MICs Of Itraconazole Research Articles

Combinatory effect of ALA-PDT and itraconazole in the treatment of cutaneous protothecosis

BackgroundAs a rare subcutaneous infection, protothecosis is easily misdiagnosed. Similar to other subcutaneous infection, there is no unified standard for treatment, for cases not suitable for surgery, clinicians often use antifungal drugs based on their experience, and the course of treatment varies from several months to several years. Based on the fact that there are few relevant materials and researches on photodynamic therapy (PDT), we conducted a study based on a clinical case that used oral itraconazole combined with 5-aminolevylinic acid photodynamic therapy (ALA-PDT) to treat a patient with cutaneous protothecosis caused by Prototheca wicherhamii. MethodsDifferent concentrations of ALA and different light doses were used to investigate the effects of ALA-PDT on the growth inhibition of P. wickerhamii in vitro with Colony-counting Methods. And we used transmission electron microscopy (TEM) to visualize the structural changes and the effects of ALA-PDT treating on cellular structures of the P. wickerhamii. Futher, we performed the susceptibility test of P. wickerhamii to itraconazole before and after ALA-PDT in vitro. ResultsWe have successfully treated a patient with cutaneous protothecosis caused by P. wickerhamii by using combination therapy in a total of 9-week course of treatment. In vitro, ALA-PDT can inhibit the growth of P. wickerhamii when the ALA concentration was 5 mg/mL (P < 0.01), and this effect became stronger as the concentration of ALA or light dose is increased. Using TEM, we confirmed that ALA-PDT can disrupt the cell wall structure and partition structure of P. wickerhamii, which may contribute to its inhibitory effect. Further studies showed that the MIC of itraconazole for P. wickerhamii was decreased after ALA-PDT. ConclusionsALA-PDT combined with oral itraconazole can be used to treat cutaneous protothecosis. Accordingly, ALA-PDT can destroy the cell wall and partition structure of P. wickerhamii leading to an inhibitory effect on it in vitro, and the effect is enhanced with the increase of ALA concentration and light dose. Also, the sensitivity of P. wickerhamii to itraconazole is observed increased after ALA-PDT. So our study provides a theoretical basis for the promising treatment against cutaneus protothecosis.

Clinicomycological Study of Dermatophyte Infections in Patients Attending Dermatology Outpatient Department in a Tertiary Care Hospital

Context: Dermatophytosis is a fungal disease characterized by the infection of skin, hair, and nails. There is an upsurge of complicated superficial dermatophytosis and an increase in the incidence of chronic, relapsing, recurrent cases in India that are also often unresponsive to conventional doses of drugs. Aims: The aim of this study was to study the prevalence of dermatophyte species in the particular study area and it is susceptibility to itraconazole, terbinafine, and fluconazole. Settings and Design: All dermatophytes isolates obtained were subjected to 10% KOH mount, fungal culture, and antifungal sensitivity testing for fluconazole, itraconazole, and terbinafine. Materials and Methods: Newly diagnosed cases of dermatophytosis without treatment for the past 3 months were included in the study. KOH preparation was observed and subjected to fungal culture. Then, the susceptibility of the dermatophytes to oral antifungals (itraconazole, terbinafine, and fluconazole) was done. Results: Out of 28 patients with tinea, 14 males and 12 females were tested KOH positive. Out of them, 23 patients showed positive for dermatophyte growth. In them, 15 isolates were Trichophyton mentagrophytes and 8 isolates were Trichophyton rubrum. Antifungal drug susceptibility testing data indicated that, for the two species, MIC of itraconazole ranged between 0.03 and 16 μg/ml, terbinafine between 0.03 and 16 μg/ml, and fluconazole between 0.25 and 64 μg/ml. Conclusions: The study provided information regarding the prevalent species of dermatophyte in our study area and it is high sensitivity to itraconazole compared to terbinafine and the lowest for fluconazole. This gives us guidance as to which antifungal to use empirically in our clinical practice to treat tinea infection.

Two different types of tandem sequences mediate the overexpression of TinCYP51B in azole-resistant Trichophyton indotineae.

Trichophyton indotineae is an emerging dermatophyte that causes severe tinea corporis and tinea cruris. Numerous cases of terbinafine- and azole-recalcitrant T. indotineae-related dermatophytosis have been observed in India over the past decade, and cases are now being recorded worldwide. Whole genome sequencing of three azole-resistant strains revealed a variable number of repeats of a 2,404 base pair (bp) sequence encoding TinCYP51B in tandem specifically at the CYP51B locus position. However, many other resistant strains (itraconazole MIC ≥0.25 µg/mL; voriconazole MIC ≥0.25 µg/mL) did not contain such duplications. Whole-genome sequencing of three of these strains revealed a variable number of 7,374 bp tandem repeat blocks harboring TinCYP51B. Consequently, two types of T. indotineae azole-resistant strains were found to host TinCYP51B in tandem sequences (type I with 2,404 bp TinCYP51B blocks and type II with 7,374 bp TinCYP51B blocks). Using the CRISPR/Cas9 genome-editing tool, the copy number of TinCYP51B within the genome of types I and II strains was brought back to a single copy. The azole susceptibility of these modified strains was similar to that of strains without TinCYP51B duplication, showing that azole resistance in T. indotineae strains is mediated by one of two types of TinCYP51B amplification. Type II strains were prevalent among 32 resistant strains analyzed using a rapid and reliable PCR test.

