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Onychomycosis Workup

  • Author: Antonella Tosti, MD; Chief Editor: Dirk M Elston, MD  more...
Updated: Jun 30, 2016

Laboratory Studies

The clinical features of onychomycosis may mimic a large number of other nail disorders. Therefore, laboratory diagnosis of onychomycosis must be confirmed before beginning a treatment regimen. A negative mycological result does not rule out onychomycosis, because direct microscopy may be negative in up to 10% of cases and culture in up to 30% of cases.

Direct microscopy

A 20% potassium hydroxide (KOH) preparation in dimethyl sulfoxide (DMSO) is a useful screening test to rule out the presence of fungi. Before obtaining a specimen, the nails must be clipped and cleansed with an alcohol swab to remove bacteria and debris. The preparation does not require heating or prolonged incubation if DMSO is a component of the KOH solution.

In distal lateral subungual onychomycosis, a specimen should be obtained from the nail bed by curettage. The onycholytic nail plate should be removed and the sample should be obtained at a site most proximal to the cuticle, where the concentration of hyphae is greatest.

In proximal subungual onychomycosis, the overlying nail plate must initially be pared with a No. 15 blade. Then, a sample of the ventral nail plate may be taken. A No. 15 blade may also be used to remove a specimen from the nail surface in white superficial onychomycosis.

Specimens suspected of candidal onychomycosis should be taken from the affected nail bed closest to the proximal and lateral edges.

Nail fragments must be small enough for examination under low power. Large pieces of nail plate may be pulverized prior to microscopy by using a hammer or a nail micronizer. Counterstains, such as chlorazol black E or Parker blue-black ink, may be used to accentuate the hyphae. Shemer et al reported that drilling to obtain specimens and taking the sample from a more proximal site yield better results.[18]


Direct microscopy cannot identify the specific pathogen involved in onychomycosis. A fungal culture must be used to identify the species of organism.[1] Nondermatophyte molds may be resistant to the conventional therapy used for the more common dermatophytes.[19] Therefore, 2 types of growth medium should be used, one with cycloheximide (dermatophyte test medium [DTM], Mycosel, or Mycobiotic) to select for dermatophytes and one without cycloheximide (Sabouraud glucose agar, Littman oxgall medium, or inhibitory mold agar) to isolate yeasts and nondermatophyte molds.[2]

Cultures should be obtained from pulverized nail scrapings or clippings while the patient has abstained from antifungal medication for at least 2 weeks. The specimen should be kept at room temperature with the cap placed loosely over the inoculated medium.


Other Tests

Polymerase chain reaction (PCR) assays have been developed to detect fungal DNA from infected nails.[20] A highly sensitive nested PCR assay using species-specific primer pairs based on the 28S ribosomal RNA gene has been developed. This methodology permits detection of both dermatophytes and nondermatophytes.[21]



Dermoscopy is useful to distinguish distal subungual onychomycosis from traumatic onycholysis. In distal subungual onychomycosis, the proximal border of the onycholytic area is jagged owing to the presence of yellow-white spikes that project into the proximal nail plate. See the image below. This patterned has been reported as the "aurora borealis" pattern.[22]

Dermoscopy of distal subungual onychomycosis showi Dermoscopy of distal subungual onychomycosis showing irregular margin of the onycholytic area with spikes projecting into the proximal nail plate, reported as the "aurora borealis" pattern. Handyscope at 20X.

Histologic Findings

Histologic examination of the nail is a very useful alternative to culture or KOH testing. Nail clippings may be sent to the laboratory for diagnosis in a formalin-filled container, or, as a last resort, an incisional nail biopsy (by punch or scalpel) may be performed to help confirm the diagnosis. Staining in the laboratory should be performed with periodic acid-Schiff stain (PAS) or methenamine silver stain to reveal fungal elements. A comparison of diagnostic methods revealed that a nail biopsy and staining with PAS is the most sensitive technique available to diagnose onychomycosis.[23] Examining formalin-fixed, PAS-stained specimens has a higher probability (a higher negative predictive value) than KOH examination in determining that a patient is disease free if the test results are negative.

