Fungal Screening Test in Ankara, Turkey

The fungal test is a microscopic and cultural examination method performed to diagnose fungal infections on the skin, nails, or mucosal surfaces. The test identifies the infectious agent and allows appropriate antifungal treatment to be planned. Early diagnosis reduces the risk of spread and chronicity.

Direct microscopic examination is the first step of the fungal test process. A skin scraping, nail sample, or mucosal specimen is prepared with a potassium hydroxide (KOH) solution and examined. This method quickly shows the presence of fungal hyphae or spores.

The culture method provides definitive identification of the fungal species. The specimen is inoculated into special media and incubated under certain temperature conditions. During this process, the colony type and color characteristics that grow are evaluated, and antifungal susceptibility tests may then be applied.

The points to be considered before and after the fungal test are important for the accuracy of the diagnosis. Antifungal cream or medication should not be used before the test. Results are generally available within a few days, and treatment is individually arranged according to laboratory findings.

What Is a Fungal Test?

A fungal test is a laboratory analysis performed to make a diagnosis when a fungal infection is suspected on the skin, hair, or nails. A sample is taken from the infected area and microscopic examination, culture, or molecular tests are applied. Dermatophyte, yeast, or mold species are identified. The type and spread of the infection are determined by this test in order to guide the correct treatment. Antifungal treatment is planned according to the results.

When we say “fungal infection,” are we always talking about the same thing?

Actually, no. Superficial fungal infections, which are one of the most common problems encountered in dermatology, target the upper hard layer (keratin tissue) of the skin, hair, and nails. However, the fungal family causing these infections does not consist of a single group.

We can roughly divide these infections into three main groups:

• Dermatophytes: These are fungi specialized in digesting keratin and are responsible for most of the classic fungal infections known as “tinea.” Trichophyton, Microsporum, and Epidermophyton are the best-known members of this group.
• Yeasts: Yeasts such as Candida species can cause infection especially in moist fold areas (armpits, groin, under the breasts) and in the nails.
• Other Fungi (Non-Dermatophytes): The best known of these are Malassezia species. This yeast-like fungus is actually normally present on all of our skin, but under certain conditions it multiplies and causes the condition called Tinea (Pityriasis) Versicolor, which generally leads to light or dark colored patches on the trunk (also popularly known as ‘sam yeli’).

Treatment protocols change radically according to the organism causing the infection. A shampoo used for Malassezia may not heal an athlete’s foot caused by Trichophyton. Therefore, knowing which “enemy” we are facing is of vital importance in order to choose the right weapon.

Where are fungal infections most commonly seen?

Infections caused by dermatophytes take different names according to where they are located on the body. These are the most commonly encountered types of fungal infections:

• Tinea pedis (Athlete’s foot)
• Tinea cruris (Jock itch)
• Tinea corporis (Body ringworm)
• Tinea capitis (Scalp fungus or ringworm of the scalp)
• Onychomycosis or Tinea unguium (Nail fungus)

Especially nail fungus increases significantly with age and may affect nearly half of individuals over the age of 70.

Why is it risky to diagnose fungus just by looking at it?

In dermatology, many diseases imitate one another. Eczema, psoriasis, some allergic reactions, or bacterial infections can very easily be confused with fungal infections in appearance.

An experienced eye can often arrive at a preliminary diagnosis by looking at the clinical appearance. If the infection is very small, in a localized area, a superficial cream treatment may be tried. However, if the condition has become chronic (has been continuing for a long time), has spread over a very wide area, involves the hair, palms, or soles, or if systemic (oral pill) antifungal treatment is being considered, laboratory confirmation is an absolute necessity.

This is such an important rule that international dermatology guidelines clearly recommend, “Do not prescribe oral antifungal treatment for suspected nail fungus without confirming the fungal infection in the laboratory.” Laboratory confirmation both guarantees the correct treatment and prevents serious complications due to misdiagnosis.

How does incorrect treatment make a fungal infection even worse?

One of the situations we encounter most often in the clinic and that makes things the most difficult is the problem of “Tinea Incognito,” that is, “Hidden Fungus.”

This condition develops as follows: Let us say you have an itchy, red rash. Thinking it is eczema (or on a neighbor’s recommendation), you applied a strong cortisone cream on it. At first, that cream quickly relieves the itching and redness thanks to the cortisone it contains, because cortisone suppresses the immune response (inflammation) in that area. You think the cream is “helping.”