The Domestic Isolation of Terbinafine- and Itraconazole-Resistant Trichophyton indotineae in Chinese Mainland.

Trichophyton indotineae, a new species of dermatophytes, has become a significant concern in treating dermatophytosis due to the high level of terbinafine resistance reported in India and even worldwide. This study aimed to report the terbinafine- and itraconazole-resistant T. indotineae in Chinese mainland, by identifying the phylogenetic classification of the isolate strain, and detecting the drug resistance, gene mutation and expression. The skin scales of the patient were cultured on SDA and the isolate was authenticated by DNA sequencing and MALDI-TOF MS. Antifungal susceptibility testing was performed following the M38-A2 CLSI protocol to examine the MICs values of terbinafine, itraconazole, fluconazole, etc. The strain was screened for mutations in the squalene epoxidase (SQLE) gene by Sanger sequencing and detected the expression of CYP51A and CYP51B by qRT-PCR. A multi-resistant ITS genotype VIII sibling of the T. mentagrophytes complex (T. indotineae) was isolated in Chinese mainland. The strain harbored high terbinafine MIC of > 32μg/mL and itraconazole MIC of 1.0μg/mL, which was identified a mutation in the squalene epoxidase gene with amino acid substitution (Phe397Leu, mutation 1191C > A). In addition, overexpression of CYP51A and CYP51B was observed. With multiple relapses, the patient finally achieved clinical cure by itraconazole pulse therapy and topical clotrimazole cream for 5weeks. The first domestic strain of terbinafine- and itraconazole-resistant T. indotineae from a patient in Chinese mainland was isolated. Itraconazole pulse therapy can be an effective method for the treatment of T. indotineae.

MYCOLOGICAL AND CLINICAL PROFILE ALONG WITH ANTIFUNGAL SUSCEPTIBILITY PATTERN OF DERMATOPHYTOSIS IN A TERTIARY CARE HOSPITAL FROM WESTERN INDIA

Introduction:Dermatophytosis is a common superficial mycosis causing significant cutaneous morbidity. In recent times, the prevalence of dermatophytosis is increasing. The dermatophyte infections spread easily and rapidly especially in low socioeconomic classes and thus warrant early therapy.Dermatophyte infections are commonly treated by topical antifungal drugs like clotrimazole, terbinafine, ketoconazole. But severe and chronic form of dermatophytosis requires treatment with systemic antifungal drugs like itraconazole, griseofulvin and terbinafine.There is emergence of antifungal resistant strains due to incongruous use of antifungals and poor antifungal policy. There are limited studies related to antifungal susceptibility testing (AFST). So present study was undertaken to determine mycological, clinical profile and antifungal Susceptibility testing ofdermatophytosis. Material &amp; Methods: A prospective study was conducted on patients with superficial fungal infections over a period of 11 months (October 2018 to August 2019). Various samples like skin scrapings, scales, hair and nail clippings were processed by standard fungal culture methods. AFST was performed by using E-test strips (HiMedia) of fluconazole, itraconazole and terbinafine on Sabourauds dextrose agar plates and interpreted according to CLSI (M38A). Results: A total of 25 (23.8%) dermatophytes were isolated from 105 (skin 57, Nail 41, scales 11, Hair 6) samples. Out of 25 culture positive patients, 18 presented as tineacorporis, 3 as tineacruris, 3 onchomycosis, 1 each as tineacapitis&amp;tinea incognito. T. tonsurans was most common dermatophyte 40% (N10), followed by T. rubrum 36% (N9), T. mentagrophytes 12% (N3) and M. canis 8% (N2) and T. megninii 4% (N1). AFST of all 25 isolates revealed that 21 isolates were sensitive to itraconazole (0.023 to 0.75 mcg/ml) wheras a single isolate of T. rubrum and T. tonsurans each were resistant. Two isolates of T.tonsurans showed lower MICs for itraconazole (0.023mcg/ml). For terbinafine (0.002-0.008mcg/ml), 14 isolates (56%) showed resistance with MICs &gt;32 mcg/ml. For fluconazole (range 0.5-4 mcg/ml) only 3 isolates showed MIC in range while22 were resistant MICs &gt;256mcg/ml. The results were communicated with dermatologists and appropriate changes were made in patient therapy. Conclusion: The emergence of resistant dermatophytesemphasises the need of antifungal drug susceptibility tests, antifungal stewardship and strong antifungal policy to enable the clinician to start suitable antifungals to avoid antifungal resistance and treatment failure.

Afu-Emi1 Contributes to Stress Adaptation and Voriconazole Susceptibility in Aspergillus fumigatus.

