|Year : 2019 | Volume
| Issue : 4 | Page : 388-392
In vitro susceptibility of dermatophytes to oral antifungal drugs and amphotericin B in Uttar Pradesh, India
Satyendra Kumar Singh1, Dheeraj Kumar Patwa1, Ragini Tilak2, Arghya Das2, Tej Bali Singh3
1 Department of Dermatology and Venereology, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
3 Division of Biostatics, Department of Community Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Web Publication||7-Jun-2019|
Dr. Satyendra Kumar Singh
Department of Dermatology and Venereology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Dermatophytosis is a major public health problem in our country. Although resistance to conventional oral and topical antifungal agents is being increasingly encountered, the sensitivity pattern of dermatophytes has not been systematically analysed.
Aims: We aimed to determine the sensitivity pattern of dermatophyte isolates to amphotericin B and six oral antifungal drugs.
Materials and Methods: Patients with dermatophytosis attending the outpatient department of dermatology were enrolled in the study. Samples were collected for mycological examination and in vitro antifungal sensitivity testing was done by broth microdilution as per the Clinical and Laboratory Standard Institute M38-A standards.
Results: A total of 804 patients were enrolled. Specimens from 185 patients (23%) were both KOH and culture positive, and 44 of these isolates (41 Trichophyton mentagrophytes and 3 Trichophyton rubrum) were subjected to sensitivity testing. Minimum inhibitory concentrations (MIC) of itraconazole, ketoconazole, voriconazole and amphotericin B were comparable. The median MIC to fluconazole was higher than the other tested drugs. Dermatophytes were most susceptible to ketoconazole and voriconazole, followed by itraconazole, amphotericin B, fluconazole and griseofulvin. A high incidence of resistance was found to terbinafine and the difference was statistically significant in comparison to fluconazole, itraconazole, voriconazole, ketoconazole (P = 0.001) and griseofulvin (P = 0.003). The strains were more sensitive to amphotericin B as compared to griseofulvin (P = 0.02) and terbinafine (P < 0.001).
Limitations: This was a hospital-based study and may not reflect the true pattern in the community. Only a few of the isolates were selected for study. The clinical response of patients, whose isolates were studied for in vitro sensitivity of the antifungals, was not studied.
Conclusions: The sensitivity pattern of dermatophytes to various antifungals including amphotericin B, ketoconazole, voriconazole and itraconazole were determined. The studied isolates were least susceptible to terbinafine.
Keywords: Amphotericin-B, antifungal, dermatophytes, in vitro, resistance, voriconazole
|How to cite this article:|
Singh SK, Patwa DK, Tilak R, Das A, Singh TB. In vitro susceptibility of dermatophytes to oral antifungal drugs and amphotericin B in Uttar Pradesh, India. Indian J Dermatol Venereol Leprol 2019;85:388-92
|How to cite this URL:|
Singh SK, Patwa DK, Tilak R, Das A, Singh TB. In vitro susceptibility of dermatophytes to oral antifungal drugs and amphotericin B in Uttar Pradesh, India. Indian J Dermatol Venereol Leprol [serial online] 2019 [cited 2019 Jun 26];85:388-92. Available from: http://www.ijdvl.com/text.asp?2019/85/4/388/255285
| Introduction|| |
Dermatophytoses are a major public health problem and are commonly encountered in the dermatology clinics. These infections are frequently ignored, and rampant self-medication with inappropriate drug combinations has lead to resistance and frequent failure of treatment. For many years, griseofulvin was the only approved systemic antidermatophytic agent. But today, it is not widely used due to griseofulvin-resistant isolates of dermatophytes and existence of strains with elevated minimum inhibitory concentration levels to griseofulvin., Resistance to griseofulvin was first reported in 1969 and terbinafine resistance was documented in 2003. Decreased clinical responses to currently available oral and topical antifungal agents with frequent clinical failures and relapses has necessitated updosing of antifungals and finding any other antifungal agent, which is not generally used for dermatophytoses.
| Materials and Methods|| |
The study was conducted on patients with dermatophytosis attending the dermatology outpatient department at S.S. Hospital, Institute of Medical Sciences, Banaras Hindu University, Varanasi, a tertiary health-care system, from January 2016 to June 2017. Ethical clearance was obtained from the Institutional Ethical Committee. Samples were collected after informed consent and subjected to microscopy and culture. Only samples that were both KOH and culture-positive were selected for study.
