CAMP Test: Principle, Procedure, Results

Last updated on June 18th, 2021

CAMP test is used for the presumptive identification of Group B beta-hemolytic streptococci, Streptococcus agalactiae.

CAMP test is effective for the “prompt and reliable” identification of Streptococcus agalactiae in the clinical lab, as results could be observed in as little as 18 hours and require few manipulations. CAMP test rarely gives false positives with other Streptococcus. This hemolytic phenomenon was first described in 1944 by Christie, Atkins, and Munch-Petersen, and CAMP test is an acronym of their names.

The hemolytic activity of the beta-hemolysin produced by most strains of Staphylococcus aureus is enhanced by an extracellular protein produced by group B streptococci. Interaction of the beta-hemolysin with this factor causes “synergistic hemolysis,” which is easily observed on a blood agar plate. This phenomenon is seen with both hemolytic and non-hemolytic isolates of group B streptococci.

Reverse CAMP Test

A reverse CAMP test is a reaction whereby hemolysis by the beta-hemolysin of staphylococci is inhibited through the production of phospholipase C or D by organisms such as S. agalactiae, Listeria, Corynebacterium spp., and Clostridium perfringens.

An arrow of no hemolysis is formed at the junction of the organism being tested with the staphylococci if the reverse CAMP test is positive.

Quality control

Test each lot of beta-lysin reagent or disks with a positive and negative control by streaking them in a line parallel to the test organism.

Organisms

1. S. agalactiae ATCC 12386-CAMP test positive
2. Streptococcus pyogenes ATCC 19615-CAMP test negative
3. Periodically use an in-house laboratory strain of Arcanobacterium haemolyticum to demonstrate the reverse CAMP test for training purposes.

Procedure for CAMP test

Standard Method

1. Down the center of a blood agar plate, make a single straight line streak of beta-hemolysin producing Staphylococcus aureus
2. Taking care not to intersect the staphylococcal streak, inoculate a streak of the test organism (beta-hemolytic streptococci to be identified) perpendicular to the staphylococcal streak. Make these streaks in such a way that, after incubation, the growth of the two organisms will not be touching.
3. The streptococcal streak should be 3 to 4 cm long.
4. Inoculate known group A and B streptococcal strains similarly on the same plate as negative and positive controls respectively.
5. Label the location of each streak on the back of the plate.
6. Incubate the plate at 35°C in ambient air for 18-24 hours.

Disk method

1. Place disks containing beta-lysin of S.aureus on a warmed blood agar plate.
2. Streak microorganisms 2 to 3 mm from the edge of the disk.
3. Incubate the plate overnight at 35°C in a CO2 incubator.

Spot rapid method

1. Place 1 drop or a 10 μl loopful of CAMP liquid reagent next to a presumptive S. agalactiae colony growing on a blood agar plate. Do not worry if the liquid touches or even engulfs the colony.
2. Incubate the plate right side up, to prevent the spot CAMP reagent from running over the plate’s surface, for 20 min at 35°C.
3. Examine with transmitted light for a zone of enhanced hemolysis next to the colony.
4. Reincubate for up to 30 min if the reaction is initially negative. Use a hand lens if necessary for examining the plate.
5. Refrigeration may enhance reaction after incubation.

Results and Interpretations

• CAMP Test: A positive CAMP test is the formation of a distinct arrowhead of hemolysis at the intersection of the staphylococcus and test organism streaks. If any gram-positive, catalase-negative, beta-hemolytic cocci, which is bacitracin-resistant, trimethoprim-sulfomethoxazole-resistant shows such arrowhead hemolysis, it can be reported as group B. streptococci (Streptococcus agalactiae).
• A positive reverse CAMP test or phospholipase D is indicated by a distinct arrow of no hemolysis at the intersection of the two hemolytic organisms. C. perfringens is reverse CAMP or phospholipase D positive.
• In the disk test, a positive result is indicated by a distinct crescent- or arc-shaped zone of complete hemolysis at the intersection of the disk of beta-lysin and the isolate.
• In the rapid spot test, the presence of clear enhanced hemolysis only where the diffused hemolysis overlaps is a positive result.
• A lack of enhanced hemolysis near the colony being tested is a negative test.

Limitation of CAMP test

1. Some group A streptococci will be CAMP test positive if the test plate is incubated in a candle jar, in a CO₂  atmosphere, or under anaerobic conditions. Therefore, ambient air incubation should be used.
2. S. pyogenes can give a reaction that may be interpreted as positive. When there is confusion check for
   pyrrolidonyl-β-naphthylamide (PYR) test. S. pyogenes is PYR positive whereas S. agalactiae is PYR negative.
3. CAMP test has a 98% sensitivity for detecting S. agalactiae so isolates with a negative CAMP test could still be S. agalactiae and may require further testing.
4. If the agar is too thin or hemolyzed, the reaction may be very weak.

Other CAMP test positive organisms

1. Listeria monocytogenes
2. Rhodococcus equi
3. Vibrio cholerae (certain strains)
4. Certain species of Corynebacterium.

References and further readings

• Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
• Koneman’s Color Atlas and Textbook of Diagnostic Microbiology
• Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814