Invasive aspergillosis (IA) is the second most common invasive fungal disease and is associated with high mortality rates. Aspergillus fumigatus is the predominant causal agent of this life-threatening infection. Triazoles are still the cornerstone of antifungal treatment, and voriconazole remains the first-line choice. However, voriconazole resistance has been increasingly reported, which results in significantly higher mortality rates for IA and is particularly problematic. In the present study, we report the identification and functional study of a protein with previously unknown function that is encoded by the gene designated Afu-emi1 (AFUA_1G07360). High-throughput gene replacement technology was applied to construct the knockout ΔAfu-emi1 strain and a revertant strain. The MICs for azoles, including posaconazole, itraconazole, and voriconazole, were evaluated via the broth microdilution method and E-tests, which revealed that disruption of Afu-emi1 resulted in 4-fold increased susceptibility to voriconazole. Colony growth in the presence of oxidants, namely, H2O2 and menadione, and osmotic pressure-altering agents, namely, NaCl and d-sorbitol, was measured. The Afu-emi1 mutant strain exhibited a significant growth defect under oxidative and osmotic stress. The reactive oxygen species (ROS) production levels with or without voriconazole pretreatment were determined, and the Afu-emi1 mutant strain exhibited significantly lower ROS production levels. The effects of Afu-emi1 disruption on voriconazole susceptibility, growth under stress, and ROS production were restored in the revertant strain. In addition, the expression of cyp51A, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1b in the ΔAfu-emi1 strain was significantly reduced. In conclusion, deletion of the gene Afu-emi1 resulted in increased voriconazole susceptibility, attenuated ability for oxidative and osmotic stress adaptation, decreased ROS production, and downregulation of cyp51A, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1b expression, suggesting that Afu-Emi1 is an important regulator of stress adaptation and cyp51A and efflux pump expression in this medically important fungus. IMPORTANCE Voriconazole is the first-line choice for IA, a life-threatening disease. Therefore, voriconazole resistance has become particularly problematic. Disruption of Afu-emi1 resulted in increased susceptibility to voriconazole, a significant growth defect under oxidative and osmotic stress, and downregulation of target enzyme Cyp51A and efflux pump expression, suggesting that Afu-Emi1 is an important regulator of stress adaptation and cyp51A and efflux pump expression in this medically important fungus. Targeting Afu-Emi1 might help to enhance azole therapeutic efficacy and impede azole resistance.

A Clinical-Mycological Study of Dermatophytosis in Western India with Focus on Antifungal Drug Resistance as a Factor in Recalcitrance.

Dermatophytosis have assumed epidemic proportions in India. Antifungal drug resistance solely cannot explain disease magnitude and changing epidemiology. Aim of this study was to analyse clinical-mycological aspects of dermatophytosis, and estimate contribution of drug resistance in clinical recalcitrance. This single-centre observational, cross-sectional, descriptive study was done in tertiary centre of western India after ethical approval, enrolling dermatophytosis patients of all ages and sex. After history and examination, KOH mount and culture in modified SDA medium was done. Culture positive isolates were subjected to E-strip antifungal susceptibility method to test MIC for Terbinafine, Itraconazole, Fluconazole and Griseofulvin. Total 300 patients were included, with mean age of 33.83±27.5 years and male-to-female ratio of 1.22:1; tinea corporis et cruris being commonest, 39.33% (n=118). Only 11.67% (n=35) were treatment naïve, having classical annular morphology. History of topical steroid abuse was found in 81.67% (n=245), with pseudoimbricate lesions in 70.61% (n=173). 86.67% (n=260) had KOH positivity while 83.33% (n=250) had culture positivity: Trichophyton mentagrophytes 45.6% (n=114), followed by Trichophyton rubrum in 34.4% (n=86). A total of 265 patients fit into definition of recalcitrance, from which 12.45%, i.e., 33 isolates showed in-vitro fluconazole resistance. 14.33% (n=43) cases were chronic, 37% (n=111) persistent, 46% (n=138) recurrent while 17% (n=51) had relapse in their disease course. Steroid abuse was the commonest denominator. Role of antifungal resistance in recalcitrant dermatophytosis remains debatable. Stopping steroid abuse, which is often the commonest culprit, with adherence to standard antifungal therapy remains the paradigm in management.

In Vitro Antifungal Activity of Azoles and Other Antifungal Agents Against Pathogenic Yeasts from Vulvovaginal Candidiasis in China.

Vulvovaginal candidiasis (VVC) is a public health issue worldwide. Little is known of the optimal treatment of recurrent VVC (RVVC) has not been established. Through the in vitro antifungal susceptibility profiling of VVC isolates, we hope to foster significant improvements in the control and treatment of this disease. Candida isolates from VVC patients were collected from 12 hospitals in 10 cities across China. Species were identified by phenotype analysis and DNA sequencing. Species were identified by phenotype analysis and DNA sequencing. Susceptibilities to 11 drugs were determined by Clinical and Laboratory Standards Institute broth microdilution. 543 strains were isolated from those VVC patients enrolled in this study, of which, 15.7% were from RVVC. The most commonly identified species was C. albicans (460, 84.71%), and the most commonly non-albicans Candida spp. (NAC) was C. glabrata (47, 8.66%). NAC also included C. Krusei, Meyerozyma Guillermondii, Meyerozyma Caribbica, C. Tropicalis, C. Parapsilosis, and C. Nivariensis. Most C. albicans isolates were susceptible to caspofungin (99.8%), followed by fluconazole (92%) and voriconazole (82.6%). The proportion of C. albicans strains with wild type (WT) MICs that were susceptible to amphotericin B and caspofungin were 98%, followed by posaconazole at 95%, itraconazole at 86%, fluconazole at 74% and voriconazole at 54%. The fluconazole MICs for C. albicans were lower than those for NAC (P < 0.05), while the itraconazole MICs showing no significant difference (P > 0.05). The susceptible rate of uncomplicated VVC to fluconazole was 92%. The proportion of WT strains to fluconazole in RVVC was much lower than that in other types of VVC (67 vs. 77%, P < 0.05). However, the proportions of WT strains to itraconazole in RVVC was over 85%, which was much higher than that to fluconazole (87 vs. 67%, P < 0.05). C. albicans was still the predominant pathogen for VVC in China, while C. glabrata was the main species in NAC. Fluconazole could still be used as an empirical treatment for uncomplicated VVC. However, fluconazole may not be the first choice for the therapy of RVVC. In such cases, itraconazole appears to be the more appropriate treatment. As for VVC caused by NAC, nonfluconazole drugs, such as itraconazole, may be a good choice.