What is actually happening is this: In that area where the defense system is suppressed, the underlying fungal infection virtually has a “celebration.” The fungus, which is normally being limited by the immune system, begins to multiply uncontrollably and spread rapidly when this pressure is removed.

Even worse, the infection loses its typical “ring-shaped” appearance with distinct borders, its appearance changes completely, and it turns into a shapeless, widespread redness. Now it becomes much more difficult to diagnose it as a fungal infection just by looking at it. The fungus has literally “hidden itself.” This causes serious loss of time until the correct diagnosis is made and the infection becomes much more severe. Therefore, a lesion suspected to be fungal should never have a cortisone cream applied to it unconsciously.

Why are ringworm (Tinea Capitis) tests so urgent?

When fungal infections involving the scalp (Tinea capitis) are seen especially in children, the cost of delay in diagnosis can be very severe.

This condition has a very severe, inflammatory form known as “Kerion.” Kerion presents as a painful, swollen, crusted plaque on the scalp covered with pus-filled pimples. This is an excessive immune response developing against the fungus.

If Kerion is not recognized quickly and treated aggressively (generally with oral medications), it causes permanent damage to the hair follicles in that area. After the inflammation heals, it leaves a deep scar in that area and hair will never grow there again. In other words, it results in permanent baldness.

To prevent this irreversible damage, the diagnostic process in suspected scalp fungus must be both correct and as fast as possible.

Why is it necessary to stop medications before having a fungal test?

The reliability of all laboratory tests fundamentally depends on meticulous compliance with the preparation (pre-analytical) rules before the test.

This is the most common reason for a “false negative” laboratory result (that is, when you have fungus but the test shows ‘no’). Whether cream, spray, nail polish, or pills, the fungal medications you use remain in these hard tissues (especially the nail) for a long time.

When we take a sample from that area and inoculate it into a special medium in the laboratory (for the culture test), the medication residue in the sample prevents the fungus from multiplying and growing. The fungus is there, but because of the medication it cannot develop and the result comes back ‘negative.’

To avoid this situation, it is essential to comply with a “drug discontinuation” or “washout” period for the sake of the reliability of the test. These periods vary according to the type of test and the medication used, but they are especially important if a culture test is to be performed.

What are the medication discontinuation periods for a fungal test?

These periods are determined according to the rate at which the medication is eliminated from the body or tissue. The required waiting periods for a reliable culture test are as follows:

• Topical Antifungals (Creams, Sprays, Nail Polishes): They should not have been used for at least 1 month before the test.
• Oral (Systemic) Antifungals (Pills): This period is much longer. It takes months for the medication to be completely cleared from keratin tissue (especially the nail). Therefore, it may be necessary to wait between 3 and 6 months after stopping the systemic medication.

Failure to comply with these waiting periods delays the definitive diagnosis and, if the initial suspicion is wrong, may cause unnecessary and ineffective treatments to continue for months.

How is a sample taken for a fungal test?

The accuracy of the test depends on taking the sample from the correct place and in the correct way. The aim is to reach the area where living fungal cells are most concentrated.

Before sampling, the area is generally wiped with an alcohol pad and allowed to dry. This is to get rid of environmental bacteria and molds (contamination) on the surface.

Sampling methods vary according to the area.

• From the Skin (Body, Groin, Athlete’s Foot): The sample is taken not from the healing center of the lesion, but definitely from the outer spreading, active, red border. This active border is where live fungi are most concentrated. The scales shed from this area are collected by scraping with a scalpel or the edge of a slide.
• From the Hair (Tinea Capitis): Simply cutting the hair strands is not enough. The infection is at the root of the hair. Therefore, the sample must be taken by epilation (pulling out from the root). Broken hair shafts and hair roots (bulb) are collected by pulling them out with tweezers.
• From the Nail (Onychomycosis): In nail fungus, the infection is generally under the nail, in the nail bed. Therefore, simply cutting a piece from the tip of the nail is not enough. It is necessary to reach the deepest (proximal) point where the nail separates from the bed, where the live fungus is located. Generally, the upper part of the nail is cut or filed a little and the subungual debris underneath is collected by scraping. This is the most valuable sample.

All these collected samples are placed into a sterile and dry container. If a culture test is to be performed, the samples should not be refrigerated in order not to lose viability; they should be kept at room temperature and, if possible, delivered to the laboratory within 24 hours.

