Onychomycosis, commonly known as toenail fungus, is a widespread infection affecting the nail plate, nail bed, or both. While the condition often presents with noticeable visual changes like discoloration, thickening, and crumbling of the nail, these signs are not unique to a fungal infection. Definitive diagnosis requires a laboratory test to examine the material at a cellular level. This microscopic confirmation is the only way to positively identify the infectious agent and ensure the selection of an appropriate treatment plan.
The Need for Microscopic Confirmation
Visual inspection alone is insufficient for a conclusive diagnosis because many non-fungal conditions can mimic the appearance of onychomycosis. Nail trauma, for example, can cause discoloration and thickening that looks identical to a fungal infection. Psoriasis, lichen planus, and a condition called onychogryphosis, which involves a thickened, curved nail, all present with similar clinical features.
Relying solely on visual symptoms can lead to misdiagnosis and the prescribing of ineffective antifungal medication. These medications are often taken orally for months and carry potential side effects, making it important to confirm the pathology before starting a long-term treatment regimen. Microscopic analysis confirms the presence of fungal elements, allowing the healthcare provider to differentiate between a true mycosis and other nail disorders and ensuring the patient receives the correct therapy.
Preparing the Sample for Viewing
The process of preparing the sample, known as a Potassium Hydroxide (KOH) preparation, is designed to isolate the fungal structures from the surrounding human tissue. The sample is typically collected by scraping debris from beneath the nail plate, particularly from the area closest to the cuticle where the fungus is most actively growing. This subungual material contains both human keratin and the fungal elements.
The collected scrapings are placed on a glass slide and mixed with a drop of potassium hydroxide solution, usually at a concentration of 10% to 20%. The strong alkaline nature of the KOH works to chemically dissolve the keratin, which makes up the bulk of the nail and skin cells. Crucially, the rigid cell walls of the fungi are resistant to the KOH and remain intact. Applying gentle heat or adding a clearing agent like dimethyl sulfoxide (DMSO) can speed up this dissolving process.
Once the keratin has cleared, the slide is ready for examination under a microscope. Sometimes, counterstains like chlorazol black E or Parker blue-black ink are added to the preparation. These stains bind to the fungal cell walls, making them stand out with greater contrast against the now-clear background material, enhancing visibility.
Key Structures Seen Under the Microscope
When viewing a positive sample under the microscope, the most common finding is the presence of dermatophytes. These fungi, such as Trichophyton rubrum, are responsible for the majority of toenail infections and appear as thread-like filaments called hyphae. These hyphae are described as septate, meaning they are divided by internal cross-walls that look like parallel lines, giving them a characteristic long, branching, and refractile appearance, sometimes described as a “railroad track” pattern.
Interspersed among the hyphae are small, rectangular or barrel-shaped structures known as arthroconidia. These spore-like segments form when the hyphae fragment and are considered highly diagnostic for a dermatophyte infection. The microscopist looks for these elements resting in the cleared, dissolved keratin background.
Infections caused by yeasts, such as Candida albicans, present a distinct morphology under the microscope. Instead of the septate filaments of dermatophytes, yeast cells appear as small, oval or round budding cells. They often form structures called pseudohyphae, which are chains of elongated cells that look like hyphae but lack true septa and have constrictions at the joints, differentiating them from the more uniform filaments of dermatophytes.
Non-dermatophytic molds, such as species of Aspergillus or Fusarium, are less common causes of onychomycosis but may also be seen. Their hyphae can sometimes be distinguished by being wider or having irregular, non-parallel branching patterns. While microscopic examination can confirm the presence of a fungus and categorize it (dermatophyte, yeast, or mold), a fungal culture or advanced molecular testing is often necessary to identify the specific species to guide precise antifungal treatment.