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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 6  |  Issue : 3  |  Page : 105-112

Patterns of culture positivity and antifungal sensitivity in dermatophytosis


Department of Dermatology, MGM Medical College and Hospital, Navi Mumbai, Maharashtra, India

Date of Submission18-Jan-2020
Date of Acceptance21-Jan-2020
Date of Web Publication16-Mar-2020

Correspondence Address:
Dr. Shylaja Someshwar
Dr. Shylaja Someshwar, Department of Dermatology, MGM Medical College and Hospital, Kamothe, Navi Mumbai 410209, Maharashtra.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mgmj.mgmj_4_20

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  Abstract 

Background: Antifungal drug resistance and a lack of clinical response in 20% of cases of dermatophytosis calls for an evaluation of resistant dermatophytes using a standardized simple and reproducible in vitro assay, to allow clinician to select the appropriate antifungal agent based on the susceptibility of the isolate to the antifungal agent. Objective: The objective of this study was to clinically correlate the patterns of antifungal susceptibility of dermatophyte isolates and study resistance patterns and cross-resistance among antifungal agents. Materials and Methods: A study was conducted with 60 patients. At visit 1, the patient was started on empiric treatment with terbinafine 250mg daily for 2 weeks. Skin scraping for potassium hydroxide (KOH) mount and fungal culture and antifungal susceptibility testing was collected. After 2 weeks of therapy, the treatment was either continued or changed to a sensitive antifungal agent depending on the susceptibility testing results. The patient was clinically reevaluated every fortnightly. The patient completed a total of 4 weeks of the susceptible antifungal agent. The outcome of therapy was assessed by means of a skin scraping for KOH mount examination. Results: There were 44 males and 16 females. Maximum belonged to the age-group 30–40 years. Maximum patients had tinea corporis (51.6%) followed by tinea cruris (20%). Conclusion: The study provided information regarding clinico-mycological correlation of terbinafine, which revealed terbinafine to be the most effective antifungal agent clinically as well as mycologically.

Keywords: Antifungal culture and sensitivity, dermatophytosis, itraconazole, terbinafine


How to cite this article:
Ghuse V, Someshwar S, Jerajani H. Patterns of culture positivity and antifungal sensitivity in dermatophytosis. MGM J Med Sci 2019;6:105-12

How to cite this URL:
Ghuse V, Someshwar S, Jerajani H. Patterns of culture positivity and antifungal sensitivity in dermatophytosis. MGM J Med Sci [serial online] 2019 [cited 2020 Apr 6];6:105-12. Available from: http://www.mgmjms.com/text.asp?2019/6/3/105/280757




  Introduction Top


Dermatophytosis refers to the superficial dermatophyte infection of the skin. It occurs most commonly with organisms of the genera Trichophyton, Microsporum, and Epidermophyton. Tinea corporis includes infection of the glabrous skin excluding scalp, beard, face, palms, soles, and groin. Tinea cruris includes infection of the genital, pubic, perineal, and perianal areas.[1] In the selection of the appropriate antifungal agent, a number of considerations are necessary, including isolated species, minimal inhibitory concentration (MIC), cure rate, cost, complications, drug interactions, convenience, compliance, and the age, health, and medical history of the patient.[2]

Identification of dermatophytic species and susceptibility testing need to be performed to provide information to the clinician to select an appropriate antifungal agent useful for treating a particular fungal agent. Different antifungal agents have been recommended to control dermatophytes.[3] Several methods have been developed for testing antifungal agents against this group of pathogens. The Clinical and Laboratory Standards Institute (CLSI) document M38-A2 Reference method for broth dilution antifungal susceptibility testing for filamentous fungi (cutaneous fungi) describes a standard approved reference method for antifungal susceptibility testing of dermatophytes by broth dilution.[4]

The main aim of this study was to determine the in vitro activity of the seven antifungal drugs used most commonly in our outpatient department (OPD) to treat dermatophytic infections:
  1. Griseofulvin (GF)


