ABSTRACT cutaneous fungal infections, a reduced response of


the outer layers of the skin, nails and hair without tissue invasion and are
often caused
by dermatophytic molds, candida & non dermatophytic molds.
Although not dangerous, they are important as a public health problem particularly
in the immunocompromised. There are limited studies on the efficacy of
antifungal agents against dermatophytes in North India, therefore, a study was
conducted to test the efficacy of 5 systemic antifungal agents viz. Voriconazole,
Itraconazole, Terbinafine, Fluconazole & Griseofulvin  using Microbroth dilution technique. Three
different species of dermatophytes which were isolated from the clinically
suspected cases were Trichophyton mentagrophytes, T.rubrum and M.gypseum.
According to the obtained results, Itraconazole and Voriconazole showed the
lowest MIC range while Fluconazole and Griseofulvin had the highest MIC range
for most fungi tested.  Despite several treatment options being
available for cutaneous fungal infections, a reduced response of the latter in
the current scenario has led to an increasing need for determining an antifungal
susceptibility profile for specific fungal strains.
This will enable the clinician to select an appropriate antifungal agent with
minimal side effects to avoid antifungal resistance & treatment failure.


Dermatomycosis, Antifungal resistance, Microbroth dilution










            Dermatomycoses or superficial fungal infections have shown a monumental increase both in
incidence and prevalence in the recent past. Typically, these infections affect the outer layers of the skin, nails
and hair without tissue invasion and are often caused by dermatophytic molds belonging to genera Trichophyton,
Microsporum and Epidermophyton & sometimes by Pityriasis versicolor, candida & non
dermatomycotic molds.1,2  Dermatophytes have not only adapted
themselves to animal and human parasitism through evolution but have also developed host specificity ascribed to
difference in the composition of keratin.3
Based on their host specificity dermatophytes are classified into three
ecological groups namely geophiles (soil), anthropophiles
(man) and zoophiles (animals).

For a long time, the sole anti?dermatophytic agent approved for
systemic treatment was griseofulvin.4 Lately however, it has
fallen out of favour due to rise in Griseofulvin?resistant isolates of dermatophytes and existence of
strains with elevated MIC levels to Griseofulvin.5-8 Consequently,
and triazoles with higher efficacies, less side effects
and a shorter duration of treatment have become the mainstay of management of
most superficial mycoses.

The emergence of
innumerable antifungal-resistant strains and their widespread distribution are
the result of many years of underuse, overuse, and misuse of antifungal
medication (both topical and oral) and a weak or non-existent antifungal policy
and poor infection control.9 Taking medications by consulting
internet instead of doctors, self medication, prescription by quacks and
non-dermatologists, non-completion of treatment, easy availability of over the
counter drugs and excessive use of anti-fungal pesticides on crops are the main
reasons for antifungal resistance.

For in vitro detection of resistance to
antifungal agents, there are
guidelines given by CLSI (Clinical and Laboratory Standard Institute) document
M?38A for broth microdilution method for
filamentous fungi, in which MIC (Minimum Inhibitory Concentration) is
calculated. Depending on that, a particular drug with a higher MIC is considered
relatively resistant.10

studies related to antifungal susceptibility patterns are extremely scarce.
Furthermore, the development of an elaborate antifungal profile might
contribute to a decreased transmission and impact of resistant fungal strains
in the near future.11 Effective treatment depends on various factors
including duration of treatment, appropriate dosage and frequency of

A research might contribute in controlling
antifungal resistance. Therefore a study to determine the antifungal
susceptibility profile of 5 antifungal agents including Fluconazole,
Itraconazole, Voriconazole, Terbinafine, and Griseofulvin as per CLSI protocol
M38-A, against dermatophytes using Microbroth dilution technique, was conducted in the Department of Dermatology
of a tertiary care center of North India.














A prospective study was conducted in the
Department of Dermatology of a tertiary care center over a period of two years.
A total of 240 patients were included. An approval from the institutional
ethics committee was taken. Data was collected in a predesigned format. For
patients with sufficient scales, specimen collection, processing, microscopy
and culture were done and antifungal susceptibility testing was carried out as
per CLSI guidelines by the microbroth dilution technique.

