Chapter 3 Diagnostic techniques
1. What is the most sensitive office laboratory test for diagnosing dermatophyte infections of the skin?
Microscopic examination of a potassium hydroxide (KOH) preparation of scrapings taken from the affected area is the most sensitive office laboratory test. A study of 220 specimens examined by both KOH and culture demonstrated positive KOH preparations and positive cultures in 45% of samples, a positive KOH preparation and negative culture in 52% of samples, and a negative KOH preparation and a positive culture in only 3% of samples. However, cultures can be useful because other studies have shown a 5% to 15% increase in positive specimens by culturing KOH-negative materials. Moreover, it is important to realize that the diagnostic accuracy of the KOH preparation depends on the skill of the individual performing the test.
2. How is a KOH examination performed?
The highest rate of recovery of organisms occurs in specimens taken from the tops of vesicles, the leading edges of annular lesions, or deep scrapings from the nails suspected to be infected with fungi. The site should be swabbed with an alcohol pad or water and scraped with a no. 15 blade. In some instances, such as the scalp or nail, a curette may be more effective. The moist corneocytes are then easily transferred from the blade to a glass slide. One or two drops of KOH (10% to 20%) are added, and a coverslip is applied to the specimen. The KOH preparation is gently warmed, but not boiled, and then examined under the microscope. It is important to focus back and forth through the material so that the refractile hyphae can be visualized. Fungal hyphae can be recognized by their regular cylindrical shapes with branching and the presence of septae that may demonstrate a subtle greenish hue (Fig. 3-1). A pencil eraser or pen cap gently applied to the surface of the coverslip may enhance keratinocyte breakdown, especially for clumped specimens. If no organisms are observed initially, waiting 10 to 15 minutes may aid visualization.
3. What laboratory tests are useful for diagnosing tinea capitis?
Testing for fluorescence in the affected area using a Wood’s light is the quickest technique. If the hair fluoresces yellow-green, then a fungal infection is likely. However, lack of fluorescence does not exclude tinea capitis, because Trichophyton tonsurans accounts for 80% to 95% of scalp ringworm infections in the United States, and it does not fluoresce. Therefore, examination of KOH-treated infected hair is more sensitive and can also be rapidly performed. The best results are obtained when broken-off hairs are examined, because these are the ones infected by hyphae and arthrospores. Most dermatophytes, such as T. tonsurans, grow within the hair shaft (endothrix), and a few minutes are required to let KOH break down the hair shaft and visualize the infection. Finally, the diagnosis can also be proved by fungal cultures. The easily broken, infected hairs are embedded in the media. The specimen can be obtained using a no. 15 blade, curette, or hemostat.
4. What is a Wood’s light or lamp? How is it useful in skin diseases?
A Wood’s light produces invisible long-wave ultraviolet radiation, or “black light,” at a wavelength of 360 nm. When this light strikes the surface of the skin or urine, fluorescence is produced in some disorders. This fluorescence is best observed in a completely dark room. The Wood’s lamp is useful in diagnosing cases of several skin conditions: tinea capitis (see preceding text), tinea versicolor (dull yellow fluorescence), erythrasma (coral red fluorescence; Fig. 3-2), and Pseudomonas infections of the skin (green fluorescence). It is also useful as a screening test in porphyria cutanea tarda as the urine fluoresces a coral red color (Fig. 3-3). The Wood’s light may also be used in certain disorders of pigmentation. In patients with hyperpigmentation, it is used to localize the site of the pigment because it accentuates superficial epidermal pigment, whereas deeper dermal pigment is unchanged. It is also used in patients with vitiligo because it demonstrates complete depigmentation. Finally, it can be used to delineate the borders of melanocytic lesions, such as lentigo maligna, prior to surgery.