Tea contamination by mycotoxins and azole-resistant mycobiota – The need of a One Health approach to tackle exposures

Despite tea beneficial health effects, there is a substantial risk of tea contamination by harmful pathogens and mycotoxins. A total of 40 tea samples (17 green (raw) tea; 13 black (fermented) tea; 10 herbal infusions or white tea) were purchased from different markets located in Lisbon district during 2020. All products were directly available to consumers either in bulk (13) and or in individual packages (27). Bacterial analysis was performed by inoculating 150 μL of samples extracts in tryptic soy agar (TSA) supplemented with 0.2 % nystatin medium for mesophilic bacteria, and in Violet Red bile agar (VRBA) medium for coliforms (Gram-negative bacteria). Fungal research was performed by spreading 150 μL of samples in malt extract agar (MEA) supplemented with 0.05 % chloramphenicol and in dichloran-glycerol agar (DG18) media. The molecular detection of the Aspergillus sections Fumigati, Nidulantes, Circumdati and Flavi was carried out by Real Time PCR (qPCR). Detection of mycotoxins was performed using high performance liquid chromatograph (HPLC) with a mass spectrometry detector. Azole resistance screening was achieved following the EUCAST guidelines. The highest counts of total bacteria (TSA) were obtained in green raw tea (81.6 %), while for coliform counts (VRBA) were found in samples from black raw tea (96.2 %). The highest fungal counts were obtained in green raw tea (87.7 % MEA; 69.6 % DG18). Aspergillus sp. was the most prevalent genus in all samples on MEA (54.3 %) and on DG18 (56.2 %). In the raw tea 23 of the samples (57.5 %) presented contamination by one to five mycotoxins in the same sample. One Aspergillus section Fumigati isolate from green tea beverage recovered form itraconazole-Sabouraud dextrose agar (SDA) medium, presented itraconazole and posaconazole E-test MICs above MIC90 values. Our findings open further discussion regarding the One-Health approach and the necessary investment in researching biological hazards and azole-resistance associated with the production and consumption of tea (in particular green tea).

Gradient diffusion strips for detecting azole resistance in Aspergillus fumigatus sensu lato.

Studies comparing gradient diffusion strips (GDSs) and the EUCAST E.Def 9.4 microdilution method are scarce, thwarted by a low number of isolates, and restricted to selected antifungal agents. We evaluated the performance of GDSs to detect azole resistance in A. fumigatus, including cryptic species. A. fumigatus sensu stricto (n=89) and cryptic species (n=52) were classified as susceptible or resistant to itraconazole, voriconazole, posaconazole and isavuconazole (EUCAST E.Def 9.4; clinical breakpoints v10). A. fumigatus sensu stricto azole-resistant isolates had the following cyp51A gene mutations: TR34 -L98H (n=24), G54R (n=5), TR46 -Y121F-T289A (n=1), F46Y-M172V-N248T-D255E-E427K (n=1), F165L (n=1) and cyp51A gene wild type (n=3). GDSs (ETEST®, bioMèrieux, Marcy-l'Etoile, France and Liofilchem®, Roseto degli Abruzzi, Italy) MICs were obtained by following the manufacturer's guidelines. For A. fumigatus sensu stricto, itraconazole MICs >1.5mg/L, voriconazole >0.38 mg/L, posaconazole >0.75 mg/L, and isavuconazole >0.5mg/L correctly separated resistant from susceptible isolates with two exceptions. Considering the aforementioned cut-off MICs, sensitivity/specificity values of GDSs to detect azole resistance were: itraconazole (97%/100%), voriconazole (97%/100%), posaconazole (97%/100%) and isavuconazole (93.3%/100%). For cryptic species isolates, voriconazole MICs >1mg/L and isavuconazole >0.75 mg/L separated resistant isolates from susceptible isolates with 15 and 27 exceptions, respectively. Considering the aforementioned cut-off MICs, sensitivity/specificity values were as follows: voriconazole (68.1%/100%) and isavuconazole (25%/100%). For itraconazole and posaconazole, it was not possible to establish cut-off values. We set tentative cut-off MIC values to correctly spot resistant Aspergillus fumigatus sensu stricto isolates using GDSs. The performance against cryptic species was poor.

Molecular epidemiology and clinical-laboratory aspects of chromoblastomycosis in Mato Grosso, Brazil.

