Analysis of ERG 11 Expression in Clinical Isolates of Dermatophytes in Patients with Resistant Tinea Infection

Background: Dermatophytes are keratinophilic groups of microorganisms which invade keratinized tissue. Isolate and identify the dermatophyte species from samples collected from patients suffering from onychomycosis, tinea corporis and tinea cruris; and perform in vitro microbroth antifungal susceptibility testing. Further, the expression of Ergosterol 11 (ERG 11) amongst the isolates from responders and non-responders to antifungal treatment was studied by Real-time PCR. Methods: A total of 120 dermatophytosis cases attending a dermatological clinic in a tertiary care hospital were included in the study. Microscopy of KOH mount was positive in 90 isolates while 60 were culture positive. Results: The most


Introduction
Dermatophytes, a group of keratinophilic filamentous fungi thriving on the keratin substrate, are the etiological agents responsible for causing superficial fungal infection in human and animals with an estimated global prevalence of approximately 20 percent as per the World Health Organization (WHO) report.Predominant in the tropical and subtropical countries; especially in the developing countries like India, the hot and humid climate is favorable to the acquisition and maintenance of the disease. 1 Routine procedures for dermatophyte species identification is based on the conventional phenotypic method of microscopy and culture.
Morphological and physiological characteristics can frequently vary, often influenced by temperature variation, and type of medium, further hampering strain identification.[4][5] In the past few years, chronic dermatophytosis has become a public health problem.Recalcitrant

Materials and Methods
The present study was conducted on a total of 120

RNA isolation
Total RNA from culture isolates obtained from skin and nail were extracted by TRIzol TM Reagent (Invitrogen Bio services India Pvt.Ltd) method.
Isolates were mixed in TRIzol TM Reagent, 1 ml of RNA express reagent was added in the sample, total lysate cells were obtained, which was further mixed by micropipette to form a homogeneous lysate and incubated for 5 min at room temperature.200µl of chloroform per ml of RNA express reagent was added and centrifuged at 12,000 rpm for 15 min at 4°C to obtain a colorless upper aqueous phase containing RNA which was separated in another 1.5 ml Eppendorf tube.RNA was washed by adding 500 µl of Isopropanol.The Eppendorf tube was centrifuged at 12,000 rpm for 10 min at 4°C and supernatant was discarded.1 ml of 75% ethanol was added to the pellet and vortexed.This was followed by centrifugation at 10,500 rpm for 5 min at 4°C.The supernatant was discarded, and the pellet was suspended in RNase free water, which was incubated at 55-60°C for 15 min.
The isolated RNA was stored at -80°c immediately.Of the 60 isolates, 15 isolates were identified as  Time PCR data and by applying 2 −ΔΔC T method was 18.18-fold change and up-regulated between the two groups as depicted in Table II and Figure   C.It was also seen that the level of ERG 11 expression was significantly different between the responders and the non-responder groups (t = 5.617, p<0.001) as shown in Table III.As documented in other studies, males appeared to be more exposed to acquire dermatophytic infection (58.3 %) as compared to females (41.7 %) which is in accordance with the other researchers worldwide. 19Interestingly we found T.