  2. Miconazole (MIZ)


  3. Terbinafine (TBF)


  4. Clotrimazole (CTZ)


  5. Fluconazole (FLZ)


  6. Ketoconazole (KTZ)


  7. Itraconazole (ITZ)



  Materials and methods Top


A quasi-experimental study was conducted on 60 patients in the OPD of a tertiary care center situated in the periphery of Mumbai, the capital of Maharashtra state in India, who were clinically diagnosed as tinea corporis, tinea cruris, and tinea faciei with age >18 years, potassium hydroxide (KOH) positive for dermatophytes, and with >5% body surface area involvement. Pregnant or lactating women, patients with a history or clinical evidence of severe systemic disease, patients on immunosuppressants, and patients with a history of hypersensitivity to azoles or allylamine antifungals were excluded from the study. A detailed history was taken and clinical examination was carried out. Baseline clinical score was calculated for all individuals. At visit 1, the patient was started on empiric treatment with TBF 250mg daily for 2 weeks. Skin scraping for KOH mount and fungal culture and antifungal susceptibility testing was collected. After 2 weeks of therapy, the treatment was either continued or changed to a sensitive antifungal agent depending on the susceptibility testing results. The patient was clinically reevaluated every fortnightly. The patient completed a total of 4 weeks of the susceptible antifungal agent. The outcome of therapy was assessed by means of a skin scraping for KOH mount examination and clinical scores at 42 days (final treatment visit) and 72 days (point of cure) after starting treatment. Clinical and mycological testing at day 42 revealed complete cure (clinical and mycological). At day 72, the patients were reevaluated clinically and mycologically to detect relapse. We included the following antifungal agents in our study: TBF, ITZ, GF, FLZ, MIZ, KTZ, and CTZ.

Ethical considerations: The study design was approved by the Institutional Ethics Review Committee. Written informed consent was obtained from each subject before enrolling in the study.


  Results Top


Most of the individuals, 16 of 60 individuals were in the age-group of 31–40 years. This was followed by 15 individuals in the age-group of 21–30 years, 14 in the age-group of 10–20 years, and 7 each in the age-group of 41–50 and 51–60 years. A single patient was found to be in the age-group of 61–70 years. Of the total of 60 patients in our study, 44 (73.3%) were males and 16 (26.7%) were females. The male:female ratio was 2.75:1. More men than women presented to our center with dermatophyte infections.

Most patients, 26 (43.3%) had a 1–2 month long duration of disease followed by 14 (23.3%) for 3–5 months, 10 (16.7%) for 6–12 months, 6 (10%) for less than 1 month, and 4 (6.7%) for more than 1 year.

Majority of the patients (29, 48.3%) were found to have used only an over-the-counter (OTC) agent in the past, whereas 14 (23.3%) had not taken any kind of treatment. Of the total patients, 13 (21.7%) had used a known antifungal agent, whereas 4 (6.7%) had used both antifungals as well as OTC agents. Majority of the patients, 53 (88.3%) did not give a history of dermatophytic infection in the immediate family.

Family history was found to be present in 5 (8.3%) patients, whereas the conjugal transmission was found in 2 (3.3%).

Majority of patients (23, 38.3%) had isolated dermatophytic infection of the buttocks (tinea corporis) followed by groin (12, 20%), whereas 7 (11.7%) had both buttocks and groin involved [Figure 1] and [Figure 2]. The face was involved only in 1 (1.7%) patient. There were no patients with scalp and scalp hair involved.
Figure 1: Therapeutic response in a patient of tinea cruris

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Figure 2: Therapeutic response in a patient of tinea corporis

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Of the 60 patients, 5 (8%) were lost to follow-up in the fourth visit and additional 3 patients (total: 8 [13%]) were lost to follow-up in the fifth visit of the study.

After treatment of 4 weeks, clinical scores drastically reduced to zero (100%) in all these patients. The clinical score remained zero in all (100%) the patients who followed up even at visit 5 as there was no recurrence of lesions at visit 5.