What is the KOH (Potassium Hydroxide) test and how does it show fungus?

This is the most commonly used, fastest, and most cost-effective test in the clinic. It generally gives results within 15–30 minutes.

The procedure is simple: The skin scraping, nail debris, or hair strand taken is placed on a glass slide. A drop of Potassium Hydroxide (KOH) solution is placed on it and it is covered with a small glass coverslip.

The task of KOH is this: It dissolves and, as it were, “cleans” our skin cells, keratin tissue, and other cellular debris. However, the wall structure of fungal cells is different and very durable; they are not affected by KOH. After waiting for a while (sometimes it is heated slightly), when viewed under the microscope, the fungal structures (hyphae or yeasts) that remain standing among the dissolved skin cells become clearly visible.

This test is the fastest way to say, “Yes, there is a fungal infection here.”

Which type of fungus does the “Meatballs and Spaghetti” appearance indicate?

The KOH test not only shows the presence of fungus but sometimes also gives a clue about its type.

• Dermatophyte Infections (Body, Nail, Foot): Under the microscope, branched, septate filaments (hyphae) are generally seen.
• Candida (Yeast) Infections: Budding round yeast cells and sometimes structures called “pseudohyphae” attached to them are seen.
• Tinea (Pityriasis) Versicolor: The microscopic appearance of the Malassezia fungus causing this infection is so typical that it is almost diagnostic. This appearance is called the “meatballs and spaghetti” (or “bananas and grapes”) appearance because short, curved hyphae (filaments) and small yeast clusters are seen together.

The sensitivity of the KOH test (its rate of detecting infection) is over 70%, which is even higher than the traditional culture test.

What is the biggest limitation of the KOH test?

Although the KOH test is very useful, its biggest disadvantage is that it cannot identify the fungal species (name).

That is, “filaments (hyphae)” are seen under the microscope, it is understood that this is a dermatophyte, but it cannot be said whether these filaments belong to Trichophyton rubrum, Trichophyton mentagrophytes, or a Microsporum species. If a serious treatment such as oral medication is being planned, we want to know which species we are fighting. Therefore, after a positive KOH test, additional tests (culture or PCR) are generally required to determine the species.

Can Wood’s Lamp (Purple Light) examination detect every fungus?

No, it cannot. A Wood’s lamp is a device that emits long-wave ultraviolet (UV) light. It is also used in dermatology for some pigment disorders (such as vitiligo) or bacterial infections.

Its role in fungal diagnosis is quite specific. Some fungal species produce a fluorescent glow under UV light because of the special metabolites (waste products) they produce.

• Tinea Capitis (Scalp Fungus): Some fungi of the Microsporum genus (although they are no longer seen as often as before) produce a bright blue-green glow in the hair.
• Pityriasis Versicolor: Lesions caused by the Malassezia fungus generally show a yellow-orange glow.

However, the biggest limitation of this test is this: The vast majority of dermatophytes, especially Trichophyton rubrum, which is responsible for the overwhelming majority of nail, foot, and body fungus cases today, DEFINITELY DO NOT GLOW under Wood’s light.

Therefore, not seeing a glow when examined with purple light does not mean that the patient does not have a fungal infection. A negative Wood’s lamp examination does not rule out the diagnosis of fungus.

Why is the fungal culture test known as the “gold standard”?

Fungal culture has been accepted as the “gold standard” in fungal diagnosis for decades. There is only one reason for this: It not only shows the presence of fungus, but also allows us to identify its exact species (genus and name) by growing a living organism in the laboratory.

The sample taken is inoculated into specially prepared media in the laboratory for fungal growth (they also contain antibiotics so that bacteria cannot grow). If there are live fungal spores or hyphae in the sample, they begin to multiply in this medium and form a colony. Then, by looking at the appearance, color, and microscopic structure of this colony, a definite diagnosis such as “This is Trichophyton rubrum” is made.

This definitive species identification is critically important especially in treatment-resistant cases or to choose which medication will be more effective.

What are the disadvantages of the fungal culture test?

Although the culture test is the “gold standard” for species identification, it has very serious disadvantages that make its use in practical clinical settings difficult. Because of these problems, it has begun to be replaced by modern methods today.

