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  Table of Contents  
Year : 2019  |  Volume : 10  |  Issue : 3  |  Page : 225-233  

Antifungal drug susceptibility testing of dermatophytes: Laboratory findings to clinical implications

1 Department of Dermatology, Venereology, and Leprology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Web Publication17-May-2019

Correspondence Address:
Shivaprakash M Rudramurthy
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/idoj.IDOJ_146_19

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How to cite this article:
Dogra S, Shaw D, Rudramurthy SM. Antifungal drug susceptibility testing of dermatophytes: Laboratory findings to clinical implications. Indian Dermatol Online J 2019;10:225-33

How to cite this URL:
Dogra S, Shaw D, Rudramurthy SM. Antifungal drug susceptibility testing of dermatophytes: Laboratory findings to clinical implications. Indian Dermatol Online J [serial online] 2019 [cited 2021 Oct 24];10:225-33. Available from: https://www.idoj.in/text.asp?2019/10/3/225/258583

Dermatophytes are the most commonly encountered fungi in humans and other vertebrates that spread through direct contact with infected humans, animals, and soil.[1] Infections due to these agents are usually restricted to the stratum corneum and are generally referred as 'tinea' or 'ringworm' (tinea capitis; tinea barbae; tinea corporis; tinea cruris; tinea manuum; tinea pedis and tinea unguium).[2],[3] Dermatophytes belong to 3 closely related genera- Trichophyton, Microsporum and Epidermophyton.[4] Worldwide, several studies have documented a varied prevalence rate of dermatophytosis ranging from 14-26.8% in North America, East Asia and Europe, and 5-31.6% in Africa (Ethiopia, Kenya, Nigeria, and Tanzania).[5],[6],[7] The regional variations are mainly due to differences in the lifestyle, socioeconomic conditions, underlying risk factors, and environmental factors of different geographic areas.[1] Epidemics of dermatophytosis have also been reported in the area of overcrowding and poor hygienic conditions.[4],[8],[9],[10] In 2005, World Health Organization (WHO) reported a prevalence of up to 19.7% for tinea capitis in the general population of developing countries.[11] High prevalence rates of tinea pedis and onychomycosis have been recognized in certain occupational groups like a marathon runner (22-31%), miners (21-72.9%), and soldiers (16.4-58%).[12],[13] Trichophyton species are the major causative agents responsible for dermatophytosis with a prevalence rate of 70-90% for onychomycosis and 53-86% for rest of the tinea infections.[14],[15] Of these, Trichophyton rubrum is the key etiological agent followed by T. mentagrophytes complex, Microsporum canis, and M. gypseum.[16],[17],[18] In India, we are presently noticing a significant rise in number of dermatophytosis cases with chronic recalcitrant disease, atypical presentations, frequent relapses, and treatment failures.[19],[20],[21],[22] Though the reason for this phenomenon is not yet clear, it is assumed that unchecked availability of cheap and irrational fixed-dose corticosteroid–antifungal–antibacterial combinations sold over the counter in India and in-vitro resistance to common antifungals (to some extent) is playing a pivotal role. Due to recent increase in the reports of antifungal drug resistance in dermatophytes, many groups have suggested to perform the antifungal drug susceptibility testing especially for the dermatophytes isolated from chronic/recurrent/recalcitrant cases or those with atypical presentations. Clinical successful treatment does not always correlate with the MIC (minimum inhibitory concentration) value of antifungals (in-vitro) [Table 1]. The discordance between the in-vivo and in-vitro resistance in fungi has been illustrated by the “90–60 rule,” which states that infections due to susceptible strains respond to appropriate therapy in 90% of cases, whereas infections due to resistant strains respond in approximately 60% of patients.[23] The clinical breakpoints (CBP) for different antifungals against dermatophytes, has not been defined due to lack of clinical correlation and pharmacokinetic/pharmacodynamic (PK/PD) studies [Table 1]. This manuscript provides a comprehensive update on the antifungal drugs susceptibility testing and its application in treating dermatophytosis.
Table 1: Common Definitions used in Antifungal Drug Susceptibility Methods[59]

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   Resistance in Dermatophytes Top

