Pachyonychia congenital is an infrequent genodermatosis, characterized by nail dystrophy,

hyperkeratosis of the palms and soles, follicular keratosis and leukoplakia. It was seen in two
male patients aged 12 and 35 years respectively. The younger patient had nail changes,
palmo-planter keratoderma, eye changes, hypotrichosis and mental retardation, while the
elder one had minimal nail changes, keratoderma and leukoplakia.

Prior to obtaining a nail sample, the nails are cleansed by swabbing them with alcohol; this helps eliminate
bacterial growth, which may inhibit dermatophyte growth even on media containing antibiotics. The
possibility of cultures being overgrown by contaminating, environmentally derived mold spores is also
diminished with this technique. In patients with suspected DLSO, a small curette or nail clippers are used
to cut away the nail plate. A curette is then used to scrape the debris, as proximally as possible, from the
nail bed. We have found a 2 mm curette to be an invaluable instrument for this purpose. The undersurface
of the nail plate may also be scraped. For an optimal yield, the procedure may take several minutes. In
the case of superficial onychomycosis, a scalpel blade or sharp curette is used to scrape debris from the
nail surface. In contrast, in PSO, the healthy portion of the nail is pared down with a scalpel blade so that
a curette can be used to extract material from the proximal nail bed.

Once a nail sample is obtained, the common diagnostic procedures used for the identification of
pathogenic organisms include microscopic examination of KOH mounts and culture. Microscopy helps to
establish the presence or absence of fungal filaments, although it cannot identify the specific pathogen.
The traditional KOH preparation can be enhanced with the addition of the fluorescent dye calcofluor white,
which, in studies of human tissue, is highly specific for fungal elements (it does not, however, work on
dematiaceous fungi); with this technique, a fluorescent microscope is required (21). Visualization may also
be enhanced with fungal cell wall or cytoplasm stains such as chlorazol black E and Parker's blue-black ink
(22).

Fungal cultures allow for identification of the genus and species of the pathogen causing onychomycosis.
Since dermatophytes, non-dermatophyte molds, and Candida species can all cause onychomycosis, it is
necessary to culture samples on two different media. Each media should contain Sabouraud's glucose agar
and antibiotics such as chloramphenicol and gentamicin; however, one media should also contain
cycloheximide, which inhibits many non-dermatophyte molds. Antibiotics such as chloramphenicol and
gentamicin are added to the media to inhibit bacterial growth.

To ensure accurate diagnosis, both microscopic examination and culture should be carried out prior to
treatment (Table 1). If microscopic examination of the nail specimen demonstrates fungal filaments, then
the diagnosis of onychomycosis is suggested, although the identity of the causative organism could be a
dermatophyte or a non-dermatophyte. In some instances, the experienced viewer may be able to hazard
an educated guess of the causative organism of onychomycosis. This is possible mainly where fungi
invading the nail have sporulated within nail cracks and fissures. For example, in some Scopulariopsis
brevicaulis infections, the characteristic lemon-shaped conidia of this fungus can be seen in large numbers
in nail scrapings or curettings. In addition, highly experienced laboratory workers may be able to
recognize the thick, glassy cell walls characteristic of Scytalidium filaments (23).

It is important when diagnosing non-dermatophyte onychomycosis to distinguish between contaminants

and fungi genuinely growing in the nail. Classically, when a mold grown from cultured nail material is
under consideration as the etiologic agent of onychomycosis, confirming that the mold has this status has
required demonstration of morphologically compatible fungal filaments invested in the nail tissue, and
consistent outgrowth of the same mold from at least one additional specimen taken at a later time point.
In practice, it is not always possible for patients to return to the clinic for re-sampling of the nail in order
to fulfill the second criterion (24). English (24) proposed to circumvent this problem by counting the
number of nail fragments ("inocula") that grew the same mold from a single specimen plated out in
culture tests. She advocated declaring a mold etiologic if microscopic examination demonstrated
compatible filaments, if growth of the mold occurred in the absence of concomitant dermatophyte growth,
and if the mold grew out from at least five of 20 inocula plated out. Gupta et al. (25) showed that this
proposal was not soundly based. With statistical testing it was determined that, when a single nail
specimen was considered, a count of 11 or more culture-positive inocula out of 15 plated, in combination
with a positive KOH result, was associated with a greater than 70% probability that the non-dermatophyte
grown was the etiologic agent. The probability was considerably higher when the mold in question was an
Acremonium species.