Chromoblastomycosis is a disease caused by melanized fungi, primarily belonging to the genera Fonsecaea and Cladophialophora, mainly affecting individuals who are occupationally exposed to soil and plant products. This research aimed to determine the clinical, epidemiological and laboratory characteristics of chromoblastomycosis in the state of Mato Grosso, Brazil. Patients diagnosed with chromoblastomycosis treated at the Júlio Müller University Hospital, Cuiabá, Brazil, from January 2015 to December 2020, whose isolates were preserved in the Research Laboratory of the Faculty of Medicine of the Federal University of Mato Grosso. Isolates were identified by partly sequencing the Internal Transcribed Spacer (ITS) and β-tubulin (BT2) loci. AFLP fingerprinting was used to explore the genetic diversity. Susceptibility to itraconazole, voriconazole, 5-fluorocytosine, terbinafine and amphotericin B was determined by the broth microdilution technique. Ten patients were included, nine were male (mean age=64.1 years). Mean disease duration was 8.6 years. Lesions were mainly observed in the lower limbs. Predominant clinical forms were verrucous and scarring. Systemic arterial hypertension and type II diabetes mellitus were the predominant comorbidities. Leprosy was the main concomitant infectious disease. Fonsecaea pedrosoi was the unique aetiological agent identified with moderate genetic diversity (H= 0.3934-0.4527; PIC= 0.3160-0.3502). Antifungal agents with the highest activity were terbinafine, voriconazole and itraconazole. Chromoblastomycosis is affecting the poor population in rural and urban areas, mainly related to agricultural activities, with F. pedrosoi being the dominant aetiologic agent. All isolates had low MICs for itraconazole, voriconazole and terbinafine, confirming their importance as therapeutic alternatives for chromoblastomycosis.

P093 A novel GAL4-like transcriptional regulator modulate the azoles sensitivity of Exophiala dermatitidis

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectives Exophiala dermatitidis is an emerging opportunistic pathogen among the ever-increasing numbers of immunocompromised hosts. Understanding its transmission mechanism, pathogenicity, and potential azole-resistant mechanism is important for better understanding of this fatal pathogen and benefits future diagnosis, and treatment.By using a PCR-based fragment fusion approach, we applied phosphotransferase (HPH) resistance gene which amplified from pAN7-1 as a selective gene for generating replacement cassettes. Combined with electroporation transformation and screening on selection medium containing 50 μg/ml HMB, we obtained the gene knockout strain Δ GAL4, a novel GAL4-like transcriptional factor (XP_009154426.1) encoding gene (GeneID:20 306 783) accurately deleted.MethodsGrowth curve determination: Take 200 μL of 2 × 10⁴ CFU/ml bacterial suspension and 200 μL liquid sabourand medium in EP tubes. Shaking at 200 rpm 37°C for 0 h, 1 h, 2 h, 3 h, 4 h, 8 h, 12 h, 16 h, 18 h, 24 h, 30 h, 36 h, 48 h. Then add 200 μL cultured bacterial suspension to a 96-well plate and repeat three times. Meanwhile set up blank medium control wells. By using an enzyme marker to measure the absorbance at 504 nm, analyzing with SPSS, and then graphing with GraphPad software.Morphological observations: Activated strains on Sabourand microcultures and incubated at 37 °C for 3d. Observe morphology by using fluorescent microscopy to compare the different strains of wild and knockout strains, and using lactic acid cotton phenol blue/fluorescent dye filming observe the morphology of conidia.E-test drug sensitivity method: Spread fresh bacterial suspension of ΔGAL4 and ATCC34100 containing 1 × 10⁶CFU/ml spores on RPMI 1640 agar plates with sterile cotton swabs, and place drug sensitivity test strips [itraconazole (Itraconazole Liofilchem, ICZ 92148), posaconazole (Posaconazole Liofilchem (PCZ 92152), voriconazole (Mérieux, VRC 532800)] in the center plate, after sealing with a parafilm, incubating in 37°C for 36-48 h, and read the values at the tangent of the drug susceptibility strip and the antibacterial circle.ResultsThe results revealed that the ΔGAL4 showed almost no difference in growth and morphology compared with wild strain, but its sensitivity to azoles was altered. We applied E-test to evaluate the MIC changes of azoles. The MIC of itraconazole, voriconazole, and posaconazole against ΔGAL4 was 0.5 μg/ml, 0.125 μg/ml, and 0.25 μg/ml; against ATCC34100 was 2 μg/ml, 0.19 μg/ml, and 0.38 μg/ml, respectively. △GAL4 MIC suggests that ∆GAL4 showed more sensitivity to azoles compared to its wild-type strain.ConclusionThe present results suggested that a novel GAL4-like transcriptional factor may play an important role in azole sensitivity of E. dermatitidis, which may be through modulating efflux pumps or Cyp51A. Further investigation of this GAL4 related modulating pathway is needed.