Potassium hydroxide preparation (KOH mount)
The specimen was placed on a slide and a few drops of 30% KOH solution was added. A cover slip was placed and the slide was warmed over a flame and left for 15 min (longer in the case of nails) for clearing of the specimen. The slide was then examined for the presence of fungal elements.
Culture media (Sabouraud dextrose agar) with cycloheximide (0.05 g/L) and chloramphenicol (0.005 g/L) were used for culturing the specimens. All cultures were incubated at 28°C in a bio-oxygen demand (BOD) incubator for 4 weeks. Plates and tubes were examined every day during first week and every 2 days thereafter. Re-inoculation was done if any bacterial or saprophytic fungal contamination was detected. The day of appearance of the dermatophyte colony was noted for assessing the rate of growth of the isolates.
The rate of growth, colony morphology and pigment on the obverse and reverse were noted. A lactophenol cotton blue mount was prepared from suspect colonies and examined microscopically. Presence of any conidia, type, size, shape and any other special structures such as spiral or racquet hyphae was noted. Dermatophyte identification was as per standard procedure. The urease test was performed when needed. A portion of the specimen was saved for re-examination or re-inoculation.
Sensitivity was performed using the broth microdilution method as per CLSI (Clinical and Laboratory Standard Institute) M38-A standards (see appendix 1 for details).
| Results|| |
A total of 804 patients were enrolled. Of the 804 specimens, 678 (84.3%) were KOH positive, 194 (24.1%) culture positive and 185 (23%) were both KOH and culture positive. These 185 strains were stored for further study. Forty four of these 185 isolates (41 strains of Trichophyton mentagrophytes and 3 strains of Trichophyton rubrum) were revived after repeated culture and minimum inhibitory concentration testing was done by broth microdilution on these pure isolates only. The details are summarized in the flowchart in [Figure 1].
MIC range and median MICs for T. mentagrophytes and T. rubrum are shown in [Table 1]. Resistance patterns of Trichophyton mentagrophytes isolates are presented in [Table 2]. The MIC levels above which strains are considered resistant for the tested drugs is also displayed in [Table 2].
|Table 1: Results of in vitro sensitivity testing of 41 strains of T. mentagrophytes and 3 strains of T. rubrum (in μg/ml)|
Click here to view
|Table 2: Distribution of sensitive and resistant isolates of drugs for T. mentagrophytes (n=41)|
Click here to view
Most strains of T. mentagrophytes were resistant to terbinafine (33/41, 65.9%) and griseofulvin (20/41, 48.8%). There was a statistically significant difference in the susceptibility of T. mentagrophytes isolates to amphotericin B compared with griseofulvin (P = 0.02) and terbinafine (P < 0.001). However the susceptibility of T. mentagrophytes to amphotericin B was similar to that of fluconazole, itraconazole, voriconazole and ketoconazole (P = 0.80, 0.41, 0.27 and 0.25, respectively). Although dermatophytes were more sensitive to voriconazole and ketoconazole than itraconazole, this difference was not statistically significant (P = 0.76 and P = 0.29, respectively). A large number of strains were resistant to terbinafine and was statistically significant in comparison to fluconazole, itraconazole, voriconazole, ketoconazole (P = 0.001) and griseofulvin (P = 0.003).
