CAMP Test – Principle, Procedure, Result

CAMP Test

• CAMP Test was first discovered by four researchers Christie, Atkins, Munch, and Peterson in 1944, that’s why this test is also known as Christie–Atkins–Munch-Peterson test.
• CAMP test is used to identify Streptococcus agalactiae from other Group B β-hemolytic streptococci. They form a substance called CAMP factor which enlarges the area of hemolysis formed by the β-hemolysin elaborated from Staphylococcus aureus.
• This test is also known as “group b strep camp test”, because it help to identify the Streptococcus agalactiae which is a member of Group B Streptococci.
• Streptococcus agalactiae is the only beta-hemolytic Streptococcus that shows a positive CAMP test.
• The CAMP test mainly recognizes the CAMP factor which is a diffusible, heat-stable, extracellular protein produced by Group B Streptococcus that enhances the hemolysis of sheep erythrocytes by Staphylococcus aureus. The CAMP factor acts synergistically with the beta hemolysin produced by S. aureus to induce enhanced hemolysis of sheep or bovine RBCs but not human, rabbit or horse RBCs.
• S. agalactiae is responsible for invasive diseases in humans and mastitis in cows.
• In some laboratories the CAMP (Christie, Atkinson, Munch, Peterson) test is used to confirm whether bacteria have enhanced staphylococcus beta-lysis activity test, which has long been considered as a key, confirmed test for the identification of GBS
• Some PCR-based assays are also used to target the cfb gene, which encodes the CAMP factor.

Purpose of CAMP Test

• To identify the Streptococcus agalactiae.
• To distinguish the capability of an organism to generate the CAMP factor.

CAMP test principle

A diffusible, thermostable, extracellular protein produced by the Group B β-hemolytic streptococci, Streptococcus agalactiae, which interacts synergistically with the beta-hemolysin produced by Staphylococcus aureus, this results in a zone of enhanced lysis of sheep or bovine erythrocytes. 

The standard CAMP test is based on the elaboration of two toxins during growth to produce a typical arrowhead or flame-shaped area at the intersection of the two organisms when they are placed perpendicular to each other.

The rapid test uses an extract of Staphylococcal beta-lysin that acts immediately with the CAMP factor previously dispersed in the medium around the S. agalactiae colony. The hemolysis generated by beta-hemolytic strains of Staphylococcus aureus is magnified by the extracellular protein formed by Group B Streptococci. The intercommunication of the beta-hemolysin of Staphylococcus aureus with the CAMP factor of Group B Streptococci ends in synergistic hemolysis, resulting in an arrowhead zone of hemolysis.

A positive CAMP reaction is shown by enhanced hemolysis within 30 minutes to 1 hour of adding a drop of CAMP factor reagent. Many Gram-positive rods (Listeria monocytogenes) and S. agalactiae, can be identified by using this test.

Requirement for CAMP test

• Media: Blood Agar
• Reagent: Beta-lysin reagent.
• Culture of aureus
• Commercial reagents: Disks containing beta-lysin of aureus.
• Sterile wooden applicator sticks or bacteriologic loops
• Distilled water
• Petri dish and slide

CAMP test procedure

The CAMP test can be performed by three distinct methods such as; standard test on blood agar plates, Rapid CAMP test and the disk method.

Standard CAMP test Method

1. Streak the beta-lysin-producing S. aureus  on the blood agar plate by using an inoculating loop in a straight line across the center of the plate.
2. Take the test organism/unknown microorganism and steak it in the same manner perpendicular to the S. aureus , and leave 1cm space between the two streaks ( avoiding the touching of the organism to the previously streaked area). 
3. Streak the positive control organism parallel to and approximately 1 inch from the unknown organism.
4. Label each streak on the back of the plate.
5. Incubate the plate overnight at 35°C in a CO2 incubator.