P107 Spectrum of Dermatophyte infections and drug susceptibility pattern of Dermatophytes in patients visiting to tertiary care hospital in Chhattisgarh state of India

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectives1. To isolate and identify various species of Dermatophytes from clinical specimens 2. To perform and analyze the antifungal susceptibility testing of isolated Dermatophytes for commonly used antifungal agents; terbinafine and itraconazole.MethodsA prospective study was conducted from December 2019 to October 2021. Clinical specimens (skin, hair, and nail) from suspected cases of dermatophytosis were received and processed in the department of microbiology. All the samples were subjected to microscopic examination and culture by standard techniques. Their clinico-demographic profile was obtained. Specimen were processed for KOH and fungal culture. Dermatophytes were identified by studying macroscopic and microscopic characteristics of the isolates. The conidium-forming dermatophyte isolates were processed for antifungal susceptibility testing for terbinafine and itraconazole by Microbroth dilution testing following the CLSI M-38A2 guidelines.ResultsTotal 248 patients with male predominance (68%) were noted in the above-mentioned study period. Predominance of study population belonged to rural area. Maximum numbers of cases were from the age group 21-30 years. Majority of patients belong to poor socioeconomic status. Out of 248 samples, 178 (72%) had a positive KOH mount amongst which 72% had positive culture results. Amongst 2 4881% were skin scraping, 17% were nail, and 1.6% hair samples were processed. Out of culture-positive samples 52% were Dermatophytes. The most clinical form of dermatophytosis was combination of both Tinea cruris and T. carporis (31%) followed by T. cruris (22%), and T. corporis (17%) for which skin scraping was processed. The most common isolate was Trichophyton tonsurans (73%) followed by T. mentagrophytes (10%), and T. verrucosum. Onychomycosis was diagnosed in 17% patients of which 59% were positive by KOH 49% were culture positive.11.5% isolates from nails were dermatophytes.Antifungal susceptibility testing was done by Microbroth dilution method and analyzed the range. The MIC range of major isolates, i.e., T. tonsurans showed MIC ranges against terbinafine <0.03-4 μg/ml and itraconazole 0.03-2 μg/ml. Trichophyton mentagrophyte for terbinafine <0.12-4 μg/ml and for itraconazole 0.12-2 μg/ml. Four isolates of T. tonsurans had higher MIC values for terbinafine and two isolates had higher MIC for itraconazole. One isolate of T. mentagrophytes had higher MIC values of itraconazole, and one another isolate had higher MIC for terbinafine.ConclusionThis study highlights the change in pattern of causative agents of dermatophytosis. The present study showed the predominance of T. tonsurans. More extensive studies are needed to evaluate the cut-off range of antifungal susceptibility testing of dermatophytes with clinical follow-up to see the response of respective antifungals and to guide the therapy.

P083 Susceptibility pattern of fungal isolated from patients with otomycosis

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesAntifungal resistance is posing several new concerns to clinicians. Increased rates of azole and echinocandin resistance in various non-albicans Candida species and azole resistance in A. fumigatus may arise due to clinical or environmental exposure to these drugs. The study evaluated the antifungal susceptibility for clinical fungal isolates causing otomycosis.MethodsA total of 89 Aspergillus isolates containing A. niger (58 isolates), A. flavus (19 isolates), A. fumigatus (12 isolates), and 25 Candida isolates containing C. parapsilosis (14 isolates), C. orthopsilosis (6 isolates), and C. albicans (5 isolates) collected from individuals with confirmed otomycosis during October 2020-November 2021 were tested for antifungal susceptibility testing (AFST). AFST of ketoconazole, voriconazole, tioconazole, amphotericin B, miconazole, fluconazole, nystatin, and itraconazole was conducted using the broth microdilution method based on CLSI (M38-A2, M27-A3) protocols. Conidia of molds and colonies of yeasts were harvested from fungal cultures on SDA incubated at 35°C; the turbidity of the suspension was then adjusted to OD630 nm = 80%-82%T for molds and 75%-77%T for yeasts.ResultsMainly, all antifungals examined were effective against most Aspergillus isolates, aside from tioconazole (GM = 5.54767 μg/ml) and nystatin (GM = 2.10151 μg/ml). Terbinafine (GM = 1.69824 μg/ml) had minimal in vitro effects (Table 1). Nystatin (GM = 2.94853 μg/ml) and itraconazole (GM = 1.08673 μg/ml) showed higher GM MICs against all Candida species isolates. Conversely, amphotericin B (GM = 0.07129 μg/ml) in Aspergillus, ketoconazole (GM = 0.02570), and voriconazole (GM = 0.03686 μg/ml) in Candida showed the highest antifungal activity (Table 2). Regarding the CLSI-M59 document for ECV, one A. niger (MIC 8 μg/ml), A. flavus (MIC 2 μg/ml), and A. fumigatus (MIC 2 μg/ml) isolates were non-wild type against itraconazole. A total of 3 A. niger non-wild type isolates with MIC 4 μg/ml against voriconazole were inspected (Table 1). Three C. albicans isolates with high itraconazole MICs (two 8 μg/ml and one 16 μg/ml) were observed (Table 2). Even though the MIC50 of Aspergillus niger for tolnaftate was 0.37 μg/ml, 9 isolates with high MICs (16 μg/ml) were found.ConclusionThe foremost commonest yeast isolates in this study, C. parapsilosis, exhibit significant sensitivity to various antifungals, including ketoconazole, voriconazole, tioconazole, amphotericin B, miconazole, fluconazole, and itraconazole. However, contrary to other studies, nystatin had high MICs and is not recommended as an effective drug. Since the pattern of antifungal susceptibility is varied among the cryptic species of Aspergillus sections, we recommend that physicians request a drug susceptibility testing before antibiotic therapy to prevent the development of resistance.