All three isolates of T. rubrum were sensitive to itraconazole, voriconazole and ketoconazole but resistant to both terbinafine and amphotericin B [Table 3].
|Table 3: Distribution of sensitivity and resistance of drugs for T. rubrum (n=3)|
Click here to view
| Discussion|| |
Dermatophyte infections have dramatically increased during this decade by misuse of topical corticosteroids cream alone or in combination with topical antibacterial and antifungal agents. The rising trend of resistance among dermatophytes leading to poor response and frequent relapses is of serious concern and has been attributed to inappropriate treatments with steroid combination creams, improper dosages of antifungals and lifestyle changes. Resistance of dermatophytic infections to all antifungals (except voriconazole and amphotericin B) has been reported in the literature.,,,,
Resistance to fluconazole is well documented,, and was noted in 11 (26.8%) strains of T. mentagrophytes and 1 (33.3%) strain of T. rubrum in our study. The MICs of fluconazole ranged from 0.25 to >64 μg/ml which was similar to that reported in earlier studies.,,,,
The MICs of itraconazole ranged from 0.06 to >16 μg/ml which was similar to that reported by Ataides et al. Most other studies noted a narrow range of MIC (0.01–4 μg/ml),,,,, but a wider range of MICs (0.06–32 μg/ml) has been observed by Gupta et al. in Canada., All strains of T. rubrum were susceptible to itraconazole but 7/41 (17.1%) strains of T. mentagrophytes were resistant. An earlier study published from our institution reported a lower (6%) incidence of resistance to itraconazole but Magagnin et al. observed resistance to itraconazole in 42.3% of the strains they studied.
All strains of T. rubrum were susceptible to voriconazole. However, 14.6% strains of T. mentagrophytes were resistant to this drug. MICs of sensitive isolates ranged from 0.002 to 0.06 μg/ml, similar to the reports of Deng et al. but a wider ranges of 0.031–16 μg/ml have been previously reported.
MICs of ketoconazole in our study was in the range of 0.06 to >16 μg/ml. Six strains (14.6%) of T. mentagrophytes were resistant to ketoconazole but all strain of T. rubrum were susceptible. A high incidence (53%) of resistance to ketoconazole was observed by Magagnin et al.
A high incidence of resistance to griseofulvin and terbinafine was noted in our study. Twenty (48.8%) strains of T. mentagrophytes and 1 (33.3%) of T. rubrum were resistant to griseofulvin and 33 strains (65.9%) of T. mentagrophytes and all the three strains (100%) of T. rubrum to terbinafine. Similar findings have been reported previously.,,,,,,,
MICs of amphotericin B ranged from 0.06 to >16 μg/ml, which is higher than that noted in a previous study. Ten strains (24.4%) of T. mentagrophytes and all three strains (100%) of T. rubrum were resistant. However, Yenisehirli et al. found amphotericin B to be more active than itraconazole and ketoconazole.
In our study, dermatophytes were most sensitive to ketoconazole and voriconazole and least to terbinafine. The sensitivity pattern of T. mentagrophyte in decreasing order was voriconazole and ketoconazole > itraconazole > amphotericin B > fluconazole > griseofulvin > terbinafine while that for T. rubrum was ketoconazole = itraconazole = voriconazole > fluconazole = griseofulvin > terbinafine = amphotericin B. T. rubrum was least sensitive to terbinafine and amphotericin B.
We did not attempt to correlatein vitro sensitivity to the clinical response of the patients. This hospital-based study may not reflect the true pattern in the community. These are some limitations of this study.
| Conclusions|| |
Knowledge of sensitivity to antifungals prevalent in the local area can help determine choice of drugs for dermatophytoses. With such a high degree of resistance to terbinafine and griseofulvin, these may not be appropriate choice for the treatmet of tinea in our area.
The authors would like to acknowledge the staff members, especially Mr Vidyasagar of the Department of Microbiology for all kinds of support.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
This study was funded by grants from both the Microbiology and Dermatology Departments for purchase of microtiter plates, disposables, reagents and RPMI-1640 solution.
