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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 16  |  Issue : 1  |  Page : 16-21

Isolation and identification of fungus associated with skin and nail scalps of patients in a tertiary care teaching hospital


1 Department of Gastroenterology, IMS and SUM Hospital, Siksha “O” Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India
2 Department of Microbiology, IMS and SUM Hospital, Siksha “O” Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India
3 Department of Skin and VD, IMS and SUM Hospital, Siksha “O” Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India
4 Medical Research Laboratory, IMS and SUM Hospital, Siksha “O” Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India

Date of Web Publication11-Mar-2019

Correspondence Address:
Mahesh Chandra Sahu
Medical Research Laboratory, IMS and SUM Hospital, Siksha “O” Anusandhan (Deemed to be University), Bhubaneswar - 751 003, Odisha
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/am.am_80_18

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  Abstract 


Background and Objectives: Ringworm of the scalp is starting at now a disease of overall criticalness and a general prosperity teaching care hospitals. It is otherwise known as dermatophytes, which attack and create on dead animal keratin. This assessment was intended to recognize the regular dermatophyte causing parasitic infection with both minute just as social strategies. Materials and Methods: This is a Prospective study which was carried out for the period 1 year and included the patients with tinea cruris and tinea corporis, tinea unguium, tinea pedis, and tinea barbae which are seen in the patients between 6 to 70 years of age. Dermatophytes were confirmed with microscopic examination of skin and nail tests taken from the influenced lesion of the body. Both cotton blue and potassium hydroxide grouping of 10%– 30% were utilized to see under magnifying micrioscope. All information were broken down with SPPS 20 programming. Results: Out of 78 patients, the recurrence of ringworm infection among those patients attending in the department of Skin and VD, IMS and SUM Hospital was Trichophyton rubrum (73%), Trichophyton mentagrophytes (53%), Epidermophyton floccosum (26.82%), and Trichophyton verrucosum (7.31%). T. rubrum was the most widely recognized etiological fungus in tinea corporis, and this positioned first in the other type of ringworm infection. Recurrence of dermatophytes infection partner with various clinical examples; for example, nail pieces and skin swabs from the infected area. Conclusions: The greater part of the tinea corporis patients were infected with T. rubrum then comes the disease of Trichophyton violaceum, Trichophyton tonsurans, and T. mentagrophytes. The most astounding recurrence of disease was among the patients matured 20– 30 years. It is additionally essentially noticed that this infection takes a somewhat serious turn in patients of cutting edge in older age (60– 80 years).

Keywords: Dermatophytes, lactophenol cotton blue, potassium hydroxide, potato dextrose agar, sabouraud dextrose agar


How to cite this article:
Uthansingh K, Sahu MK, Debata NK, Behera D, Panda K, Sahu MC. Isolation and identification of fungus associated with skin and nail scalps of patients in a tertiary care teaching hospital. Apollo Med 2019;16:16-21

How to cite this URL:
Uthansingh K, Sahu MK, Debata NK, Behera D, Panda K, Sahu MC. Isolation and identification of fungus associated with skin and nail scalps of patients in a tertiary care teaching hospital. Apollo Med [serial online] 2019 [cited 2019 Jul 16];16:16-21. Available from: http://www.apollomedicine.org/text.asp?2019/16/1/16/253873




  Introduction Top


Dermatophytes are the most common infection in the human body. Mostly affected areas are scalp, skin, and nails of the body. These dermatophytes are closely related to fungi that have the capacity to invade the keratinized tissues of above.[1] It includes 3 genera, i.e., Epidermophyton, Microsporum, and Trichophyton. These fungi colonize in the keratin tissues and are repeatedly restricted to the nonliving cornified layer of the upper cell layer of the tissues. Dermatophytes are also associated with secondary bacterial infections leading to systemic skin infections. According to WHO, the prevalence rate of superficial mycotic infection worldwide has been found to be 20%–25%. Dermatophytic infections are commonly encountered in more than 50% of patients attending dermatology outpatient departments in South India.[2] The tinea infections are prevalent globally, but they are common in tropics and in geographical areas with higher humidity, overpopulation, and poor hygienic living conditions. The clinical appearance and the causative types of shallow parasitic infection shift with geographic locale, financial conditions and propensities.[3] It is transmitted by either direct contact or indirect contact (fomites). An increasing frequency of dermatophytosis has been observed during last two decades especially in immunocompromised patients such as AIDS, diabetes mellitus, cancer, and organ transplantation patients. Dermatophytoses generally respond well to topical antifungal therapy although systemic therapy would be required for extensive infections or for infections affecting the nails or scalp. The present study was conducted to isolate and identify dermatophytes from skin, hair, and nail samples of clinically suspected cases of dermatophytosis by potassium hydroxide (KOH) and culture on sabouraud dextrose agar (SDA) and potato dextrose agar (PDA) media.


