Dermatologic diagnostics: What you can do in clinic (Proceedings)

Parasitology

Deep skin scrape: Identifies Demodex mites. Clip hair from area, apply a thin film of mineral oil to site, squeeze skin to extrude mites, scrape area with a dull number 10 scalpel blade or a skin spatula, collect material on blade and transfer to a microscope slide, repeat scraping until capillary bleeding is noted (or your scrape is not deep enough). Make sure you have enough mineral oil on the slide, apply coverslip, then systematically examine area under cover slip for the presence of mites (note life stage- adult versus larva versus nymph versus egg, note if adults are alive or dead). Lower the condenser to increase contrast. Examine on 10X at low light.

Superficial skin scrape: Identifies Demodex mites that live in/on superficial skin layers (Demodex gatoi in cats, short Demodex mite of dogs- may be called Demodex cornei), Sarcoptes scabiei, Notoedres, Otodectes cynotis, Cheyletiella spp., lice, Lynxacarus radovsky (cat fur mite), etc.

Clip hair if needed from a large area, apply a thin film of mineral oil to skin surface, scrape skin surface with a dull number 10 scalpel blade or skin spatula, collect all material and transfer to a microscope slide. Scrape multiple large areas.

Hair plucks for mites: Identifies Demodex mites. Select site, gently grasp base of hairs with hemostats and pull in direction of hair growth, place hairs in mineral oil on slide, place coverslip on slide, and examine under 10X. Mites may be seen at base of hairs in keratin debris.

Tape preps for mites: Identifies skin surface dwelling Demodex mites and other superficial mites (Sarcoptes scabiei, Notoedres cati, Otodectes cynotis, Cheyletiella spp., lice, Lynxacarus radovsky, etc.) Use a small piece of clear packing tape to collect skin surface debris and hairs from multiple sites on the animal. Put a drop of mineral oil on slide, place tape on slide sticky surface down, and examine under 10X.

Ear swabs for mites: Identifies Otodectes cynotis and Demodex mites. Collect debris from ear canal on cotton swab and roll onto slide with a small amount of mineral oil, apply coverslip and examine under 10X.

Fecal flotation: Occasionally identifies Demodex gatoi in cats- they ingest the mites while grooming themselves.

Hand lens examination: can identify lice and Cheyletiella

Flea combing: best way to find fleas and/or flea dirt!

Cytology

Cytology is done primarily to identify infectious organisms, in particular bacteria (cocci versus rods) and yeast (Malassezia).

Tape impressions: Tear off a small piece of clear packing tape (we use 3M super strength mailing tape). Firmly press tape onto affected area and remove, repeating process until the tape is no longer sticky. Attach one end of tape to the bottom edge of a microscope slide and dip the tape into the three solutions that make up diff-quick stain, rinse tape, detach from bottom edge of slide, then place tape sticky surface down onto slide surface, blot on paper towel to dry, then examine microscopically. Tape impressions are good for drier areas or crevices (skin folds, interdigital area). Do not heat fix tape.

Slide impressions: Press slide surface directly onto skin to collect exudates, debris, etc. Good for easily accessible surfaces with moist exudates. Let dry, stain with diff-quick, then examine.

Sticky slide impressions: These are slides that come with a thin layer of adhesive on one side of the slide. Press the sticky side of the slide to the skin surface, diff quick, then examine. Good for drier areas, but difficult to get into crevices.

Superficial skin scrape: If there is a significant amount of greasy surface debris or purulent exudate, this material can be collected with a dull scalpel blade or skin spatula and smeared onto a slide. Heat fix if desired, stain with diff-quick, then examine.

Pustule cytology: Identifies infectious agents (bacteria), inflammatory cells (neutrophils, eosinophils, lymphocytes, macrophages), acantholytic cells (seen most often with Pemphigus foliaceus). Open the surface of the pustule with a 25 gauge needle (held close to parallel with the pustule surface), then either press a slide to the pustule contents or collect the pustule contents with the needle and smear contents on the slide. Stain with diff-quick, then examine. If pustules are not present, but crusts are, do impression smears of the crusts and the raw skin/exudates underneath them.

Ear cytology: Identifies infectious organisms, inflammatory cells, occasionally neoplastic cells. Collect exudates fro canals with a cotton swab, roll onto a slide, heat fix if desired, stain with diff-quick, then examine.

Is it ringworm?

Wood's lamp: Examination of the haircoat with a Wood's lamp (Long wave Wood's Lamp UVL-56 Lamp 365 nm, Fisher scientific) can be helpful in the diagnosis of dermatophytosis provided it is done correctly. Unfortunately, both false positive and false negative results are obtained quite often. A Wood's lamp emits long wave uv radiation through a nickel or cobalt glass filter. Battery operated Wood's lamps are inadequate. The wood's lamp should be allowed to warm up for at least 10 minutes before it is used. The lights in the exam room should be turned off and the suspect lesions should be exposed to the Wood's light for at least 3-5 minutes. The lamp should be held within several inches of the lesion for best results. A positive Wood's lamp reading is when the hair shafts fluoresce an apple green to blue green color. Crust, scale, topical medications, lint, and carpet fibers can all fluoresce and cause a false positive reading. A negative Wood's lamp examination does not rule out dermatophytosis since only some strains of Microsporum canis fluoresce.