The most common species isolated was Trichophyton mentagrophytes [Figure 3] in 57 (95%) skin samples, whereas Trichophyton rubrum [Figure 4] consisted of only 3 (5%) isolates.
Figure 3: Lactophenol Cotton Blue (LPCB) mount Trichophyton mentagrophytes showing scattered round micro conidia and terminal rat tail spiral filaments

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Figure 4: LPCB mount Trichophyton rubrum showing micro conidia simulating a “bird on fence” appearance

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Antifungal susceptibility testing [Figure 5A] and [5B] was carried out with seven drugs [Figure 6] on the isolates of all the 60 patients.
Figure 5: (A) Sensitivity plates at day 1. (B) Control plates at day 4 of sensitivity testing showing turbidity

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Figure 6: Sensitivity plates of seven antifungal agents

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Minimum inhibitory concentration (MIC) obtained of each drug was noted [Table 1]. Mycological cure at visit 4 was obtained in 55 (91.7%) patients, whereas 5 (8.3%) were lost to follow-up. No relapse was observed in 47 (78.3%), whereas the rest 13 (21.7%) were lost to follow-up at visit 5.
Table 1: Minimal inhibitory concentration of antifungal agents

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  Discussion Top


Our study was an attempt to identify correlations between MIC and clinical outcome before MICs could independently be carried out to make decisions regarding the class of a drug to be used for the treatment in the future. Although a reference method for dermatophytes is not available, a good correlation between the in vitro data, using broth microdilution method, and the clinical outcome has been shown earlier.[5] Lately, it was reported that correlations of MICs with clinical outcomes have not been determined.[6] Our study showed a good correlation between the MIC of TBF and GF with clinical outcomes as the clinical scores drastically reduced to zero after treatment with the sensitive (low MIC) drug.

In our study, of the 60 patients, only 16 were females. Therefore, the male-to-female ratio was 2.75:1. This was higher than the sex ratios in other studies. The male-to-female ratio of 1.57:1 was observed in a study conducted in Gujarat.[7] A higher incidence of dermatophytosis in males than in females has been reported both in India and abroad.[8] Philpot[9] suggested that males may be more vulnerable to infection due to higher exposures in the army, schools, and sporting activities. This is especially true for tinea cruris. The higher incidence in young males could be due to greater physical activity and increased sweating.

Most of the individuals in our study were in the age-group of 30–40 years (26.7%) followed by the age-group of 20–30 years as observed in the majority of studies.[10] Our institute being in an industrial setting had a number of male patients of laborer class attending the OPD and contributing to the particular age-group.

Most cases in our study had a clinical diagnosis of tinea corporis, involving buttocks, extremities, face, trunk, and abdomen (31 of 60; 51.6%), and 12 (20%) patients had only tinea cruris. The major clinical type was tinea corporis followed by tinea cruris. This is in contrast to the studies by Sardari et al.[11] and Verma et al.,[12] where it has been reported that tinea cruris was the most common clinical type.

There was no history of contact with infected family members in the majority of our patients (53 of 60; 88.3%), whereas 5 of 60 (8.3%) presented with a history of infected family members (other than the spouse), which was in contrast to a study conducted at Calicut, wherein a history of contact with infected family members and nonfamily members was observed in 16.6% and 2.6%, respectively. There was one case of conjugal transmission.[13] Our study found two cases of conjugal transmission.

Duration of infection was 1–2 months in the majority (26 of 60; 43.3%) of the patients, whereas only 4 (6.7%) had the disease for 1 year. Twenty-nine (48.3%) patients had used only OTC preparations, whereas 4 (6.7%) had used both OTC preparations as well as antifungal agents in the past. Of 60 patients, 14 (23.3%) had not taken treatment of any kind.

Only two species were isolated and identified, that is, T. mentagrophytes (57 of 60; 95%) and T. rubrum (3 of 60; 5%) in the skin samples of our study population. T. mentagrophytes was the most common species isolated in our study. These findings were consistent with the findings of studies conducted in Gujarat (42.62%),[14] northwest India (37.9%),[15] central India (25%),[16] and Chennai.[17]

Antifungal susceptibility testing, using the CLSI M38-A2 guidelines (2008)[4] for filamentous fungi, was carried out for the isolated species. All patients were found to be clinically and mycologically responsive to therapy with TBF, except one. With TBF, a failure of therapy corresponding to in vitro resistance, as depicted by a high MIC (1.00 µg/mL) was found. The patient was also found to be clinically unresponsive to TBF and was switched to treatment with GF, which was the next antifungal agent of choice in this patient due to the lowest MIC (0.125 µg/mL) obtained with this drug.

In our study, antifungal susceptibility results of dermatophytes were evaluated according to MIC ranges and geometric means, MIC50, MIC80, and MIC90. No ideal data were observed concerning the breakpoint of dermatophytes, which indicates the susceptibility as sensitive, resistance, or intermediate sensitivity.