  Materials and Methods Top


This study was carried out on 78 clinically suspected cases of dermatophytosis irrespective of gender, attending the outpatient Department of Skin and VD, IMS and SUM Hospital, Bhubaneswar, Odisha from March 2013 to February 2014.

All the clinical samples were collected from the Skin and VD Department, IMS and SUM Hospital, Bhubaneswar. Two types of samples were taken for this study such as skin scraping and nail clipping collected with a sterile swap stick and fairly blunt scalpel for scraping the skin or nail of affected patients. For preliminary diagnosis, the microscopic examination was carried out in the Department of Microbiology, IMS and SUM Hospital, Bhubaneswar. Dermatophytes were identified by the presence of translucent, nonpigmented, septate mycelium and arthrospores. Then, the remaining samples were cultured in SDA medium, and whenever the growth observed in the culture tubes, it was cultured by PDA media. Sterilized equipments were used to avoid contamination by nonpathogenic fungi and bacteria. The equipment and the media were used for the collection of specimens from the patients and the isolation and identification of the etiologic agents. Materials such as ultraviolet lamp, wood lamp, epilating forceps, nail clippers, scalpel, scissors, inoculating needle, sterile test tubes, and  Petri dish More Detailses, clean slides, and sterile cottons were used for collection and culture the samples.

Culture media

For the isolation and identification of the etiologic agents, SDA and PDA media were used. Both these culture media for the growth of fungi had a high concentration (20 gm/L, Himedia, Mumbai) of either glucose or dextrose, and it contained mycological peptone. The medium was in low pH 5.0, which inhibited the growth of most bacteria. Antibacterial agents – gentamicin (50 mg/l of medium) were added to augment the antibacterial effect.

Process of materials for culture

Clinical materials were collected from the patients suffering from various types of dermatomycoses. Proper collection of clinical materials was important for both the method of identification. Following types of specimens were included.

Skins scrapings

Scrapings were obtained from the lesions involving the skin with a sterile scalpel after carefully washing the site with 70% alcohol. Scrapings taken from active border areas of lesions were placed in sterile petri dish for laboratory examination and culture. In some lesions, vesicles were present which were carefully clipped off with small sterile scissors and successful microscopic. and cultural examination was made.

Nail clippings

Ringworm infected nails [Figure 1] were found to be thickened and deformed. Clippings of nail, especially near the bed of the nail were collected in a sterile petri dish for microscopic examination and culture. The patients suffering from tinea capitis and tinea favosa were examined under normal light for areas without hair, scaling, crust formation, hair stumps, and erythema. The tinea capitis patients were then subjected to Wood's light examination in a dark room to determine the fluorescence. The basal portion of the hair or the hair tufts was collected as the fungus is usually found in this area.
Figure 1: Clinical symptom of patients with dermatophytes

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Microscopic examination of clinical materials

The clinical material so obtained was dissolved in two drops of 10% KOH on a glass slide. If the nail clippings were thick, 20% KOH was used. After putting the coverslip, the slide was heated for few seconds over spirit lamp taking care that the material should not boil. The preparation was observed under the low power of the microscope in reduced light. The presence of mycelia fragments and the distribution of spores inside the hair were noted. For detailed microscopic morphology of the etiologic agent in clinical material, the slide preparation was studied under the high power of the microscope, and the observations were recorded [Figure 2].
Figure 2: Direct microscopic methods with potassium hydroxide