There is not much data available regarding in-vitro drug resistance to dermatophytes, but recently many reports suggest that resistance is on rise.[24],[25] Though, few reports suggest a good correlation between in-vitro resistance and treatment failure, there is no conclusive evidence to implicate in-vitro resistance with therapeutic failure in dermatophyte infections.[26] But relapse/recalcitrant infection after completion of recommended therapy in different presentations of dermatophyte infections is now well known. Resistance/recurrence after griseofulvin therapy in patients with T. rubrum and T. tonsurans is known since 1960's.[27],[28] A study from North India also showed that there were non-responders to griseofulvin therapy among the tinea capitis patients.[17] With the emergence of treatment failure with griseofulvin, allylamines became the preferred choice of treatment.[29] Mukherjee et al., in 2003 first reported T. rubrum strain exhibiting primary resistance to terbinafine and later Osborne et al., showed single missense amino acid substitution at L393F and F398L leading to terbinafine resistance.[30],[31],[32] This missense substitution in T. rubrum also contributes to cross-resistance to the other antifungals in this class (allylamines). Recently, Yamada et al., showed the presence of amino acid substitution at one of the four positions (Leu 393, Phe 397, Phe 415, His 440) of the squalene epoxidase protein in 17 isolates with a higher MIC to terbinafine.[33] Rudramurthy et al., in 2018 from India, reported high terbinafine resistance in 17% of T. interdigitale and 14.3% of T. rubrum isolates, with few strains exhibiting F397L mutation.[25] Another recent study from India also reported L393F and F397L mutations with higher terbinafine MIC's in isolates.[24]

It has been reported that repeated exposure to azole antifungals may be responsible for the development of azole resistance in dermatophytes.[34] T. rubrum can develop resistance to azoles, amorolfine and terbinafine after prolonged exposure to sub-inhibitory concentrations of these drugs leading to treatment failures and consequently contributing to persistence and chronicity of the infections.[35],[36] Azole resistance in dermatophytes has been reported as high as 19% worldwide.[37] Azambuja et al., found high MIC values for fluconazole and itraconazole (66.7% and 25% respectively) in 100 isolates of T. rubrum obtained from the patients with onychomycosis.[38]

Off late, there is an alarming trend of recalcitrant dermatophyte infection [22] in India, which could be related to inadequate treatment regimen or discontinuation of medication, difficulties in eliminating predisposing factors and sources of re-infection. However, very few reports have addressed the issue of resistant mechanisms operating in dermatophytes.[39] In vitro antifungal drugs susceptibility testing of dermatophytes may help significantly in the management of the patients, especially cases presenting with therapy failure,[40] when the disease has failed to respond to an empiric regimen and whenever prolong therapy is required.[23] Antifungal susceptibility testing also helps to understand the epidemiological pattern of drug resistance in any given region, and thus may help to choose more efficacious antifungal agents for standard treatment. Antifungal susceptibility data may also help in future to determine the clinical breakpoints or epidemiological cutoff (ECV)/ECOFF values that may assist in effective management. At present, the correlation between clinical outcome; and in-vitro dermatophytes' drug susceptibility patterns and MICs is not clearly understood.[41] Moreover, all species of dermatophytes may not have the same pattern of drug susceptibility.

   Antifungal Susceptibility Testing (AFST) Methods Top

Antifungal drug susceptibility testing is not routinely performed with dermatophytes due technical difficulties, expertise required and suboptimal reproducibility. However, the recent epidemic like scenario particularly in Indian subcontinent and emerging isolated case reports of recalcitrant tinea from Europe have greatly enhanced interest among microbiologists and dermatologists. Different techniques have been evaluated to test the antifungal susceptibility of dermatophytes such as agar disc diffusion, agar dilution and macro- and micro broth dilution methods.[40] The standard guidelines by Clinical Laboratory Standards Institute (CLSI), European Committee on Antimicrobial Susceptibility Testing (EUCAST) and British Society of Antimicrobial Chemotherapy (BSAC) are available for testing antifungal susceptibility of yeasts and molds [Table 2]. In 2008, CLSI included a method for antifungal susceptibility testing for dermatophytes [42] in their document on AFST of molds. Many modifications of this standard guideline has been proposed to improve the results.[43],[44] Standardization of antifungal susceptibility testing for dermatophytes is generally difficult as there are many variable critical parameters that needs to be considered while performing the test such as inoculum size (i.e., number of conidia/spores), incubation temperature and duration, media to be used, and time and percentage of growth inhibition for end point detection.[45],[46]
Table 2: Antifungal susceptibility testing techniques

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Standardization of media

Norris et al., evaluated four different culture media with 18 clinical dermatophyte isolates of 3 different species (T. mentagrophytes, T. rubrum, and T. tonsurans).[45] RPMI 1640 (chemically defined media) and Sabouraud's dextrose agar (chemically non-defined media) supported the growth of all the isolates but antibiotic medium #3 (Penassay; Difco Laboratories, Michigan) (chemically non-defined media), yeast nitrogen base (chemically defined media) with 0.5% dextrose showed a consistent growth of the isolates tested in the experiment. This study recommended RPMI 1640 as a superior media as its chemical composition is defined. Thus this study formed the basis for the establishment of future development of antifungal susceptibility testing [45] Though Sabouraud's dextrose broth and RPMI 1640 shows a similar type of efficiency for the growth of different species of dermatophytes, very few studies have been done to evaluate antifungal susceptibility testing against dermatophytes. McVeigh and Morton (MVM) is also a chemically defined medium but the interpretation of the results becomes difficult due to the non-transparency of media.[47] Thus various studies concluded that RPMI 1640 should be used as a standard media for a determination of antifungal susceptibility testing of dermatophytes.[44],[46],[47]

