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MALDI-TOF, molecular detection and strain typing

15 min

Skills you'll build4 skills
  • Recall how MALDI-TOF matches a protein spectrum to identify an organism
  • Select a suitable isolated colony for MALDI-TOF identification
  • Tell NAAT target detection from active infection, carriage, or leftover DNA
  • Tell isolates of one strain from isolates that only share a species, using strain typing

At the bench: Reconcile organism identification with specimen source and observed growth

Try first

Try first

What does MALDI-TOF mass spectrometry compare with its reference library to identify a bacterial colony?

Keep going to see the reasoning.

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

MALDI-TOF identification

A colony is spread on a target spot and covered with an acidic matrix. Gram-positive organisms and fungi are first extracted with formic acid. The laser ionizes the colony's abundant ribosomal proteins, and lighter ions reach the detector first. The resulting spectrum, roughly 2 to 20 kDa, is matched against a reference library.1

MALDI-TOF identifies most Enterobacterales, nonfermenters, staphylococci and anaerobes quickly. The library cannot distinguish the spectra of some organisms. Shigella and E. coli are one pair, and Streptococcus pneumoniae and its closest mitis-group relatives are another. These identifications need supplemental tests before a species is reported.1,2

Pure culture for MALDI-TOF

Each spot gives one spectrum, so MALDI-TOF needs growth in pure culture.1 Pick one well-isolated colony. A colony touching another colony type gives a blended spectrum. The result can be a low-confidence call, or the name of only one organism in the mix. Check that the identification agrees with the Gram stain and colony. When it does not, subculture to purity and spot again.1

Interpreting a detected NAAT result

A nucleic acid amplification test (NAAT) detects its target sequence. That DNA or RNA can come from infection, from colonization or carriage, or from organisms left after treatment. A C. difficile NAAT, for example, can be positive in a person without infection.3 The laboratory reports the target as detected. The specimen source and the patient's symptoms determine its significance.3,4

Strain typing

Two patients can carry the same species, even with the same antibiogram, without sharing a strain. Strain typing shows whether isolates are related. Whole-genome sequencing has largely replaced pulsed-field gel electrophoresis, because it compares isolates down to single nucleotides. Typing is done in reference and public health laboratories, and its result is interpreted with the epidemiology of time and place.5

Follow the bench demonstration

Sources for this step
References
  1. Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th ed. Elsevier; 2021.
  2. Association of Public Health Laboratories. Identification Guide: Isolation and Identification of Shigella species and Enteroinvasive Escherichia coli from Culture-Independent Diagnostic Test Positive Specimens. APHL; July 2025. Accessed September 27, 2026. https://www.aphl.org/aboutAPHL/publications/Documents/FS-Shigella-EIEC-CIDT-Guide.pdf
  3. Centers for Disease Control and Prevention. Clinical testing and diagnosis for C. diff infection. March 6, 2024. Accessed September 27, 2026. https://www.cdc.gov/c-diff/hcp/diagnosis-testing/index.html
  4. Miller JM, Binnicker MJ, Campbell S, et al. Guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2024 update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis. Published online March 5, 2024. doi:10.1093/cid/ciae104
  5. Carroll KC, Pfaller MA, Landry ML, McAdam AJ, Karlowsky JA, Patel R, Pritt BS, eds. Manual of Clinical Microbiology. 13th ed. ASM Press; 2023.

Watch one

Read each decision and its explanation.

Three patients on one intensive care unit grow methicillin-resistant Staphylococcus aureus (MRSA) within 2 weeks. All three isolates have the same antibiogram. Infection prevention asks whether this is transmission. Whole-genome sequencing at the public health laboratory reports that patients A and B carry closely related isolates in one cluster. Patient C's isolate belongs to a different lineage.

How do you interpret the result?

  1. Start with what the routine results show: the same species with the same susceptibility pattern, which does not prove a shared strain.

    MRSA is common, so a shared species and antibiogram can occur by chance.

  2. Check the epidemiology: all three were on one unit within 2 weeks.

    Transmission needs a link in time and place as well as in the genome.

  3. Read the typing: A and B are closely related, and C is a different lineage.

    Sequencing compares isolates at single-nucleotide level.

  4. Combine the two: A and B are consistent with transmission on the unit. C shares only the species and antibiogram.

    Transmission is supported when the genome and the epidemiology agree.

Patients A and B carry one strain, consistent with transmission. Patient C carries an unrelated MRSA strain and is not part of that cluster.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A molecular assay detects C. difficile in a stool specimen. What does the positive result show?

A positive molecular result can occur in a person without infection. Interpret it within the diagnostic pathway.

Asymptomatic carriage can give a positive molecular result. Interpretation depends on symptoms and on appropriate specimen selection.

