Identification of Dermatophytes Isolated from Tinea Capitis Patients and Their in vitro Susceptibility to Terbinafine

INTRODUCTION

Tinea capitis is a common dermatophyte infection of the scalp hair and mainly happening at a young age¹. Its clinical manifestations are determined through hair invasion by the dermatophyte and the host's immune response, ranging from mild scaling with little hair loss to severe inflammatory and pustular presentations with scarring alopecia². Tinea capitis caused by Trichophyton (T), and Microsporum (M) dermatophytes genera can dissolve keratinized tissue ¹.

Antifungal drugs that are used to treat dermatophytosis direct the ergosterol synthesis pathway. Azoles inhibit lanosterol 14-α-demethylase, leading to the aggregation of sterol precursors, resulting in disruption of plasma membrane structure and  function³.  In  contrast,  terbinafine  belongs  to

allylamines inhibits squalene epoxidase enzyme, which isinvolved in the initial steps of ergosterol biosynthesis ⁴. This inhibition results in the aggregation of squalene and fungal growth inhibition ⁵.

However, the effect of the drug is different according to the infectious agent. Terbinafine was reported to be higher in treating T. tonsurans and T. violaceum, while griseofulvin was higher in treating M. canis and other Microsporum species ^6, 7. Lipozencic et al. ⁸ showed that the use of terbinafine for a long duration does not enhance its activity against Microsporum species, concluding that terbinafine not recommended as a first- line in treating tinea capitis caused by Microsporum species

Therefore, the importance of early identification of dermatophyte species and their antifungal susceptibility profiles is mandatory for establishing ways to control the spread of dermatophytosis ⁹.

This study was conducted to identify species and in vitro antifungal susceptibility profile of terbinafine on dermatophytes, accompanied by sequencing of the highest minimal inhibitory concentration (MIC) isolates.

PATIENTS AND METHODS

A descriptive cross-sectional study was conducted at Al-Azhar Hospital outpatient clinic, Al Azhar University (Assiut), and Assiut University Mycology Center (AUMC), in the period from the 1st of July 2019 to the 15th of July 2020.  The study protocol was reviewed and approved by the Ethical Review Committee of Al Azhar Faculty of Medicine (Assiut). All participants have signed informed consent.

Study Participants

Fifty patients suspected clinically as tinea capitis infection were asked to participate in the study. Data was taken from the patients, including age, sex, presenting symptoms, and signs. Patients who received antifungal therapies in the previous two weeks were kept out from the study.

Sample Taking and Transport

The suspected tinea capitis lesions were cleaned with 70% ethyl alcohol, then hair, including the roots, was plucked for hair samples, a sterile scalpel was used to collect scales from the scalp. Samples were transported in sterile plastic petri dishes and were divided between microscopic examination and culture.

Microscopic Examination and Culture of Collected Samples

Direct microscopic examination was done to all samples using 20% potassium hydroxide (KOH) and lactophenol cotton blue (LCB) followed by gentle heating for 3-4 seconds. Every sample was inoculated on Sabouraud'sdextrose agar (SDA) plates (Himedia Company, India). Chloramphenicol (0.5 gm/L) was added to prevent bacterial contamination and to prevent saprophytic fungi cycloheximide (0.5 gm/L) was added. Incubation of plates at 28ºC for at least six weeks was done ¹⁰.

In vitro Susceptibility Testing to terbinafine

Antifungal activity study was performed in vitro against six isolates of Microsporum canis and six of T. violaceum using modified agar cup diffusion method. The isolates were subcultured on SDA at 28°C, and dermatophyte suspension was prepared in Sabouraud's broth by smoothly scraping the colonies' surface with a sterile cotton swab then vortex and permitted to settle. The final inoculum concentration was adjusted to 1 × 10³ to 3 × 10³ CFU/mL. SDA was poured into Petri dishes at 50–70 °C, and it was left to solidify under ultraviolet rays insterile category II laminar flow for 15 min. Standardized inoculum 300ul was streaked by cotton swab over the agar medium. Cylindrical plugs were removed from the agar by a sterile cork borer. Terbinafine was obtained as a powder (Sigma-Aldrich), a stock was made by dilution with Dimethyl sulfoxide (DMSO) kept at -70 °C. 50ul of final concentrations in distilled water ranged from 4ug/ml to 0.001ug/ml were added to the wells. The plates were incubated at 28 °C, and then the inhibition zones were measured in millimeters¹¹.

Molecular Identification

 DNA extraction:following the manufacturer's instructions, DNA was extracted by QIAamp DNA Mini Kit extraction kit (QIAGEN,  Germany).

Sequencing

The extracted product was sent to SolGent lab (Daejeon, South Korea). Before sequencing, using two universal fungal primers, Internal transcribed spacer (ITS1) (forward) and ITS4 (reverse), the ribosomal RNA(rRNA) gene was amplified by PCR reaction. Primers used have the following composition: ITS1 (5' - TCC GTA GGT GAA CCT GCG G - 3'), and ITS4 (5'- TCC TCC GCT TAT TGA TAT GC -3') ¹². The PCR reaction was prepared using Solgent EF-Taq (SolGent, Daejeon, South Korea).

RESULTS

The current work was a descriptive cross-sectional study of 50 patients having clinically suspected tinea capitis infections. Out of these 50 patients, 21 were diagnosed with tinea capitis scaly type while 15 patients were having tinea capitisblackdot type with scales. Blackdot without scales was noted in 10 patients, and four patients were having kerion.

Data analysis showed that 43 were males (86%) and 7 were female (14%). The youngest was 1.4 years old, and the eldest was 14 years old male patients with mean age 6.1± 2.67 years old. Tinea capitis infections were found to be more prevalent in males than in females.

Out of 50 samples, fungi were demonstrated in 68 % of cases by direct microscopy and 78% by culture.  Both of them were positive in 48% of the samples. Dermatophyte species isolated from tinea capitis infection were 16 (32%) isolates of  M. canis and 23(46%)  isolates of T.violaceum, as shown in (Table 1).

Table 1: Showing the number and (%) of tested and positive patients with fungal growth (KOH: potassium hydroxide, T:Trichophyton, M:Microsporium).

On examination, it was observed that scales and +ve hair pulling were the most common signs in tineacapitis. Also, 100% of kerion cases were presented with a secondary bacterial infection. Most of the pa­tients were presented with a single lesion (n=32, 64%) where­as multiple lesions were noted in 36%. The type of the lesions was mainly black dot in 50% ( n= 25); however, scales were common in both types as shown in (Table 2).

Table 2: Showing clinical profile in different clinical presentations (N=number, 2ry= secondary)