5. Name common culture media used for isolating dermatophytes.
Dermatophyte test media (DTM) and Sabouraud’s dextrose agar, with or without antibiotics (e.g., Mycosel agar, Mycobiotic agar), are the two most common types of culture media used. Many dermatologists prefer DTM because it has the advantage of a color indicator that changes the media from yellow to red when a dermatophyte is present. DTM is 95% to 97% accurate in differentiating dermatophytes from nondermatophytes. Sabouraud’s dextrose agar is a standard in mycology laboratories and also in many dermatologists’ offices. It consists of dextrose (energy source), peptone (protein source), and agar (for a firm surface). Antibiotics can be added to suppress bacterial contaminants, and cycloheximide is added to suppress yeasts and nondermatophytes. Plain Sabouraud’s agar is an especially good culture medium for Candida albicans.
Head E: Laboratory diagnosis of the superficial fungal infections, Dermatol Clin 2:93–108, 1984.
6. Describe a simple test for tinea versicolor other than a KOH preparation.
Clear cellophane tape preparations are an excellent diagnostic testing material because the organism is found in the upper stratum corneum. First, the skin is scraped to ensure there is adequate scale. The tape is applied over the scale and then mounted on a glass slide and examined under the microscope. Clusters of short hyphae and yeasts are seen producing a “spaghetti and meatballs” pattern. Methylene blue may also be added to the slide, selectively staining the organism and thus enhancing visualization (Fig. 3-4). It is important to note that Malassezia globosa and M. furfur, the most common etiologic agents of tinea versicolor, cannot be cultured on any of the routine fungal media kept in most laboratories.
7. What is a Tzanck preparation or smear?
A Tzanck smear is a standard technique for the rapid diagnosis of herpes simplex virus (HSV) or varicella-zoster virus (VZV) infections. It cannot distinguish between these two agents, nor can it distinguish between HSV subtypes (HSV type 1 or 2). It is performed by scraping the base of a fresh blister with a scalpel blade and then spreading the adhering cells and material onto a glass slide. The slide is then stained with a Giemsa, Wright, or Sedi stain. The typical multinucleated giant cells or atypical keratinocytes with large nuclei are then easily visualized (Fig. 3-5).
8. What is the best method of diagnosing scabies?
The best method is to scrape a burrow and demonstrate the parasite inside it. A classic burrow appears as an irregular, linear, slightly elevated lesion, best found on the flexor wrists, fingerwebs, and genitalia. Eighty-five percent of adult male patients with scabies will have mites on the hands or wrists. Occasionally, the mite can be seen with the naked eye as a small dot at one end of the burrow. Following the application of mineral oil, on either the blade or skin, the burrow is scraped vigorously with a no. 15 scalpel blade, but not so vigorously as to draw blood. The mineral oil is collected from the skin and blade and transferred to a glass slide, which is then examined under the microscope. The diagnosis is established by identifying either the fecal pellets (scybala), eggs, or the mite itself (Fig. 3-6).
Figure 3-6. A positive scraping for scabies showing an immature mite, eggs, and numerous fecal pellets.
(Courtesy of James E. Fitzpatrick, MD.)
Hay RJ: Scabies and pyodermas—diagnosis and treatment, Dermatol Ther 22:466–464, 2009.
9. How do you diagnose mite bites acquired from an animal?
Clinically, patients present with pruritic red bumps, most commonly on the arms, breasts, and abdomen. The most common sources of these animal mites are cats infested with Cheyletiella species. This nonburrowing mite exhibits “bite and run” tactics, so it is not likely to be found on the patient’s body. The key to making the diagnosis is to have the animal examined by a veterinarian who is familiar with these parasites. The diagnosis is established by a cellophane tape preparation taken from the cat, dog, or rabbit, demonstrating either the six-legged larval form or the eight-legged adult. Unlike the scabies mite, Cheyletiella has well-developed, clawlike mouth parts.
