The Diagnosis of Nail Fungus Infection Revisited

IN THE ARTICLE written by Lawry et al,¹ "Methods for Diagnosing Onychomycosis: A Comparative Study and Review of the Literature," the authors conclude that pathology periodic acid–Schiff stain (PATHPAS) is the most sensitive method for diagnosing onychomycosis. Although we disagree that PATHPAS is the most sensitive diagnostic tool, we agree with their premise that the methods for diagnosing onychomycosis clearly need to be improved. In the past decade, there have been significant advances in the development of effective and safe drugs for onychomycosis. However, diagnostic methods lag behind therapeutic advances.

Onychomycosis is very common in the United States and has probably increased in prevalence during the past several decades.^{2 - 3} Indeed, about one half of all patients presenting to dermatologists' offices for nail disorders have onychomycosis.^{2 - 3} One recent study showed the incidence of onychomycosis in a population in northeast Ohio to be 13.7% and almost 50% in those more than 70 years old.⁴ Another, multistate study noted similar prevalence rates.²

In the past, treatment outcomes were unsatisfactory because of the poor effectiveness of griseofulvin and the common practice of empiric treatment without diagnostic tests.⁵ Treatment outcomes are now better for a number of reasons, including the Food and Drug Administration's approval of itraconazole and terbinafine for the treatment of onychomycosis, an explosion of research and published articles on onychomycosis and its therapy, and physician and consumer education, including suggested treatment methods for difficult cases.^{6 - 9} Although treatment outcomes have improved, there have been only a few advances in the diagnostic methods for onychomycosis.^{10 - 15}

The diagnosis of onychomycosis can be confirmed by direct microscopy (potassium hydroxide [KOH] examination), fungal cultures, and routine histopathologic examination with periodic acid–Schiff stain (PATHPAS) of nail clippings or nail biopsy specimens. Correct collection of the specimen is critical to optimize results. The following abbreviated suggestions on collection of specimens are not meant to be all inclusive, and one may look elsewhere for more detail.^{10 - 12}

Distal Lateral Subungual Infection. A 1- to 2-mm curette, 2-mm dental spatula, or 2-mm nail elevator may be used to obtain crumbling subungual debris from under the distal edge of the nail. It is important to remember that the most viable hyphae tend to be those in the region of the most proximal involvement of the nail apparatus.^{10 - 12 ,14 - 15}

Superficial Nail Plate Involvement. Superficial thin scrapings may be obtained by a curette, a No. 15 scalpel blade, or a small nail elevator.¹²

Proximal Subungual Involvement. A nail drill or scalpel blade may be used to carefully obtain the debris.¹²

Do not use whole nail clippings for KOH examination or fungal cultures. If, for some reason, whole nail clippings are obtained, they should be ground down in a nail micronizer.¹²

Ample subungual debris should be obtained for KOH examination and fungal cultures.^{11 - 12 ,14 - 16} The most common form of onychomycosis is distal-lateral subungual onychomycosis, which is a nail bed disease and only secondarily a nail plate disease. In the article by Lawry et al,¹ the specimens were all obtained from the nail plate, which would result in false-negative results in those patients with early stages of the disease, involving only the nail bed.

Direct microscopic examinations using KOH are an easy bedside test in the hands of an experienced dermatologist. A single positive result from a KOH test, revealing septate hyphae in a patient with clinical changes consistent with onychomycosis, confirms the diagnosis. However, the identification of the pathogen can only be determined by fungal culture.

It is our suggestion that the practitioner consider having at least documentation of a positive KOH test result on the medical record for these reasons: (1) The safety of the patient is most important, and because of the extended treatment regimen required, mycological confirmation is suggested. (2) The treatment is not inexpensive. (3) In this litigious society, the reason for therapy should be satisfactorily documented.

False-negative results may occur because (1) inappropriate material or an insufficient quantity was obtained for analysis; (2) outdated or defective KOH solution was used; (3) inadequate time was spent on examining the specimen; and (4) repeated studies are not done even if the clinical impression is strong but the first KOH test result is negative.^{10 - 11 ,16}

False-negative results were likely in the study by Lawry et al¹ because the specimen collection technique used only the nail plate. Direct microscopy using a standard published technique and subungual debris is a highly sensitive test. In one study, nails with a KOH-positive result were found in 96% (1327) of 1450 nail specimens obtained from patients with clinical onychomyosis.¹⁷ In another study, of 2065 nail specimens received in a laboratory, direct microscopy showed positive results in 1707 (82.7%) and negative results in 358 (17.3%).¹⁸

