Diagnostic Procedures | Clinical Dermatology | AccessDermatologyDxRx

KOH examinations (Table 4-1) are a cost-effective method for the detection of superficial fungal skin infections. In the hands of an experienced clinician this test has a high level of specificity and sensitivity. However, cotton or nylon fibers from clothing and socks can mimic fungal hyphae, a mosaic artifact created by cell walls can also mimic fungal hyphae, and air bubbles can mimic spores. Some of these false positives can be reduced by the use of special stains such as Chicago Sky Blue⁴ or Chlorazol Black E. Fungal cultures are another option for detection of fungal infections. Dermatophyte Test Media (DTM), a modified Sabouraud agar contains an indicator dye that turns red within 7 to 14 days in the presence of viable dermatophytes (Figure 4-3).

Table 4-1.Potassium hydroxide (KOH) examination for superficial fungal infections.

Table 4-1. Potassium hydroxide (KOH) examination for superficial fungal infections.

Equipment needed

#15-scalpel blade (for skin and nails), small curette (for nails), tweezers or needle holder, and swab or gauze (for scalp and hair)
Microscope, glass slides, coverslip, and 20% KOH solution (plain or with dimethylsulfoxide [DMSO] or dyes such as Chlorazol Black E or Chicago Sky Blue)
If specimens are submitted for culture, a sterile urine container or Petri dish is needed for transport of the specimens to the laboratory. Modified Sabouraud agar, such as Dermatophyte Test Medium (DTM) or Mycosel^TM or Mycobiotic^TM, should be available if specimens will be directly placed onto fungal media

Techniques for specimen collection

Skin: Select an area of scale from the edge of the lesion. Clean the area with an alcohol pad. Gently scrape off the scale using a #15-scalpel blade onto a glass slide or sterile urine container. If the lesions are vesicular (eg, vesicular tinea pedis), trim off the roof of a vesicle with iris scissors and submit that as a specimen
Nails: Scrape out subungual debris using a small metal skin or ear curette or a #15-scalpel blade
Scalp: Pluck involved hairs with a needle holder or tweezers; cut the hair saving the proximal 1 to 2 cm of the hair fiber and bulb. Scalp scales can be collected for culture using a bacterial culturette swab that has been premoistened by the transport media or sterile gauze or a sterile tooth brush. The involved areas of the scalp should be vigorously rubbed with these devices

Examination of specimens

Place the skin scales or proximal hair fibers on a glass slide and cover the specimen with 2 to 4 drops of 20% KOH solution (plain or with DMSO or Chlorazol Black E or Chicago Sky Blue) to partially dissolve keratin. Apply a coverslip
Let the slide sit for 20 min
Lower the condenser on the microscope and decrease the intensity of the light
Scan the entire preparation at low power (10 ×) magnification looking for branching septate hyphae (Figure 4-1) or pseudohyphae and spores (Figure 4-2). These may be slightly refractile. Switch to high power (40 × magnification) to confirm findings
Be aware of the many causes of false-positive examinations, such as clothing fibers, hair fibers, and the cell walls of keratinocytes, which can look like hyphae. Also, air bubbles and oil droplets which can look like spores

Figure 4-1.

Potassium hydroxide (KOH) preparation. Fungal hypha with branches.

Figure 4-2.

Chicago Sky Blue and potassium hydroxide (KOH) preparation. Pseudohyphae and spores in tinea versicolor infection.

Figure 4-3.

Dermatophyte Test Media (DTM). Change in color of medium from yellow to red indicating the presence of a dermatophyte fungus.

Indications for a KOH examination or fungal culture include the presence of scaly annular plaques, areas of scale and/or alopecia on the scalp (primarily in preadolescents), vesicular lesions on the feet, and thickened nail plates.

A video demonstrating specimen collection techniques and preparation of the specimen for KOH examination can be found at www.LangeClinicalDermatology.com.

TZANCK TEST

The Tzanck test (Table 4-2) is a cost-effective method for the detection of cutaneous herpes simplex and herpes zoster infections;¹ however, there can be significant rates of false positives and false negatives. Therefore, it is being replaced in many clinics and hospitals by PCR tests that have higher rates of sensitivity and specificity.

Table 4-2.Tzanck test.

Table 4-2. Tzanck test.

Equipment needed

#15-scalpel blade and alcohol pad
Microscope, glass slide, and coverslip
Stain for specimen (Giemsa, Wright, toluidine blue, or methylene blue)

Technique for specimen collection

Select an intact vesicle(s) and swab with an alcohol pad
If there are no intact blisters, a crusted lesion or an erosion could be sampled
Remove the roof of the blister or the crust with a #15 blade
Gently scrape the base of the blister with the #15 blade
Smear a thin film of the collected contents on to a microscope slide
Let the slide air dry

Examination of specimen

Apply 2 to 4 drops of one of the above stains and leave the stain on for the amount of time recommended by the manufacturer
Rinse the slide with water and allow it to dry completely
Scan the entire specimen first at low power (10 × magnification) looking for cells (keratinocytes) with large nuclei
Switch to high power (40 × magnification) or oil immersion to confirm the presence of multinucleate giant cells (Figure 4-4) which represent infected keratinocytes

Figure 4-4.

Positive Tzanck smear. Giant, multinucleated keratinocytes in herpes simplex infection.

Indications for a Tzanck test include vesicles on an erythematous base, primarily on the central face, genitals, or in a unilateral dermatome. The Tzanck test can also be done in many other vesicuobullous disorders such as pemphigus and bullous impetigo, but the findings may be difficult to interpret.⁵

A video demonstrating specimen collection techniques and preparation of the Tzanck smear can be found at www.LangeClinicalDermatology.com.

