Dear Colleagues,

HOW IS FUNGUS SEARCHED IN THE FOOT?

Most of the search for fungi takes place by means of scrapings of volar horn treated with KOH for softening and clearing, followed by microscopic observation of coverslipped slides, either unstained or stained with ink or similar dyes.

Occurring several times a day in a dermatologic office, besides time-consuming, KOH preparation could also interrupt the flow of the examination of the now busy medical practices of modern times.

IS THERE ANOTHER WAY TO DO IT?

A better way of fungus search is the use of the VOLAR CORNEAL BIOPSY (VCB), i.e., the use of the cornified scrapings or clippings from the foot (or hand) for histologic examination, instead of a KOH preparation.

In my area of the United States (Metropolitan Boston), more clinicians, particularly podiatrists, are often using this method that has arisen parallel to the use of the nail plate biopsy, already described in these letters.

There is an advantage to this referral that does not come readily to the front of the argument and it is simply the convenience of delegation of the duty into the pathologist in search for greater accuracy in the diagnosis of tinea pedis using the more stable and better stained sections prepared histologically from the harvested horn.

WHY YOU SHOULD TRY IT?

Granting the advantage that immediate result for the KOH in the office speeds the therapy for positive cases, the risk of false negatives increases as well. The benefit of the greater sensitivity of the VCB surpasses any perceived disadvantage.

Although KOH is a time-honored technique, it pays to concentrate in newer techniques in other areas of the dermatologic practice that may be more beneficial to both patients and doctors than practicing the traditional routine of a KOH exam. As in onychomycosis, the results of PAS-stained tissue sections are better than direct examination.

HOW IS IT DONE?

Simply, in fact: after the scraping is done, formalin fixation is unnecessary. Bagging the fresh specimen and sending it to the laboratory, even mailing it, suffices.

With relatively clean sample-taking, not even in aseptic conditions, a belated fungal culture from fresh portions of the specimen could be performed several days after the specimen has been received by the laboratory. This step can be done in nails that at first are PAS-negative to see if the culture achieves a “rescue” and a diagnosis of tinea pedis can still be made, in spite of cultures being less sensitive than PAS stain to detect fungi, at least in nail clippings.

NOW LET’S TALK ABOUT RESULTS…

The soundness of initial air-dry fixation to keep intact the histologic features of horns and even epithelia is assured. There is practically no decay in the histology of squamous epithelia, from epidermis to matrical or lectural epithelium.

Formalin fixation of the samples after arrival to the lab is more than sufficient to insure adequate processing. This “nonviable” tissue ‑the horn from a foot‑ is so full of information when it is processed as a biopsy that one wonders that the volar corneal biopsy has not been used more often or in earlier medical practice. Besides containing no epidermis or only a little (according to the vigor applied to the curettage or scraping), the histologic sections are in equal terms to those of deeper viable tissues derived from the punch or shave technique.

The tissue is embedded is sectioned and stained with both hematoxylin and eosin and, more importantly, PAS stain (one without diastase digestion is sufficient). A Gomori Methenamine Silver (or Grocott stain) can be complementary but it is generally unnecessary.

Fig. 1, Volar Horn, Tinea Pedis, GMS Stain

The morphology of the fungi will allow, like in nail plate biopsies, the suggestion of class, genus and even species based on the morphologic characteristics of the isolate.

WHAT OTHER MICROBES CAN BE PICKED UP?

Fig. 2, Subungual Horn, Filamentous Corynebacteria, PAS Stain

Not only fungi can be identified. It is not uncommon that Corynebacterium minutissimum, the agent of the underestimated pitted keratolysis of the soles, can be detected. The defining thin granules and filaments making the diphtheroid will stand up in the PAS stain. There is no need to perform a Brown-Brenn (Gram) stain but one could follow for confirmation of the Gram-positive character of the bacterium.

WHAT OTHER DISEASES CAN BE DIAGNOSED?