COVID-19-associated pulmonary aspergillosis (CAPA) in Iranian patients admitted with severe COVID-19 pneumonia.

PurposeBacterial or virus co-infections with SARS-CoV-2 have been reported in many studies; however, the knowledge on Aspergillus co-infection among patients with COVID-19 was limited. This study was conducted to identify and isolate fungal agents and to evaluate the prevalence of pulmonary aspergillosis (CAPA) as well as antifungal susceptibility patterns of Aspergillus species in patients with COVID-19 admitted to Shahid Beheshti Hospital, Kashan, Iran.MethodsThe study involved 119 patients with severe COVID-19 pneumonia referred to the Shahid Beheshti Hospital, Kashan, Iran. A total of 17 Aspergillus spp. that were isolated from COVID-19 patients suspected of CAPA were enrolled in the study. CAPA was defined using ECMM/ISHAM consensus criteria. The PCR amplification of the β-tubulin gene was used to identify the species. The antifungal activities of fluconazole, itraconazole, voriconazole, amphotericin B against Aspergillus spp. were evaluated according to the Clinical and Laboratory Standards Institute manual (M38-A3).ResultsFrom the 119 patients with severe COVID-19 pneumonia, CAPA was confirmed in 17 cases (14.3%). Of these, 12 (70.6%) were males and 5 (29.4%) were females; the mean age at presentation was 73.8 years (range: 45–88 years; median = 77; IQR = 18). Aspergillus fumigatus (9/17; 52.9%), Aspergillus flavus (5/17; 29.4%), Aspergillus oryzae (3/17, 17.6%), were identified as etiologic agents of CAPA, using the molecular techniques. Voriconazole and amphotericin B showed more activity against all isolates. Moreover, the MIC of fluconazole, itraconazole varied with the tested isolates. For 3 clinical isolates of A. fumigatus, 2 isolate of A. flavus and 3 A. oryzae, the MIC of fluconazole and itraconazole were ≥ 16 µg/mL.ConclusionsWe observed a high incidence (14.3%) of probable aspergillosis in 119 patients with COVID-19, which might indicate the risk for developing IPA in COVID-19 patients. When comparing patients with and without CAPA regarding baseline characteristics, CAPA patients were older (p =0 .024), had received more frequent systemic corticosteroids (p = 0.024), and had a higher mortality rate (p = 0.018). The outcome of CAPA is usually poor, thus emphasis shall be given to screening and/or prophylaxis in COVID-19 patients with any risk of developing CAPA.

Phenotypic Variants of Azole-Resistant Aspergillus Fumigatus that Co-exist in Human Respiratory Samples are Genetically Highly Related

Respiratory specimens obtained from patients with chronic forms of aspergillosis contain phenotypic variants of azole-resistant Aspergillus fumigatus (ARAF) that co-exist in the airway. Here we aimed to study whether phenotypic variants of ARAF that co-exist in clinical specimens were genetically distinct. A panel of six phenotypic variants of ARAF cultured from two sputum samples collected from two patients with chronic aspergillosis were included. Preliminary identification of all isolates was obtained using MALDI–ToF mass spectrometry and confirmed by AsperGenius® real-time PCR assay. Antifungal susceptibility testing was determined using EUCAST E.Def 9.3 microbroth dilution. Genomic DNA libraries were constructed with the Illumina TruSeq Nano kit. Prepared whole-genome libraries were sequenced on an Illumina HiSeq 2500. Whole genome data were converted into presence/absence of a SNP with respect to the Af293 reference genome. Colonies of ARAF that co-existed in one respiratory sample demonstrated marked phenotypic diversity. Two cyp51A polymorphisms were found among azole-resistant isolates: TR34/L98H/T289A/I364V/G448S was consistently present in four variants with a pan-azole resistant phenotype and TR34/L98H was detected in two variants (itraconazole MIC > 16 mg/L). WGS typing showed that despite marked phenotypic variation, each sample contained a population of highly genetically related azole-resistant A. fumigatus variants. Our SNP analysis suggest that mechanisms additional to genetic-based variation are responsible for phenotypic diversity. Our data demonstrate that the phenotypic variants of ARAF that co-exist in clinical specimens are highly clonal and strongly suggest their origination from a single common ancestor.

Nannizzia incurvata in Hue city - Viet Nam: Molecular identification and antifungal susceptibility testing.

Nannizzia incurvata, a species belonging to the Nannizzia gypsea complex, is considered a neglected pathogen. To detected N. incurvata isolates from dermatophytosis patients in Hue city - Viet Nam, and test the antifungal susceptibility of this species. Moreover, fungal capability to produce hydrolytic enzymes was evaluated. Patients' samples were collected and cultured on Sabouraud-chloramphenicol-cycloheximide medium. Dermatophytes isolates were initially macroscopically and microscopically identified. ITS PCR-RFLP and ITS rDNA sequences were performed to determine and confirm species. An ITS Neighbor-Joining phylogenetic tree evaluated the genetic relationship among isolates. Fungal hydrolytic enzymes were examined, including lipase, phospholipase and protease. Antifungal susceptibility testing was carried out by the disk diffusion method. MICs of itraconazole, voriconazole, and terbinafine against these isolates were determined by the broth microdilution method. Twelve isolates of N. gypsea complex were preliminary morphologically identified. PCR-RFLP and ITS-rDNA sequencing identified and confirmed dermatophytes as N. incurvata strains, respectively. An evident polymorphism among isolates was highlighted in the phylogenetic tree. All isolates showed the activity of lipase, phospholipase, and protease production. Overall, all N. incurvata isolates were susceptible to itraconazole, voriconazole, clotrimazole, miconazole, and terbinafine. Few isolates were susceptible to griseofulvin, and none of them were susceptible to fluconazole. There was a presence of polyclonal N. incurvata isolates in dermatophytosis patients from Hue city, identified by PCR-RLFP and confirmed by ITS sequencing. We confirmed PCR-RLFP as a reliable technique to identify this species. Azole and terbinafine are the optimal choices for N. incurvata treatment except for fluconazole.