Standardization of Inoculums Size, Incubation Time and Temperature

The initial inoculum size required to start the susceptibility testing is a critical factor while determining the minimum inhibitory concentrations (MIC). Any variation in the inoculum size leads to variable result during interpretation. Thus, an intra and interlaboratory comparison becomes important to validate the inoculums size.[44] Apart from standardization of culture media, Norris and co-researchers also tested 3 different inoculums size: 103, 104 and 105 conidia/ml. Four antifungal drugs such as griseofulvin, itraconazole, terbinafine and fluconazole against 18 dermatophytes isolates tested revealed 103 conidia/ml as an optimum inoculum for antifungal susceptibility testing. There was no difference in the MICs of itraconazole and terbinafine with higher inoculums size but higher MICs were observed for fluconazole and griseofulvin.[45] Effect of temperature and incubation time for antifungal susceptibility testing varies according to studies. Norris et al., checked two different temperature conditions i.e. 30°C and 35°C in which no significant difference in their growth was noticed.[45] The studies reported that micro broth dilution method requires incubation at 35°C and should be read at the end of 72-96 hour.[48],[49],[50] Though the incubation period of 3-4 days is generally accepted to read the results, the time duration may exceed for a slow growing dermatophyte. Hence, final reading should be read on the basis of presence of growth in the control well.[51] A multi-center study tested 60 dermatophyte isolates against 3 antifungals (clotrimazole, itraconazole and terbinafine) with different incubation time (3, 7, 14 days) and temperature (28°C and 37°C). Significantly better and reproducible results were obtained after 7 days at 28°C.[52] In contrast, Perea et al., determined that sufficient growth of dermatophytes while performing antifungal susceptibility testing not only depends on the incubation time but also on nature of the solvent used to dissolve the drugs. The shorter time duration (48 to 72 h) was required when water was used as a solvent whereas the incubation time increased to 10-14 days' when polyethylene glycol was used as solvent.[53]

   Reliability of In-Vitro Antifungal Susceptibility Testing Top

In 2008, antifungal susceptibility testing protocol for dermatophytes was approved for the first time by CLSI, which was further modified in 2010.[44] Reproducibility of the endpoint should be considered as a principal factor for the detection of any resistance. The experts recommend multicenter studies to develop, and validate accurate optimal conditions for performing antifungal susceptibility testing of dermatophytes.[44],[51] Ghannoum and co-researchers in 2004 conducted a multicenter study including six laboratories to evaluate the reproducibility of antifungal susceptibility testing results. In this study, the activities of seven antifungal agents (ciclopirox- olamine, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine and voriconazole) were examined against 5 different species of dermatophytes (T. rubrum, T. mentagrophytes, T. tonsurans, E. floccosum and M. canis). MIC for all the isolates was determined by microbroth dilution method according to CLSI (previously known as NCCLS) M38-A standard. The MIC for all the isolates were determined using the endpoint of 50% and 80% inhibition of growth compared to control. The MIC data generated at different laboratories for all the dermatophytes isolates were analyzed. MIC values read at 50% inhibition compared with control growths showed an agreement of 92-100% whereas for 80% growth inhibition the agreement was 88 to 99%. Thus, the study concluded that CLSI M38-A standard guidelines for testing antifungal susceptibility of dermatophytes gave reproducible results.[54] Another collaborative study was conducted to define the specific inoculum sizes, incubation temperatures and other procedural end points for performing antifungal susceptibility testing by broth microdilution test against dermatophytes for clotrimazole, itraconazole, and terbinafine. A total of 60 isolates of six different species of dermatophytes including T. mentagrophytes, T. rubrum, T. tonsurans, M. gypseum, M. canis and E. floccosum were evaluated. The study concluded that the optimal condition for in- vitro antifungal susceptibility of dermatophytes requires incubation at 28°C for 7 days with 104 CFU/ml inoculum density. The MIC of all the drugs should be determined by 100% growth inhibition.[52] Other than terbinafine, clotrimazole, and itraconazole, newly introduced antifungal drugs including sertaconazole, luliconazole and lanoconazole, amorolfine, bifonazole, and miconazole have also been evaluated by in- vitro antifungal testing against dermatophytes.[55],[56],[57] ME1111 is a newer antifungal agent mainly used as a topical agent for the treatment of onychomycosis. Ghannoum and co-workers evaluated and standardized the activity of the ME 1111 antifungal agent by performing CLSI M38- A2 methodology against three isolates (T. mentagrophytes, T. rubrum, and E. floccosum) along with ATCC strain of T. rubrum and T. mentagrophytes as quality control. Evaluation of their results showed the interlaboratory agreement of more than 90% for the MIC's read with 80% inhibition as end point and it reduced to 76.2% when 100% inhibition was taken as criteria to read endpoint. At least on the basis of the above-mentioned studies it is clear that broth microdilution test of CLSI as per the M38-A2 protocol is the standard guideline for performing antifungal susceptibility testing against dermatophytes [Table 3]. Although methods are available for the performance of susceptibility testing, clinical interpretation of the MIC values or the breakpoints to consider whether the agent tested is susceptible or resistant clinically is yet to be defined clearly.[58] In the clinical setting, for better management of patients, clinical breakpoint (CBP) play an important role.[59] CBP depends upon several factors like the MIC distribution, pharmacokinetic/pharmacodynamic (PK/PD) data of the antifungals, and the most importantly, the outcome of disease.[59] Due to paucity of data on clinical outcome with the antifungal susceptibility data, it is hard to decide the CBP for a particular species. Therefore, in such situations, epidemiological cutoff value (ECV) may be determined for any given species and antifungal agent. This is the MIC value that is provisionally used to differentiate the wild-type isolates (generally considered as susceptible) from non-wild-types (generally considered as resistant isolates). This provisional value may help to choose appropriate antifungals while treating the infection. However, it is pertinent to mention that ECV's are not determinant of successful treatment, it only predicts and separate the population into two categories- wildtype or non- wildtype.[59]
Table 3: Review of antifungal susceptibility testing techniques (CLSI) for dermatophytes

