Leucine Aminopeptidase (LAP) Test: Principle, Procedure, Results

A blood culture flags positive. The Gram stain shows Gram-positive cocci in pairs and short chains, and the catalase test is negative, so you are somewhere in the crowded family of streptococci, enterococci, and their look-alikes. Then the vancomycin disk shows the organism growing right up to the edge of the zone: it is vancomycin resistant. In a hospitalized patient, the first worry is vancomycin-resistant Enterococcus (VRE).

Leucine Aminopeptidase (LAP) test is used to identify catalase-negative, Gram-positive cocci. Specifically, it helps differentiate Aerococcus and Leuconostoc from Streptococcus, Enterococcus, Lactococcus, and Pediococcus.

LAP is used for the preliminary characterization of Streptococcus-like organisms, and it is only one of a battery of tests for identifying catalase-negative, Gram-positive cocci. It is almost always read next to the PYR test, and the streptococcal identification panel (API Rapid Strep) includes a LAP reaction as well. Because nearly all of these cocci are LAP positive, the informative result is usually the negative one: a negative LAP points you toward Aerococcus or Leuconostoc rather than the larger LAP-positive group.

Principle

The LAP disk is a rapid test for detecting the enzyme leucine aminopeptidase. A disk impregnated with L-leucine-beta-naphthylamide serves as the substrate. Leucine aminopeptidase cleaves the substrate and releases beta-naphthylamine, which then condenses with the cinnamaldehyde reagent to produce a red color. No color means the enzyme is absent.

Quality control

Before use, perform QC on each new lot or shipment of LAP disks and color reagent. Because the test is performed infrequently and most Gram-positive cocci are LAP positive, verify potency by testing the positive control each time a negative result is obtained with a test organism.

1. Enterococcus faecalis ATCC 29212 — LAP positive
2. Aerococcus viridans ATCC 11563 — LAP negative

Procedure

1. Before inoculation, slightly dampen the LAP disk with reagent-grade water. Do not supersaturate the disk.
2. Using a wooden applicator stick, rub a small amount of several colonies from an 18 to 24 hour pure culture onto a small area of the LAP disk.
3. Let the disk stand at room temperature for 5 minutes. This is the time the enzyme needs to act on the substrate; no reagent is added yet.
4. After this reaction time, add 1 drop of cinnamaldehyde reagent and read the color.

Image: Disk showing a positive LAP test

Expected results

1. Positive: development of a deep red to reddish-purple color within 3 minutes after adding the cinnamaldehyde reagent.
2. Weak reaction: pink.
3. Negative: no color change, or development of a slight yellow color.

LAP Test Results

The LAP reactions of some catalase-negative, Gram-positive cocci are:

• LAP negative: Leuconostoc
• LAP positive: Streptococcus, Enterococcus, Lactococcus, and Pediococcus
• LAP variable: Aerococcus (A. viridans, the negative control strain, is typically LAP negative)

Read alongside PYR, LAP places an unknown into a genus quickly:

Limitations

1. LAP is one of several tests for identifying catalase-negative, Gram-positive cocci. It is most helpful when kit tests give an uncommon identification, or when the Gram stain does not show the expected cocci in chains.
2. An insufficient inoculum can produce a false-negative result. When a negative is obtained, confirm disk potency with the positive control before reporting.

How to Remember

• The negative is the news. Almost every catalase-negative Gram-positive coccus is LAP positive, so a positive tells you little. A negative is the flag that narrows you to Aerococcus or Leuconostoc. When you read a LAP disk, ask yourself "is this the boring expected positive, or the useful negative?"
• Two-enzyme spot panel. Think of LAP and PYR as a pair you read together. PYR splits off Enterococcus and S. pyogenes (both PYR positive); LAP splits off Aerococcus and Leuconostoc (LAP negative). The single organism that is PYR positive and LAP negative is Aerococcus, which is the one clean corner of the grid worth memorizing.
• Same red, different reagent. LAP and PYR both end in a red color from the same released beta-naphthylamine, which is exactly why students mix them up. Anchor the difference on the reagent: LAP uses cinnamaldehyde; PYR uses dimethylamino-cinnamaldehyde (DMACA). Different enzyme, different substrate, same colored finish line.

Key exam facts in one table

Where Students Get Confused

• LAP vs PYR. This is the number-one trap. Both are rapid enzyme spot tests, both use a beta-naphthylamide substrate, and both finish in a red color. They are different tests detecting different enzymes (leucine aminopeptidase vs pyrrolidonyl arylamidase) with different reagents (cinnamaldehyde vs DMACA). Do not treat them as interchangeable.
• Thinking a positive is diagnostic. Because most of these cocci are LAP positive, a positive rarely narrows anything. The discriminating value is in the negative.
• The "5 minutes" vs "3 minutes." These are two different clocks. The 5 minute wait is the enzyme acting on the substrate before reagent is added. The 3 minute window is how fast the red color should appear after cinnamaldehyde is added. They are not contradictory.
• The "variable" Aerococcus. Students want a clean plus or minus. Aerococcus is genuinely variable by species, which is why A. viridans serves as the negative QC strain while other Aerococcus can read positive. Interpret LAP for Aerococcus alongside PYR, not on its own.
• Which vancomycin-resistant coccus is which. Leuconostoc and Pediococcus are intrinsically vancomycin resistant and can be mistaken for VRE. LAP helps separate them: Leuconostoc is LAP negative (and produces gas from glucose), while Pediococcus is LAP positive (no gas).

References

1. Leber AL, editor. Clinical Microbiology Procedures Handbook. 4th edition. Washington, DC: ASM Press; 2016.
2. Procop GW, Church DL, Hall GS, et al. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th edition. Philadelphia: Wolters Kluwer; 2017.
3. Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th edition. St. Louis: Elsevier; 2022.