10. How do you diagnose lice infestation?
Lice infestation is caused by Pediculus humanus capitis (head louse), P. humanus corporis (body louse), or Phthirus pubis (pubic louse). Lice can be identified by eye or by using a hand lens, but they can be very difficult to locate. If body lice infestation is suspected, examination of the seams of clothing is more likely to be diagnostic than is examining the skin. Lice will appear as brownish-gray specks. Head and pubic lice are more often seen in hairy areas and are often found with their mouth parts embedded in the skin with outstretched claws grasping hairs on either side. Usually more numerous are nits (eggs), which are white-gray oval structures smaller than 1 mm in size and are firmly attached to the hair shaft. Eggs that are near the junction of the hair shaft and the skin are indicative of active or recent infection. Since hair casts are white and may resemble nits, it is sometimes necessary to examine possible nits under the microscope to prove their identity (Fig. 3-7).
11. What is the diagnostic test of choice for a patient presenting with a suspected syphilitic chancre on his penis?
Dark-field examination of the chancre is the most specific test for the diagnosis of syphilis. This test is typically positive unless the patient has applied or ingested antibiotics. In addition to primary syphilis, dark-field microscopy can also be used to diagnose all of the mucocutaneous lesions of secondary syphilis. However, it is less reliable for examining specimens from the mouth or rectum because of the high prevalence of commensal, nonpathogenic treponemes in these locations that may be mistaken for Treponema pallidum, the agent of syphilis. The best specimens for dark-field examination are serous fluid expressed from the bases of the chancre following cleaning with sterile saline and clean gauze. The specimen then should be immediately evaluated for the organism’s characteristic corkscrew morphology and flexing, hairpin motility. If a patient is suspected of having a syphilitic chancre and a dark-field evaluation is negative, it should be repeated at least once before the diagnosis is excluded. Fluorescent antibody microscopy, although not widely available, offers a sensitive alternative and has the advantage that it does not require live organisms and it can be done on fixed slides. This technique utilizes antibodies to T. pallidum.
12. How is secondary syphilis diagnosed?
As with primary syphilis, the most specific test is dark-field microscopy. Special stains on skin biopsies are diagnostic as well. More often, screening tests for syphilis such as nontreponemal serologic tests are utilized, usually a rapid plasma reagin (RPR) or a Venereal Disease Research Laboratory (VDRL) test is ordered. Nontreponemal serologic tests for syphilis detect antibodies to reagin, a cholesterol-lecithin-cardiolipin antigen that cross-reacts with antibodies present in the sera of patients with syphilis. These antibodies are not specific for syphilis and should always be confirmed by a specific test for syphilis, usually a fluorescent treponemal antibody–absorption (FTA-ABS) or the microhemagglutination–T. pallidum (MHA-TP) test. Nontreponemal serologic tests for syphilis are positive in almost all cases of secondary syphilis. Adequate treatment causes the titer to decline to low titers or nonreactivity.
13. How long do serologic tests for syphilis remain positive?
Nontreponemal test antibody titers (e.g., VDRL) are reported quantitatively in the form of the highest positive titer. This titer correlates with disease activity and treated patients may demonstrate negative results or very low titers. More than 50% of patients will be seronegative 1 year after treatment. A patient with a positive treponemal test (e.g., FTA-ABS) is less likely to revert to negative and only 24% of patients will be seronegative 1 year after treatment; both treated and untreated patients may demonstrate positive serologic tests for the rest of their lives.
14. In patients with symptomatic gonococcal urethritis, how efficacious is a Gram stain of the exudate in comparison to a culture utilizing selective media for gonococcus?
A Gram stain of a urethral discharge in symptomatic males is an excellent method of diagnosing gonorrhea. A positive Gram stain showing multiple neutrophils, some containing clusters of gram-negative diplococci with the sides flattened toward one another, is cited as having a sensitivity of 98%. With such a Gram stain, a culture is expensive and adds little diagnostic yield (about 2%). Cultures are usually done in males with a urethritis and a negative or nondiagnostic Gram stain of the urethral exudates. In women suspected of having gonorrhea, the site of choice for obtaining specimens is the endocervix. However, gram-stained smears are relatively insensitive (30% to 60%), and their interpretation is difficult. A culture on selective media is essential to diagnose gonorrhea in women.
Holder NA: Gonococcal infections, Pediatr Rev 29:228–234, 2008.
