Diagnostic and Therapeutic Procedures

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.