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Culture of the clinical specimens

Sabouraud dextrose agar with gentamicin medium was used for the isolation of the dermatophytes from the clinical material. The agar slants were inoculated by placing skin scrapings or nail clippings with the help of a sterile needle on the slant surface of the medium. All the tubes inoculated were incubated at room temperature at 30°C for 3–4 days. Slants were examined every 4–6 days [Figure 3] and [Figure 4]. If any saprophytic fungus appeared, the suspected colony of dermatophyte was transferred to other slants then inoculated the specimen as soon as possible after it is received in the laboratory. Place the specimen onto the center of the agar with sterile forceps. Press carefully to ensure firm contact with the agar surface. Replace the cap but do not tighten completely. Incubate the inoculated media at 30°C for 4 days (do not incubate cultures at 37°C). Examine the culture daily for a change in the color of the medium and evidence of fungal growth. Expected cultural response on dermatophyte test medium at 30°C after 2–7 days.  Escherichia More Details coli inhibition, partial to complete are mycelial fungi which possess keratolytic properties that allow them to invade skin, nails, and hair [Table 1]. Dermatophytes test medium incorporates antibiotics that suppress the growth of saprophytic fungi and contaminating bacteria while allowing the growth of dermatophytic fungi. Dermatophytes are presumptively identified by gross colonial morphology and the production of alkaline metabolites which cause a color change in the medium from yellow to red.
Figure 3: Difference dermatophytes cultured on slants

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Figure 4: Difference dermatophytes cultured on slants with reverse

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Table 1: Generic subdivisions of dermatophytes

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Identification of the growth of the dermatophytes (microscopic after culture)

The skin sample applied for KOH mounting and observed in the low to high magnificent microscope, i.e., in ×10, ×40, and ×100. By the application of KOH, the fungal elements seen were only the fungal hyphae [Figure 2]. The remaining sample was used for cultural growth by means of SDA medium. The sample along with the SDA media was kept in the BOD in 30°C for 3–4 days. After that, the colony morphology was observed [Table 2]. The colony morphology were seen as white fluffy growth, velvety red pigment on reverse which is known to be Trichophyton species, white fluffy with reddish in the middle, yellow pigmentation were also seen sometimes when the colony morphology was seen as black in color, it was known to be contaminated otherwise the above all colonial morphology showed the +ve growth [Figure 3]. The fungus which seemed to be positive was taken for microscopic examination [Figure 4]. For microscopic examination, well-grown tubes, sterile scalpel, lactophenol cotton blue, clean slide, and coverslip were taken. Applied 2–3 drops of lactophenol cotton blue on the clean slide and a little amount of fungus from the cultured tube were taken. By the help of botanical needle, the materials were spread over the slide so that the clear examination could possible. After teasing the whole material, they were covered with coverslips and were taken to microscope for fungal identification. It was observed that in two cases, the Trichophyton agent was the most common one because after growth and microscopic examination in the skin as well as in nail samples, Trichophyton rubrum and Trichophyton mentagrophytes were observed in the microscope. It was also observed that in T. rubrum there were pencil-shaped macroconidia. T. mentagrophytes were also seen, i.e., like clusters of macroconidia. Septate spindle-shaped macroconidia were observed. Interestingly, T. mentagrophytes were spiral hyphae which were seen with macroconidias; so, they were confirmed to be Microsporum canis and Microsporum audouinii. Epidermophyton species were also seen, i.e., yellowish green powdery in cultural morphology and club-shaped macroconidia in microscopic examination. All the microscopic observations after using lactophenol cotton blue were taken, which confirms the various dermatophytic organism represented in the [Figure 5].
Table 2: Microscopic and colony morphology of the dermatophytes

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Figure 5: Identification of dermatophytes with cotton blue stains