Fig. 3, Volar Horn, Dyshidrotic Dermatitis, PAS Stain

Even after all stains are negative for organisms, there is the added certainty that a pseudomycotic horn may be that of dyshidrotic dermatitis or, even, psoriasis.

Fig. 4, Volar Horn, Probable Psoriasis, PAS Stain

This is an additional bonus to the examination:

the negative horn of eczematous dermatitis could be as useful to management of a patient as is the assertion of the presence of tinea pedis.

TAKE-HOME MESSAGE

The transformation of a KOH examination into a minibiopsy of corneal elements of the skin is another nonexfoliative cytologic procedure that is processed as if it was an invasive biopsy of the skin. Cytology and histology meet at the foot!

This underutilized volar corneal biopsy may increase the range of the diagnosis of tinea pedis and may ultimately help in the prevention of tinea unguium associated with and probably following a longstanding case of tinea pedis.

FROM A DERMATOPATHOLOGIST TO YOU!

Finally there goes a desideratum. The above arguments go to stimulate the use of a dermatopathologist in the resolution of some aspects of the dermatologic practice other than the traditional tissue biopsy. Strengthening the binomial partnership of dermatologists and pathologists may be very rewarding to the success of each other’s practice.

Cordiali saluti,

Aldo González-Serva, MD

IS THERE A SAVING GRACE TO SHALLOW SHAVE BIOPSIES?
Fortunately, the horn of diverse conditions will frequently reflect the nature of the epidermis or other deeper foot structures that have induced the thickening of the cornified layer. Thus, even in the absence of viable tissues, the pathologist may be able to surmise the diagnosis on the basis of the horn. While this is an indirect approach that is not fool-proof, it will be enough on most occasions to help the clinician decide what to do next.

WHICH IS THE FIRST TELLTALE SIGN THAT SAVE THE SHALLOW BIOPSY?
At the least, the milieu that surrounds the volar horn without viable tissues will tell if a malignancy lurks nearby. In clinically benign conditions, a good “horn milieu” will reinforce the diagnosis of benignancy and may even temper the need for a repeat biopsy, at least for a prudential lapse. A good milieu is horn without evidence of necrotic debris or blood cells and showing only limited parakeratosis and relative homogeneity of its structure. It will be seen in most warts, corns or other benign keratodermas. A bad milieu, in contrast, will show horn that is heterogeneous, degenerated, massively parakeratotic, probably impetiginized and often associated with necrotic debris. All these signs reflect serious abnormalities of the underlying tissues, even if they do not appear under the microscope.

HOW IS THE HORN OF BENIGN LESIONS?
The most common diagnostic horn will be that of the plantar verruca, whose cornified layer will be thickened. This trait is more easily discerned if normal horn is also present. The abnormal warty horn will be orthokeratotic (devoid of nuclear residues) or, most commonly, parakeratotic (bearing residues of the nuclei of generating keratinocytes). The parakeratosis will be uniform, practically symmetric, without dramatic changes in its composition and lacking the accoutrements of a bad milieu.

The callus (hypertrophic lichen simplex chronicus), being a response to persistent blunt rubbing, will display a thickened horn that is almost without exception orthokeratotic. It will be basophilic, as is normal in volar skin biopsies stained with hematoxylin and eosin stain.

The heloma durum (corn) will contain a broad column of parakeratosis as if it were a cylindrical clavus buried among more normal horn or stuck in horn resembling that of a callus.

WHAT HAPPEN TO THE HORN ABOVE MALIGNANT CONDITIONS?
In malignant conditions, two important entities dramatically modify the horn. One is verrucous carcinoma (epithelioma cuniculatum); the other is malignant melanoma.

Verrucous carcinoma is generally exophytic and hyperkeratotic. That excessive horn is markedly abnormal. Besides being irregularly parakeratotic, the nuclear ghostly contours are dissimilar, often laid in the bowl-shaped streams that reflect the recesses and sinuses that characterize verrucous carcinomas.

