KOH Preparation Test - Introduction, Principle, Procedure, Result Interpretation, Advantages, Disadvantages

Introduction to KOH preparation test:

KOH preparation test is a simple, rapid, accurate, virtually painless, non-invasive traditionally recommended procedure for the diagnosis of fungal infections of skin or nails. This test is also called a potassium hydroxide skin lesion exam, fungal smear, or skin scraping.

Common skin conditions which are usually diagnosed using the KOH preparation test include ringworm, athlete's foot, jock itch, and nail infections by fungi. This is usually done after a clinical examination followed by a Wood lamp examination that uses ultraviolet light to inspect the skin.

Although culture is the standard test used to determine fungal infection, it is only moderately superior to KOH preparation as it is expensive and takes 6 weeks to achieve the results. However, a negative or inconclusive KOH preparation test result should still prompt a culture. Biopsy or periodic acid-Schiff stain may also be necessary to confirm the diagnosis.

Principle of KOH preparation test:

Samples such as keratinized tissues or nail materials (non-fungal cells) are digested by KOH and wet preparations. As KOH clears the tissue and cellular debris without impacting fungal cells, the KOH preparation is examined under the microscope for the presence of fungal particles.

KOH preparation test can confirm fungal infection by yeast (Candida albicans) and dermatophytes including Epidermophyton (skin and nail infections), Trichophyton (skin, nail, and hair infections), and Microsporum (skin and hair infections).

Sample collection for KOH preparation test:

For the KOH preparation test, samples are collected from affected areas with the help of a needle or scalpel, placed on a slide containing potassium hydroxide, and examined under a microscope for the presence of fungal particles.

Depending upon the desired samples to be collected, different instruments are used for sample extraction and collection.

• Skin scales/scrappings are obtained by scraping the surface of the margin of the lesion using a sterile blunt scalpel

• Parts of crusts are removed with the help of sterile scissors

• Infected nail pieces are snipped using sterile scissors, placed into a tube containing a few drops of KoH solution, heated, and then left overnight

• Infected hair is plucked with forceps

• The samples (or wet preparation) are placed on the surface of culture media and incubated at 30°C for 4 weeks

Procedure of KOH preparation test:

The procedure of the KOH preparation test for fungal infection diagnosis is as follows.          

1. Place the specimen on a clean, grease-free slide

2. Add one drop of 10-20% KOH solution to it

3. Place glass cover on the mix and press gently to remove air bubbles

4. Use gauze/blotting paper to blot the excess solution

5. Examine the KOH preparation under the microscope again after 5 to 20 mins (The slide can be slightly heated to speed up the action of KOH)

6. Place the slide on the microscopic stage and examine it with low-power (10x objective) and then under 40x objective for fungal bodies, budding yeast, or yeast with pseudohyphae

* Dimethyl sulfoxide can be added to the slide for enhanced artifact clearing.

* Lactophenol cotton blue can be used for easier visualization of fungi.

* If calcofluor-white stain is added, fungi become fluorescent which makes identification easier under the fluorescence microscope.

Result Interpretation of KOH Preparation Test:

Abnormal results indicate the presence of fungal infection while normal results mean that there is no presence of fungi in the specimen.

In skin or nail specimens, chains of spores or branching hyphae are seen. In a hair specimen, parallel rows of spores are observed inside (endothrix) the hair shaft. The infected hair shaft may also contain air space. However, skin test does not distinguish active infection.

The presence of fungi in the sample does not definitely identify the fungi species/genus but rather only shows fungal presence. It is unnecessary to identify the genus/species of fungi present in the sample as broad antifungal treatment is prescribed.

If it is necessary to identify the fungi, a fungal culture can be performed to identify the specific type of fungus. Since culture takes approximately 6 weeks, it is mostly done to determine the fungal strain to develop a more precise treatment plan.

Advantages of the KOH preparation test:

• rapid diagnostic procedure

• readily available or accessible

Disadvantages of the KOH preparation test:

• Following the KOH reaction with host tissues, background artifacts may be produced which may resemble hyphae or budding forms of the fungal body

• Clearing of larger specimens may require an extended time.