Its biggest disadvantages are:

• It Gives Results Very Slowly: Fungi are very slow-growing organisms. It is necessary to wait at least 3 to 4 weeks for a culture to be reported as “negative” (that is, no growth occurred). This is a very long period if oral treatment is to be started.
• Its Sensitivity Is Low: Surprisingly, the rate at which culture detects infection (sensitivity) is not very high. In some large analyses, this rate has been found to be around 40–50% on average. In other words, culture may come back “negative” in one out of every two patients who actually have fungus.
• It Is Open to False Negatives: If the patient used medication before the test (without complying with the washout period), the result comes out directly negative.
• It Is Open to Contamination: Especially in nail samples, molds from the environment that are not related to the disease (contaminants) may grow very quickly in the culture medium, cover the real slow-growing dermatophyte, and prevent it from being seen.

How did PCR (DNA test) change fungal diagnosis?

The slowness and low sensitivity of the culture test enabled molecular methods such as PCR (Polymerase Chain Reaction) to rapidly come to the forefront in dermatology.

PCR is basically a “fungal DNA test.” This method does not need the fungus in the sample to be alive or to grow; it directly searches for the genetic material (DNA) of the fungus. If there is even a very small amount of fungal DNA in the sample, the PCR device brings this DNA to a detectable level by copying (amplifying) it millions of times.

PCR technology has virtually created a revolution in fungal diagnosis because it eliminates all the disadvantages of culture.

What are the advantages of the PCR test in fungal diagnosis?

The PCR test has overwhelming advantages over culture, especially in conditions requiring serious treatments such as nail and scalp fungus:

• It Is Very Fast: While it is necessary to wait 3–4 weeks for culture, the PCR test gives results within 1 to 3 days (and even within 16–24 hours in optimized systems).
• It Has Very High Sensitivity: The sensitivity of PCR is between 85% and 100%. It detects many cases that culture misses.
• It Is Less Affected by Medication Residues: Culture requires a live and “growable” fungus; medication residue prevents this. PCR, on the other hand, only needs DNA. Even if medication has been used, if fungal DNA is present in the sample (even if it is not alive), PCR can detect it (However, the ideal is still the medication-free period).
• It Makes Definitive Species Identification: Advanced PCR panels can simultaneously search for the DNA of the 15–20 most common fungal species (both dermatophytes, yeasts, and molds) and give a clear species definition such as “Yes, T. rubrum positive.”
• It Is Not Affected by Contamination: PCR finds only the DNA of the specific pathogen it is searching for and distinguishes it from environmental molds.

For these reasons, in cases where a rapid and reliable species identification is required (especially if oral medication is to be started), PCR is rapidly replacing culture, which used to be the “gold standard.”

Why is the PCR test not used to understand whether treatment is finished?

This is the most important and only clinical limitation of the PCR test, and it must definitely be known. PCR is not a reliable method for “healing control” (cure test) after treatment.

The reason is this: PCR cannot distinguish between live and dead fungal DNA.

You used the treatment (for example a 3-month nail fungus medication) and the fungus died. However, the DNA fragments of that dead fungus continue to remain in that tissue until your nail fully grows out and is cut off (which may take 6–12 months). If a PCR test is performed for control during this period, the test finds that dead DNA and the result comes back “false positive.” In other words, although you have actually recovered, the test may say “there is still an infection.”

Therefore, the best method to understand whether the treatment has been successful is the clinical appearance (healthy nail growth) or, if necessary (which is rarely needed), the traditional culture test.

How does your doctor decide which fungal test to choose?

An effective fungal diagnosis requires these tests to be used in a strategic order. The clinician’s aim is to confirm the diagnosis as quickly as possible, but to determine the exact species for systemic treatment when necessary.

The general approach is usually as follows:

• First Step (Rapid Screening): KOH direct microscopy is almost always the first choice. It is fast, inexpensive, and immediately answers the question “Is there fungus or not?” If the suspicion is Pityriasis Versicolor or Tinea Capitis, Wood’s lamp may also provide a quick clue.
• Second Step (Definitive Diagnosis and Species Identification): If KOH is positive and oral medication (systemic treatment) needs to be started because of the location (nail, hair) or severity of the infection, definitive species identification is essential.

Speed or Tradition?: At this point, PCR and Culture face each other. If the patient’s condition is urgent (if there is a risk of permanent damage such as Kerion) or if treatment needs to be started quickly (nail fungus), the speed of PCR (1–3 days) provides an overwhelming advantage over the slowness of culture (3–4 weeks). The high sensitivity of PCR (85%+) also eliminates the risk of “false negatives” caused by the low sensitivity of culture (40%+).