Classification of dermatophytes SlideShare

Isolation, Identification, and In Vitro Antifungal Susceptibility Testing of Dermatophytes from Clinical Samples at Sohag University Hospital in Egypt

Abstract

1. Introduction

Dermatophytes are a group of closely related filamentous fungi able to damage and utilize keratin found in the skin, hair, and nails [1]. They are classified into three genera: Microsporum, Trichophyton, and Epidermophyton. Dermatophytosis is an infection produced by dermatophytic fungi in the keratinized tissues [2]. These cutaneous mycoses affect 20% to 25% of the worlds population [3]. Clinically, dermatophytosis can be classified depending on the site involved. These include Tinea capitis [scalp], Tinea corporis [non-hairy skin of the body], Tinea ungium [nail infection], Tinea cruris [groin], Tinea pedis [athletes foot], and Tinea barbae [bearded areas of the face and neck] [4]. The dermatophytosis transfers from animal [zoophilic dermatophytes] and soil [geophilic dermatophytes] to man or through direct infection by personal contact [anthropophilic dermatophytes] [5]. The hot and humid climate in tropical and subtropical countries like Egypt makes dermatophytosis a very common fungal skin infection. Generally, they are superficial infections, but in immunocompromised patients they are experienced as a disseminated disease. The gross appearance of the lesion includes an outer ring of active, progressing infection with central healing within the ring accompanied with itching, redness, scaling, or fissuring of the skin [6]. If the infection involves the scalp, areas of scaling with dull gray hair stumps up to a highly inflammatory reaction with folliculitis, kerion formation, and alopecia [7]. More aggressive infections may lead to an abscess or cellulitis. Areas infected by dermatophytes may become secondarily infected by bacteria. Symptoms typically appear between 4 and 14 days following exposure [8]. The diagnosis of a dermatophytic infection is mostly done clinically, but often confused with other skin infections due to the topical application of steroid ointments and creams, leading to further misdiagnosis and mismanagement [9]. Hence, there arises the need for the correct, efficient, and rapid laboratory diagnosis of dermatophytes. The laboratory identification of dermatophytes is typically based on the macroscopic observation of colony morphology [pigmentation, growth rate, texture, etc.] grown on selective media, followed by a microscopic examination of conidia [10]. Another important point to consider is that resistance to antifungals has started appearing in dermatophytes. The establishment of a reference antifungal susceptibility testing method may allow the clinician to select the appropriate therapy for the treatment of infections caused by dermatophytic fungi. The evaluation of in-vitro susceptibility testing has been hampered due to the lack of reliable in-vitro techniques for testing antifungal agents against dermatophytes. The agar-based disk diffusion [ABDD] susceptibility method for dermatophytes is quick, easy, and inexpensive and does not require specialized equipment, making it a good option [11]. However, data on disk diffusion methods for dermatophytes are scarce [12]. Given this background, our study focused primarily on how to isolate, identify, and detect the in-vitro sensitivity pattern of various dermatophytes isolates, from clinically suspected cases of dermatophytosis to the most commonly used antifungal agents, using the ABDD susceptibility method.

2. Material and Methods

3. Results

A total of 135 clinically suspected cases of skin and hair dermatophytosis were included in the study. Dermatophytes were demonstrated in 110 cases [81.5%]71 [64.5%] males and 39 [35.5%] females-through direct microscopy and/or culture. The age of the study group ranged from 5 to 56 years [mean age 30.5 years], with the maximum number of patients falling in the 1630 years age group [32.7%], followed by 515 years [30%]. A considerable number of the patients [39.1%] were from rural areas and engaged in occupations related to agriculture, and 22.7% of patients were students. The patients were clinically diagnosed and mycologically confirmed to have tinea capitis [49 cases; 44.5%] presented with hair loss, scaling, and/or black dots [Figure 9]; tinea corporis [30 cases; 27.3%] presented with pruritic, characteristic annular progressive scaly plaque with a burning sensation and healed center [Figure 10]; tinea pedis [16 cases; 14.5%] presented with moist peeling irritable skin in the cleft between the toes [Figure 11]; tinea cruris [9 cases; 8.2%] presented with pruritus and erythematous patches with sharp border in the groin; and tinea barbae [6 cases; 5.5%] presented with red plaques, pustules, and hair loss in moustache and beard [Table 2].