T. mentagrophytes (American type culture collection [ATCC] 9533) and T. rubrum (ATCC 28188) were included as quality control (QC) strains in our study, which showed MICs well within the range for antifungals mentioned in the CLSI M38-A2 guidelines.

Overall, considering the MICs obtained for each of the drugs, TBF was found to be the most active agent against dermatophytes as most patients (58 of 60; 96.7%) were found to be sensitive up to the concentration of 0.0313 µg/mL. The MIC90 of TBF was the least (0.031) for both isolated species. This is consistent with the reports from various studies in which TBF was found to be the most active drug, with the MIC90 ranging between 0.01 and 0.07mg/mL.[18] In another study by Gupta and Kohli,[19] TBF was found to have MIC ± standard error of mean as 0.04 ± 0.02, and MIC of 0.03 µg/mL was observed in a study conducted in Brazil.[20]

Among the seven drugs, ITZ was found to be the second most active agent and the most active azole antifungal. Similar results have been verified by other researchers.[21]

In our study, FLZ was the least potent drug with the highest MIC, similar to other studies. In FLZ, MIC >64 μg/mL is attributed to resistance to this drug.[22]

We can thus conclude that the order of in vitro activity in our study is TBF > ITZ > GF > KTZ > MIZ > CTZ > FLZ.

No side effects were found in any of the patients.

The evaluation of in vitro susceptibility showed that the antifungal drugs tested, with the exception of FLZ, displayed good activity against the dermatophytes. It is worth mentioning that ITZ, KTZ, and TBF had the lowest MIC values. Similar results have been verified by other authors, which showed that these drugs had low MICs against dermatophytes.[19]

We were unable to observe variance of susceptibilities between species, as has been described before for dermatophytes,[23] due to isolation of only two species, of which, there were only three isolates of T. rubrum.

These low MICs found for TBF, GF, and ITZ can help to explain the promising results obtained for the treatment of dermatophytosis with these antifungal agents.[5] One of the patients responded clinically to TBF despite an MIC of 0.0625 µg/mL. This discordance between in vivo and in vitro data is illustrated by the “90–60 rule,” which maintains that infections due to susceptible strains respond to appropriate therapy in almost 90% of cases, whereas infections due to resistant strains respond in almost 60% of cases.

The determination of the in vitro susceptibility may prove helpful to predict the ability of a given antifungal agent to eradicate dermatophytes. Although a reference method for dermatophytes is not available, a good correlation between the in vitro data, using both microdilution method, and clinical outcome has been shown.[5]

These results can allow clinicians for appropriate therapy with a high probability of successful results. However, we need in vitro tests correlated with clinical studies to develop interpretive break points for dermatophyte susceptibility testing. Evaluation of the results obtained for susceptibility and resistance is greatly improved when break points are determined.[24]


  Conclusion Top


Our study successfully noted the desired objectives. All the patients attained clinical cure and effective treatment at 48 and 72 days, respectively. There was no case of relapse (clinical or mycological) when evaluated at day 72 after starting treatment. On the basis of in vitro antifungal susceptibility testing studies, the MICs obtained were well in range as mentioned in the CLSI M38-A2 (2008) guidelines. Clinico-mycological correlation revealed TBF to be the most effective antifungal agent. To the best of our knowledge, perhaps this study is the first of its kind showcasing clinico-mycological (in vitro and in vivo) correlation and in vitro cross-resistance among azoles in the treatment of cutaneous dermatophytic infections. This study has provided an overview of the development of in vitro resistance in dermatophyte species and the management of cases clinically unresponsive to treatment. Identifying correlations between MIC and clinical outcomes is an ongoing challenge, and further research is needed before MICs are independently used to make decisions regarding the class of drug to be used for the treatment of cutaneous dermatophytosis.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/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

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Margaret S, Lee B, Nellie K. Oral antifungal agents. In: Fitzpatrick’s Dermatology in General Medicine. 7th ed. New York: McGraw-Hill; 2008. p. 2211-7.  Back to cited text no. 1
    
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Rand S. Overview: The treatment of dermatophytosis. J Am Acad Dermatol 2000;43:S104-12.  Back to cited text no. 2
    