Direct examination of hairs: Direct examination of hairs for the presence of fungal arthrospores can also be used as an aid in the diagnosis of dermatophytosis. The procedure can be cumbersome and may not be time or cost effective. Hairs are plucked and placed in a clearing solution (KOH or chlorphenolac) to dissolve debris and extraneous material. This allows better visualization of the hair shafts. Hairs can be examined in mineral oil but this requires a trained eye. The hair shafts are examined for the presence of fungal hyphae within the hair shaft as well as arthrospores. Microsporum canis produces ectothrix spores (surrounding the shaft).

Fungal culture: Fungal culture is the gold standard for the diagnosis of dermatophytosis. This is a simple technique that can easily be done in office. You will need sterile hemostats for plucking hairs, toothbrushes, and fungal culture plates. Use either Sab Duet plates or Derm Duet plates (www.bactilab.com). Sab Duet plates contain dermatophyte test medium on one side and plain Sabouraud's medium on the other. Derm Duet plates contain dermatophyte test medium on one side and rapid sporulating medium on the other. Do not use the small glass jars for culturing. You will not be able to obtain a sample of the colony growth for identification. To obtain samples, pluck hairs from the periphery of lesional areas or brush lesional areas with a new toothbrush, then gently press hairs or the toothbrush onto the culture medium. If no obvious lesions are present or if you are monitoring response to therapy, the entire coat is combed with the toothbrush. Swiffers are also useful for whole body cultures and for culturing the environment. Cultures should be kept in a dark area at 75-80°F for at least 21 days. Keep a small dish of water in the area with the culture plates to prevent dehydration of the media and store plates upside down to prevent moisture accumulation on the surface of the media. A reptile heating pad works nicely to keep the temperature elevated.

Culture plates must be examined daily to every other day. Dermatophyte test medium is Sabouraud's dextrose agar with cycloheximide (inhibits growth of saprophytic fungi), gentamicin (antibiotic), chlortetracycline (antibiotic), and phenol red (pH indicator) added. Dermatophytes use the protein in the medium first. This causes the pH to become alkaline and the medium turns red. Dermatophytes produce colony growth that is white to light tan in color. They are never heavily pigmented. Saprophytic fungi use the carbohydrate in the medium first, then when the carbohydrate is gone, they will use the protein and the medium will turn red later. Therefore, you must look for white to tan colony growth together with red color change. This will usually occur within 7-14 days, but can take longer for some strains of dermatophytes or if they have been treated. Unfortunately, a few strains of Microsporum canis have been identified that did not turn the medium red and there are some saprophytes that do. Therefore, it is safest to examine any white colony growth on the plate. Plates can quickly be overgrown with saprophytic fungi so subculturing suspicious colonies may be necessary.

Once a suspicious colony is identified, clear tape should be applied to the growth, then the tape is applied to a slide with a drop of lactophenol cotton blue and examined for macroconidia and microconidia. Microsporum canis colonies are white and fluffy with a yellow to orange-brown underside. The macroconidia are spindle shaped with a knob on the end, walls are thick and rough, and six or more cells should be present. Microsporum gypseum produces a flat to grainy, buff to cinnamon colony with a pale yellow to tan underside. The macroconidia are ellipsoid, thin-walled, and have up to six cells. Trichophyton mentagrophytes produces flat, white to cream colored colonies with a brown to tan underside. Macroconidia are rare, cigar shaped, and thin walled. Microconidia are single or in grape like clusters. The website www.doctorfungus.org has excellent images of all the dermatophytes. Histopathology can also be used to diagnose dermatophytosis; however, fungal culture will still be needed to identify the species of dermatophyte involved. If you are unsure of your fungal identification, send samples to the lab for confirmation until you are more comfortable. Be sure to check that the lab identifies the dermatophyte to species level.

Bacterial culture and sensitivity testing (aerobic)

This can be done on a limited basis in the office, but I recommend sending these samples to an outside laboratory. There are some important points to discuss about obtaining samples. Samples from intact pustules or furuncles are ideal- open the pustule with a sterile needle, then collect exudates on a culturette. If pustules/furuncles/erosions/ulcers are crusted over, good samples may be obtained from under the crust. It can be helpful to do cytology from under the crust to look for bacteria. Culturing the skin surface is often unrewarding, unless skin surface cytology reveals a uniform population of bacteria. Caution should be used when interpreting results of bacterial growth on cultures from crusts and the skin surface. If deeper infection is present, skin biopsies obtained using sterile technique may be the best samples for culture.