Disk Method of CAMP test

1. Place the disk on a warmed Blood agar plate.
2. Streak the microorganism about 2 to 3 mm from the edge of the disk.
3. Incubate the plate overnight at 35°C in a CO2 incubator.

Rapid CAMP test

1. Take the test kit from the freezer and allow it to thaw.
2. Place one drop of CAMP spot test reagent next to a characteristic colony ( S. agalactiae colony) grown for 18-24 hours on a blood agar plate.
3. Incubate the plate aerobically or in 5-10% CO2 at 37°C for 20-30 minutes. It will prevent the spot CAMP reagent from running over the plate’s surface.
4. Observe the plate with transmitted light for a zone of enhanced hemolysis next to the colony.
5. Reincubate for up to 30 min if reaction is initially negative.
6. Use a hand lens if necessary for examining the plate.
7. Refrigeration may enhance reaction after incubation.

Quality Control of CAMP test

For the Quality Control of CAMP test two organisms are selected as a positive and negative control.

Streptococcus agalactiae (Positive Control of CAMP test)

• Incubation: Incubate for 24-48 hours at 33-37°C in the air with 5% CO2.
• Results: CAMP test positive; formation of arrowhead hemolysis at the intersection of the streaks.

Streptococcus pyogenes (Negative Control of CAMP test)

• Incubation: Incubate for 24-48 hours at 33-37°C in the air with 5% CO2.
• Results: CAMP test negative; β-hemolysis with no enhanced arrowhead.

Result of CAMP test

• CAMP test Positive: Streptococcus agalactiae –  A CAMP test Positive results enhanced hemolysis which is indicated by an arrow head-shaped zone of beta-hemolysis at the junction of the two organisms.
• CAMP test Negative: Streptococcus pyogenes — A CAMP test Negative results beta-hemolysis without enhanced arrowhead formation.

List of CAMP test positive Organisms

• Streptococcus agalactiae.
• Rhodococcus equi
• Listeria monocytogenes
• Propionibacterium avidum/granulosum,
• Actinomyces neuii
• Turicella otitidis
• Corynebacterium glucuronolyticum
• Corynebacterium colyeae
• Corynebacterium imitans, and some strains of Corynebacterium striatum and Corynebacterium afermentans group.

CAMP Test Uses

• The Christie, Atkins, and Munch-Peterson (CAMP) test is used to differentiate group B streptococci (Streptococcus agalactiae– positive) from other streptococcal species.
• Used to distinguish the Listeria monocytogenes, it also produces a positive CAMP reaction.
• Used to detect the ability of an organism to produce CAMP factor.

CAMP Test Limitation

• A false-positive result can be formed if the incubation times are extended or elevated incubation temperatures.
• If the plate is incubated in a candle jar in an atmosphere or under anaerobic conditions, some Group A Streptococcal will give a CAMP test positive result. Therefore, ambient air incubation should be done.
• If Rhodococcus equi replaces S. aureus, then L. ivanovii will show a positive CAMP reaction.
• The reaction cabe be feeble tf the agar is too thin or hemolyzed.
• 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.
• 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.

References

• Guo, D., Xi, Y., Wang, S. et al. Is a positive Christie-Atkinson-Munch-Peterson (CAMP) test sensitive enough for the identification of Streptococcus agalactiae?. BMC Infect Dis 19, 7 (2019). https://doi.org/10.1186/s12879-018-3561-3
• https://en.wikipedia.org/wiki/CAMP_test
• https://www.researchgate.net/publication/316432013_camp_test_for_identification
• https://www.onlinebiologynotes.com/camp-test-principle-requirements-procedure-and-result-interpretation/
• Bailey and Scott’s Diagnostic Microbiology.
• https://microbenotes.com/camp-test-principle-procedure-and-result-interpretation/
• https://microbiologynotes.com/camp-test-principle-purpose-procedure-result-and-limitation/
• https://microbiologyinfo.com/camp-test-principle-uses-procedure-result-interpretation/