Bacterial culture

Bacterial culture is a frequently performed diagnostic test. Purulent material draining from a pustule or nodule can easily be obtained with a bacterial swab. If the material is dry or crusted, wetting the swab with water prior to rubbing the area increases the likelihood of obtaining material to grow an organism. The results of a Gram stain performed by the microbiology laboratory are usually available within a few hours, while the final bacterial culture result with drug susceptibilities may not be known for 48–72 h. The laboratory should be notified when Gram-negative or anaerobic bacterial infections are suspected, so that specific culture media may be used.

Bacteria causing certain infections such as necrotizing fasciitis may not be easily cultured from superficial skin samples, so that a skin biopsy for frozen section may be necessary for a rapid diagnosis (see ‘ Skin biopsy ’, below). A bacterial stain such as Brown–Brenn performed on the histologic sections can differentiate Gram-positive from Gram-negative organisms.

KOH preparation

Potassium hydroxide (KOH) preparations are useful to identify the presence of fungi and yeast. They can be performed on samples of skin, hair, and nails. The skin should be scraped such that adequate material is available for microscopic examination ( Box 6.1 ). If the KOH preparation is negative or equivocal, the skin scales may be placed onto fungal culture plates or sent for culture in an appropriate container, such as a sterile urine cup or wax paper envelope (see ‘ Fungal culture ’, below).

A scalp sample may be obtained by wetting a cotton-tipped applicator with water and rubbing the suspected area of involvement. Another technique is to use a toothbrush or gynecologic viral collection brush. The material from the swab is plated directly onto the fungal culture plate. Hairs can be plucked for culture, but this is not usually recommended in infants because of the pain caused by pulling hairs. Usually enough hairs can be gathered by scraping the scalp with a glass slide or #15 blade.

For nail samples, a sharp instrument, such as a Skeele curette, is used to collect debris from underneath the nail plate and this material can be stabbed directly onto the fungal plate. Nail clippings can be obtained by using a nail nipper. In young infants with soft nails, a cuticle nipper is sometimes sharp enough. The clippings can either be placed directly on the culture medium or in a sterile cup for the laboratory to plate. In addition, nail clippings can be placed in formalin and sent directly to pathology so that a periodic acid Schiff (PAS) stain can be performed; the results are usually known within 2–3 days compared with 30 days, which is the standard for fungal cultures in a microbiology laboratory. This method has been shown to be more reliable than KOH preparation and fungal culture in detecting the presence of organisms; however, fungal culture remains the gold standard for identifying the organism and determining drug susceptibilities.

Fungal culture

Mycosel™ agar and mycobiotic agar are two types of fungal culture media that are frequently used. They contain Sabouraud dextrose agar with chloramphenicol and cycloheximide to decrease bacterial overgrowth.

For deep fungal infections of the skin, a skin biopsy is necessary so that material from the dermis, and sometimes the subcutaneous fat, can be cultured, since the organism is not present in the superficial epidermal scales (see ‘ Skin biopsy ’, below).

Direct fluorescent antibody test for diagnosis of herpes virus infection

Direct fluorescent antibody (DFA) testing uses mouse monoclonal antibodies to detect herpes viruses such as herpes simplex virus (HSV) 1 and 2 and varicella-zoster virus (VZV). Specimens are best obtained from the base of a ruptured vesicle, erosion, or ulcer. The likelihood of a positive result is much lower if the lesions are crusted or already healing. A Dacron® swab with a plastic shaft is used to rub the lesion and the swab is placed in viral transport medium. A calcium alginate swab should not be used because the chemicals are toxic to the virus. The laboratory can prepare the slide after cytospin preparation; however, a slide can also be prepared at the bedside by careful rubbing of the swab onto the slide, which is allowed to air dry prior to transport to the laboratory.

Viral culture

Using a Dacron® tipped swab, the fluid from an intact vesicle is absorbed and the swab is rubbed over the base of the lesion prior to placement in viral transport medium (buffered isotonic saline solution, often with antibiotics added to prevent bacterial contamination). The results are usually available in 2–3 days for herpes simplex viruses and 7–14 days for varicella-zoster virus.

Viral culture has historically been the gold standard for isolating viral pathogens, such as herpes viruses; however, this is likely to change with improved polymerase chain reaction (PCR) techniques.

Polymerase chain reaction (PCR) test for diagnosis of herpes virus infection

Polymerase chain reaction (PCR) is now used by many laboratories to definitively identify herpes virus infections as well as other viral infections. Compared with viral culture, this test is more sensitive and the result is available more rapidly. The swab obtained for viral culture that has been placed in viral transport medium can be used for PCR.