SCABIES SCRAPING

A scabies scraping (Table 4-3) can be used to confirm the presence of the scabies mite, which is less than 0.5 mm and invisible to the naked eye. The specimens are examined for the mite (Figure 13-6), eggs, and/or fecal material (scybala) (Figure 13-7). If these are present in the preparation, they can be easily detected. False negatives can occur because there are typically few mites present in scabies infestations and the chance of finding them in any one lesion is low, with the exception of burrows (Figure 4-5) which are short, wavy lines typically seen on the wrists, finger webs, feet, and penis.⁶

Table 4-3.Scabies scraping.

Table 4-3. Scabies scraping.

Equipment needed

#15-scalpel blade, alcohol pad, and mineral oil
Microscope, glass slide, and coverslip

Technique for specimen collection

Select a burrow (Figure 4-5) or papule(s) that have not been excoriated and swab the lesion(s) with an alcohol pad
Apply a small amount of mineral oil on the scalpel blade or on the lesion(s). This step is optional
Using firm pressure scrape the burrow or papules with a #15-scalpel blade and smear the contents on a glass slide

Examination of specimens

Cover the contents with 2 to 4 drops of mineral oil and apply a coverslip. Do not use KOH as this may dissolve the mite's fecal material
Scan the entire specimen at low power (10 ×) for the presence of mites, eggs, and/or fecal material
If needed, switch to high power (40 × magnification) to confirm findings

Figure 4-5.

Scabies burrow. Thin white line above toe web.

The indications for scabies scraping include intensely pruritic papules in patients of any age on the hands, feet, extensor extremities, or genitals.

WOOD'S LIGHT EXAMINATION (BLACK LIGHT)

A Wood's light is a handheld mercury vapor bulb (ultraviolet light 360 nm) which is helpful in the diagnosis of several conditions (Table 4-4).⁷ The examination should take place in a dark room with the Wood's light held about 10 cm from the skin. Patients should not look directly into the light during the examination. A Wood's light examination is most helpful in pigment disorders and certain infections. Lesions with increased melanin in the epidermis appear darker than surrounding skin. In contrast, lesions with increased melanin confined to the dermis do not appear darker than surrounding skin. A Wood's light examination also can be used to detect porphyrins in the urine.

Table 4-4.Wood's light findings in selected skin disorders.

Table 4-4. Wood's light findings in selected skin disorders.

Disease

Color with Wood's Light Examination

Vitiligo, postinflammatory depigmentation, halo nevus, tuberous sclerosis (ash leaf macules)

Bright white to off-white

Melasma (melanin in epidermis), lentigo, freckles, café au lait spots

Darker than surrounding skin

Melasma (melanin in dermis), Mongolian spot

No change from surrounding skin

Tinea capitis due to Microsporum species

Blue-green

Tinea (pityrosporum) versicolor

Yellow-white or copper-orange

Pityrosporum folliculitis

Blue-white around hair follicles

Erythrasma (Figure 4-6)

Coral red (porphyrins produced by Corynebacterium minutissimum)

Pseudomonas

Green (pyocyanin)

Porphyrins in urine, blood, and teeth

Red-pink

Figure 4-6.

Erythrasma in medial and anterior thighs. Coral red fluorescence with Wood's light examination due to porphyrins produced by Corynebacterium minutissimum.

PATCH TESTING

Skin patch testing is used to detect allergens responsible for allergic contact dermatitis which is a type IV delayed hypersensitivity reaction. The Thin-layer Rapid Use Epicutaneous (TRUE) Test (SmartPractice, Phoenix, Arizona; Mekos Laboratories A/S, Hillerød, Denmark) is a commercially available patch test kit that is Food and Drug Administration (FDA) approved.⁸ It consists of 36 common allergens and allergen mixes in a gel coating on polyester sheeting. The tests are placed on the patient's back and taped in place and left on for 48 hours. The results are read at 48, 72, and 96 hours after application. The presence of erythema, papules, and/or vesicles indicates a positive test (Figure 4-7). However, clinical correlation is needed to confirm that positive reactions are indeed the cause of the patient's dermatitis.

Figure 4-7.

Positive reaction to balsam of Peru in a patch test. Erythema and papules present at 48 hours after application of patch tests.

DERMOSCOPY

A dermatoscope is a handheld device with a 10 × lens, light-emitting diode (LED) lighting, and polarizing filters that allow for visualization of skin structures in the epidermis and papillary dermis such as melanin and blood vessels. It is most commonly used to diagnose melanocytic lesions (eg, benign nevi, atypical nevi, and melanoma) (Figures 18-5 and 18-6), basal and squamous cell carcinomas, and benign tumors (eg, angiomas, seborrheic keratoses, and sebaceous gland hyperplasia).⁹ The first step in dermoscopy of tumors is distinguishing melanocytic tumors from nonmelanocytic tumors and the second step involves reaching a specific diagnosis using various algorithms.^10,11 The 3-point algorithm for melanocytic tumors is discussed on page 165 in Chapter 18. Studies have shown that even a 1-day training course in dermoscopy improves primary care clinicians' detection of skin cancers.¹² Training courses are regularly offered by organizations such as the International Dermoscopy Society and the American Academy of Dermatology.

Dermoscopy is also useful in the diagnosis of several other cutaneous disorders including scabies, lice, hair and scalp disorders, and several inflammatory disorders (eg, psoriasis and lichen planus).¹³ It also can be used to detect nail fold capillary abnormalities in connective tissue diseases.¹⁴

REFERENCES