15. What is the best way to diagnose allergic contact dermatitis?
The diagnostic test of choice is a properly applied and correctly interpreted patch test. Contact dermatitis is divided into irritant or allergic subtypes. Allergic contact dermatitis is immunologically mediated and is an acquired sensitivity that affects only certain individuals. Irritant contact dermatitis is not immunologically mediated and is due to chemical damage of the skin. For example, excessive hand washing or exposure to battery acid will involve almost everyone exposed in such fashion.
Goossens A: Recognizing and testing allergens, Dermatol Clin 27:219–226, 2009.
Key Points: Diagnosis of Fungal Infections
1. Microscopic examination of KOH-treated clinical material (epidermal scale, hair, nails) is more sensitive in experienced hands than are cultures for establishing the diagnosis of dermatophyte infections.
2. The Wood’s light is very specific but not sensitive for establishing the diagnosis of tinea capitis because the majority of cases are produced by Trichophyton tonsurans, a species that does not fluoresce.
Key Points: Biopsy Techniques
1. Shave biopsies are often poor choices for melanocytic lesions such as dysplastic nevi or melanoma, as they may fail to allow full and complete microscopic assessment of the lesion.
16. How are patch tests applied?
The suspected allergen is usually placed in an appropriate vehicle at an appropriate concentration. The patch test allergens are usually purchased in prepared forms, but less common allergens can be prepared individually. The allergens are placed in special wells that are taped against the skin of the back for 48 hours and then removed. It is important to instruct patients to keep the testing area completely dry. This skin area is then examined 24 to 72 hours after the patch is removed for a reading. A strong positive reaction has erythema, infiltration, papules, and vesicles. A bullous reaction is extremely positive. Interpretation of patch tests and correlation with the clinical disease are complex and usually performed by dermatologists.
17. In what diseases is a skin biopsy helpful?
A skin biopsy with routine hematoxylin-eosin staining is the best diagnostic technique for many cutaneous neoplasms that cannot be diagnosed visually. It is also helpful in many inflammatory skin disorders, especially those in which a specific diagnosis cannot be made from clinical examination, blood tests, or scrapings of the skin. The skin biopsy is a common office procedure, and specimens may also be submitted for more advanced studies such as immunofluorescence, electron microscopy, special stains, cultures, or polymerase chain reaction studies.
18. When are shave biopsies indicated?
The choice of the biopsy technique requires knowledge of basic dermatology, most specifically, where in the skin the pathology is likely to be located. A shave biopsy is usually the most superficial of the skin biopsies and particularly useful when the lesion is in or close to the epidermis. A shave biopsy is best for pedunculated, papular, or otherwise exophytic lesions. It is particularly useful for diagnosis of basal cell and squamous cell carcinomas, seborrheic keratoses, warts, intradermal nevi, and pyogenic granulomas. Shave biopsies are often poor choices for biopsies of melanocytic lesions such as dysplastic nevi or melanoma. Unlike punch biopsies, shave biopsies only require a clean, nonsterile field and do not require sutures.
19. What are the indications for punch biopsies?
A punch biopsy utilizes a round knife that takes a cylinder of tissue including the epidermis, dermis, and often the subcutaneous fat. Although punch biopsies from 2 to 10 mm in diameter can be used, the most common diameter is 4 mm. A larger punch biopsy may be useful if the specimen is to be divided for culture or other procedures. When splitting the biopsy, the specimen is cut through the dermal rather than the epidermal side to reduce crush artifact. A larger punch biopsy of the skin is also helpful in the diagnosis of cutaneous T-cell lymphoma and for scalp biopsies, where a generous specimen is often necessary to establish the diagnosis. The surgical defect left by punch biopsies may be allowed to heal-in secondarily, but most dermatologists close the defect with a single suture.
