Trichophyton mentagrophytes - Laboratory diagnosis

Laboratory diagnosis of Trichophyton mentagrophytes

Laboratory diagnosis of Trichophyton mentagrophytes is done by demonstration of fungal particles such as hyphae, and spores in the specimen.

Samples

• Hair

• Skin

• Nail scrapping

Microscopy

Microscopy (direct microscopy) is performed following KOH preparation to demonstrate fungal particles in the specimen. Spiral hyphae are characteristic of Trichophyton mentagrophytes.

KOH preparation

The procedure of KOH preparation for fungal infection diagnosis is as follows.          

Procedure

1. the specimen is placed on a clean, grease-free slide and a drop of 10-20% KOH solution is added to it

2. the sample is covered with a coverslip and immediately viewed under a microscope

3. the KOH preparation is examined again after 20 mins

4. in skin or nail specimens, chains of spores or branching hyphae are seen

5. in hair specimen, parallel rows of spores are observed inside (endothrix) the hair shaft

* infected hair shaft may also contain air spaces

* skin test does not distinguish active infection

Hair perforation test

The hair perforation test is done mostly to distinguish Trichophyton rubrum from Trichophyton mentagrophytes, which are morphologically similar.

Trichophyton rubrum causes only surface erosion of the hair shaft while from Trichophyton mentagrophytes infection results in wedge-shaped perforation.

Procedure

1. hair specimens (5-10mm in size) are placed in a Petri dish with 20ml distilled water and autoclaved

2. sterile 10% yeast extract (2-3 drops) is added to the Petri dish

3. the hair strands are inoculated with small fragments of test fungi that have been grown on SDA

4. the inoculated hair strands are incubated at 25°C

5. for up to a month, the inoculated hair strands are examined weekly after the addition of Lacto Phenol Cotton Blue (LPCB)

Wood’s lamp examination

Wood’s lamp examination of Trichophyton mentagrophytes is done by the use of wood’s lamp. This device is used mostly in the diagnosis of fungal infection of the scalp.

Wood’s lamp glass consists of barium silicate containing about 9% nickel oxide. It transmits long-wave UV light with a peak of 365nm. As a result, a distinct fluorescence can be detected on hair infected by dermatophytes.

In Trichophyton mentagrophytes infection, Wood's lamp (blacklight) examination shows bright green to yellow-green fluorescence of hairs.

Culture

The culture of Trichophyton mentagrophytes is done as a diagnostic step.

The specimen is inoculated onto Sabouraud’s Dextrose Agar (SDA) incorporated with chloramphenicol and cycloheximide and incubated at 25 to 30°C aerobically for 1 to 3 weeks.

The fungi are identified on the basis of colony morphology, color, pigment production, and the presence/absence of microconidia or macroconidia.

The fresh culture was extracted from the agar plate using cellophane tape and placed on a slide containing a drop of Lacto Phenol Cotton Blue (LPCB). The preparation is viewed under a microscope to demonstrate the presence/absence of microconidia or macroconidia.

Trichophyton mentagrophytes colonies may range from granular to powdery. They produce abundant grapelike clusters of subspherical microconidia on terminal branches. In 30% of isolates, the hyphae are spiral while the thin-walled microconidia are smooth, club-shaped, and multiseptate. Each microconidium may measure 6 to 8 µm in diameter and 20 to 50 µm in length.

Urease test

A urease test is done for distinguishing Trichophyton rubrum (urease negative) from Trichophyton mentagrophytes (urease positive).

Procedure

1. a sterile Christensen’s urea agar is taken and inoculated with test fungi

2. the test tube is incubated at 25°C for 5 days

3. if the color of the agar changes to pink in color, it is urease positive

Molecular test

The molecular test used for diagnosis of Trichophyton mentagrophytes is:

• PCR-reverse line blot test

• real-time PCR test

• multiplex PCR test

• PCR-ELISA test

• MALDI-TOF test