Conflicts of interest
There are no conflicts of interest.
If any suspected dermatophyte colony was mixed with contaminating colonies, the colony was subcultured again on Sabouraud dextrose agar with gentamicin. Sensitivity was performed using the broth microdilution method as per CLSI (Clinical and Laboratory Standard Institute) M38-A standards. 8 Stock solutions of concentration 1 mg/ml were made in normal saline for fluconazole and in 100% dimethyl sulfoxide for the other drugs. Double dilutions (from 0.25–64 μg/ml for fluconazole and from 0.06–16 μg/ml for the others) were prepared in RPMI-1640 (HiMedia) with L-glutamine buffered at pH 7.0 with 3-(N-morpholino) propanesulfonic acid, monosodium salt (but without sodium bicarbonate). Fungal colonies grown on potato dextrose agar after 7 days were used for in vitro sensitivity testing. The slant was flooded with 1 ml of sterile normal saline. Colonies were scraped gently with the help of sterile loop. The heavy particles were allowed to settle for 3–5 min. The upper homogeneous suspension containing mixture of nongerminated conidial and hyphal fragments was mixed for 15s with vortex. The turbidity was measured using a spectrophotometer at 530 nm and adjusted to final optical density range of 0.09–0.11 or visually containing standard 1,000,000 cells/ml of fungi counted on Neubauer's chamber. Stock inoculums suspension was diluted at 1:50 in RPMI-1640 medium. This test was performed in round-bottomed 96-well microdilution trays. Columns 1–9 were filled with double dilutions of 100 μl of respective antifungal drugs in rows in each well. Column 10 was a sterility control, containing 200 μl of RPMI-1640 and column 11 acted as a growth control (drug-free), having 200 μl of pure conidial suspension. Now, 100 μl of conidial suspension was filled in 1–9 well of serially diluted drugs. This tray was incubated at 30°C for 48–96 h of incubation and minimum inhibitory concentrations were determined and read visually. The growth in each well was compared with that of drug-free growth control and negative control. For most of the drugs, the minimum inhibitory concentration end-point criterion for fungi was the lowest drug concentration, showing 80% inhibition for azole and 90% inhibition of growth for terbinafine and amphotericin B.
| References|| |
Aboueisha AM, El-Mahallawy H. Public health significance of dermatophytes in Ismailia and Port Said provinces, Egypt. Med Mycol J 2013;54:123-9.
Poluri LV, Indugula JP, Kondapaneni SL. Clinicomycological study of dermatophytosis in South India. J Lab Physicians 2015;7:84-9.
] [Full text]
Gupta AK, Cooper EA. Update in antifungal therapy of dermatophytosis. Mycopathologia 2008;166:353-67.
Yenişehirli G, Tunçoǧlu E, Yenişehirli A, Bulut Y.In vitro
activities of antifungal drugs against dermatophytes isolated in Tokat, Turkey. Int J Dermatol 2013;52:1557-60.
Chadeganipour M, Nilipour S, Havaei A.In vitro
evaluation of griseofulvin against clinical isolates of dermatophytes from Isfahan. Mycoses 2004;47:503-7.
Lenhart K. Griseofulvin-resistant mutants in dermatophytes 1. The frequency of spontaneous and UV-induced mutants. Mykosen 1969;12:655-60.
Mukherjee PK, Leidich SD, Isham N, Leitner I, Ryder NS, Ghannoum MA, et al.
Clinical Trichophyton rubrum
strain exhibiting primary resistance to terbinafine. Antimicrob Agents Chemother 2003;47:82-6.
Clinical and Laboratory Standard Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi. Approved Standard. CLSI Document M38-A2. 2nd
ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
Kothiwala RK, Kumar R, Bohara DS, Rawat L, Meherda A, Chawla L, et al
. Hot trends of steroid modified Tinea at tertiary care hospital in India. Int Multispecialty J Health 2017;7:263-7.