Melanoma, although not generally a hyperkeratotic disorder, is one with marked abnormalities of the pigmentary system. This hyperpigmentation (absent, though, in amelanotic melanoma) will bleach into the horn. Coarse dark-brown melanin granules will permeate the corneocytes irregularly and asymmetrically, mirroring the abnormal growth of the melanoma in the undisclosed epidermis.

A SUMMATION
Not everything is lost when an acral shave biopsy misses the deeper tissues. The superficial horn will often tell the tale! Everyone should keep in mind the importance of using this ‘shallow’ information in most benign cases or in inducing a new biopsy in suspicious cases in the near future.

©Aldo González-Serva, MD
Dermatopathologist
Boston, MA, USA

©Aldo González-Serva, MD
Boston, MA, USA
September 8, 2009

PAS IS NOT ALWAYS A DIAGNOSTIC PANACEA FOR DYSTROPHIC NAILS

The increasing use of the nail plate biopsy (NPB) stained with PAS in search of fungi has dramatically highlighted that many dystrophic nail plates thought as diagnostic of onychomycosis are fungus-free.

This brings chagrin to both the clinician and the pathologist. To the clinician, the concern is if a false negative is at play and, if not, what to do with this case of pseudo-onychomycosis. To the pathologist, the quandary is whether, in spite of careful screening fungi, the search for fungi has failed. After frantic minutes of examining the fruitless sections, further teeth-grinding ensues: “Has my skill to pick up a fungal needle from a keratinous haystack waned?” Was the sample not representative? What are the clinicians expecting tinea going to think of their pathologist for giving them so many “negatives”?

NEGATIVE PAS MAY BE REALLY NEGATIVE AFTER ALL

It is now obvious that many of the NPBs deemed negative by pathologists continue to be negative when a subsequent fungal culture from the same specimen is requested. This means that there is something such as pseudo-onychomycosis, indeed, unless some cases of tinea are so subtle to require rare molecular techniques for detecting the fungal genome.

Besides a modicum of genuine false-negatives that probably exists, it is evident that the nail field produces a limited range of clinical and histologic responses to various stimuli and conditions.

ENTITIES THAT MIMIC ONYCHOMYCOSIS

Well known are the similarities of psoriasis and onychomycosis, both producing a psoriasiform onychitis with intracorneal subungual microabscesses atop mounds of parakeratosis. In onychomycosis, however, fungal hyphae will be some microns away from the microabscess.

Tinea unguium will also be accompanied in some cases of an abundant spongiotic fluid, resembling serum that gets collected, sometimes in gigantic pools, within the abnormal subungual horn.

But not all spongiosis is fungal-induced. Current dermatology treatises speak of spongiotic disease of the nails without carving a specific niche for the term ‘eczematous onychitis’. It makes sense that the foot, so rich on expressions of atopy, is going to involve the nail field. Contact dermatitis, dishydrotic dermatitis and stasis dermatitis, to name the main ones, are common occurrences in the feet. Why not accepting that the same diseases could be expressed preferentially yet less distinctly in the nails than on the sole?

Therefore, psoriasiform onychitis on the biopsy, minus the presence of fungi on the PAS (repeated) or in nail cultures (following a negative NPB) should be considered an entity related to contact, atopic (dishydrotic) or stasis dermatitis, i.e., eczematous onychitis. If id reactions to tinea pedis are found in distant body regions (trunk or upper extremities), is not conceivable that an id reaction may occur in the nail field?

COROLLARY

The time to expand our range of pseudomycotic nail conditions is here. The first step is being confident that a thoroughly examined yet negative NPB stained with PAS is the basic tool to rule out onychomycosis. After that it is the opening of our minds that less understood signs of other diseases can be a source of crumbly, dystrophic and pseudomycotic nails.

THE KOH METHOD FOR DETECTION OF NAIL FUNGI HAS BEEN BESTED: KNOW BY WHAT! POTASSIUM HYDROXIDE PREPS TODAY: IS IT WHAT YOU NEED?