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   Alternative Susceptibility Testing Methods Top

Disk diffusion method

Macro broth dilution and micro broth dilution methods are generally laborious and need expertise to perform in laboratories compared to the antimicrobial susceptibility testing by disc diffusion and 'E' test method. According to the standard guideline of CLSI, disc diffusion test and E-test are not recommended for dermatophytes antifungal susceptibility testing.[44] But, studies are available comparing disc diffusion with broth microdilution methods.[60],[61] Niewerth et al., in 1998, compared two methods of antifungal susceptibility testing to test four different species against five topical antifungal agents (griseofulvin, itraconazole, sertaconazole, terbinafine, and ciclopirox olamine) and found discrepancy in the result obtained from these two methods. The agar dilution method yielded higher MIC value than the broth dilution method.[62] In contrast, Macura et al., reported that disk diffusion technique for antifungals susceptibility for dermatophytes was much simpler and easy to perform in routine clinical settings and provided as consistent results as broth dilution method.[63] Karaca et al., compared these two antifungal susceptibility testing technique using four species of dermatophytes against a large number of antifungal agents (itraconazole, fluconazole, ketoconazole, miconazole, sulconazole, oxiconazole, bifonazole, griseofulvin, ciclopirox olamine and terbinafine). Similar results were obtained from disk diffusion method when compared with the micro- broth dilution methods. So disk diffusion method may be considered as an alternative to gold standard dilution method.[64] E- Test methods are mainly based on agar diffusion method and are used to determine the MIC of fastidious, slow growing or nutritionally deficient microorganisms. Castro Mendez and co-researchers compared the two agar based methods; E test and disk diffusion method and CLSI broth dilution method (CLSI M38-A) against three antifungal drugs (fluconazole, itraconazole, voriconazole). The results obtained with this disk diffusion method had low correlation with the results obtained from CLSI broth microdilution method for azoles. E- test and broth dilution methods showed agreement of 45.6% for fluconazole, 19.5% for itraconazole and 52.1% for voriconazole.[65] Although, an agar-based method is much simpler and easier to perform than broth dilution method, further research is essential before incorporating this technique in routine laboratory practice to test dermatophytes susceptibility [44],[51] [Table 3].