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Bokhari FM. Antifungal activity of some medicinal plants used in Jeddah Saudi Arabia. Mycopathologia 2009;7:51-7.  Back to cited text no. 3
    
4.
CLSI. M38-A2. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard. 2nd ed. Wayne, PA: CLSI; 2008.  Back to cited text no. 4
    
5.
Korting HC, Ollert M, Abeck D. Results of German multicenter study of antimicrobial susceptibilities of Trichophyton rubrum and Trichophyton mentagrophytes strains causing tinea unguium. German Collaborative Dermatophyte Drug Susceptibility Study Group. Antimicrob Agents Chemother 1995;39:1206-8.  Back to cited text no. 5
    
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Favre B, Hofbauer B, Hildering KS, Ryder NS. Comparison of in vitro activities of 17 antifungal drugs against a panel of 20 dermatophytes by using a microdilution assay. J Clin Microbiol 2003;41: 4817-9.  Back to cited text no. 6
    
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Kanwar AJ, Mamta, Chander J. Superficial fungal infection. In: Valia RG, Valia AR, editors. IADVL Textbook and Atlas of Dermatology. 2nd ed. Mumbai, India: Bhalani Publishing House; 2001. p. 215-58.  Back to cited text no. 8
    
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Philpot CM. Some aspects on the epidemiology of tinea. Mycopathologia 1997;3:62.  Back to cited text no. 9
    
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Khosa RK, Girgla HS, Hajini GH, Sharma BM, Singh GM. Study of dermatomycoses. Int J Dermatol 1981;20:130-2.  Back to cited text no. 10
    
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Sardari L, Sambhashiva RR, Dandapani R. Clinico-mycological study of dermatophytes in a coastal area. Indian J Dermatol Venereol Leprol 1983;49:71-5.  Back to cited text no. 11
    
12.
Verma BS, Vaishnav VP, Bhat RP. A study of dermatophytosis. Indian J Dermatol Venerol Leprol 1970;36:182.  Back to cited text no. 12
    
13.
Gupta AK, Chaudhary M, Elewski B. Tinea corporis, tinea cruris, tinea nigra, and piedra. Dermatol Clin 2003;21:395-400, v.  Back to cited text no. 13
    
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Thaker SJ, Mehta DS, Shah HA, Dave JN, Mundhava SG. A comparative randomized open label study to evaluate efficacy, safety and cost effectiveness between topical 2% sertaconazole and topical 1% butenafine in tinea infections of skin. Indian J Dermatol 2013;58:451-6.  Back to cited text no. 14
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Agarwal US, Saran J, Agarwal P. Clinico-mycological study of dermatophytes in a tertiary care centre in northwest India. Indian J Dermatol Venereol Leprol 2014;80:194.  Back to cited text no. 15
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Sahai S, Mishra D. Change in spectrum of dermatophytes isolated from superficial mycoses cases: First report from central India. Indian J Dermatol Venereol Leprol 2011;77:335-6.  Back to cited text no. 16
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Kumaran G, Jeya M. Clinico-mycological profile of dermatophytic infections. Int J Pharm Bio Sci 2014;5:1-5.  Back to cited text no. 17
    
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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.  Back to cited text no. 18
    
19.
Gupta AK, Kohli Y. In vitro susceptibility testing of ciclopirox, terbinafine, ketoconazole and itraconazole against dermatophytes and nondermatophytes, and in vitro evaluation of combination antifungal activity. Br J Dermatol 2003;149:296-305.  Back to cited text no. 19
    
20.
Araújo CR, Miranda KC, Fernandes Ode F, Soares AJ, Silva Mdo R. In vitro susceptibility testing of dermatophytes isolated in Goiania, Brazil, against five antifungal agents by broth microdilution method. Rev Inst Med Trop Sao Paulo 2009;51:9-12.  Back to cited text no. 20
    
21.
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. 21
    
22.
Santos DA, Hamdan JS. In vitro activities of four antifungal drugs against Trichophyton rubrum isolates exhibiting resistance to fluconazole. Mycoses 2007;50:286-9.  Back to cited text no. 22
    
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Howyda ME, Ahmed MA, Ahmed A. Antifungal susceptibility patterns of dermatophytes clinical isolates from dermatophytosis patients before and after therapy. Egypt J Med Microbiol 2010;19:41-6.  Back to cited text no. 24
    


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