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  Results Top


A total of 78 samples were obtained from patients with suspected dermatophytes (superficial mycoses). Diagnosis was confirmed by microscopic examination (KOH mounting) was in 22 cases (28.28%) and the causal agents were isolated in culture were in 41 cases (52.56%). From the total isolates identified by culture growth, dermatophytes species were the most common, accounting for 41 cases (52.56%) of all fungal infections. Aspergillus species and Candida albicans were the next most commonly isolates agents with 16 cases (24.61%) and 9 cases (13.85%), respectively. The frequency of ringworm infection among patients attending the Dermatology Clinic (Skin and VD Department of IMS and SUM Hospital) were T. rubrum 30 (73%), T. mentagrophytes 20 (53%), Epidermophyton floccosum 11 (26.82%), and Trichophyton verrucosum 3 (7.31%) [Table 3]. According todermatophytes infection, tinea corporis was the predominant clinical form of all ringworminfection seen, in which 5% of cases were of children below 15 years of age. Tinea pedis wasfound more frequently on adults than females as in males. With the exception of tinea pedis andtinea cruris in which T. rubrum was the predominant isolate. T. rubrum was the most common etiological organism in tinea corporis or ranked 1st in the other form of ringworm infection. Recurrence of dermatophytes infection in relationship with skin swabs from different parts of body.[4] Infection of glabrous skin such as chest, neck, and back was most commonly associated with the lipophilic M. audouinii. The infection was found to occur more frequently in adult males (30) than in females (11). Super facial candidiosis was mainly due to C. albicans (9 cases) and most cases were male. The commonly affected sites were axillaries (19 cases), hand (15 cases), feet (4), and face (3). Nondermatophytic fungi isolated from dermatophytic sample are summarized in [Table 4]. True dermatophytes such as T. rubrum, T. mentagrophytes, E. floccosum, and T. verrucosum were isolated during that period. Dermatophytes found in males and females are also estimated in this short-term research program and it was identified.
Table 3: Frequency distribution of dermatophytes identified

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Table 4: Dermatophytes found in male and in female patients in this study

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  Discussion Top


The incidence of dermatophytic infection is undoubtedly very high. However, neither the medical profession nor the research workers paid any attention toward the investigations of these important human infections. In the present studies, a total of 78 cases of dermatomycoses were studied. Among these 78 cases, 41 or 52.56% were identified as positive. In the present study, clinically suspected cases of tinea infections were examined over a period of 12 months, among which the most common tinea infections were tinea capitis (49 cases), followed by tinea corporis (30), tinea pedis (16), tinea cruris (9), and tinea barbae (6 cases). Our results agreed with those of Achterman and White,[4] who reported that – although dermatophytosis is found throughout the world – developed 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 (5–30 years), which may be due to increased physical activity and increased opportunity for exposure. Indeed, Madhavi et al.[5] found that tinea infections were more common in the 16-year-old to 45-year-old age group. In our study, a higher incidence of tinea infection of the capitis variety in particular in schoolgoing 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.[6] 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.[7] 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.,[8] who reported that T. rubrum (73%) was the most common isolate, followed by T. mentagrophytes (17%), Trichophyton violaceum, and finally E. 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,[9] 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 Oyeka[10] 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 Okafor's[11] 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 KOH was used as a clearing agent for the direct demonstration of fungi in skin or hair scrapings,[12] but the addition of dimethyl sulfoxide (DMSO), as described by Rebell and Taplin,[13] 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.[14] It also prevents the rapid drying of the fluid and, thus, is a better option. Our findings suggested that 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 nonviability of fungal elements in some cases, inadequacy in sampling due to very small lesions, and possible nonreported partial treatment with antifungal electronic physician page 2565 agents. On the other hand, Tampieri[15] 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.[16] 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. 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. 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 < 0.05) for the primary isolation of dermatophytes from clinical samples. However, of much concern was our finding that the culture of dermatophytes on Dermasel agar was more specific with better topography, texture, color, and colony surface and reverse; macroconidia and microconidia are typical for the species when studied microscopically, and less growth of contaminant bacteria and saprophytic fungi is common. This elevates the importance of Dermasel agar as a selective medium for the isolation of dermatophytes and, thus, the treatment of infection. There were reports concerning the failure of topical, as well as systemic antimycotic treatment of dermatophyte infections,[17] thereby making it essential to evaluate and standardize simple and reproducible in vitro assays to determine the antifungal activity of various drugs against dermatophytes. Studies evaluating the in vitro activity of antifungal agents are rare, particularly in filamentous fungi.[18],[19]