A detection assay does not establish susceptibility to drugs or mechanisms it does not assess.

Hint
  1. Ask what a NAAT detects.
  2. Think about people who carry C. difficile with no diarrhea.

Review Molecular detection: target selection and amplification

Problem 2 of 3 · Hints available

A urine plate grows a lactose-fermenting gram-negative rod. Most of its colonies touch small white colonies of a second type. Which material goes on the MALDI-TOF spot?

A sweep picks up both colony types. The blended spectrum can give a low-confidence call or name only one of the two.

Spotted a mixed colony for MALDI-TOF

MALDI-TOF reads one spectrum from each spot, so material from a mixed colony gives a blended spectrum. The result can be a low-confidence call, or the identity of only one organism in the mix reported as though it were the whole isolate.

One colony that touches no other gives one organism's spectrum. When every colony touches another, a subculture gives isolated colonies to spot.

MALDI-TOF matches one spectrum per spot. Material from two organisms blends their spectra.

Spotted a mixed colony for MALDI-TOF

MALDI-TOF reads one spectrum from each spot, so material from a mixed colony gives a blended spectrum. The result can be a low-confidence call, or the identity of only one organism in the mix reported as though it were the whole isolate.

Hint
  1. One spot gives one spectrum.
  2. Look for material that contains only one organism.

Review Mass spectrometry (MALDI-TOF)

Problem 3 of 3 · On your own

A lactose-negative, nonmotile isolate from stool gives "Escherichia coli" on MALDI-TOF with a high score. How is it reported?

A high score cannot separate Shigella from E. coli, because the library does not resolve their spectra. A nonmotile, lactose-negative stool isolate could be Shigella.

Accepted a MALDI-TOF call its library cannot resolve

MALDI-TOF matches a mainly ribosomal-protein spectrum to a reference library. It cannot separate organisms whose spectra the library does not resolve, such as Shigella and E. coli, or S. pneumoniae and its mitis-group relatives. Those calls need supplemental testing before a species is reported.

MALDI-TOF does not reliably separate the two. Motility, lysine decarboxylase, acetate or mucate reactions and serology complete the identification.

Nonmotile, lactose-negative E. coli strains exist. The supplemental tests and serology determine the identification.

Review Mass spectrometry (MALDI-TOF)

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Over 10 days, three patients on ward 6 East grow carbapenem-resistant Klebsiella pneumoniae with the same antibiogram.
  • A fourth patient on the ward, with no symptoms, has a rectal screening swab. Its NAAT detects the KPC carbapenemase gene.
  • For patient 3, the first MALDI-TOF spot gave "no reliable identification". The colony picked was touching a small white colony.
Decision 1 of 3

What do you do about patient 3's spot?

A low-confidence result is not an identification. The blended material caused it.

An antibiogram does not identify an organism. The isolate needs its own reliable identification.

The picked colony touched another type, which blends the spectrum. A pure subculture gives a clean spectrum.

The same mixed material gives the same blended spectrum.

Spotted a mixed colony for MALDI-TOF

MALDI-TOF reads one spectrum from each spot, so material from a mixed colony gives a blended spectrum. The result can be a low-confidence call, or the identity of only one organism in the mix reported as though it were the whole isolate.

Review Mass spectrometry (MALDI-TOF)

Decision 2 of 3

How is the fourth patient's screening result reported?

The swab shows a resistance gene in the gut of a patient with no symptoms. Detection does not show infection or name the organism carrying the gene.

Reported target detection as active infection

A molecular assay detects its target nucleic acid, which can come from colonization, carriage, or residual DNA after treatment. Report the target as detected. Whether it means infection depends on the specimen source and the patient's symptoms.

The report states what the assay found and from which specimen. Infection prevention and the clinician act on it in the context of the patient's condition.

The screen is a validated test with its own result. Withholding the report delays the precautions it is meant to start.

Review Molecular detection: target selection and amplification

Decision 3 of 3

Infection prevention asks whether the three K. pneumoniae isolates are one outbreak strain. What does the laboratory advise?

A shared species and antibiogram can occur in unrelated strains. They cannot show transmission.

Called transmission from a shared species name

Two patients can carry the same species without sharing a strain. Calling transmission from the species name can start an outbreak response where none exists or hide a real one. Strain typing, such as whole-genome sequencing read with the epidemiology, shows whether isolates are related.

Spread over 10 days on one ward fits transmission. Timing alone neither proves nor rules it out.

Strain typing shows whether the isolates are closely related. Interpreted with the ward timeline, it shows whether they are one outbreak strain.

Review Molecular epidemiology and strain typing

This case depends on the difference between a species identification and a strain. Species, antibiogram and gene detection each describe what was found. A reliable identification required a pure isolate, and only strain typing can show whether the patients' isolates are linked.

For reference

Sources checked