The most widespread method to quickly detect fungal infection has traditionally been the use of KOH to soften and clear skin or nail scrapings, followed by direct or dye-mediated microscopic search for fungi.

When unstained, playing with the condenser and the iris of the microscope will bring forth from the coverslipped preparation the refractile hyphae or other fungal mycelia.

When a stain is used, a common one being ink, it will highlight the fungal wall and will make easier the detection of the fungi and the certainty that artifacts are not misinterpreted as hyphae or spores.

In the case of tinea corporis or pedis, the fungal elements may be relatively frequent or at least regularly distributed throughout the horny sample. In cornified material from the nail, in contrast, the fungal load may be low and the fungal elements could be irregularly distributed among the nail keratins, some of which may not have been procured by the clippings.

In addition, the practice of the KOH by the clinician will require some interruption of the flow of patients in the office for preparation and interpretation of the sample.

A NAIL PLATE BIOPSY STAINED WITH PAS IS BETTER

In consideration to the caveats of KOH examination, the alternative has arrived: the nail plate biopsy (NPB) processed with PAS stain. This progressive trend of substitution of a tedious KOH exam for a referral of the specimens to a histology lab and the reading by a colleague dermatopathologist is here to stay.

ADVANTAGES OF THE NAIL PLATE BIOPSY

Besides producing a permanent record of the nail search, a NPB will afford the examination of the specimen by one or many observers at times that do not hampered the flow of patients through the clinic.

In addition, more tissue is available to examination, as heaps and clumps of nail keratins are eliminated by thin sections on which no confusion is allowed.

Furthermore, the PAS will offer greater distinctiveness and contrast to the fungi than the tenuous demonstration gained by direct observation or the use of inks in a KOH preparation.

An additional advantage of the NPB is the possibility of performing, if needed in apparently negative PAS-stained nails, a high-contrast stain such as Gomori Methenamine Silver (GMS) on which rare and previously unperceived hyphae can be picked up more readily.

THE REAL CLINCHER FOR NAIL PLATE BIOPSIES WITH PAS STAIN

Besides the above advantages, the most important reason to use a NPB stained with PAS is the high rate of return of positive cases in nails suspected to harbor fungi. It is clear now that many other conditions of the nail look clinically like onychomycosis yet biopsies and cultures are negative.

Surprisingly high is the yield of NPB with PAS for fungi, clearly now in reported percentages that are greater than even fungal cultures. This counterintuitive failure of cultures may depend on degeneration of hyphae, which would become unable to grow in vitro while still keeping their tinctorial avidity for PAS stain. Among recent statistics informed in the literature is 33-92% positivity for PAS, rounding up on 48% success in identifying fungi vs. 25.83-59% % for fungal culture and even a higher sensitivity than KOH examination (25-82.5%). Our personal series of 2007, including 2651 nail plate biopsies stained with PAS, had the highest sensitivity reported in a large series of 65.54%! (92% positivity rate was in a series of 105 cases only…). No doubt that the most cost-benefit ratio is obtained from the nail clipping sent to a pathologist for PAS staining.

WHAT IMPROVEMENTS CAN BE EXPECTED ON NAIL PLATE BIOPSIES

A current limitation for NPBs is the lack of certainty about the speciation of the observed fungi. However, when a septate regular hyphae is seen, it is almost certain that we are dealing with a dermatophyte and so can be reported to the clinician. Yeasts are also easy to discriminate but molds are somewhat less certain to be unequivocally identified. At least, they can be suggested when irregular or globular mycelia are noted in the PAS-stained biopsy.

COROLLARY

A small revolution in the care of nail diseases has been brought by getting pathologists as your clinical partners. If you want fast results and expeditious therapy for mycotic nails, no doubt that the way is clipping away…

©Aldo González-Serva, MD

Dermatopathologist

Boston, MA, USA