CAMP Test: Principle, Procedure, and Results

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

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

Principle of CAMP Test

The basis of the CAMP test is the enhanced hemolytic activity of beta-hemolysin-producing strains of Staphylococcus aureus by an extracellular protein ( CAMP factor) produced by group B streptococci. Synergistic hemolysis in the blood agar plate results from the interaction of the beta-hemolysin with the factor. Both hemolytic and non-hemolytic strains of group B streptococci show this phenomenon. 

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

• S. agalactiae ATCC 12386-CAMP test positive
• Streptococcus pyogenes ATCC 19615-CAMP test negative.
• 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

• Firstly, make a single straight line streak of beta-hemolysin producing Staphylococcus aureus down the center of a blood agar plate, 
• Then, inoculate a streak of the test organism (beta-hemolytic streptococci to be identified) perpendicular to the staphylococcal streak. Take care not to intersect the staphylococcal streak. 

Note: Make these streaks in such a way that, after incubation, the growth of the two organisms will not be touching.

• The streptococcal streak should be 3 to 4 cm long.
• Similarly, inoculate known group A and B streptococcal strains on the same plate as negative and positive controls.
• Ensure labeling the location of each streak on the back of the plate.
• Finally, incubate the plate at 35°C in ambient air for 18-24 hours.

Disk method

• Firstly, place disks containing beta-lysin of S.aureus on a warmed blood agar plate.
• Then, streak microorganisms 2 to 3 mm from the edge of the disk.
• Finally, incubate the plate overnight at 35°C in a CO2 incubator.

Spot rapid method

• At first, place one drop or a 10 μl loopful of CAMP liquid reagent next to a presumptive S. agalactiae colony growing on a blood agar plate. 

Note: Do not worry if the liquid touches or engulfs the colony.

• Then, 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.
• After that, examine the plate with transmitted light for a zone of enhanced hemolysis next to the colony.
• Again, re-incubate for up to 30 min if the reaction is initially negative. Use a hand lens if necessary for examining the plate.
• Refrigeration may enhance reaction after incubation.

Results and Interpretations

• CAMP Test: A positive 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, bacitracin-resistant, trimethoprim-sulfamethoxazole-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 negative test is a lack of enhanced hemolysis near the colony being tested.

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 the pyrrolidonyl-β-naphthylamide (PYR) test. S. pyogenes is PYR positive, whereas S. agalactiae is PYR negative.
3. It 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. The reaction may be very weak if the agar is too thin or hemolyzed.

Other CAMP test positive organisms

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

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.

If the reverse CAMP test is positive, an arrow of no hemolysis is formed at the junction of the organism being tested with the staphylococci.

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