Data analysis

mean (GM), MIC range, MIC50 and MIC90 were obtained for all the isolates
tested. MIC50 and MIC90 being the lowest drug concentration, showing 50% and
90% inhibition of growth, respectively. MIC value of antifungal drugs for
different species were compared by one-way ANOVA using SPSS version 16 software
and a p-value < 0.05 was considered statistically significant.                           RESULTS A total of 240 clinically suspected cases of superficial fungal infections were selected for microbiological diagnosis. The total number of positive cultures was 59. Final strain identification revealed 41(69.49%) dermatophytes. As shown in figure 1, among the dermatophytes, Trichophyton genus represented 97.6% of the isolates, with T. mentagrophytes being the commonest that is 25 (60.98%), followed by T. rubrum being 15 (36.58%) and Microsporum gypseum being 1 (2.44%). The MIC (Minimum Inhibitory Concentration) distribution, MIC50, MIC90, Geometric Mean (GM) of Fluconazole for T.mentagrophytes, T.rubrum and M.gypseum is as shown in Table 1. Table 1: In vitro susceptibility of dermatophytes to Fluconazole     Concentration of Fluconazole (µg/ml)   T.mentagrophytes (n = 25) T.rubrum (n = 15) M.gypseum (n = 1) GM 52.48 51.20 64 MIC 50 64 64 64 MIC 90 128 128 128 Range 32 – 64 32 – 64 NA GM=Geometric Mean The MIC (Minimum Inhibitory Concentration) MIC50, MIC90, Geometric Mean (GM) of Griseofulvin for T.mentagrophytes, T.rubrum and M.gypseum is as shown in Table 2.   Table 2: In vitro susceptibility of dermatophytes to Griseofulvin   Concentration of Griseofulvin (µg/ml)   T.mentagrophytes (n = 25) T.rubrum (n = 15) M.gypseum (n = 1) GM 1.33 4.67 0.06 MIC 50 0.5 4 0.06 MIC 90 1 8 0.125 Range 0.03 – 16 2 – 8 NA GM=Geometric Mean             The MIC (Minimum Inhibitory Concentration) MIC50, MIC90, Geometric Mean (GM) of Terbinafine for T.mentagrophytes, T.rubrum and M.gypseum is as shown in Table 3.   Table 3: In vitro susceptibility of dermatophytes to Terbinafine   Concentration of Terbinafine (µg/ml)   T.mentagrophytes (n = 25) T.rubrum (n = 15) M.gypseum (n = 1) GM 1.33 3.41 0.03 MIC 50 0.5 0.5 0.03 MIC 90 1 1 0.06 Range 0.03 – 16 0.03 – 8 NA GM=Geometric Mean             The MIC (Minimum Inhibitory Concentration) MIC50, MIC90, Geometric Mean (GM) of Itraconazole for T.mentagrophytes, T.rubrum and M.gypseum is as shown in Table 4.         Table 4: In vitro susceptibility of dermatophytes to Itraconazole   Concentration (µg/ml)   T.mentagrophytes (n = 25) T.rubrum (n = 15) M.gypseum (n = 1) GM 0.05 0 .06 0.03 MIC 50 0.03 0.03 0.03 MIC 90 0.06 0.06 0.06 Range 0.03 – 0.125 0.03 – 0.125 NA GM=Geometric Mean             The MIC (Minimum Inhibitory Concentration) MIC50, MIC90, Geometric Mean (GM) of Voriconazole for T.mentagrophytes, T.rubrum and M.gypseum is as shown in Table 5.   Table 5: In vitro susceptibility of dermatophytes to Voriconazole   Concentration of Voriconazole (µg/ml)   T.mentagrophytes (n = 25) T.rubrum (n = 15) M.gypseum (n = 1) GM 0.04 0.04 0.03 MIC 50 0.03 0.03 0.03 MIC 90 0.06 0.06 0.06 Range 0.03 – 0.125 0.03 – 0.125 NA GM=Geometric Mean             The results of micro dilution tests for most strains were read after 7 days at 28 °C. Mean MICs of antifungal drugs did not show statistically significant differences between various species (p > 0.05).

can be seen in the tables above, the MIC50 and MIC90 values of Fluconazole
(Table 1) and Griseofulvin (Table 2) for all three strains of dermatophytes
isolated were found to be higher and those of Itraconazole (Table 4) and
Voriconazole (Table 5) were found to be in the lower bracket and those for
Terbinafine were found to lie in the intermediate range.