The Dermasel agar culture was able to detect 110 cases [81.5%] from the 135 clinically suspected cases of skin and hair dermatophytosis whereas the culture on SDA was able to detect 107 cases [79.3%]. No statistically significant difference was evident between the two media [P< 0.05] for primary isolation of dermatophytes from the clinical samples. Ninety-seven cases [71.9%] were positive in both the microscopic examination and culture, 13 cases [9.6%] were negative in microscopy but culture positive, 6 cases [4.4%] were positive in microscopy and negative in culture, and 19 cases [14.1%] were negative in both microscopy and culture. Thus, the culture is more accurately confirmatory than the microscopic mount. The sensitivity and specificity of the 10% KOH/40% DMSO mount examination was calculated as 88.2% and 76%, respectively, when compared with reference standard culture results [Table 3]. A total of 110 isolates of dermatophytes was identified by macroscopic and microscopic morphological characteristics. The isolates belonged to two genera: Microsporum [92 cases]the most prevalent cause of dermatophytosis in our study, accounting for 83.6% of isolatesand Trichophyton [18 cases; 16.4%]. The isolates included four species: [M. canis {zoophilic} 58 [52.7%], M. gypseum {geophilic} 23 [20.9%], M. audouinii {anthropophilic} 11 [10%], and T. mentagrophytes {zoophilic} 18 [16.4%] [Table 4]. The results of the susceptibility testing to antifungal drugs were as follows: Clotrimazole105 [95.5%] susceptible and 5 [4.5%] of intermediate susceptibility; Miconazole93 [84.5%] susceptible, 12 [10.9%] of intermediate susceptibility and 5 [4.5%] resistant; Griseofulvin51 [46.4%] susceptible and 59 [53.6%] resistant; and Fluconazole104 [94.5%] resistant and 6 [5.5%] of intermediate susceptibility. Based on these data, Clotrimazole was the most effective antifungal drug and Fluconazole had the poorest activity [Table 5]. In general, different species of dermatophytes showed similar patterns of susceptibility to antifungal agents tested [Table 6].