In conclusion, various techniques are available for antifungal susceptibility testing of dermatophytes but only broth microdilution technique is currently accepted to determine in-vitro susceptibility of dermatophytes. As this technique is laborious and need expertise, only few mycology laboratories can perform this test. In the present scenario of increasing resistance to the dermatophytes, there is a need to perform antifungal drug susceptibility tests at least in cases with chronic/recurrent dermatophytosis or treatment failure/relapse. As there is no CBP defined as of yet, there is urgent need to establish ECV for dermatophytes and this value may guide the clinician while managing recalcitrant/resistant dermatophytosis.[74]

   References Top

Ames I. Dermatophytosis. Inst Int Coop Anim Biol 2013;3:1-13.  Back to cited text no. 1
Maraki S. Epidemiology of dermatophytoses in Crete, Greece between 2004 and 2010. G Ital Dermatol Venereol 2012;147:315-9.  Back to cited text no. 2
Abd Elmegeed AS, Ouf SA, Moussa TA, Eltahlawi SM. Dermatophytes and other associated fungi in patients attending to some hospitals in Egypt. Braz J Microbiol 1973;46:799-805.  Back to cited text no. 3
Weitzman I, Summerbell RC. The dermatophytes. Clin Microbiol Rev 1995;8:240-59.  Back to cited text no. 4
Ndako JA, Osemwegie OO, Spencer THI, Olopade, BK, Yunusa GA, Banda J. Prevalence of Dermatophytes and other associated Fungi among school children. GARJMM 2012;1:49-56.  Back to cited text no. 5
Kannan P, Janaki C, Selvi GS. Prevalence of dermatophytes and other fungal agents isolated from clinical samples. Indian J Med Microbiol 2006;24:212-5.  Back to cited text no. 6
[PUBMED]  [Full text]  
Alemayehu A, Minwuyelet G, Andualem G. Prevalence and etiologic agents of dermatophytosis among primary school children in Harari Regional State, Ethiopia. J Mycol 2016;1489387:5.  Back to cited text no. 7
Venkatesan G, Singh AJAR, Murugesan AG, Janaki C, Shankar SG. Trichophyton rubrum – The predominant etiological agent in human dermatophytoses in Chennai, India. African J Microbiol Res 2007;1:9-12.  Back to cited text no. 8
Chakrabarti A, Sharma SC, Talwar P. Isolation of dermatophytes from clinically normal sites in patients with tinea cruris. Mycopathologia 1992;120:139-41.  Back to cited text no. 9
Rezaei-Matehkolaei A, Rafiei A, Makimura K, Gräser Y, Gharghani M, Sadeghi-Nejad B. Epidemiological aspects of dermatophytosis in Khuzestan, southwestern Iran, an Update. Mycopathologia 2016;181:547-53.  Back to cited text no. 10
Organization WH, Others. Epidemiology and management of common skin diseases in children in developing countries. Geneva World Heal Organ. 2005;54.  Back to cited text no. 11
Bell-Syer SEM, Khan SM, Torgerson DJ. Oral treatments for fungal infections of the skin of the foot. Sao Paulo Med J 2014;132:127.  Back to cited text no. 12
Achterman RR, White TC. A foot in the door for dermatophyte research. Heitman J, editor. PLoS Pathog 2012;8:e1002564.  Back to cited text no. 13
Balakumar S, Rajan S, Thirunalasundari T, Jeeva S. Epidemiology of dermatophytosis in and around Tiruchirapalli, Tamilnadu, India. Asian Pacific J Trop Dis 2012;2:286-9.  Back to cited text no. 14
Garg A, Venkatesh V, Singh M, Pathak KP, Kaushal GP, Agrawal SK. Onychomycosis in central India: A clinicoetiologic correlation. Int J Dermatol 2004;43:498-502.  Back to cited text no. 15
Peerapur BV, Inamdar AC, Pushpa PV, Srikant B. Clinicomycological study of dermatophytosis in Bijapur. Indian J Med Microbiol 2004;22:273-4.  Back to cited text no. 16
[PUBMED]  [Full text]  
Singal A, Rawat S, Bhattacharya SN, Mohanty S, Baruah MC. Clinico-myocological profile of tinea capitis in North India and response to griseofulvin. J Dermatol 2001;28:22-6.  Back to cited text no. 17
Yadav P, Singal A, Pandhi D, Das S. Clinico-mycological study of dermatophyte toenail onychomycosis in New Delhi, India. Indian J Dermatol 2015;60:153-8.  Back to cited text no. 18
[PUBMED]  [Full text]  
Majid I, Sheikh G, Kanth F, Hakak R. Relapse after oral terbinafine therapy in dermatophytosis: A clinical and mycological study. Indian J Dermatol 2016;61:529-33.  Back to cited text no. 19
