Superficial fungal infection occurs when dermatophytes, yeast or other fungi invade the outermost layer of skin, known as the stratum comeum epidermidis. When this type of infection is suspected, a potassium hydroxide (KOH) examination should be performed on a sample of skin scraping. This simple, inexpensive office test can confirm the diagnosis, permitting prompt, definitive treatment, or it can provide evidence leading to the consideration of other diagnoses.
Types of Infection
Superficial fungal infections are usually caused by dermatophyte species and are classified by their location on the body. Tinea faciei usually presents as an enlarging, scaly, annular patch on the face. Tinea corporis appears as a similar patch or patches on the trunk or extremities. Tinea capitis may present as scaly patches of alopecia or as inflammatory boggy nodules or pustules on the scalp. Tinea cruris, sometimes referred to as "jock itch," may involve one or both medial thighs and may extend to the buttocks and posterior thighs or into the suprapubic area. Tinea pedis may give the appearance of dry skin on the soles, maceration between the toes, or vesicles or bullae on the soles or toes. Onychomycosis is fungal infection of the nails.
The lesions of eczema or psoriasis may appear quite similar to the scaly patches of a superficial fungal infection. Parapsoriasis, pityriasis rosea, lichen planus and eczematous drug eruptions can also be mistaken for fungal infections.
Superficial fungal infections may be caused by yeast organisms such as Candida albicans or Malassezia furfur. C. albicans can cause thrush (oral candidiasis), angular cheilitis, perliche (candidal infection at the corners of the mouth), intertrigo (infection between opposed surfaces of the skin), balanitis and vaginitis. M. furfur is responsible for tinea versicolor. This infection is usually manifested by fawn-colored or hypopigmented scaly patches on the trunk and the proximal extremities. The hypopigmented patches resemble pityriasis alba and may occasionally be mistaken for vitiligo.
The materials needed for a KOH preparation include (1) a 10 to 20 percent KOH solution, (2) Parker black or blue-black Super Quink ink, (3) a no. 15 scalpel blade, (4) a microscope slide and coverslip, (5) a methanol burner and (6) a microscope Figure 1). Sampling
When obtaining a sample from the patient, it is important to collect material that is most likely to give a positive result. If the scraping is from a ringworm lesion (a dermatophyte infection), the sample should be collected from the outer rim of the lesion. Scrapings from the center of the lesion more of ten give a false-negative result. When obtaining a specimen from an apparent lesion of tinea versicolor, the scraping should be taken from the scaling patches or scaly margins of larger patches that are characteristic of this infection. If a superficial candidal infection is likely, the moist, macerated, cheesy material on the skin or mucosal surface provides the best sample.
When obtaining nail samples, it is best to look for white areas, since these are the most likely sites of active infection. I If no white areas can be detected, sampling of the subungual debris at the free end of the nail is preferred.
With candidal infection of the skin around the nails (paronychia), pus that contains yeast forms may be obtained by compressing the swollen, erythematous paronychial tissue or by incising the area with a no.II scalpel blade. This material should be placed on a microscope slide and covered with a coverslip.
If tinea capitis is suspected, a sample of the base of the patient's hair and/or scales from the affected scalp should be obtained. Wood's lamp examination is of little value since Trichophyton tonsurans, the organism that most commonly causes tinea capitis in children, does not produce fluorescence. (2) Scalp infections caused by T. tonsurans are also called black-dot ringworm. The short hairs that give the blackdot appearance should be sampled and examined for endothrix spores. Because of inflammation surrounding the hair follicle, the hair can usually be scraped with a scalpel blade or easily removed with forceps or tweezers. The scrapings and/or hair should be placed on a microscope slide and protected with a coverslip.(3)
Two important points should be kept in mind when collecting samples for KOH preparation. First, a large amount of scale should be collected to ensure a thorough examination and increase the chance of demonstrating the organism if it is present.(4) Second, a coverslip should be placed on the microscope slide immediately after collection so that the specimen does not blow away as the slide is carried from the examining room. Technique
After an appropriate area has been chosen, a no. 15 scalpel blade is used to scrape scales from the lesion onto a microscope slide. The slide is held perpendicular to the skin below the area being scraped.(5) When scraping the scales for a sample, the scalpel blade should be held with its sharp edge trailing behind so that the skin is not lacerated Figure 2). When an adequate amount of material has been obtained, a coverslip is immediately placed on the slide.
