CAMP Test – Its Principle, Procedure, and Limitations

Introduction

The authors of the test (Christie, Atkinson, Munch, and Peterson) are abbreviated as CAMP. Since its discovery in diagnostic microbiology by Christie et al 1944, the CAMP test has been frequently used for the presumptive diagnosis of Streptococcus agalactiae (an important source of newborn infections).

The camp test is based on “lytic phenomenon” between Staphylococcus aureus and Streptococcus agalactiae (group B). The phenomenon was initially detected on milk cultures being tested for hemolytic Streptococcus during a scarlet fever outbreak. Streptococcus colonies were only surrounded by zones of full hemolysis when they were streaked near to colonies of β-hemolytic Staphylococcus.

The CAMP reaction is a co-hemolysis phenomenon that consists of a zone of complete hemolysis. The hemolysis is observed when S. agalactiae which produces the CAMP factor, is streaked on sheep blood agar next to Staphylococcus aureus, which secretes sphingomyelinase.

Further research revealed that the increased hemolysis was caused by the growth of a heat-stable, filterable agent Streptococcus agalactiae (Group B Streptococcus). When grown alongside, colonies of β-hemolytic Staphylococcus. non-group B Streptococcus did not demonstrate this elevated hemolysis.

Munch-Petersen used this phenomenon to create a test for detecting Streptococcus agalactiae (group B), a common cause of mastitis in milk samples. He observed that when two streptococcus colonies grow 5 mm from the β-producing staphylococcus and 5 to 6 mm from each other, the lysed area may become half-moon shaped.

Purpose

The CAMP test is used to distinguish Streptococcus agalactiae (group B) from Streptococcus pyogenes (group A) (CAMP negative) and non-group B Streptococcus (CAMP negative).

Principle

Red blood cells (erythrocytes) from sheep contain up to 51% sphingomyelin, but erythrocytes from other mammalian species support the CAMP response to varied degrees depending on the sphingomyelin composition of their cell membranes.

S. aureus sphingomyelinase and CAMP factor (a protein released by S. agalactiae), are responsible for the CAMP reaction. Sphingomyelinase first hydrolyzes sphingomyelin to ceramide, making erythrocytes susceptible to the lytic activity of factor CAMP.

This synergistic interaction produces a zone of enhanced and visible hemolysis between the two cultures. The synergistic zone is not detected in Streptococcus groups A, C, and G.

Requirements

  1. Media: The CAMP test is carried out on standard sheep blood agar plates with trypticase soy agar +5% sheep blood.
  2. Streptococcus aureus culture
  3. Beta lysin discs
  4. CAMP reagent
  5. Positive control: S. agalactiae colony 
  6. Negative control: Streptococcus pyogenes culture
  7. Apparatus: Petri plates, inoculation loop, Bunsen burner, incubator

Methods for CAMP test

The following methods are used to perform the CAMP test:

  1. Standard CAMP test
  2. Rapid CAMP test
  3. CAMP test by disc method

Procedure

Standard CAMP test:

  1. To inoculate Staphylococcus aureus onto a sheep blood agar plate, make a narrow streak down the center of the plate with a loop or the edge of a needle. It is best to use a strain of S. aureus known to produce a high quantity of β-toxin.
  2. The test organism (group B Streptococcus) is streaked at right angles to the S. aureus in a straight-line inoculum. The Streptococcus streak should be within 2 mm of the S. aureus streak but not contacting it.
  3. The plates are incubated for 24 hours at 37°C. To limit the number of false positives, incubation in ambient air is suggested.

Results

Positive result

An ‘arrowhead-shaped’ increased zone of β-hemolysis in the area between the two cultures with the “arrow point” towards the S. aureus streak indicates a positive result.

Negative result

In a CAMP negative reaction, there is no increased zone of β-hemolysis.

Figure 1: figure represents the standard CAMP test results. A represents the S. aureus, B is the marked arrow represents the CAMP test results. K+ is positive control (S. agalactiae), S11 represents sample which is showing positive results and K- is the negative control (S. pyogenes) (Effendi et al., 2018).

