CAMP Test- Principle, Procedure, Types, Results, Uses, Limitations

What is CAMP Test?

CAMP (Christie–Atkins–Munch-Peterson) test is a biochemical test used to differentiate Streptococcus agalactiae from other beta-hemolytic Streptococci.

• S. agalactiae is the only member of the Group B Streptococci which is separated from other beta-hemolytic Streptococci by the demonstration of a large area of hemolysis when grown with Staphylococcus aureus.
• This test is used for the presumptive identification of Group B Streptococcus (Streptococcus agalactiae) which is the only beta-hemolytic Streptococcus that yields a positive CAMP test.
• S. agalactiae is an important pathogen that causes invasive diseases in humans and mastitis in cows.
• The CAMP test detects the CAMP factor, which is a heat-stable protein produced by Group B Streptococci as a result of the synergetic association between Streptococcus agalactiae and Staphylococcus aureus.
• The CAMP factor was named CAMP factor for the initials of the authors of the article that first described the phenomenon.
• A reverse CAMP test is another biochemical test that uses a similar principle except that the positive test is demonstrated by the formation of an arrow of no hemolysis at the intersection of the organism being tested and the Staphylococci.

Objectives of CAMP Test

• To differentiate Streptococcus agalactiae from other beta-hemolytic Streptococci.
• To detect the ability of an organism to produce the CAMP factor.

Principle of CAMP Test

• Streptococcus agalactiae produces a diffusible, thermostable, extracellular protein that interacts synergistically with the beta-hemolysin produced by Staphylococcus aureus, resulting in a zone of enhanced lysis of sheep or bovine erythrocytes.
• The standard CAMP test relies on the elaboration of two toxins during growth to form a typical arrowhead or flame-shaped clearing at the juncture of the two organisms that are streaked perpendicular to each other.
• The rapid disk test utilizes an extract of staphylococcal beta-hemolysin that interacts directly with the CAMP factor already diffused in the medium around the S. agalactiae colony.
• The hemolysis produced by beta-hemolytic strains of Staphylococcus aureus is enhanced by the extracellular protein produced by Group B Streptococci.
• The interaction of the beta-hemolysin of Staphylococcus aureus with the CAMP factor of Group B Streptococci results in synergistic hemolysis, resulting in an arrowhead zone of hemolysis.
• A positive CAMP reaction is indicated by enhanced hemolysis within 30 min of adding the CAMP factor reagent.
• The test is useful in the identification of both S. agalactiae and many Gram-positive rods, including Listeria monocytogenes.

Microorganism Tested

• Gram-positive cocci in pairs and chains that are catalase-negative, with medium-sized, smooth, whitish colonies with a small halo of beta-hemolysis. Nonhemolytic isolates also may be tested, since the CAMP factor can still be present.
• Gram-positive rods, as part of their identification.
• Gram-positive cocci in clusters, as part of the identification of staphylococci to the species level, if Staphylococcus intermedius is used as the strain that supplies the hemolysin.

Media, Reagent, and Supplies Used

Media

• Blood Agar

Reagent

1. Beta-lysin reagent

• Culture of aureus
• Commercial reagents: Disks containing beta-lysin of aureus

Supplies

1. Sterile wooden applicator sticks or bacteriologic loops
2. Distilled water
3. Petri dish and slide

Procedure of CAMP Test

The CAMP test can be performed either via the standard test on blood agar plates or the disk method.

Standard Method

1. S. aureus is streaked on the blood agar in a straight line across the center of the plate.
2. The unknown microorganism is then streaked in the same manner perpendicular to the staphylococcus while avoiding the touching of the organism to the previously streaked area.
3. The positive control organism is streaked parallel to and approximately 1 inch from the unknown organism.
4. Each streak on the plates is labeled on the back of the plate.
5. The plate is incubated overnight at 35°C in a CO2 incubator.

Disk Method

1. The disk is removed from the vial and placed on a warmed Blood Agar Plate.
2. The microorganism is streaked about 2 to 3 mm from the edge of the disk.
3. The plate is incubated overnight at 35°C in a CO2 incubator.

Quality Control

As a form of quality control for the CAMP test, two different organisms can be taken as a positive and negative control.

 Result and Interpretation of CAMP Test

Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

• A positive result in the standard assay is the formation of a distinct arrowhead of hemolysis at the intersection of the Staphylococcus and test organism streaks.
• A positive reverse CAMP or phospholipase D is indicated by a typical arrowhead of no hemolysis at the junction of the two hemolytic organisms.
• In the case of the disk test, a positive result is demonstrated by a distinct arc-shaped zone of complete hemolysis at the point of interaction of the disk with beta-lysin and the test microorganism.
• A lack of enhanced hemolysis near the colony being tested is a negative test.

Reporting result

• A test organism that gives a positive CAMP test and has the morphologically and biochemical characteristics (catalase-negative, Gram-positive cocci in pairs and chains) of Streptococcus species is reported as Streptococcus agalactiae.
• The following Gram-positive rods are CAMP test positive: Rhodococcus equi, monocytogenes, Propionibacterium avidum/granulosum, Actinomyces neuii, Turicella otitidis, Corynebacterium glucuronolyticum, Corynebacterium coyleae, Corynebacterium imitans, and some strains of the Corynebacterium striatum and Corynebacterium afermentans group.

Uses of CAMP Test

• The CAMP test is used to differentiate Streptococcus agalactiae from other Streptococcal species.
• The test is used to identify Listeria monocytogenes, which is a CAMP positive organism.
• It can be used to determine the ability of an organism to produce the CAMP factor.

Limitations of CAMP Test

• Increased nonspecific hemolysis at the intersections (a “matchstick” effect) may be seen with other streptococci, but only group B streptococci will produce a definite arrowhead. Verify typical group B streptococcus, colony morphology, and hemolysis.
• Isolates with a negative CAMP test could still be S. agalactiae and require further testing.
• pyogenes can give a reaction that may be interpreted as positive. When there is a question, S. pyogenes is pyrrolidonyl-β-naphthylamide (PYR) positive, but S. agalactiae is PYR negative.
• The CAMP test separates L. monocytogenes, the human pathogen, from most other Listeria species.
• If the agar is too thin or hemolyzed, the reaction may be feeble.

References and Sources

• Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook, 3.17.1.1–3.17.48.3. DOI:10.1128/9781555818814.ch3.17.1.
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