[PUBMED]  [Full text]  
Ahmed S, Jeelani S, Lanker A, Qayoom S, Sameem F. Relapse of cutaneous fungal infection in healthy individuals-A rising concern. Br J Med Med Res 2016;11:1-8.  Back to cited text no. 20
Sahoo AK, Mahajan R. Management of tinea corporis, tinea cruris, and tinea pedis: A comprehensive review. Indian Dermatol Online J 2016;7:77-86.  Back to cited text no. 21
[PUBMED]  [Full text]  
Dogra S, Uprety S. The menace of chronic and recurrent dermatophytosis in India: Is the problem deeper than we perceive? Indian Dermatol Online J 2016;7:73.  Back to cited text no. 22
[PUBMED]  [Full text]  
Verma S, Madhu R. The great indian epidemic of superficial dermatophytosis: An appraisal. Indian J Dermatol 2017;62:227-36.  Back to cited text no. 23
  [Full text]  
Singh A, Masih A, Khurana A, Singh PK, Gupta M, Hagen F, et al. High terbinafine resistance in Trichophyton interdigitale isolates in Delhi, India harbouring mutations in the squalene epoxidase gene. Mycoses 2018;61:477-84.  Back to cited text no. 24
Rudramurthy SM, Shankarnarayan SA, Dogra S, Shaw D, Mushtaq K, Paul RA, et al. Mutation in the squalene epoxidase gene of Trichophyton interdigitale and Trichophyton rubrum associated with allylamine resistance. Antimicrob Agents Chemother 2018;62:1-9.  Back to cited text no. 25
Vandeputte P, Ferrari S, Coste AT. Antifungal resistance and new strategies to control fungal infections. Int J Microbiol 2012;1-26.  Back to cited text no. 26
Michaelides P, Rosenthal SA, Sulzberger MB. A Case demonstrating clinical and in vitro resistance. Archo Dermatol 1961;83:988-90.  Back to cited text no. 27
Berry CZ. Recurrence of Trichophyton rubrum infection during treatment with griseofulvin. Arch Dermatol 1960;81:982.  Back to cited text no. 28
Newland JG, Abdel-Rahman SM. Update on terbinafne with a focus on dermatophytoses. Dermatology 2009;2:49-64.  Back to cited text no. 29
Mukherjee PK, Leidich SD, Isham N, Leitner I, Ryder NS, Ghannoum MA. Clinical Trichophyton rubrum strain exhibiting primary resistance to terbinafine. Antimicrob Agents Chemother 2003;47:82-6.  Back to cited text no. 30
Osborne CS, Leitner I, Favre B, Ryder NS. Amino acid substitution in Trichophyton rubrum squalene epoxidase associated with resistance to terbinafine. Antimicrob Agents Chemother 2005;49:2840-4.  Back to cited text no. 31
Osborne CS, Leitner I, Hofbauer B, Fielding CA, Favre B, Ryder NS. Biological, biochemical, and molecular characterization of a new clinical Trichophyton rubrum isolate resistant to terbinafine. Antimicrob Agents Chemother 2006;50:2234-6.  Back to cited text no. 32
Yamada T, Maeda M, Alshahni MM, Tanaka R, Yaguchi T, Bontems O, et al. Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene. Antimicrob Agents Chemother 2017;61:e00115-17.  Back to cited text no. 33
Gupta AK, Kohli Y. Evaluation of in vitro resistance in patients with onychomycosis who fail antifungal therapy. Dermatology 2003;207:375-80.  Back to cited text no. 34
Hryncewicz-Gwóźdź A, Kalinowska K, Plomer-Niezgoda E, Bielecki J, Jagielski T. Increase in resistance to fluconazole and itraconazole in Trichophyton rubrum clinical isolates by sequential passages in vitro under drug pressure. Mycopathologia 2013;176:49-55.  Back to cited text no. 35
Ghelardi E, Celandroni F, Gueye SA, Salvetti S, Senesi S, Bulgheroni A, et al. Potential of ergosterol synthesis inhibitors to cause resistance or cross-resistance in Trichophyton rubrum. Antimicrob Agents Chemother 2014;58:2825-9.  Back to cited text no. 36
Ghannoum M. Azole resistance in dermatophytes: Prevalence and mechanism of action. J Am Podiatr Med Assoc 2015;106:79-86.  Back to cited text no. 37
Azambuja CV, Pimmel LA, Klafke GB, Xavier MO. Onychomycosis: Clinical, mycological and in vitro susceptibility testing of isolates of Trichophyton rubrum. An Bras Dermatol 2007;89:581-6.  Back to cited text no. 38
Martinez-Rossi NM, Peres NTA, Rossi A. Antifungal resistance mechanisms in dermatophytes. Mycopathologia 2008;166:369-83.  Back to cited text no. 39
