9-11 bacterial,¹² fungal,¹³ and viral^14,15 microorganisms, and parasites.¹⁶ In addition, it has applications for in vitro chemical diagnostics,¹⁷ food-borne pathogen identification,¹⁸ biopharmaceuticals,¹⁹ and in agriculture and ecology.^20,21 MALDI-TOF enjoys several advantages that have made it popular, including being an untargeted and high-throughput method with ease and speed of sample preparation and data acquisition, amenability to automation, and low sample cost. The initial procurement of a MALDI-TOF instrument is relatively expensive and samples cannot be identified if a reference spectrum is not available within the database.

However, its advantages have prevailed and MALDI-TOF is the central element in numerous established and developing assays. “It’s an important new tool in the toolbox,” says Dr. Minogue, of the Diagnostic Systems Division at the United States Army Medical Research Institute of Infectious Diseases, who has recently added MALDI-TOF to his lab’s arsenal of instruments. “Proteomics-based, it is an orthogonal approach to nucleic acid detection complementing numerous extant and emerging applications.”

Microbial identification by MALDI-TOF goes from bench to bedside

One powerful application of MALDI-TOF that has made the leap from research to the clinic is for the identification of microorganisms from infectious diseases in patients. A microorganism colony cultured from a patient sample is placed onto a MALDI “target” plate, treated with formic acid, embedded within matrix, and the laser is aimed at the target plate to begin sample collection. The detected biomolecules are not identified from the resultant spectrum; rather, the unique pattern produced by proteins and other biomolecules from the microorganism is matched to a spectrum of a known microorganism from a database, like fingerprints are matched to identify individuals. Each spectrum contains peaks unique to specific genera, species, and strains and the MALDI-TOF machine uses an algorithm to produce a value on the level of confidence in its identification.

The application of MALDI-TOF to clinical microbiology has a relatively short history spurred by the introduction of commercial instruments with curated databases. Since a seminal report by Mellmann et al. in 2008,²² followed closely by Seng et al. in 2009,²³ the method has been rapidly assimilated as a clinical diagnostic tool with regulatory approval for certain types of microorganisms that are more readily identified.

MALDI-TOF can identify a multitude of microorganisms

Since the advent of commercial machines, numerous labs around the globe have used MALDI-TOF and validated its accuracy for identifying various microorganisms. Robin Patel, M.D., Chair of the Division of Clinical Microbiology in the Department of Laboratory Medicine and Pathology at Mayo Clinic, recognized the potential of MALDI-TOF and procured a machine for her lab. “In just under a week, we convinced ourselves that it worked very well, needed to be implemented into the clinical laboratory, and had generated enough data to submit an abstract to an international scientific meeting,” she recalls. She and her lab have since spearheaded efforts for microbial identification by MALDI-TOF at Mayo Clinic.^1,24-27

It is presently as effective for the identification of aerobic bacteria as other methods,¹ and can identify gram-positive^28,29 and gram-negative species²⁴ from various clinical sources and repositories as well as from patient isolates.²⁵ MALDI-TOF is also suitable for identifying anaerobes and even supersedes traditional methods of identification.^1,26,30 The simple on-target plate formic acid treatment is also appropriate for identifying yeasts,^1,27 and some MALDI-TOF databases are cleared by regulatory bodies for yeast identification.

The identification of mycobacteria, which includes important pathogens such as  Mycobacterium tuberculosis, has proven more challenging due to their thicker cell walls.¹ Studies have examined various lysis techniques to extract more protein content from mycobacteria to determine whether identification accuracy by MALDI-TOF may be improved.^31,32 The difficulty is also present for filamentous fungi, whose cell walls are also more troublesome to disrupt than bacteria and require protein extraction prior to analysis. Nevertheless, tailored extraction methods are proving effective,^33,34 and progress toward identification of filamentous fungi for clinical purposes is ongoing.

Commercial machines have clinical datasets that have been validated and approved for clinical diagnostic use, such as for identification of bacteria and yeasts. Research datasets, so called research use only (RUO) contain reference spectra for microorganisms whose identification have not been approved by regulatory bodies.¹ In addition, in order to expand the number of infections that can be identified, it is also possible to construct custom in-house libraries.

This is particularly useful for microorganisms that are more prevalent in certain regions of the world where it might be necessary to detect strains or species not adequately represented in commercial databases. Also, identification of rarer or more obscure organisms may be improved by custom built spectrum libraries. The field may potentially benefit from a central repository of spectra similar to nucleic acids database of genes and mRNA. “It has proven to be more challenging with MALDI-TOF mass spectra than with nucleotide sequences because the data can be affected by details of the conditions used to generate the data, including processing of the sample, alongside individual instruments and their associated software,” Dr. Patel says. If experimental variability can be standardized, it may pave the way for repositories.

Pushing boundaries

MALDI-TOF has swept through the microbial diagnostic playing field, bringing accurate, rapid, and low sample cost microorganism identification from patients’ infections. Despite its numerous advantages, it could improve certain aspects to expand diagnostic capability even further. One aspect, discussed above, is increasing representation for less common microorganisms in commercial databases.

Another facet, which is critical for selecting an antibiotic to treat an infection, is determining the antimicrobial susceptibility of the microorganism. Dr. Minogue and his lab recently employed MBT-ASTRA, a method to identify microbial vulnerability to antibiotics.³⁵ Previous techniques relied on time-consuming culturing, but MBT-ASTRA shortens the process to just a few hours by recording MALDI-TOF spectra on colonies grown on antibiotics at various time points and quantifying the total protein content relative to an internal standard. The total protein content increases if the colonies are not susceptible and grow. Unlike microbial identification, which is broadly applicable, MBT-ASTRA is presently limited to a few test microorganisms but may become more extensive. “It is contingent on the instrument’s sensitivity and whether it will be able to discern small differences in growth rate in, for instance, slow growing bacteria to make this a generalized method,” Dr. Minogue commented. “Kits or standardized procedures will need to be developed to help researchers select the antibiotic level and time points to yield optimal results.”

Another important aspect from a clinical perspective is decreasing the total time of the procedure to identify microorganisms sooner and begin treating patients earlier. Although sample collection and identification are rapid from a colony, MALDI-TOF presently still requires culturing the microorganism from samples taken from patients to isolate colonies. Therefore, direct testing of microorganisms from clinical samples such as urine, cerebrospinal fluid or blood could shave valuable time off from the lengthy culturing step.¹ This may be complicated by proteins contributed by the biofluids that obscure spectral features from the microorganism. However, “depletion of biomolecules or capture of the microorganism from samples may help enhance the signal-to-noise ratio to pave the way for this potential application,” Dr. Minogue explained. Positive blood cultures, which are blood samples that have been incubated on instruments that “sense” bacterial growth, can be plated to solid media and incubated to produce a “film” of growth within hours to a day that is amenable to MALDI-TOF. “This is quick and inexpensive and leverages the remarkable breadth of coverage of MALDI-TOF mass spectrometry,” says Dr. Patel, whose lab performs this on a daily basis.

All of the established clinical MALDI-TOF assays currently available and the scope of budding applications is making it an indispensable tool to the microbiologist and to improve medical care and treatment for patients. It has stepped into its role within a span of only a decade and continued research may further its uses into the next decade, which it is anticipated will yield more applications.

References

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