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 Dermatol Venereol 2014;28:633-40.
Scholz R, Meinhof W. Susceptibility of Trichophyton rubrum
to griseofulvin. Mycoses 1991;34:411-4.
Barros ME, Santos Dde A, Hamdan JS.In vitro
methods for antifungal susceptibility testing of Trichophyton
spp. Mycol Res 2006;110:1355-60.
Gupta AK, Kohli Y, Batra R.In vitro
activities of posaconazole, ravuconazole, terbinafine, itraconazole and fluconazole against dermatophyte, yeast and non-dermatophyte species. Med Mycol 2005;43:179-85.
Fernández-Torres B, Carrillo AJ, Martín E, Del Palacio A, Moore MK, Valverde A, et al
. In vitro
activities of 10 antifungal drugs against 508 dermatophyte strains. Antimicrob Agents Chemother 2001;45:2524-8.
Favre B, Hofbauer B, Hildering KS, Ryder NS. Comparison ofin vitro
activities of 17 antifungal drugs against a panel of 20 dermatophytes by using a microdilution assay. J Clin Microbiol 2003;41:4817-9.
Adimi P, Hashemi SJ, Mahmoudi M, Mirhendi H, Shidfar MR, Emmami M, et al
. In vitro
activity of 10 antifungal agents against 320 dermatophyte strains using microdilution method in Tehran. Iran J Pharm Res 2013;12:537-45.
Mahajan S, Tilak R, Kaushal SK, Mishra RN, Pandey SS. Clinico-mycological study of dermatophytic infections and their sensitivity to antifungal drugs in a tertiary care center. Indian J Dermatol Venereol Leprol 2017;83:436-40.
] [Full text]
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.
Santos DA, Hamdan JS.In vitro
activities of four antifungal drugs against Trichophyton rubrum
isolates exhibiting resistance to fluconazole. Mycoses 2007;50:286-9.
Magagnin CM, Stopiglia CD, Vieira FJ, Heidrich D, Machado M, Vetoratto G, et al.
Antifungal susceptibility of dermatophytes isolated from patients with chronic renal failure. An Bras Dermatol 2011;86:694-701.
Ataides FS, Chaul MH, El Essal FE, Costa CR, Souza LK, Fernandes OF, et al.
Antifungal susceptibility patterns of yeasts and filamentous fungi isolated from nail infection. J Eur Acad Dermatol Venereol 2012;26:1479-85.
Indira G.In vitro
antifungal susceptibility testing of 5 antifungal agents against dermatophytic species by CLSI (M38-A) micro dilution method. Clin Microbial 2014;3:145-9.
Galuppi R, Gambarara A, Bonoli C, Ostanello F, Tampieri MP. Antimycotic effectiveness against dermatophytes: Comparison of twoin vitro
tests. Vet Res Commun 2010;34 Suppl 1:S57-61.
Gupta AK, Kohli Y.In vitro
susceptibility testing of ciclopirox, terbinafine, ketoconazole and itraconazole against dermatophytes and nondermatophytes, andin vitro
evaluation of combination antifungal activity. Br J Dermatol 2003;149:296-305.
Ghannoum M, Isham N, Sheehan D. Voriconazole susceptibilities of dermatophyte isolates obtained from a worldwide Tinea capitis clinical trial. J Clin Microbiol 2006;44:2579-80.
Deng S, Zhang C, Seyedmousavi S, Zhu S, Tan X, Wen Y, et al.
Comparison of thein vitro
activities of newer triazoles and established antifungal agents against Trichophyton rubrum
. Antimicrob Agents Chemother 2015;59:4312-4.
Sowmya N, Appalaraju B, Srinivas CR, Surendran P. Antifungal susceptibility testing for drmatophytes isolated from clinical samples by broth dilution method in tertiary care hospital. J Med Res 2015;1:64-7.
[Table 1], [Table 2], [Table 3]