For a diagnosis of Candida onychomycosis, microscopic examination of the nail specimen should
demonstrate the presence of pseudohyphae. In our experience, the most common Candida species that
cause onychomycosis are C. albicans and C. parapsilosis. It should be noted that the presence of
onycholysis and paronychia of a nail does not necessarily indicate a Candida infection. When onycholysis is

present, it is important to clip or pare the affected nail, as proximally as possible, when obtaining material
for examination. When paronychia is present, it may be difficult to obtain sufficient material for
microscopic examination.

While histological examination may also be used to obtain nail plate and bed specimens, this technique
has been infrequently used due to the potential risk of permanent nail dystrophy (26), although
permanent nail dystrophy is exceptional when biopsy is properly done. Typically, the distal-most portion of
the nail plate is clipped and submitted for histopathological evaluation using the Periodic Acid-Schiff (PAS)
stain. The results of this technique can be obtained quickly and the record is permanent (27). PAS stain
demonstrates the presence of certain polysaccharides, specifically glycogen and mucoproteins, which are
present in the walls of the fungal hyphae. A positive PAS stain is observed when the fungal hyphae appear
bright red (28). According to Lawry (29), PAS testing is 85% sensitive in diagnosing onychomycosis, and
94% sensitive when combined with Sabouraud's culture. Suarez (26) also recommends the combination of
PAS staining and Sabouraud's culture as the best method for increasing the odds of detecting fungal
infection in the nail unit.

Non-Routine Techniques

Immunohistochemistry and dual flow cytometry are other techniques used, although less frequently, to
diagnose onychomycosis (30,31). Immunohistochemistry is useful when identifying mixed infections (31).
With this procedure the nail sample is exposed to antibodies specific to certain fungi: if the fungi
correspond to the antibodies applied, they will be labeled and rendered visible by direct
immunofluorescence, immunoperoxidase, or avidin-biotin complex methods (31). Flow cytometry
differentiates fungi on the basis of molecular differences and provides information on the fungal load, as
well as on the family to which the fungus belongs (30,31). These methods, however, are not readily
available in most laboratories.

The conventional methods for identifying fungal cultures grown from nails are based on observation of
phenotypic characteristics, which can be influenced by external factors (e.g., temperature variation,
growth medium) as well as by minor strain-specific genetic differences, particularly among dermatophytes
(32). Nucleic acid-based identification techniques, when used to assay phenotype-neutral genetic markers,
are generally not susceptible to variations of this nature (32). Polymerase chain reaction (PCR) carried out

with appropriate primers can provide sensitive and specific detection of particular DNA sequences, such as
those of etiologic dermatophyte species (33). A shortcoming of the DNA-based technique is that multiple
steps may be necessary to determine the causative species. Also, where molds and yeasts are concerned,
qualitative PCR cannot distinguish contaminating fungi from etiologic agents, and the possible use of
quantitative PCR in such cases has not been investigated. Furthermore, molecular biology methods are not
in routine use in most laboratories.

A confocal microscope uses reflected light to section living tissue optically at various depths to examine
and image layers of the nail unit in vivo without the addition of stains or dyes (34). The depth that the
confocal microscope can optically penetrate is limited only by light penetration into the tissue and the
reflectivity of the structures being observed (34). Confocal microscopy may be a fast and reliable method
of diagnosing onychomycosis (35), though it has very limited ability to distinguish between dermatophyte
and mold infections.