Dermatophytes

Dermatophytes are filamentous, keratinophilic fungi of the Ascomycota (order Onygenales, family Arthrodermataceae). They are the only fungi that regularly use keratin as a nutrient source and the only group that causes dermatophytosis. Infection is confined to keratinised tissue: the stratum corneum, hair and nails. Living tissue is not invaded in an intact host. Deep dermatophytosis occurs in CARD9 deficiency and in severe cellular immunodeficiency.

Dermatophytes are not skin commensals. Asymptomatic carriage occurs, especially of Trichophyton tonsurans on the scalp and Trichophyton rubrum on feet, but a positive culture from a carrier still represents a transmissible organism.

Dermatophytosis is the commonest fungal infection worldwide and affects an estimated 20 to 25 percent of the global population at any time. Tinea pedis and onychomycosis dominate in adults, and tinea capitis dominates in prepubertal children, particularly in sub-Saharan Africa.

Taxonomy was revised on multilocus sequence data. Clinical laboratories still report three traditional genera, but several species have moved.

Species complexes matter clinically. Trichophyton mentagrophytes and Trichophyton interdigitale are separated by sequencing and ecology, and the Trichophyton mentagrophytes complex also contains Trichophyton indotineae, which is terbinafine resistant, and a genotype designated VII that causes genital and pubic disease transmitted by sexual contact. Trichophyton rubrum is a complex that includes Trichophyton violaceum, Trichophyton soudanense and Trichophyton megninii by genomic criteria, though they remain distinct clinical entities.

Ecological niche predicts the source of infection, the inflammatory response and the public health response.

Infection follows these steps:

• Attachment: arthroconidia adhere to corneocytes within hours, using fungal adhesins and host fibronectin. Minor trauma, maceration and occlusion assist entry.
• Germination and penetration: conidia germinate and hyphae penetrate the stratum corneum, growing between and through cells.
• Keratin digestion: secreted subtilisins, fungalysins and dipeptidyl peptidases cleave keratin. Sulphite efflux, through the transporter Ssu1, reduces the disulphide bonds of keratin and makes it accessible to proteases.
• Centrifugal spread: hyphae spread outward in the stratum corneum, producing an annular lesion with an active edge. In hair, they penetrate the shaft and grow down to the keratogenous zone near the bulb, where growth stops because the hair is alive.
• Host response: inflammation depends on the organism and the host.

Immune features:

• Keratinocytes and Langerhans cells sense fungal glucan and mannan, and Th1 and Th17 responses are protective. Delayed type hypersensitivity correlates with clearance, with centrifugal clearing of the lesion and a scaly active edge.
• Trichophyton rubrum produces mannan that suppresses lymphocyte proliferation and keratinocyte responses, which favours chronic infection with little inflammation.
• Zoophilic and geophilic species provoke strong neutrophilic and granulomatous responses, producing inflammatory tinea corporis and kerion.
• Dermatophytid (id) reaction is a sterile vesicular or papular eruption, commonly on the hands, in response to circulating fungal antigen at a distant inflammatory focus, most often tinea pedis. Fungal elements are absent from the id lesions.
• CARD9 deficiency produces extensive, recalcitrant and sometimes deep dermatophytosis, including invasion of lymph nodes and viscera, with Trichophyton rubrum. Autosomal dominant hyper IgE syndrome and STAT1 gain of function also cause severe, chronic disease.
• Systemic corticosteroids and topical corticosteroids alter the appearance and spread of lesions, producing tinea incognito.

Arthroconidia in shed scale and hair survive for months to years on floors, combs, hats and bedding, which explains persistent household transmission.

Hair infection is classified by the position of arthroconidia relative to the shaft. This guides organism identification and the presence of fluorescence.

The fluorescence of Microsporum species is due to pteridine produced in the infected hair. Fluorescence is therefore a feature of infected hair and not of the colony.

Specimens

• Skin: scraping from the active edge with a blade or the edge of a slide, after cleaning with 70 percent alcohol.
• Hair: plucked hairs with their roots, because cut hairs lose the infected portion. Broken stubs, black dots and scale are also collected.
• Scalp carriage or sparse disease: moistened cotton swab or sterile toothbrush rubbed over the scalp.
• Nails: clippings from the most proximal part of the diseased nail, and subungual debris scraped from beneath it with a curette. In white superficial onychomycosis, the surface is scraped.
• Specimens are collected before antifungal treatment. Recent topical therapy causes false negative microscopy and culture.