20. Describe the indications for an excisional or incisional biopsy.
Excisional or incisional biopsies are usually elliptical in shape and typically deeper than punch biopsies. An excisional biopsy is the complete removal of a lesion into the fat, followed by layered closure of the skin. It is particularly helpful in the complete removal of malignancies, such as malignant melanoma, basal cell carcinoma, and squamous cell carcinoma. Excisional biopsies can also be performed when the cosmetic result is felt to be superior to that of a punch biopsy. An incisional biopsy is the incomplete or partial removal of a lesion. If a suspected malignancy is felt to be too large to remove with simple surgery, an incisional biopsy is used to remove the thickest or clinically most worrisome portion for diagnostic pathologic examination. It is also useful for diagnosing panniculitis, sclerotic, or atrophic lesions in which it is important to compare normal adjacent skin to that of the lesion, and to lesions with active expanding borders, such as pyoderma gangrenosum.
21. Define and describe direct immunofluorescence of the skin.
Direct immunofluorescence (DIF) of the skin is a histologic stain for antibodies or other tissue proteins in skin biopsy specimens. A skin sample obtained from the patient is immediately frozen in liquid nitrogen or placed in special media to preserve the immunoreactants. Arrangements should be made to ensure proper and timely transport to the immunofluorescence laboratory. Once received, the tissue is sectioned and then incubated with antibodies to human immunoglobulins or complement components that have been tagged with a fluorescent molecule to allow their visualization. The samples are then examined with a fluorescence microscope, where fluorescence indicates that immunoreactants were deposited in the patient’s tissue. The specific immunoreactants present, and the pattern and intensity of staining, are used to determine the diseases most likely to be associated with the DIF findings.
Zillikens D: Diagnosis of autoimmune bullous skin disease, Clin Lab 54:491–503, 2008.
22. Name some skin diseases in which DIF is helpful in making a diagnosis.
Many of the immunobullous diseases are associated with specific DIF findings: bullous pemphigoid, herpes gestationis, cicatricial pemphigoid, epidermolysis bullosa acquisita, dermatitis herpetiformis, linear IgA bullous dermatosis, and the various types of pemphigus. In addition, DIF may be helpful in evaluating cutaneous and systemic lupus erythematosus, other collagen vascular diseases, vasculitis such as Henoch-Schönlein purpura (Fig. 3-8), and certain types of porphyria.
23. How does indirect immunofluorescence of the skin differ from direct immunofluorescence of the skin?
Indirect immunofluorescence studies test for the presence of circulating autoantibodies in the serum, in contrast to direct immunofluorescence studies, which test for the presence of autoantibodies deposited in the skin. The serum from the patient is incubated with an appropriate normal substrate such as monkey esophagus, rat bladder, or human skin. The substrate is incubated with fluorescein-labeled antibodies directed against the antibody in the tissue. The specimen is then examined under a fluorescence microscope. By running this test at various dilutions, the amount of circulating antibody can be determined and reported as a titer. Titers are useful in some diseases, such as pemphigus vulgaris, in determining disease activity and treatment.
24. Is ELISA ever used for the diagnosis of immunobullous disease?
Yes. In recent years the enzyme-linked immunosorbent assay (ELISA) has been increasingly used for the diagnosis and differentiation of various forms of pemphigus, based on the presence of IgG autoantibodies directed against desmoglein-1 (dsg-1) and desmoglein-3 (dsg-3), which are proteins found in the intercellular junctions between keratinocytes. The ELISA is also, but less commonly, used to detect IgG autoantibodies against BP180 and BP230, which are the major antigens associated with bullous pemphigoid. The ELISA is both more specific and sensitive when compared to indirect immunofluorescence testing.
25. How are bacterial skin cultures performed, and when are they useful?
Bacterial cultures are useful when active infection of the skin is suspected. Bacterial cultures demonstrate high yields in superficial infections such as impetigo, ecthyma, and infected ulcers, and lower yields in cellulitis. When culturing superficial infections, the involved area should first be cleaned with an alcohol pad and then thoroughly swabbed. A higher concentration of bacteria may be found at the point of maximal inflammation. The best results in cellulitis are obtained when the leading edge is injected with nonbacteriostatic saline using a 20-gauge needle mounted on a tuberculin syringe. The aspirate may be sent for culture while still in the syringe if it can be taken to the laboratory immediately, or the aspirated material may be submitted in a bacterial culturette.