Real-time PCR for Ergosterol-
mentagrophytes complex was more prevalent than T. rubrum causing dermatophytosis in Delhi, similar to other studies. 20Out of 60 patients, 43.3% of cases were naive cases presenting with the first episode of dermatophytic infection, 50% of cases were previously on antifungals for 1-2 yrs and 6.6% cases categorized as relapse.The MIC range for T. mentagrophytes and T. rubrum for different antifungals were within range of 0.125µg/ml-128 µg/ml for fluconazole; 0.0313 µg/ml-32 µg/ml for itraconazole, terbinafine and griseofulvin; 0.0625 µg/ml-16 µg/ml for voriconazole and 0.000125 µg/ml-1 µg/ml for luliconazole. To azoles are still used as the first line of management in several cases.Azoles inhibit the fungal cytochrome P-450 enzyme lanosterol 14-α-demethylase (Cyp51) encoded by ERG 11 gene, being effective against several fungi and griseofulvin amongst dermatophytes leading to clinical failure.The potential loss of efficacy of azoles has prompted many researchers to make concerted efforts to discover new drugs that might block fungal growth at different metabolic sites. 7,8Hence this study was aimed to co-relate the MIC values with the expression of ERG 11 genes by real-time PCR for fluconazole and itraconazole and to correlate the expression with MIC and clinical cure.
The purity and integrity of the RNA samples were confirmed by agarose gel electrophoresis.Nano-drop determined the concentration and purity of the samples by measuring concentration and 260/280 ratio.cDNA Preparation: -Extracted RNA was used as a template for cDNA synthesis by Superscript reverse transcriptase II (Invitrogen Bioservices India Pvt.Ltd) kit.Total RNA (2-5 µg) from each sample was reverse transcribed into cDNA. of the each reaction was 20µl; the reaction mixtures contained 2-5µl of c-DNA, 20 µl of SYBER Green Universal Master Mix, and 160 nM of each primer, and the volume was brought to 20 µl with nuclease-free water.The Real Time PCR condition had been standardized according to the melting temperature (Tm) of the primers.The Ergosterol-11 is the gene of interest and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which served as an endogenous control.The PCR program was 95°C for 10 min, followed by 40 cycles of 95°C for 10s and the annealing/extension at 60°C for 20s and 72°C for 10s.Following PCR, dissociation curve analysis was performed to verify that a single product was amplified.Real-Time PCR was carried out in a Roche Light Cycler 480-II (Roche Diagnostics, Germany).Amplification Curve for each gene was shown, and the threshold cycle (Ct) value was calculated by the software.Following Real-time PCR, dissociation curve analysis was performed to verify that a single product was amplified. 15Preparation Antifungal agents as per CLSI M-38 A: Antifungal susceptibility testing was performed according to the Clinical Laboratory Standard Institute (CLSI) 38 A2 guidelines suggested for molds.T. mentagrophytes ATCC 28185 was used as quality control.RPMI 1640 medium and MOPS (4-Morpholine propane sulfonic acid) (Sigma Aldrich, USA), pH 7.0 was used as a medium for suspension of the isolates.The antifungal drugs tested were fluconazole, itraconazole, terbinafine, griseofulvin, luliconazole and voriconazole (Sigma Aldrich, USA).The initial inoculum suspension of the isolates (i.e., 1 -3 x 10 6 cells/ml) was prepared using the spectrophotometer to match the optical density of 80 % transmittance at 530 nm wavelength.The final concentration of the inoculum (1-3 x 10 3 cells/ml) is prepared in 1: 50 dilution in RPMI.The test assay which was performed in 96 U bottom microtitre plate was incubated for 96 hours at 35°C in BOD incubator. 16Statistical analysis was done using Statistical Package for Social Sciences package (SPSS; Inc., Chicago, IL, USA; version 20.0).The analysis comprised of calculating means and proportions.The independent sample t-test was used to test the statistical significance of the data.Study Design: Cross Sectional Analytical Study.Sample Size: 60 confirmed cases.Based on this data, the sample size of 60 has been found to be statistically appropriate for this study (sample size calculated using the standard formula Z2P(1-P)/d2 for a prevalence study, where Z = Z statistic for a level of confidence (the value of Z is set at 1.96), P = expected prevalence or proportion (in proportion of one; if 45%, P = 0.45), and d = precision (in proportion of one; if 125%, d P rate of Trichophyton rubrum in dermatophytosis confirmed cases is 45% with 25% relative precision either the site with 95% confidence level is 60.8 i.e. total 60 cases is taken. 120 samples were collected which included 25 from onychomycosis patients and 95 skin scrapings from Tinea corporis and cruris patients.The mean age of patients was 32.5±11.32year (with the range from 16 to 60 years).The duration of dermatophytic infection ranged from 3 months to 10 years (9.8 months ± 5.45 months).Out of 120 samples, 90 were found to be KOH positive, of which 60 samples were culture positive.The clinical profile of patients with culture-positive isolates (60) varied from those with first dermatophytic infection (26), those with no clinical cure despite antifungal therapy (30) and those with relapse (4).
Figure-1.a:Agarose gel depicting bands of PCR products of T. mentagrophytes at 130bp.Lane 1-8 samples 1-8 of T. mentagrophytes and L is 100 base pair.