In addition to excluding other conditions (eg, psoriasis, lichen planus), the topographic distribution, the density, and the nature of the fungal elements may help guide treatment. Biopsy specimens of onychomycosis may show features of psoriasiform hyperplasia, including parakeratosis, thinned rete ridges, narrow suprapapillary plates, and dilated tortuous capillaries. As in direct microscopy, histopathologic diagnosis does not identify the species of causative pathogen.



The onychomycosis severity index (OSI) has recently been proposed to grade the severity of distal subungual onychomycosis. The OSI score is obtained by multiplying the score for the area of involvement (range, 0-5) by the score for the proximity of disease to the matrix (range, 1-5). Ten points are added for the presence of a longitudinal streaking or a patch (dermatophytoma) or for greater than 2 mm of subungual hyperkeratosis. Mild onychomycosis corresponds to a score of 1-5; moderate, 6-15; and severe, 16-35.[24]

Contributor Information and Disclosures

Antonella Tosti, MD Professor of Dermatology, Department of Dermatology and Cutaneous Surgery, University of Miami, Leonard M Miller School of Medicine

Antonella Tosti, MD is a member of the following medical societies: American Academy of Dermatology, International Society of Dermatology, European Academy of Dermatology and Venereology, Women's Dermatologic Society, Canadian Dermatology Association

Disclosure: Serve(d) as a speaker or a member of a speakers bureau for: PharmaDerm<br/>Received income in an amount equal to or greater than $250 from: Valeant; Pharmaderm.

Specialty Editor Board

Richard P Vinson, MD Assistant Clinical Professor, Department of Dermatology, Texas Tech University Health Sciences Center, Paul L Foster School of Medicine; Consulting Staff, Mountain View Dermatology, PA

Richard P Vinson, MD is a member of the following medical societies: American Academy of Dermatology, Texas Medical Association, Association of Military Dermatologists, Texas Dermatological Society

Disclosure: Nothing to disclose.

Jeffrey Meffert, MD Associate Clinical Professor of Dermatology, University of Texas School of Medicine at San Antonio

Jeffrey Meffert, MD is a member of the following medical societies: American Academy of Dermatology, American Medical Association, Association of Military Dermatologists, Texas Dermatological Society

Disclosure: Nothing to disclose.

Chief Editor

Dirk M Elston, MD Professor and Chairman, Department of Dermatology and Dermatologic Surgery, Medical University of South Carolina College of Medicine

Dirk M Elston, MD is a member of the following medical societies: American Academy of Dermatology

Disclosure: Nothing to disclose.

Additional Contributors

Richard K Scher, MD Adjunct Professor of Dermatology, University of North Carolina at Chapel Hill School of Medicine; Professor Emeritus of Dermatology, Columbia University College of Physicians and Surgeons

Richard K Scher, MD is a member of the following medical societies: Alpha Omega Alpha, American Academy of Dermatology, American College of Physicians, American Medical Association, Association of Military Surgeons of the US, International Society for Dermatologic Surgery, Noah Worcester Dermatological Society, Society for Investigative Dermatology

Disclosure: Nothing to disclose.


The authors and editors of Medscape Reference gratefully acknowledge the contributions of previous authors, Mark Blumberg, MD, MS, Gary R. Kantor, MD, and John Ratz, MD, MBA, to the development and writing of this article.

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Distal subungual onychomycosis. Onycholysis and yellow streak. Image courtesy of Dr Antonella Tosti.
Distal subungual onychomycosis. Subungual hyperkeratosis onycholysis and yellow streak. Image courtesy of Dr Antonella Tosti.
Proximal subungual onychomycosis. Proximal leukonychia. Image courtesy of Dr Antonella Tosti.
White superficial onychomycosis. Image courtesy of Dr Antonella Tosti.
Candidal onychomycosis in a patient with chronic mucocutaneous candidiasis. Total onychomycosis and paronychia. Image courtesy of Dr Antonella Tosti.
Dermoscopy of distal subungual onychomycosis showing irregular margin of the onycholytic area with spikes projecting into the proximal nail plate, reported as the "aurora borealis" pattern. Handyscope at 20X.
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