Scabies preparation

Scabies infestation is frequently seen in young infants. Sensitization after primary infestation takes 3–4 weeks before symptoms occur. With recurrent infestation, symptoms appear immediately. Performing a scabies preparation can confirm the diagnosis. Detection of evidence of scabies infestation is most likely by scraping linear burrows, vesicles, or papules that have not been excoriated. Interdigital spaces of the hands and feet, wrists, and axillae are often high-yield locations. Sometimes parents or other caregivers have papules or burrows that can be scraped as well.

The finding of mites, eggs, or feces on a scabies preparation using a microscope with a 10× objective confirms the diagnosis ( Box 6.2 ). Mites have eight legs ( Fig. 6.1 ); eggs are oval in shape and ten times smaller than mites; and feces, which are even smaller, appear as golden-brown clumps ( Fig. 6.2 ). Air bubbles are round, which helps distinguish them from eggs or feces. Dermoscopy can also be useful in the diagnosis of scabies infestation (see below).

Scabies mite.

Scabies eggs and feces.

Dermoscopy

Dermoscopy (dermatoscopy or epiluminescence microscopy) is the examination of skin lesions with a handheld dermatoscope. Most dermatoscopes utilize polarized light to eliminate skin surface reflection. Specific patterns can be seen that help confirm a suspected diagnosis. Dermoscopy can be useful in the identification of a broad set of conditions and lesions such as scabies mites or burrows; congenital and acquired melanocytic nevi including Spitz and blue nevi; and juvenile xanthogranuloma. The ‘triangle’ sign is the pigmented anterior portion of the scabies mite, which includes the head and first two pairs of legs, while ‘the jetliner with contrail’ sign represents the head of the mite along with the trailing burrow ( Fig. 6.3 ). Typical Spitz nevi have four distinct dermoscopic patterns: starburst, globular, negative network, and homogeneous ( Fig. 6.4 ). Histiocytes laden with lipid give juvenile xanthogranulomas their characteristic yellow-orange color. Dermoscopy can show this orange-yellow background with an erythematous border, the ‘setting sun’ sign. Pale yellow ‘clouds’ can also be seen, which are thought to be lipid-laden histiocytes in the superficial dermis. Blue nevi have homogeneous, structureless pigment patterns of different colors (white or blue most commonly) or a combination of colors ( Fig. 6.5 ).

Scabies dermoscopy.

Note ‘triangle’ and ‘jetliner with contrail’ signs.

(Courtesy of Ashfaq Marghoob, MD. In: Haliasos EC, Kerner M, Jaimes-Lopez N, Rudnicka L, Zalaudek I, Malvehy J, Hofmann-Wellenhof R, Braun RP, Marghoob AA. Dermoscopy for the pediatric dermatologist. Part I: Dermoscopy of pediatric infectious and inflam­matory skin lesions and hair disorders. Pediatr Dermatol 2013; 30(2):163–171.)

Spitz nevus dermoscopy.

(A) Starburst pattern. (B) Negative network pattern. (C) Globular pattern. (D) Dotted vessel pattern.

(Courtesy of Ashfaq Marghoob, MD. In: Haliasos EC, Kerner M, Jaimes N, Zalaudek I, Malvehy J, Hofmann-Wellenhof R, Braun RP, Marghoob AA. Dermoscopy for the pediatric dermatologist. Part III: Dermoscopy of melanocytic lesions. Pediatr Dermatol 2013; 30(3):281–293.)

Blue nevus dermoscopy.

Homogenous structureless pattern.

(Courtesy of Ashfaq Marghoob, MD.)

Microscopic hair examination

A number of hair shaft abnormalities can be detected by microscopic hair examination using a standard light microscope with and without a polarizing lens. Sharp iris scissors can be used to obtain hairs without the need to pluck them. A mounting medium such as Permount™, or immersion oil, and a cover slip may be used to mount the hairs on a glass slide. Only rarely is the hair bulb needed for diagnosis, such as with suspected loose anagen syndrome, in which case the hairs need to be gently pulled. Usually, an examination of scalp hair demonstrates the findings necessary for diagnosis; however, Netherton syndrome can be an exception, in which only eyebrow or eyelash hairs may have the characteristic findings. See Chapter 31 for a discussion of hair disorders.

Wood’s light examination

A Wood’s lamp is a handheld device fitted with a mercury lamp and a filter made of nickel oxide and silica. The light emitted from the device has wavelengths from 320 to 400 nm, which is the range in which melanin absorbs ultraviolet radiation. The lamp should be used in a dark room, so that ambient light does not interfere with the examination. Loss of melanin (depigmentation) appears as a ‘bright white’ area compared with surrounding normal skin. A Wood’s lamp can be useful in the identification of vitiligo, tuberous sclerosis (ash leaf macules), and incontinentia pigmenti. Also, infected hairs fluoresce bright yellow-green in tinea capitis caused by Microsporum canis . The urine of neonates with congenital erythropoietic porphyria fluoresces coral red.

