Laboratory diagnostics of mycoses.

The most important confirmatory evidence of clinical diagnosis of dermatomycosis can be obtained by scraping the lesion and by the microscopic demonstration of fungus. The procedure is very easy to perform, and does not take much time; preferably, it should be done by the physician himself.

Collection of Material

1. Skin scrapings are obtained by scraping the growing edge of the suspected lesion where it merges with the normal skin, with a scalpel, the blade of which should neither be too sharp nor too blunt. It is advisable to wet the scalpel blade with a swab of 70 per cent alcohol when the scraping is being done. It is advisable to collect the macerated separating epidermis on the periphery of the lesions for examination in dyshidrotic and intertriginous epidermophytosis of the large folds. If there are vesicles and bullae, their tops are cut off with a pair of sterile scissors and examined. In the squamous form, scales are scraped off the lesions and examined.

The edge of the scalpel blade is gently agitated in a drop of 20 per cent potassium hydroxide solution taken on a slide to release the scales adherent to it. It is advisable to prepare two coverslip preparations from each lesion, and if there are a number of lesions, it is better if specimens from two or more lesions are obtained. If vesicles are present, the domes should be snipped off with a pair of scissors, and the contents examined in a potassium hydroxide preparation.

2. Hair. Areas showing thinning or scaling of hair with broken-off stumps should be selected. The stumps of the broken-off hair should be epilated and a coverslip preparation of the material placed in a drop of 20 per cent potassium hydroxide should be microscopically examined.

3. Nails. All nails which are discoloured or lustreless or ridged with hyperkeratotic debris should be fully examined. The horny material is scraped off the nail plates with a scalpel, or cut off with a pair of scissors along the free edge of the nail plate, or the nails are cut away, and the debris from under the nail is removed. The pathological material is soaked in 20 per cent caustic alkali solution (KOH or NaOH) and examined with a 'dry system' microscope under high magnification.

4. Pus from granulomata like actinomycosis and sporotrichosis is collected aseptically and examined directly, or a smear is made and stained with Giemsa's or Gram's stain, and then examined under the microscope.

Microscopic Examination

The preparation is gently warmed, or allowed to stand for 20 minutes to half an hour. This helps to soften the keratinous tissue and allows the fungus to stand out more clearly. The preparation is first examined under the low power and then the high power dry objective of the microscope.

The components of the fungus are seen as double-contour threads of mycelium of various size and round or square spores (arthrospores). In the skin, the fungus appears as a set of parallel septate lines, which show branching at places. In some areas, the fungus may be seen segmented into a number of separate cells all in the same line. Absence of mycelia does not rule out tinea. Microscopy makes it possible to distinguish epidermophytosis from candidiasis, which is characterized by the presence of budding yeast cells in the preparation. However, the microscopic picture of the threads of the fungal mycelium in epidermophytosis, rubromycosis, and trichophytosis is similar and they are differentiated by cultural diagnosis (growth of cultures on nutrient media) in special bacteriological laboratories.

It must be clearly understood that this picture is common to all the dermatophytes infecting the skin, and the identification of their species is impossible from a coverslip preparation. Only by culturing can the different species be identified. In the hair, the fungus appears in the form of rounded spores which may be small or large in size. Furthermore, the spores may be present around the shaft of the hair. When situated thus, they are known as ectothrix spores; when present inside the hair shaft, they are called endothrix spores.

Culture. Sabouraud's medium is the one very commonly used. For culture work, the aim is to reduce bacterial contamination to the minimum.

After the affected area has been cleansed with 70 per cent alcohol, scraping is done with the usual technique; the material so obtained is removed from the scalpel with the point of a straight needle and inoculated on the surface of the medium to be used. At least two petri dishes and two slopes of the medium prepared in test tubes or screw- capped bottles are used for each case, and about 4 inoculations of the material on each medium is made. The culture is kept at room temperature or best at 26°C and examined every alternate day. No culture should be discarded as sterile unless kept for at least three weeks.

Another difficulty to overcome is to know whether a growth which is taking place is saprophytic or pathogenic. A working rule in this connection is: any growth which takes place within three days and grows rapidly is likely to be saprophytic. The growth, however, should be investigated before being so labelled. With experience in the identification of saprophytes, however, this difficulty could, in due course be overcome.

Staining. The two reagents commonly employed are: lactophenol blue for the staining of fungus smears routinely and Periodic Acid Schiff's stain (PAS) for the staining of fungus in tissues.

WOOD'S LAMP EXAMINATION

A Wood's lamp is a mercury vapour lamp with a special Wood's filter made of nickel oxide and barium silicate. It allows only certain wavelengths of ultraviolet rays (365 nm) to filter through.

In Wood's lamp light, microsporon infection of the hair is visible as a greenish fluorescence. The scalp must be thoroughly washed of all medicaments before it is examined under the Wood's lamp. It helps to pick up early cases of tinea capitis amongst contacts, especially school children, and aids in following up a treated case and declaring it cured.

The colour of the fluorescent light, seen best in a darkened room, is characteristic in the following conditions:

 

Condition	Fluorescent colours
Tinea capitis	Bright yellow green
Tinea versicolor	Golden yellow

Materials for self-checking:

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