In our study, Trichophyton genus represented
97.6% of the isolates of dermatophytes, with Trichophyton mentagrophytes
being the commonest that is 25 (60.98 %), followed by Trichophyton
rubrum 15 (36.58%) and Microsporum gypseum 1(2.44%). Similar to our
study, were the findings of the study conducted by Bhatia VK et al in the year
2014, in which Trichophyton species were implicated in 98.6%
(73/74) cases while Microsporum species was detected only in
1.35% cases. Also, none of the Epidermophyton species was
recovered by them. Further, Trichophyton mentagrophyte was also the
predominant organism (64.9%) followed by Trichophyton rubrum (35.1%).13
Trichophyton mentagrophyte was also the most common isolate in the study
conducted by Sahai et al in the year 2011.14      However, many studies have reported Trichophyton
rubrum as the commonest isolate.15-17  

Another important aspect
of this study was to carry out antifungal sensitivity testing of five commonly
used antifungal drugs Fluconazole, Terbinafine, Itraconazole, Griseofulvin and
Voriconazole. Determining the resistance pattern is especially necessary to assist clinicians in treating superficial fungal
infections more effectively.

results of micro dilution tests for most strains were read after 7 days at
28°C, when adequate growth was observed in the control well with significant
opacity. The 7 day time period has also been mentioned in the studies by Santos
et al, Gupta et al, Fernandez-Torres et al and Barros et al.18-21

            This time period was shorter (4 days
at 35°C) in the study conducted by Ghannoum et al and Mukherjee et al.22,23
This difference might be explained by the different temperatures used. Galuppi
et al reported a longer period of 14 days and incubation at 30°C.24 This
difference in the required incubation time may be due to the different volumes
of fungi inoculated into the micro plates.

findings about poor susceptibility of dermatophytes to Fluconazole (Table 1) is
compatible with the studies conducted by Favre et al, Santos et al, Barros et
al and Sarifakioglu et al.18,21,25,26 Korting et al suggested
that high values of MIC for Fluconazole may be due to technical problems, such
as interference with some ingredients of the culture media or insolubility at
high concentrations.27 The easy availability of Fluconazole at
pharmacies, self medication by patients due to its over the counter (OTC)
preparations available and a rampant practice of its irrational prescription by
quacks could be some other reasons for development of resistance to

high prevalence of resistance to Griseofulvin among dermatophytes (MIC range
0.03 – 16 µg/ml ) as found in our study (Table 2) is in accordance with the
findings of the studies by Galuppi et al and Korting et al.24,27 For
Griseofulvin, an MIC of 3 µg/ml was considered a limit of effectiveness.24

geometric mean MIC (GM) obtained in this study (1.59 µg/ml) showed that the
results of Terbinafine for all the three species of dermatophytes tested (Table
3) were significantly greater than the results obtained by Gupta et al ( 0.04
µg/ml), Favre et al (0.006 µg/ml), Deng S et al (0.03 µg/ml ), Esteban et al
(0.03 µg/ml) and Fernandez-Torres et al (0.21 µg/ml) in their studies
respectively.19,25,28,29,30 This can be explained by a wide MIC
range (0.03 – 16 µg/ml) found in our study probably due to a higher resistance
of dermatophytes to Terbinafine in our area.

According to our study, Itraconazole (MIC range 0.03 –
0.125 µg/ml) and Voriconazole (MIC range 0.03 – 0.125 µg/ml) showed the lowest
MIC ranges by the microbroth dilution technique which was also observed by
Bueno et al in their study.126 The high potency of Voriconazole and
Itraconazole against dermatophytes are in accordance with the observations made
by Favre et al in their study. 25

MIC range of Voriconazole found in our study (0.03 – 0.125 µg/ml) was found to
correspond to the lower end of the MIC range found by Deng S et al in their
study (0.031 – 16 µg/ml).28 An even lower MIC range (0.002 – 0.06
µg/ml) of Voriconazole was found
by Ghannoum et al in their study.31 The high sensitivity of
dermatophytes to Voriconazole observed in our study can be attributed to the
lower prevalence of its irrational prescription by quacks and chemists and also
to its high cost.

The MIC range of Itraconazole (by microbroth dilution method)
found in our study (0.03 – 0.125 µg/ml) was found to conform with the findings
of Deng S et al (0.031–16 µg/ml).28

In this study, Voriconazole was found to have the lowest
geometric mean while Fluconazole had the highest geometric mean value and MIC
range. Terbinafine was found to fall in the intermediate range. Therefore, it
can be interpreted that Voriconazole being the most sensitive antifungal drug for dermatophytes is a more suitable
treatment option but it must be reserved for resistant and difficult to treat
cases so as to prevent rapid development of resistance. Itraconazole is a much
more affordable antifungal drug that closely follows Voriconazole in its
effectiveness against dermatophytes, hence, it must be a preferred treatment
option for better outcome in patients suffering from dermatomycosis.
Fluconazole being the least sensitive antifungal drug against dermatophytes
must be used cautiously due to its poor effect. The clinical
significance of testing this group of fungi however remains uncertain, since
relevant breakpoints are yet to be identified and approved by regulatory
authorities. There is a need for establishing a standard method for antibiogram
of dermatophytes to facilitate the selection of drug similar to what is
routinely performed for yeasts (candida) and bacteria.





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