Summary
Dermatophyte diagnosis
Hair plucks and skin scales
The preferred diagnostic sample is collection of hair plucks and some skin scale/crust from
the margins of a few recent lesions. Ideally a light alcohol spray might be applied first to inhibit
growth of associated bacterial species. Where possible gentle plucking with artery forceps
should be performed attempting not to break the hair shafts. These samples may then be
submitted for direct microscopic and microbiologic identification of dermatophytes.
Transport in a paper envelope may be preferable to a sealed container, again to discourage
bacterial growth in the sample.
Direct Microscopy
The hair and scale sample can be examined microscopically under oil immersion. Unstained
and uncleared plain samples are generally acceptable for examination although potassium
hydroxide clearance could be used if desired and stains such as Giemsa, Congo red, Chlorazol
black E or simply blue or black writing ink may sometimes assist identification. Where
fluorescence microscopy is available then calcofluor white might also be useful to help
identify infected hairs. Infected hairs may be slightly thicker than other hairs and may be
distorted and have indistinct margins and lack of clear internal structure. Fungal hyphae
within hairs are filamentous and septated and may branch or form chains of rounded
arthroconidia on the hair surface (ectothrix infection) (Figure 2). Nevertheless, direct
microscopy is not a sensitive test with probably fewer than half of the samples from
confirmed dermatophytosis cases deemed positive microscopically.
Culture
Dermatophyte test medium (DTM) is specifically designed for ease of use and interpretation
in non-laboratory conditions, but is less well suited to in vitro growth of dermatophytes and
cannot easily be used to identify the infecting species. DTM contains antimicrobial agents to
decrease growth of contaminants but this inevitably impairs dermatophyte growth also.
Phenol red is also added as a pH indicator and works on the basis that dermatophyte species
preferentially metabolise protein within the medium creating alkaline metabolites that turn
the medium red in association with appearance of fungal colonies (Figure 3). Unfortunately,
several common environmental non-dermatophyte fungi (e.g. Aspergillus species) are also
able to metabolise proteins, leading to false positive diagnoses. However, most saprophytic
fungi will preferentially metabolise carbohydrate within the medium with acidic metabolites
maintaining the yellow colour. Daily examination for up to 10 days is important so that early
colour change is detected as dermatophytes may use carbohydrates when the protein is
exhausted and return the medium to yellow again. Conversely most saprophytic fungi have
the capacity to metabolise protein with alkaline metabolites when the carbohydrate is used
up. Characteristic pigmentation of dermatophyte colonies is generally not seen with DTM and
colonies rarely form macroconidia, making species identification hard to impossible when
using DTM.
Given the many diagnostic limitations of DTM, Sabouraud’s agar is by far the most useful
dermatophyte culture medium. This agar can be used plain or with addition of antimicrobial
agents such as chloramphenicol and actidione (cycloheximide), which are included to
suppress contaminant bacteria. However, the antimicrobial agents do also impair fungal
growth somewhat, although the effect on bacteria is far greater. Growth of dermatophytes
on Sabouraud’s medium (with or without antimicrobials) is invariably more vigorous than
when using DTM (Figure 4). The addition of niacin to culture media is commonly cited as a
requirement for successful growth of T.equinum in vitro, although studies performed in this
author’s laboratory indicated little value of this (Figure 4). Cultures should be incubated a bit
cooler than normal bacterial incubation conditions, at 27-30°C. Usually 14 days culture is
adequate for growth of most dermatophyte species on Sabouraud’s agar although some new
colonies can sometimes develop quite late (e.g. 4 weeks). Suspected dermatophyte colonies
growing on Sabouraud’s agar can be identified based on colouration, biochemical reactions
and microscopic examination. Trichophyton equinum, by far the most common equine isolate,
has a white fluffy surface with deep red reverse pigmentation although may sometimes be
more orange/yellow. In contrast, M.equinum tends to appear beige on the undersurface
(Figure 5). Microscopic examination for macroconidia is best performed by lightly brushing
the surface of the fungal colony or by applying a Sellotape strip which can then be stained
with Lactophenol cotton blue and examined under the microscope. Again the macroconidia
of T.equinum and M.equinum are easily distinguished by their shape (Figure 6).
qPCR
More recently, a real time qPCR assay for detection of dermatophyte infection in horses was
developed and validated at the Liphook Equine Hospital Laboratory and is approved by the
British Horseracing Authority for diagnosis of equine dermatophytosis. The assay was
designed to target segments from the beta-tubulin gene found in dermatophytes including
T.equinum, T.mentagrophytes, T.verrucosum and M.gypseum, and also the chitin-synthase
gene characteristic of M.equinum and M.canis. Not only has this method allowed same-day
diagnosis of dermatophytosis compared to a 10-14 day delay in reporting culture results, but
additionally it appears far more likely than culture to identify dermatophyte organisms when
present. As part of the validation process, 256 samples from horses with various forms of skin
disease were subject to both qPCR and also culture on Sabouraud’s medium. Of these
samples, 82 (32%) were qPCR positive but only 53 (21%) were culture positive, indicating that
more than a third of dermatophyte cases might be missed by culture on Sabouraud’s agar,
and almost certainly far more would be missed using DTM. It has been suggested that
dermatophyte species might occasionally be isolated from the hair coats and skin of normal
horses as a result of recent transfer from the environment [4] which is a potential concern
with highly sensitive methods such as qPCR. However, no sign of dermatophytes was found
in any of 9 samples collected from horses without signs of skin disease as part of the qPCR
validation. Importantly qPCR detects DNA whether this is from live or dead organisms, and so
this method cannot currently be used reliably to confirm effective treatment of
dermatophytosis cases.
qPCR
A real time qPCR assay was designed at Liphook to detect β-tubulin (group 1) and chitin
synthase 1 (group 2) genes. This enables detection of all common equine dermatophytes and
allows any identified organism to be grouped as either:
group 1
Trichophyton equinum
Trichophyton mentagrophytes
Trichophyton verrucosum
Microsporum gypseum; or
group 2
Microsporum equinum
Microsporum canis
Specific identification, if required, will still require growth on Sabouraud’s medium.
The assay is highly sensitive and specific and has been found to detect approximately 63%
more ringworm cases than culture on Sabouraud’s agar (and almost certainly far more if
compared with Dermatophyte Test Medium).
As this is a qPCR assay which detects specific gene segments, the assay does not differentiate
dead from live organisms and cannot be used currently to determine success of treatment.