Aktas AEA, Yigit N, Aktas AEA, Gozubuyuk SG. Investigation of in vitro activity of five antifungal drugs against dermatophytes species isolated from clinical samples using the E-test method. Eurasian J Med 2014;46:26-31.  Back to cited text no. 40
Ghannoum MA, Arthington-Skaggs B, Chaturvedi V, Espinel-Ingroff A, Pfaller MA, Rennie R, et al. An interlaboratory study of quality control isolates for a broth antifungal susceptibility method for the testing of dermatophytes. J Clin Microbiol 2006;44:4353-6.  Back to cited text no. 41
Clsi. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A. Clin Lab Stand Inst 2008;28:52.  Back to cited text no. 42
Jo Siu WJ, Tatsumi Y, Senda H, Pillai R, Nakamura T, Sone D, et al. Comparison of in vitro antifungal activities of efinaconazole and currently available antifungal agents against a variety of pathogenic fungi associated with onychomycosis. Antimicrob Agents Chemother 2013;57:1610-6.  Back to cited text no. 43
Thatai P, Sapra B. Critical review on retrospective and prospective changes in antifungal susceptibility testing for dermatophytes. Mycoses 2016;59:615-27.  Back to cited text no. 44
Norris HA, Elewski BE, Ghannoum MA. Optimal growth conditions for the determination of the antifungal susceptibility of three species of dermatophytes with the use of a microdilution method. J Am Acad Dermatol 1999;40:S9-13.  Back to cited text no. 45
Jessup CJ, Warner J, Isham N, Hasan I, Ghannoum MA. Antifungal susceptibility testing of dermatophytes: Establishing a medium for inducing conidial growth and evaluation of susceptibility of clinical isolates. J Clin Microbiol 2000;38:341-4.  Back to cited text no. 46
Santos DA, Hamdan JS. Evaluation of broth microdilution antifungal susceptibility testing conditions for Trichophyton rubrum. J Clin Microbiol 2005;43:1917-20.  Back to cited text no. 47
Baghi N, Shokohi T, Badali H, Makimura K, Rezaei-Matehkolaei A, Abdollahi M, et al.In vitro activity of new azoles luliconazole and lanoconazole compared with ten other antifungal drugs against clinical dermatophyte isolates. Med Mycol 2016;54:757-63.  Back to cited text no. 48
Ghannoum M, Chaturvedi V, Diekema D, Ostrosky-Zeichner L, Rennie R, Walsh T, et al. Multilaboratory evaluation of in vitro antifungal susceptibility testing of dermatophytes for ME1111. J Clin Microbiol 2016;54:662-5.  Back to cited text no. 49
Mohd Nizam T, Binting RA, Mohd Saari S, Kumar TV, Muhammad M, Satim H, et al.In vitro antifungal activities against moulds isolated from dermatological specimens. Malaysian J Med Sci 2016;23:32-9.  Back to cited text no. 50
Chand DV, Ghannoum MA. Susceptibility testing of dermatophytes. In: Interactions of Yeasts, Moulds, and Antifungal Agents. Totowa, NJ: Humana Press; 2009. p. 89-95.  Back to cited text no. 51
Fernández-Torres B, Cabañes FJ, Carrillo-Muñoz AJ, Esteban A, Inza I, Abarca L, et al. Collaborative evaluation of optimal antifungal susceptibility testing conditions for dermatophytes. J Clin Microbiol 2002;40:3999-4003.  Back to cited text no. 52
Perea S, Fothergill AW, Sutton DA, Rinaldi MG. Comparison of in vitro activities of voriconazole and five established antifungal agents against different species of dermatophytes using a broth macrodilution method. J Clin Microbiol 2001;39:385-8.  Back to cited text no. 53
Ghannoum MA, Chaturvedi V, Pfaller MA, Rinaldi MG, Warnock DW, Icrobiol JCLINM. Intra- and interlaboratory study of a method for testing the antifungal susceptibilities of dermatophytes. J Clin Microbiol 2004;42:2977-9.  Back to cited text no. 54
Schaller M, Borelli C, Berger U, Walker B, Schmidt S, Weindl G, et al. Susceptibility testing of amorolfine, bifonazole and ciclopiroxolamine against Trichophyton rubrum in an in vitro model of dermatophyte nail infection. Med Mycol 2009;47:753-8.  Back to cited text no. 55
Carrillo-Muñoz AJ, Tur-Tur C, Cárdenes DC, Estivill D, Giusiano G. Sertaconazole nitrate shows fungicidal and fungistatic activities against Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum, causative agents of tinea pedis. Antimicrob Agents Chemother 2011;55:4420-1.  Back to cited text no. 56
Carrillo-Muñoz AJ, Fernandez-Torres B, Guarro J.In vitro antifungal activity of sertaconazole against 309 dermatophyte clinical isolates. J Chemother 2003;15:555-7.  Back to cited text no. 57
