Collection and Processing of Skin, Hair and Nail Scrapings for Fungal Studies

Proper collection of specimen for fungal studies is very essential to increase the rate of isolation and minimize instances of false negative results. Below is a detailed guide on how to properly collect and process skin , hair and nail scrapings for the isolation of pathogenic fungi.

Skin Scrapings and Swabs

In patients with suspected tinea or ringworm any ointments or other local applications present should first be removed with an alcohol wipe. Using a blunt scalpel, tweezers, or a bone curette, firmly scrape the lesion, particularly at the advancing border. A bone curette is safe and useful for collecting specimens from babies, young children and awkward sites such as interdigital spaces. If multiple lesions are present choose the most recent for scrapings as old loose scale is often not satisfactory. Any small vellus hairs when present within the lesions should be epilated. The tops of any fresh vesicles should be removed as the fungus is often plentiful in the roof of the vesicle.

In patients with suspected candidiasis the young "satellite" lesions which have not undergone exfoliation are more likely to yield positive results if they are present. Otherwise the advancing scaly border should be scraped. When lesions in the flexures are moist and very inflamed it is more satisfactory and less painful to roll a moistened swab firmly over the surface.

In patients with suspected cutaneous manifestations of systemic pathogens scrap the lesions with a bone curette or blunt scalpel as for tinea. A biopsy may be required in some cases.

NOTE: Following the collection of skin scales all scraped lesions should be firmly rubbed with a swab moistened in BHI broth.

Skin scrapings, nail scrapings and epilated hairs where tinea is the provisional diagnosis:

1. Make a wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimen onto two slopes containing cycloheximide (actidione) i.e. one DERMASEL agar slope and one LACTRITMEL agar slope also containing chloramphenicol, gentamicin and incubate cultures at 26C. Maintain cultures for 4 weeks.
3. Where a moistened swab has also been collected from the same site as the scraping, inoculate this onto a Sabouraud's dextrose agar slope containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 26C. Maintain cultures for 4 weeks.

Skin scrapings and swabs where candidiasis is the provisional diagnosis:

A. Skin scrapings:

1.  Make a KOH mount preparation for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto Sabouraud's dextrose agar slopes containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35C. Maintain cultures for 4 weeks.

B. Skin swabs:

1. Smear swab onto heat sterilized glass slide for Gram stain.
2. Inoculate specimens onto Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35C. Maintain cultures for 4 weeks.
3. Where secondary bacterial infection is suspected, and separate swabs for routine bacteriology were not collected, the swab should first be inoculated onto a blood agar plate, followed by the Sabouraud's agar containing the antibiotics and then placed into Brain Heart Infusion Broth. All cultures should be incubated at 35C. Maintain cultures for 4 weeks.

NOTE: Where a dermatophyte is suspected or to be excluded a Sabouraud's agar slope containing cycloheximide and incubated at 26C may be included.

Scrapings from the groin, feet or nails where either a dermatophyte or Candida species may be isolated. This includes the possibility of a non-dermatophyte onychomycosis.

1. Direct Microscopy: Wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide (as for Candida) and incubate at 26C. Maintain cultures for 4 weeks.
3. Inoculate specimen onto a DERMASEL agar slope containing cycloheximide (actidione), chloramphenicol and gentamicin and incubate cultures at 26C. Maintain cultures for 4 weeks.
4. Where a moistened swab has also been collected from the same site as the scraping, inoculate this onto a Sabouraud's dextrose agar slope containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 26C. Maintain cultures for 4 weeks.

Skin scrapings from patients with suspected pityriasis versicolor:

1. Direct Microscopy: Wet mount preparation in KOH for direct microscopy along with the cellotape stripping taken at the time of collection.
2. Inoculate scrapings onto an DIXON'S agar slope for isolation of Malassezia furfur and incubate cultures at 26C. Maintain cultures for 4 weeks.
3. Inoculate specimen onto Sabouraud's dextrose agar with chloramphenicol and gentainicin but NO cycloheximide (actidione) and incubate cultures at 26C. To exclude other yeasts like Candida. Maintain cultures for 4 weeks.
4. If dermatophytes are to be excluded also inoculate onto DERMASEL agar slope and incubate cultures at 26C. Maintain cultures for 4 weeks.

Skin scrapings from patients where a systemic pathogen is suspected:

1. Direct Microscopy: Wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto:

• Sabouraud's dextrose agar with chloramphenicol and gentamicin but NO cycloheximide (actidione) and incubate duplicate cultures at 26C and 35C; and
• Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for 4 weeks.

For a detailed reference guide in examining and identifying fungal structures, see "Fungal Structures and Mycology Terminologies Essential for Identification"

Article source: Mycology Online- The University of Adelaide