Fungal culture in Cats (Felis) | Vetlexicon
felis - Articles

Fungal culture

ISSN 2398-2950


Synonym(s): dermatophyte

Overview

  • Dermatophyte test medium (DTM)  is a Sabouraud’s dextrose agar with cyclohexamide, gentamycin, chlortetracycline which inhibit non-dermatophyte growth, and phenol red as a pH indicator.
  • It is suitable for use in diagnosis of feline dermatophytosis Dermatophytosis.
  • The principle of the test is that pathogenic fungi utilize the protein in the medium and the resulting metabolites change the pH to alkaline and the medium from yellow to red.
  • Saprophytic fungi use carbohydrates first and no color change is seen. Once the carbohydrate is depleted, saprophytes will then utilize protein and a late color change is seen. Therefore it is important to observe the DTM daily and a color change either before or in association with the early visible growth of the colonies is positive.
  • The ideal is to also culture on a plain Sabouraud dextrose agar as this enables easier identification of the pathogen species. DTM inhibits formation of conidia - the best feature to identify the species; it can also make the gross colony morphologies less distinctive.

Uses

Alone

  • To aid the diagnosis of dermatophytosis in cats (and other species).
  • To identify the pathogen species.
  • To rule out the presence of dermatophytes in the fur of cats in contact with affected individuals.
  • To help identify when treatment for dermatophytosis can be stopped.

In combination

  • To differentiate dermatophytosis from other causes of skin disease in combination with history, examination, bacteriology, cytology etc.
  • To definitely diagnose dermatophytosis in combination with cytology or histopathology - definite diagnosis requires the identification of infected hairs or other tissues plus identification of dermatophyte organisms by culture or their DNA by PCR PCR (polymerase chain reaction).

Sampling

Source of test material

  • Hair, crust and scale from cats affected with suspect skin lesions. The ideal sample is hairs fluorescing under light from a Wood’s lamp Skin: wood's lamp test but not all pathogens fluoresce, eg Trichophyton mentagrophytes Trichophyton spp does not and about 50% of Microsporum canis Microsporum canis  also.
  • The modified MacKenzie toothbrush technique especially for cats without skin lesions but which are in contact with affected animals or humans or which are recovering from the disease.

Quantity of test material

Care with choosing sample site means that not many hairs are needed.  

Sample collection technique

  • Choose broken hairs from the advancing edge of a lesion, pluck them using forceps and inoculate them onto the surface of the dermatophyte test medium (DTM).
  • Or, especially when gross lesions are not present, use the modified MacKenzie toothbrush technique - rubbing the new toothbrush through the whole of the cat's fur for 2 minutes then pressing the brush into the test medium.
  • Claws and footpads are heavily contaminated with saprophytic fungi and bacteria so swabbing the skin first with alcohol and allowing it to dry before sampling is essential to avoid overgrowth of the dermatophytes in fungal media. The tips of claws could be discarded and material from inside the claw if more diagnostic.

Quality control

Precautions

Wear gloves as ringworm infection is zoonotic.

Sample storage

  • Samples should be placed in an envelope not in a plastic bag. Overgrowth of saprophytes will occur if the atmosphere is too humid.
  • Samples in plastic or glass bottles should have their lids loosened to reduce the humidity inside.

Sample transport

  • Transport to a laboratory can occur in a sample pot with a loosened lid but a paper container is better.

Tests

Methodologies

  • Culture is best at 30°​C but room temperature is acceptable. UV light reduces growth so a dark area is best.
  • A positive result may be seen in as little as 5 days but leave the culture for at least 14 days before treating as negative. The atmosphere can be too dry as well as too humid and a pan of water in the incubator will provide this ideal humidity.
  • Identifying particular dermatophytes is not often possible with DTM as macroconidia are not produced.
  • Setting up a subculture on Sabouraud’s dextrose agar will culture colonies that sporulate and mature macroconidia can then be identified using a fungal guide.
  • The most common dermatophytosis of cats is Microsporum canis.
  • Colonies of M. canis on Sabouraud’s agar  are white cotton/wool-like with an orange-yellow undersurface.
  • Once colonies are seen, take a small square of acetate tape, eg Scotch pressure sensitive tape® (3M), holding it in forceps touch it lightly to the surface of the culture then place it on a glass slide with a drop of lactophenol cotton blue and examine under x100 magnification. The macroconidia are abundant on Sabouraud’s media, for Microsporum canis they have thick walls with a terminal knob.

Availability

  • Fungal culture can be carried out in the practice situation as long as the test will be examined daily. DTM is commercially available, eg Dermaphyt® (Kruuse). Alternatively, all commercial laboratories will offer the test but samples should be submitted in paper or false-negative culture results may be seen due to bacterial overgrowth.

Validity

Sensitivity

  • Choosing sample material carefully remains the single most important determinant of success.

Specificity

  • Visual analysis of gross cultural morphology and microscopic analysis of hyphal form and macroconidial morphology is necessary for species identification and requires experience
  • These are more readily identifiable on colonies grown on plain Sabouraud medium.
  • It may be useful to submit positive DTM tests to a commercial laboratory for confirmation and identification of the species.

Technique intrinsic limitations

  • Fastidious cultural requirements.
  • Overgrowth by non-pathogenic fungi or bacteria on Sabouraud's dextrose agar plates.
  • Failure to identify species morphological characteristics on DTM.

Technician extrinsic limitations

  • Veterinarian or laboratory technician experience required.

Result Data

Normal (reference) values

  • No dermatophytes grow.

Abnormal values

  • Dermatophyte growth is seen, usually Microsporum canis. Trichophyton mentagrophytes or Microsporum gypseum.

Errors and artifacts

  • No growth may be seen due to fastidious cultural requirements not being met.
  • Lack of macroconidia on subculture may make identification difficult.
  • A dermatophyte growth does not equate to dermatophytosis as these are common contaminants. If there are typical dermatophytosis lesions this may be a reasonable diagnosis but a definite diagnosis requires identification of lesions by histopathology (which requires skin biopsy Biopsy: skin) or by microscopy of the hairs Trichography (hair plucking) (easily performed in the practice laboratory).

Further Reading

Publications

Refereed Papers

Other sources of information

  • Miller W H, Griffin C E & Campbell L (2013) Diagnostic Methods. In: Muller & Kirk’s Small Animal Dermatology. 7th edn. Elsevier Inc, USA.  pp 86-91.
  • van Cutsem J & Rochette F (1991) Mycoses in Domestic Animals. Janssen Research Foundation.