Before the specimen is viewed, a clearing agent must be added to digest the keratin material. KOH is the most appropriate clearing agent.(6-8) If KOH is used alone, it may be difficult to differentiate the cell walls of keratinocytes from the fungus. Because of this difficulty, several stains may be used to define hyphae and spores.(9-13)
Cohen(14) was the first to publish a report on the use of pen ink as a stain in KOH preparations. Modifications of Cohen's originally described stain have been reported over the past three decades. The stain we have found to be the easiest to prepare and use is a 9: 1 mixture of 10 percent KOH and either Parker blue-black Super Quink ink or Parker permanent black Super Quink ink.(11) Only these two inks can be used, because of their high content of Ink Blue PP, a dye developed by the Parker Pen Company specifically for its stability in alkaline solutions.
The KOH/ink stain can be mixed at the physician's convenience. Although the solution reportedly has a shelf life of only six months, we have found that it remains stable for years.(14) The solution must be stored in a plastic container, since glass promotes crystallization of KOH. Commercially prepared solutions of KOH with dye to stain hyphae are also available.
The best way to stain a specimen is to draw up the premixed KOH/ink stain in an eyedropper and then place one to three drops of the solution at the edge of the slide coverslip. The KOH/ink stain will be drawn between the slide and the coverslip by capillary action Figure 3). Using a methanol burner, the slide is then gently heated until it is just uncomfortably warm to the dorsum of the hand Figure 4).
Heating speeds up the digestion of keratin material and allows the specimen to be viewed immediately; without heating, the slide may not clear for several hours. It is important to avoid boiling the preparation, since this may cause crystallization of KOH and produce artifact. In KOH preparations of nails or hair, the slide may have to be warmed for a longer period to fully digest keratinous material. Again, care must be taken not to boil the preparation.
In the next step, the slide is blotted to remove excess stain and air from under the coverslip. This is most easily accomplished by placing the slide on a paper towell folding the towel over the slide and applying gentle pressure. (In staining for tinea capitis, it is important not to press too firmly because spores may easily be expressed from the hairs.) The slide is then ready for examination. Table 1 reviews the steps in slide preparation. Microscopic Features
When a KOH preparation is viewed under the microscope, the condenser should be moved as far down as possible and the diaphragm adjusted to a nearly closed position. Decreased light offers better contrast between keratinocytes and the fungal spores and hyphae.
The characteristic microscopic features of a dermatophyte infection are long, branching mycelia with septae or crosswalls. The lesions of candidiasis show pseudohyphae (nonseptate hyphae) and round or oval yeast bodies. Scrapings of tinea versicolor may demonstrate clusters of hyphae and spores that look like spaghetti and meatballs or macaroni and meatballs (Figure 5). Specimens from tinea capitis infections may show either an endothrix pattern, in which spores are seen within the hair shaft, or an ectothrix pattern, in which spores are also seen on the external surface of the hair shaft.
False-negative results with the KOH examination are common for a number of reasons, including prior treatment with topical or systemic antifungal medication, inadequate scrapings and scrapings from a low-yield area of the lesion. Obtaining fungal structures from the dry moccasin scaling of some tinea pedis infections appears to be especially difficult. Therefore, if the clinical picture is strongly suggestive of a fungal infection in the face of a negative KOH preparation, the KOH test may be repeated, a fungal culture may be obtained or treatment may be empirically based on clinical suspicion. Of course, false-negative fungal cultures can also occur.
False-positive results rarely occur and are due to observational errors. These errors occur when intercellular spaces or artifacts such as dirt, clothing fibers or KOH crystals caused by overheating are mistaken for hyphae or spores. The false-positive rate is quickly reduced with experience in performing the KOH examination.
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