Ghannoum MA, Arthington-Skaggs B, Chaturvedi V, Espinel-Ingroff A, Pfaller MA, Rennie R, et al. Interlaboratory study of quality control isolates for a broth microdilution method (Modified CLSI M38-A) for testing susceptibilities of dermatophytes to antifungals. J Clin Microbiol 2006; 44:4353-6.  Back to cited text no. 58
Lockhart SR, Ghannoum MA, Alexander BD. Establishment and use of epidemiological cutoff values for molds and yeasts by use of the Clinical and Laboratory Standards Institute M57 Standard. J Clin Microbiol 2017;55:1262-8.  Back to cited text no. 59
Mota CR, Miranda KC, Lemos Jde A, Costa CR, Hasimoto e Souza LK, Passos XS, et al. Comparison of in vitro activity of five antifungal agents against dermatophytes, using the agar dilution and broth microdilution methods. Bras Med Trop 2009;42:250-4.  Back to cited text no. 60
Singh J, Zaman M, Gupta AK. Evaluation of microdilution and disk diffusion methods for antifungal susceptibility testing of dermatophytes. Med Mycol 2007;45:595-602.  Back to cited text no. 61
Niewerth M, Splanemann V, Korting HC, Ring J, Abeck D. Antimicrobial susceptibility testing of dermatophytes--comparison of the agar macrodilution and broth microdilution tests. Chemotherapy 1998;44:31-5.  Back to cited text no. 62
Macura, A B. In vitro susceptibility of dermatophytes to antifungal drugs: A comparison of two methods. Int. J. Dermatol 1993;32:533-6.  Back to cited text no. 63
Karaca N, Koç AN. In vitro susceptibility testing of dermatophytes: Comparison of disk diffusion and reference broth dilution methods. Diagn Microbiol Infect Dis 2004;48:259-64.  Back to cited text no. 64
Méndez CC, Serrano MC, Valverde A, Pemán J, Almeida C, Martín-Mazuelos E. Comparison of E-test, disk diffusion and a modified CLSI broth microdilution (M 38-A) method for in vitro testing of itraconazole, fluconazole and voriconazole against dermatophytes. Med Mycol 2008;46:119-23.  Back to cited text no. 65
Ansari S, Hedayati MT, Zomorodian K, Pakshir K, Badali H, Rafiei A, et al. Molecular characterization and in vitro antifungal susceptibility of 316 clinical isolates of dermatophytes in Iran. Mycopathologia 2016;181:89-95.  Back to cited text no. 66
Parvaneh Adimi, Seyed Jamal Hashemi, Mahmood Mahmoudi, Hossein Mirhendi, Mohammad Reza Shidfar, Masood Emmami, et al. In-vitro activity of 10 antifungal agents against 320 dermatophyte strains using micro dilution method in Tehran. Iran J Pharm Res 2013;12:537-45.  Back to cited text no. 67
Silva LB, de Oliveira DB, da Silva BV, de Souza RA, da Silva PR, Ferreira-Paim K, et al. Identification and antifungal susceptibility of fungi isolated from dermatomycoses. J Eur Acad Dermatology Venereol 2014;28:633-40.  Back to cited text no. 68
Zalacain A, Obrador C, Martinez JP, Viñas M, Vinuesa T. Characterization of the antimicrobial susceptibility of fungi responsible for onychomycosis in Spain. Med Mycol 2011;49:495-9.  Back to cited text no. 69
Barros MEDS, Santos DDA, Hamdan JS. Evaluation of susceptibility of Trichophyton mentagrophytes and Trichophyton rubrum clinical isolates to antifungal drugs using a modified CLSI microdilution method (M38-A). J Med Microbiol 2007;56:514-8.  Back to cited text no. 70
Chadeganipour M, Nilipour S, Havaei A. In vitro evaluation of griseofulvin against clinical isolates of dermatophytes from Isfahan. Mycoses 2004;47:503-7.  Back to cited text no. 71
Eba M, Njunda AL, Mouliom RN, Kwenti ET, Fuh AN, Nchanji GT, et al. Onychomycosis in diabetic patients in Fako Division of Cameroon: Prevalence, causative agents, associated factors and antifungal sensitivity patterns. BMC Res Notes. 2016;9:1-8.  Back to cited text no. 72
Nweze EI, Mukherjee PK, Ghannoum MA. Agar-based disk diffusion assay for susceptibility testing of dermatophytes. J Clin Microbiol 2010;48:3750-2.  Back to cited text no. 73
Esteban A, Abarca ML, Cabañes FJ. Comparison of disk diffusion method and broth microdilution method for antifungal susceptibility testing of dermatophytes. Med Mycol 2005;43:61-6.  Back to cited text no. 74


  [Table 1], [Table 2], [Table 3]


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