At least 2 media for fungal cultures should be used for specimens obtained from nails: Mycosel or Mycobiotic (modified Sabouraud dextrose agar containing the antibacterial agent chloramphenicol and the antifungal agent cycloheximide) and standard Sabouraud dextrose agar containing an antibacterial agent. Cycloheximide suppresses the growth of certain relevant nondermatophyte pathogens as well as occasionally some dermatophytes, but it is necessary to eliminate most fungal contaminants.^{10 ,16}

It is best not to exclusively use Dermatophyte Test Media for nail fungal cultures.^{10 ,16} Although dermatophytes typically release alkaline metabolites that turn the media from yellow to red in less than 21 days, there are also a number of saprophytes that may cause the color to change, including some species of Aspergillus and Penicillin, some black molds, and yeast.¹⁶ Scopulariopsis, which is a common nondermatophyte nail pathogen, may also turn the media red. The red color may inhibit identification of the organism by obscuring the gross appearance of the thallus.

Fungal cultures are used to identify the genus and species of the pathogen, and consequently are specific tests. However, fungal cultures are not sensitive, with culture-positive rates from nail specimens averaging only about 50%. In one study, 58% (845) of 1454 nail cultures were positive.¹⁷ In another study, the yield of positive cultures of a nail pathogen (dermatophyte, yeast, or nondermatophyte mold) was 62.6% (1069) of 1707 specimens.¹⁸ Dermatophytes showed positive results in 976 (91.3%) of these isolates, molds in 85.5 (8%), and yeast in 7.5 (0.7%).¹⁸ Other studies had even lower culture recovery rates.^{17 - 18}

Cultures still provide the most specific identification of fungi. However, with the implementation of the new Clinical Laboratory Improvement Amendments regulations, it is difficult for a clinician's office staff to identify the specific fungus. Presence or absence of dermatophytes or yeast can be reported; however, a reference laboratory is usually required for specific identification.

Polymerase chain reaction, enzyme analysis,¹⁹ and in vivo confocal microscopy^{19 - 20} may be used, but these techniques are beyond the scope of routine practice and will not be discussed in the present article.

We enjoyed the article by Lawry et al.¹ Building on work by others,^{19 ,21 - 24} they have made a strong case for the use of PAS stains on nail clippings. However, we disagree that they have proven in their study that the results from direct microscopy and fungal cultures obtained from subungual debris are less sensitive than PAS stains of nail clippings in the diagnosis of onychomycosis. As mentioned above, onychomycosis is for the most part primarily a nail bed disease (except for superficial white onychomycosis, which is uncommon). Only in later stages does it significantly affect the nail plate. The nail-clipping technique discussed by Lawry et al might not obtain fungus in several common situations, including early stages of distal subungual or distal-lateral subungual onychomycosis, proximal subungual onychomycosis (uncommon), and dermatophytomas (proximal spikes). For KOH examination and fungal cultures, they used clippings from "The distal free edge of the nail, along with any attached subungual debris . . . No curettage or special attempt was made to obtain further subungual debris."¹

It is well accepted among those frequently dealing with nail specimens for KOH examination and fungal cultures that the greater the quantity of debris obtained and the more proximally it is obtained, the higher the yield for that test (see above). Indeed, in this article, the nail clippings for KOH examination were processed and then "washed," which may have further diminished any attached debris.

Lawry et al¹ mention that the specific fungal species were determined by "light microscopy and a subculture, if required." They then stated that "Fungal cultures are not highly sensitive or specific." We disagree. A reference laboratory may certainly make a highly specific diagnosis by culture.

It was mentioned that the PATHPAS "results are available relatively quickly."¹ How reproducible are these results among dermatopathology laboratories? Can a general pathologist or can most dermatopathologists easily handle PAS stains on nail clippings?

In summary, we feel that the article by Lawry et al¹ has well substantiated the use of PATHPAS for the diagnosis of onychomycosis in some patients, and PATHPAS may well be the most sensitive test on nail clippings. However, distal subungual onychomycosis is primarily a disease of the nail bed, not nail plate, the latter of which is infected in some patients as the disease progresses. The article also substantiated the poor effectiveness of direct microscopy and fungal cultures in the diagnosis of onychomycosis using nail clippings, which is to be expected since the nail plate is only secondarily involved. Subungual nail debris is the best material to use for direct microscopy and fungal cultures in the diagnosis of onychomycosis.