  Conclusions Top


We have to perceive and welcome the way that there is an absence of documentation and proof in relatively every risky viewpoint that we have seen with respect to this terrifying pestilence of shallow dermatophytosis in India. The path forward are huge scale methodical investigations demonstrating the relationship of topical steroids, particularly the blends and unending across the board dermatophytosis, or, in other words overwhelming assignment for operational reasons. Dermatology is a visual claim to fame, and fringe well-being laborers can be prepared to distinguish and reasonably treat dermatophytosis. Comprehensive data, instruction, and correspondence exercises, as well as stringent medication controlling laws in India are required.

Acknowledgment

Authors are grateful to Prof. Sujata Mohapatra, HOD, Botany and Biotechnology, Khallikote (Auto) College, Berhampur University and Medical Superintendent, IMS and SUM Hospital, Siksha “O” Anusandhan Deemed to be University for extended facility in research work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Alshawa K, Beretti JL, Lacroix C, Feuilhade M, Dauphin B, Quesne G, et al. Successful identification of clinical dermatophyte and neoscytalidium species by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2012;50:2277-81.  Back to cited text no. 1
    
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Grumbt M, Monod M, Yamada T, Hertweck C, Kunert J, Staib P, et al. Keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump. J Invest Dermatol 2013;133:1550-5.  Back to cited text no. 2
    
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Havlickova B, Czaika VA, Friedrich M. Epidemiological trends in skin mycoses worldwide. Mycoses 2008;51 Suppl 4:2-15.  Back to cited text no. 3
    
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Achterman RR, White TC. A foot in the door for dermatophyte research. PLoS Pathog 2012;8:e1002564.  Back to cited text no. 4
    
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Madhavi S, Rama Rao MV, Jyothsna K. Mycological study of dermatophytosis in rural population. Ann Biol Res 2011;2:88-93.  Back to cited text no. 5
    
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Jha BK, Murthy SM, Devi NL. Molecular identification of dermatophytosis by polymerase chain reaction (PCR) and detection of source of infection by restricted fragment length polymorphism (RFLP). J Coll Med Sci Nepal 2012;8:7-15.  Back to cited text no. 7
    
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Amer M, Taha M, Tosson Z, El-Garf A. The frequency of causative dermatophytes in Egypt. Int J Dermatol 1981;20:431-4.  Back to cited text no. 9
    
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Emele FE, Oyeka CA. Tinea capitis among primary school children in Anambra state of Nigeria. Mycoses 2008;51:536-41.  Back to cited text no. 10
    
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Nweze EI, Okafor JI. Prevalence of dermatophytic fungal infections in children: A recent study in Anambra state, Nigeria. Mycopathologia 2005;160:239-43.  Back to cited text no. 11
    
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Rebell G, Taplin D. Dermatophytes: Their Recognition and Identification. 2nd ed. Miami: Miami University Press; 1974. p. 124.  Back to cited text no. 13
    
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Tampieri MP. Update on the diagnosis of dermatomycosis. Parassitologia 2004;46:183-6.  Back to cited text no. 15
    
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Girgis SA, Zu El-Fakkar NM, Badr H, Shaker OA, Metwally FE, Bassim HH. Genotypic identification and antifungal susceptibility pattern of dermatophytes isolated from clinical specimens of dermatophytosis in Egyptian patients. Egypt Dermatol Online J 2005;2:14-9.  Back to cited text no. 16
    
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Galuppi R, Gambarara A, Bonoli C, Ostanello F, Tampieri MP. Antimycotic effectiveness against dermatophytes: Comparison of two in vitro tests. Vet Res Commun 2010;34 Suppl 1:S57-61.  Back to cited text no. 17
    
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    Figures

  [Figure 1], [Figure 2], [Figure 4], [Figure 3], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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