Direct microscopy

• Potassium hydroxide (10 to 20 percent) clears keratin. Adding dimethyl sulphoxide or heating improves clearing, and 20 to 40 percent potassium hydroxide is used for nails.
• Calcofluor white or chlorazol black E increases sensitivity.
• Findings in skin and nails: uniform, septate, branching, refractile hyphae of consistent width and often with arthroconidia in chains. Pseudohyphae and budding yeasts suggest Candida.
• Findings in hair identify the invasion pattern: a sheath of arthroconidia on the surface in ectothrix infection, and chains of arthroconidia filling the shaft in endothrix infection.
• Artefacts that mimic hyphae: cell borders, fibres and lipid droplets ("mosaic fungus").

Culture

• Sabouraud dextrose agar with chloramphenicol and cycloheximide at 25 to 30 degrees Celsius. Cycloheximide suppresses moulds but also inhibits some dermatophytes and non dermatophyte pathogens, so a parallel plate without cycloheximide is used where non dermatophyte moulds in nails are suspected.
• Dermatophyte test medium contains phenol red, which turns red when alkaline metabolites are produced. Contaminants can also change the colour, so microscopy is needed to confirm.
• Cultures are held for 3 to 4 weeks. Trichophyton rubrum is typically positive in 7 to 14 days. Trichophyton verrucosum, Trichophyton violaceum and Trichophyton schoenleinii are slow.
• Special media: Trichophyton agars 1 to 7 test vitamin requirements (thiamine, inositol); rice grain medium separates Microsporum canis (growth) from Microsporum audouinii (poor growth); cornmeal dextrose agar demonstrates red pigment by Trichophyton rubrum; urea agar and in vitro hair perforation separate Trichophyton mentagrophytes from Trichophyton rubrum.

Other methods

• MALDI TOF mass spectrometry: identifies common dermatophytes from cultures when the database is full. Closely related species may not be separated.
• Polymerase chain reaction: direct detection from nail, skin and hair with results in a day. It is more sensitive than culture and microscopy but cannot distinguish viable from non viable organisms, so a positive result after treatment needs clinical correlation.
• Sequencing: the internal transcribed spacer region identifies species. It is needed for Trichophyton indotineae and for terbinafine resistance work, which also requires squalene epoxidase gene analysis.
• Histopathology: periodic acid Schiff or Grocott methenamine silver shows hyphae in the stratum corneum. A neutrophil rich stratum corneum with alternating orthokeratosis and parakeratosis (the sandwich sign) strongly suggests dermatophytosis even when hyphae are scanty.
• Wood lamp: a screening aid in tinea capitis, limited by the many species that do not fluoresce.
• Dermoscopy and trichoscopy: comma, corkscrew, zigzag and Morse code hairs, and broken black dots, in tinea capitis.

Interpretation

• Positive microscopy with negative culture occurs with prior treatment, non viable organisms and slow growers. It is accepted as the diagnosis when the clinical picture fits.
• Negative microscopy with positive culture can occur with sparse elements, and the isolate is considered real if it is a dermatophyte.
• Non dermatophyte moulds in nails (Scopulariopsis brevicaulis, Fusarium, Aspergillus, Neoscytalidium) are accepted as pathogens only when microscopy shows hyphae, the same mould grows from repeated samples, and no dermatophyte grows. Candida from nails indicates paronychia more often than primary nail infection.
• A dermatophyte from a normal looking scalp is a carrier state and is treated in the context of household transmission.

Topical therapy cures glabrous skin infection. Oral therapy is necessary for scalp and hair, most nail disease, extensive or folliculitic disease, and immunocompromised patients, because topical drug does not reach the hair follicle or nail bed in effective concentration.

Terbinafine resistance is the principal clinical problem.

• Mechanism: point mutations in the squalene epoxidase gene (SQLE), including Leu393Phe and Phe397Leu, reduce binding of terbinafine. Efflux pump overexpression contributes to azole tolerance.
• Trichophyton indotineae: widespread in the Indian subcontinent, with spread to the Middle East, Europe, Southeast Asia, North America and elsewhere. It causes extensive, itchy, annular lesions of the trunk, groin and thighs that fail to respond to terbinafine at standard dose and duration. Azole resistance is also reported.
• Trichophyton rubrum: sporadic SQLE mutations in refractory onychomycosis and tinea.
• Resistance should be suspected when a standard terbinafine course of 2 to 4 weeks produces no improvement in culture confirmed disease, when the infection is extensive and relapses early, or when the patient has recently travelled to or lives in an endemic area. Confirmation needs sequencing or reference laboratory susceptibility testing. Itraconazole is the usual alternative, with dose and duration guided by response and sometimes by drug level.