Rapid CAMP test:

Protocol

  1. One drop of CAMP reagent should be placed next to a potential S. agalactiae colony developing on Blood agar.  (The colony won’t be affected if the liquid contacts it or even engulfs it.)
  2. To stop the spot CAMP reagent from running over the plate’s surface, incubate the plate with its right side up for 20 minutes at 37°C.
  3. Check the area close to the colony for a zone of increased hemolysis using transmitted light.
  4. In case the reaction is initially negative, re-incubate for up to 30 minutes.
  5. After incubation, refrigeration may improve the reaction.

Results

The appearance of clearly elevated hemolysis only when dispersed hemolysis overlapped in the fast spot test indicates a positive result.

A negative test is one that shows no increased hemolysis close to the colony being tested.

CAMP test by disc method:

  1. Place disc on a plate of warmed Blood Agar.
  2. Spread the bacteria 2 to 3 mm from the disk’s edge.
  3. Overnight, in a CO2 incubator at 37 °C, incubate the plate.

Results

A clear crescent- or arc-shaped zone of total hemolysis at the point where the isolate and beta-lysin discs converge indicates a positive result in the disc test.

Figure 2: the figure shows the positive CAMP test results by disc method

Uses of CAMP test

  • It is used to identify Streptococcus agalactiae from other beta-hemolytic Streptococcus species.
  • It is used to detect Listeria monocytogenes, which causes a positive CAMP reaction.

Limitations of CAMP test

  • A few numbers of of group A streptococci may show a positive CAMP reaction.
  • Some Group A Streptococcal isolates will be CAMP test positive whether the plate is incubated in a candle jar in an atmosphere or under anaerobic conditions. As a result, ambient air incubation should be carried out.
  • On sheep blood agar, colonies with characteristic group B streptococci morphology and narrow zone beta-hemolysis should be tested.
  • Longer incubation times or high incubation temperatures may produce false-positive results.
  • It is just sheep blood agar plates that are used. Human, horse, rabbit, or guinea pig blood plates will not produce the desired result.
  • On sheep blood agar, colonies of Listeria monocytogenes have a limited zone of beta-hemolysis and may be confused with group B beta-hemolytic streptococci if catalase and gramme stain are not done.

Quality control

  • To ensure the accuracy and dependability of the results, it is advised to include both positive and negative controls with each batch of tests. A well-known strain of Group B Streptococcus should serve as the positive control, while a non-GBS strain should serve as the negative control.
  • The blood agar plate, S. aureus culture, and GBS culture are only a few examples of the reagents that should all be of standardized quality and come from reliable suppliers.
  • It is important to use aseptic procedure when handling and preparing cultures and reagents. Before use, the sterility of cultures and reagents should be verified.
  • To guarantee optimal bacterial growth and reliable results, the incubation parameters, including temperature, duration, and ambient variables, should be standardized and controlled.
  • A qualified laboratory professional with knowledge of the test’s limitations should interpret the results. Investigate and correct any variations or unusual results.

Precautions

  • The reaction may be very weak if the agar is excessively liquid or hemolyzed.
  • Isolates that fail the CAMP test may still be S. agalactiae and must be tested further.
  • S. pyogenes can produce a positive response (S. pyogenes is positive for pyrrolidinyl–naphthylamide (PYR), whereas S. agalactiae is negative for PYR).
  • Long incubation times or high incubation temperatures may result in false positive results.

References

EFFENDI, M. H., OKTAVIANTO, A. & HASTUTIEK, P. J. J. I. T. P. J. O. V. M. 2018. Tetracycline Resistance Gene In Streptococcus agalactiae Isolated From Bivine Subclinical Mastitis In Surabaya, Indonesia. 55, 115-120.

https://en.wikipedia.org/wiki/CAMP_test

Mubashir Iqbal
Mubashir Iqbal

Mubashir Iqbal is a highly dedicated and motivated Microbiologist with an MPhil in Microbiology from the University of Veterinary and Animal Sciences. Currently, he is researching the efficacy of commercially available SARS Cov-2 vaccines to neutralize the omicron variant in Pakistan. He holds a Bachelor's degree in Microbiology and has experience in chemical and microbiological analysis of water samples, managing SOPs and documents according to standard ISO 17025. Additionally, he has worked as an internee in BSL 3, Institute of Microbiology, UVAS, where he gained experience in RNA extraction, sample processing, and microscopy.

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