Skin biopsy

A skin biopsy can be very useful in determining a diagnosis. The tissue can be examined microscopically and it can also be cultured (bacterial, viral, fungal, mycobacterial). When the lesion is <6 mm in diameter, the entire skin lesion can be removed using a punch technique for both diagnostic and therapeutic purposes. The depth of the pathologic process determines how deep the skin specimen needs to be. Usually, a punch biopsy can be performed, since epidermis and dermis will be obtained, as well as the top layer of fat in some body locations ( Box 6.3 ).

If a process or lesion involving the subcutaneous fat, such as panniculitis, is suspected, a ‘double punch’ method may be employed. One specimen is obtained and through the same defect, a second, deeper specimen is also obtained. This is often done with a larger sized punch trephine followed by a smaller one through the same defect, so that the hub does not become stuck on the epidermal rim of the initial defect. For example, a 6 mm punch trephine can be used followed by a 4 mm punch trephine to obtain the second specimen. The specimens may be placed in separate formalin bottles.

If the pathologic process appears to have multiple morphologies, it may be necessary to obtain specimens from each type of lesion seen. When a specimen is needed for tissue culture, the tissue and instruments should not come in contact with formalin because the formalin will kill any organism, rendering the culture useless. Therefore, if specimens are needed for culture as well as histology, the culture specimen should be obtained first, so that no contamination occurs.

Once the specimen has been obtained, hemostasis is usually achieved with placement of sutures. In a neonate or young infant, a 5–0 non-absorbable suture such as nylon or polypropylene can be used. Some providers prefer to place Gelfoam® (Pharmacia and Upjohn Company, Kalamazoo, Michigan) in lieu of sutures depending on the size, location and type of lesion biopsied; however, the time to place one or two sutures is minimal and can result in a wound that heals more rapidly and a final smaller scar.

Shave procedures, also known as ‘scoop biopsies’ or ‘saucerization biopsies’ can be used instead of a punch trephine for biopsy or excision of superficial lesions that involve only the epidermis and superficial dermis. Either a DermaBlade® (Personna, Verona, Virginia) or #15 scalpel blade can be used to obtain the specimen.

Electron microscopy

Electron microscopy requires extremely thin (one micro­meter, 1 µm) tissue sections and a special microscope to allow visualization of cellular ultrastructure. It is most useful in neonates to localize the cleavage plane in epidermolysis bullosa (EB). When the diagnosis of EB is suspected, twisting a new, clean pencil eraser on normal-appearing skin just prior to skin biopsy is helpful to form a microscopic cleavage plane. Specimens should be obtained using the punch biopsy method outlined in ‘Skin biopsy’, above. Instead of placing the specimen in formalin, it should be placed in glutaraldehyde fixative. With the advent of genetic testing for EB with a blood specimen, electron microscopy is not necessary in many cases, especially if the mutation in the family is already known.

Immunofluorescence

Immunofluorescence testing relies on the fluorescing of dyes to help localize immunoreactants in the skin or to determine their presence in the blood.

Direct immunofluorescence (DIF) may be useful in the diagnosis of immunobullous disorders, lupus erythematosus, and leukocytoclastic vasculitis. A skin biopsy should be taken from a perilesional location and placed in immunofluorescence transport medium (Michel’s medium: ammonium sulfate, N-ethylmaleimide, magnesium sulfate in a citrate buffer). The specimen can be placed on saline-soaked gauze, as long as it is immediately transported to the laboratory and processed. The sample is incubated with antibodies to IgG, IgA, IgM, and C3 that have been labeled with a fluorescent marker. Fluorescence indicates the presence of the specific immunoglobulin or complement component.

Indirect immunofluorescence (IIF) is most useful in the diagnosis of immunobullous disorders such as pemphigus vulgaris and bullous pemphigoid. The patient’s serum is incubated with an epithelial substrate to determine the circulating antibody titer. A modified IIF method on a skin specimen helps determine the ultrastructural level of antibodies in epidermolysis bullosa, which would be the most common use of the test in neonates. Laminin, type IV collagen, and bullous pemphigoid antigen are used, as well as other markers. Skin samples for this test are obtained in the same manner as for electron microscopy; a cleavage plane is induced with rotating pressure prior to obtaining the skin biopsy and the sample is placed in appropriate transport medium (i.e. Michel’s or Zeus’) prior to delivery to a laboratory skilled in antigenic mapping.