4. Discussion

Dermatophytosis is a major public health problem in tropical and subtropical countries, like Egypt, yet remains unresolved. Therefore, it is essential that good laboratory methods are available for the rapid and precise identification of the dermatophytes involved in order to apply appropriate treatment and prevention measures. In the present study, 110 clinically suspected cases of tinea infections were examined over a period of 12 months, among which the most common tinea infections were T. capitis [49 cases], followed by T. corporis [30], T. pedis [16], T. cruris [9], and T. barbae [6 cases]. This finding concurred with Omars [14] results, which emphasized that T. capitis is considered the major type of fungal infection in Egypt. Our results also agreed with those of Achterman and White [15], who reported thatalthough dermatophytosis is found throughout the worlddeveloped countries have high rates of tinea pedis and onychomycosis whereas developing countries, like Egypt, have high rates of tinea capitis. In the present study, most of the infections were seen in the younger age group [530 years], which may be due to increased physical activity and increased opportunity for exposure. Indeed, Madhavi et al. [2011] found that tinea infections were more common in the 16- to 45-year-old age group. In our study, a higher incidence of tinea infection of the capitis variety in particular in school-going children, resulting in increased transmission between them, would be due to increased contact, overcrowding in classrooms [a significant problem in Egypt], a lack of awareness and apathy to personal hygiene, the sharing of personal items, and exposure to soil and even animals on playgrounds. A higher incidence of dermatophytosis was seen in males than in females, which supports Sumathi et al.s [17] findings. Male predominance may be due to increased outdoor physical activities and increased sweating, which create a favorable environment for fungal infections, as well as a greater opportunity for exposure to infection than females. In our study, the most common genus of dermatophytes detected was Microsporum, followed by Trichophyton. However, Jha et al. [18] found that the most common genus was Trichophyton, followed by Epidermophyton. In the present study; M. canis [zoophilic dermatophytes] was the most common isolate [58 cases; 52.7%], followed by M. gypseum [geophilic dermatophytes; 23 cases; 20.9%], T. mentagrophytes [zoophilic dermatophytes; 18 cases; 16.4%], which could be due to patients interaction with soil and domestic animals, and finally M. audouinii [anthropophilic dermatophytes; 11 cases; 10%]. This finding did not agree with that of Kannan et al. [19], who reported that Trichophyton rubrum [73%] was the most common isolate, followed by Trichophyton mentagrophytes [17%], Trichophyton violaceum, and finally Epidermophyton floccosum. The differences between our study and previous studies stem from variations in patients environments and lifestyles. In the current study, zoophilic fungi represent 69.1% of the isolated dermatophytes [M. canis 52.7% and T. mentagrophytes 16.4%]. This result did not differ greatly from previously reported findings in Egypt [20], which could be due to the fact that a considerable number of our patients [39.1%] were from rural areas and engaged in agricultural occupations. This finding concurred with Emele and Oyekas [21] findings, which indicated that purely rural agricultural communities, coupled with the associated crowded and unhygienic rural lifestyles and low economic conditions, might have enhanced the spread of the disease among the population in these regions. Although our results demonstrated a predominance of zoophilic dermatophytes, other categories were also detected, which was similar to Nweze and Okafors [22] findings. These researchers concluded that species of dermatophytes causing different types of tinea vary from country to country and also change with time, geography, environment, climate, occupation, and lifestyles. In the present study, aqueous potassium hydroxide [KOH] was used as a clearing agent for the direct demonstration of fungi in skin or hair scrapings [23], but the addition of dimethyl sulphoxide, as described by Rebell and Taplin [24], was found to be a better preparation over plain KOH. The addition of DMSO allows for the rapid clearing of keratin and almost immediate examination of the sample without a warming of the slide [25]. It also prevents the rapid drying of the fluid and, thus, is a better option. Our findings suggested that a 10% KOH/40% DMSO test can be used for the diagnosis of dermatophytes infections in remote conditions, where a rapid and low-cost diagnosis is required. However, false positive cases were recorded in our study: 6 cases [4.4%] were positive by microscopy but negative by culture. In addition, false negative cases were recorded: 13 cases [9.6%] were negative by microscopy but positive by culture. This variation could be due to the non-viability of fungal elements in some cases, inadequacy in sampling due to very small lesions, and possible non-reported partial treatment with antifungal agents. On the other hand, Tampieri [26] reported that it seems difficult to rely on results of direct microscopy with KOH to establish the diagnosis of fungal infection as it could not detect the characteristic morphology of the three genera and it lacks sufficient sensitivity, although it is highly efficient as a screening technique before therapy is initiated because of the expense, duration, and potential adverse effects of the treatment. Given that culture is the gold standard for the isolation and identification of dermatophytes, the sensitivity and specificity of the 10% KOH/40% DMSO mount examination in our study was calculated as 88.2% and 76%, respectively, when compared with the reference standard culture results. This finding concurred with Girgis et al. [27], who found that KOH direct microscopy had a sensitivity of 88% and specificity of 74%. Culture is considered the mainstay of diagnosis because it not only isolates the organism, but also allows for the identification of the etiologic agent, thereby allowing treatment to be tailored appropriately. In our study, two media were used for culture of samples; both the Dermasel agar and SDA were supplemented with chloramphenicol and cycloheximide. The two media proved to be technically good, with no statistically significant difference between them [P