Dermatophytes and Aspergillus

Dermatophytes

Dermatophytes are fungi that infect only superficial keratinized structure (skin, hair, and nail), not inner tissues. However not all the superficial mycoses are caused by dermatophytes. The genera Trichophyton, Epidermophyton and Microsporum are the principal genera belonging to dermatophytes. The dermatophytes are also commonly known as the ringworm fungi and the infection caused by heat and humidity. For e.g. :Athletes' foot. They are characterized by pruritic papules and vesicles, broken hair, and thickened broken nails.

Most species of dermatophytes produces two types of asexual conidia, known as microconidia and macroconidia. Microconidia are small and unicellular whereas macroconidia are small and large, septate may have thick or thin walls. The macroconidia provide differentiation of dermatophytes into 3 genera.They are as follow:

1. Epidermophyton
2. Microsporum
3. Trichophyton

Epidermophyton

• Clusters of 2 or 3
• 2-4 celled (multiseptate)
• Thick walled, smooth walled
• Club-shaped
• Numerous
• Size:20-40 X 6-8mm

This genus has only one species which produces large, smooth thick macroconidia that are usually composed of 2 or 4 cells. The conidia are usually abundant and occur in a cluster of 2or3. Microconidia are not produced by Epidermophyton.

Microsporum

• Vary in number (rare to numerous)
• Spindle-shaped (ellipsoidal)
• Thick walled, rough-walled
• Multiseptate (5-10 cells)
• Size 100 * 6 – 8mm

The macroconidia of Microsporum vary in numbers from rare to numerous and are spindle shaped with thick rough walls. Each macroconidium may be divided into 5-10 cells and can be up to 100mm in length.

Trichophyton

The macroconidia of Trichophyton vary in shaped from that of a cigar to a cylinder and measure 8-50mm in length and 4-8mm wide, walls of macroconidia are smooth but may be thick or thin.

Lab diagnosis

Specimens: skin scales, crusts, nail pieces, hair

Transformation of specimens: ringworm specimens are best transported in paper packages rather than in screen capped containers to reduce humidity and multiplication of bacteria. Spores of ringworm fungus can resist drying and remain viable for several months when stored in papers.

Direct microscopy

The direct microscopical examination of sales, crusts, nail pieces or hair is the recommended method of diagnosing ringworm. In this method, specimen are first softened and cleared with a strong alkali such as 10-20% KOH. The purpose of the alkali is to digest the keratin surrounded the fungi so that the hyphae and conidia can be seen.

The addition of dimethyl sulphoxide (DMOS) to KOH enables specimens to be examined immediately or after only a few min. The calcofluor white fluorescent stain may be used especially where the lab workers lack experience and fungal preparation can be observed under fluorescent microscope. Fungal hyphae appear bright green against a dark background.

Clearing time when using KOH without DMSO

Hair 5-10 min.

Skin scales and crust: 20-30 min.

Pieces of nails may take several hours.

Culture

Culture is needed to identify the species of ringworm fungus. A typical microbiology medium such as malt agar or sabo urad's agar is used for the cultivation of dermatophytes. Incubation temperature is maintained at 28-30⁰C which is the optimum temperature for all except one of the dermatophytes, Trichophyton verrucosum ,which grow best at 37-degree centigrade. The incubation period is usually 2 to 3 weeks .Selective antibiotics for inhibition of contaminating bacteria and antifungal agents such as cycloheximide for reduction of contaminating fungi may be used in the culture media . The dermatophytes are distinguished on the basis of hyphae structure and structure of macroconidia .

Aspergillus

Aspergillus species are saprophytic molds. They exist only as molds. They have septate hyphae that from characteristic V-shaped (dichotomous branched) at 45⁰ angles. The walls are more or less parallel, in contrast to the walls of mucor and rhizopus which are irregular. They form asexual spores called conidia and the conidia of Aspergillus from radiating chains, in contrast to that of mucor and Rhizopus which are enclosed within a sporangium.

Among Aspergillus sp , Aspergillus fumigatus is the main opportunistic pathogen. Other species Aspergillus niger, Aspergillus flavus, associated with diseases.

Virulence factors and pathogenesis

Elastage production

Aspergillus Fumigatus produces 2 types of elastase enzymes which act on elastin protein that comprises approximately 30% of the lung tissue.

Catalase production

Aspergillus species also produces catalase enzymes that may be a virulence factor contributing to aspergillosis associated with chronic granulomatous diseases of childhood.

Aflatoxin

Aspergillus flavus produces aflatoxin and aflatoxin produced by this fungus are known carcinogenic hepatotoxin.

Diseases caused by Aspergillus species

Diseases caused by Aspergillus species is commonly called Aspergillosis. There are three clinical forms of aspergillosis.

• Respiratory diseases

1. Aspergillus asthma

It is an allergic bronchopulmonary aspergillosis due to hypersensitivity to Aspergillus antigen i.e. inhaled air-bone conidia. The fungus grows in the lumen of bronchioles and produces plugs of mycelium and mucous that may block the lumen.

2. Aspergilloma fungus ball

It is often called fungus ball in which fungus colonizes in the pre-existing cavities, often in the case of TB cavity i.e. in aspergilloma inhaled conidia germination in a pulmonary cavity and grows into fungus ball.

3. Disseminated (systemic) Aspergillosis

In this case, the fungus first establishes in the lungs tissues and then disseminates to different organs such as a brain, kidney, heart and other organs particularly in immunocompromised patients.

• Superficial infections

Aspergillus flavus and Aspergillus fumigatus colonize paranasal sinuses(sinusitis), external ear(otomycosis) and in some case eyes(mycotic keratitis)

Lab diagnosis

Specimens

Sputum and biopsy materials are generally used as preferred specimens.

Direct microscopy

Direct microscopy in KOH mount of exudates shows on pigments septate mycelium (3-5mm)in diameter of the fungus with characteristic branching(dichotomously branched) at 45⁰ angles. Gram`s staining may give gram positive reaction. In a case of a fluorescence microscope, calcofluor white stain may be used for direct observation of Aspergillus species.

Culture

Aspergillus fumigatus is a rapidly growing mold (2-6 days) that produces fluffy dextrose agar. Matured sporulation colonies most often exhibit the blue-green powdery appearance. Microscopically, A.fumigatus is characterized by a presence of septate hyphae with conidiophores on it. The tip of the conidiophores expands into large dome-shaped vesicles that have bottle shaped phialides covering the upper half or two-thirds of its surface. Long chains of small, spherical, rough-walled green conidia form a columnar mass to the vesicles , a culture of A.fumigatus is thermotolerant and is able to withstand temperature up to 45⁰C.

Aspergillus flavus produces yellow-green colonies on sabo urad's dextrose agar. However, A.niger beings initially as a yellow colony that soon develops a black dotted surface as conidia are produced.

Skin test

Intradermal skin test to Aspergillus antigen extracts is useful for patients suspected of allergic bronchial pulmonary Aspergillosis.

Prevention and treatment

There are no specific means of prevention. Invasive aspergillosis is treated with Amphotericin B but caspofungin may be effective in a case of invasive aspergillosis that does not respond to Amphotericin B. A.fungus ball growing in sinuses or in a pulmonary cavity can be surgically removed.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.