Gene Amplification of CYP51B: a New Mechanism of Resistance to Azole Compounds in Trichophyton indotineae.

ABSTRACTTrichophyton indotineae causes dermatophytosis that is resistant to terbinafine and azole compounds. The aim of this study was to determine the mechanisms of resistance to itraconazole (ITC) and voriconazole (VRC) in strains of T. indotineae. Two azole-sensitive strains (ITC MIC < 0.125 μg/mL; VRC MIC < 0.06 μg/mL) and four azole-resistant strains (ITC MIC ≥ 0.5 μg/mL; VRC MIC ≥ 0.5 μg/mL) were used for the investigation. The expression of MDR genes encoding multidrug transporters of the ABC family for which orthologs have been identified in Trichophyton rubrum and those of CYP51A and CYP51B encoding the targets of azole antifungal compounds were compared between susceptible and resistant strains. TinMDR3 and TinCYP51B were overexpressed in T. indotineae resistant strains. Only small differences in susceptibility were observed between TinMDR3 disruptants and parental strains overexpressing TinMDR3. Whole-genome sequencing of resistant strains revealed the creation of a variable number of TinCYP51B tandem repeats at the specific position of their genomes in three resistant strains. Downregulation of TinCYP51B by RNA interference (RNAi) restored the susceptibility of azole-resistant strains. In contrast, overexpression of TinCYP51B cDNA conferred resistance to a susceptible strain of T. indotineae. In conclusion, the reduced sensitivity of T. indotineae strains to azoles is mainly due to the overexpression of TinCYP51B resulting from additional copies of this gene.

Clinical and microbiological pattern of intertrigo

Background: Intertrigo is a common disorder affecting many flexural areas of the body with significant morbidity.&#x0D; Objectives: To determine the clinical pattern of intertrigo. To determine bacterial and fungal pathogens associated with intertrigo. To assess the antibacterial and antifungal sensitivity pattern of the responsible organisms.&#x0D; Methods: A total number of 230 patients with intertrigo attending dermatology unit were included in the study after ethical clearance. Laboratory investigations were done to identify the etiologic agent and their sensitivity pattern.&#x0D; Results: Majority of patients (75%) belonged to 20-60 years of age. Male to female ratio was 1.15:1. Remarkable proportion (52%) presented with the involvement of more than one intertriginous area. The most common site involved was toe web space (63%) in particular 4th toe web space. Of all positive bacterial cultures 45% revealed Staphylococcus aureus (MSSA+MRSA) followed by CNSA (Coagulase negative Staphylococcal aureus), diptheroids and pseudomonas. The majority of MSSA (Methicillin sensitive Staphylococcus aureus) isolates (91%) were sensitive to cloxacillin and MRSA (Methicillin resistant Staphylococcus aureus) to teicoplanin (85%). CNSA isolates showed considerable proportion of sensitivity to vancomycin (37%) and majority of diptheroids isolates were sensitive to penicillin(87%). All isolates (100%) of pseudomonas were sensitive to gentamicin. Of all positive fungal cultures, candida species constituted the majority (35%) followed by fusarium species (33%) and dermatophytes (30%). Overall fusarium species shows high value of MIC50 and MIC90 for itraconazole, fluconazole and ketoconazole. Fluconazole showed very high MIC50 and MIC90 (256 ug/ml) for dermatophytes and fusarium species whereas itraconazole and ketoconazole showed lower MIC for these fungi.&#x0D; Conclusion: Intertrigo can affect various intertriginous areas, among which toe web space is the most common region involved. Intertrigo can be caused by a variety of organisms: Staphylococcus aureus and candida species constituted the majority of cases. Majority of MSSA sensitive to cloxacillin and MRSA to teicoplanin. MIC of itraconazole, fluconazole and ketoconazole are high in fusarium species comparing to candida and dermatophytes. However, itraconazole and ketoconazole had lower MIC than dermatophytes and fusarium comparing to fluconazole.

Participation of the ABC Transporter CDR1 in Azole Resistance of Candida lusitaniae.

Candida lusitaniae is an opportunistic pathogen in humans that causes infrequent but difficult-to-treat diseases. Antifungal drugs are used in the clinic to treat C. lusitaniae infections, however, this fungus can rapidly acquire antifungal resistance to all known antifungal drugs (multidrug resistance). C. lusitaniae acquires azole resistance by gain-of-function (GOF) mutations in the transcriptional regulator MRR1. MRR1 controls the expression of a major facilitator transporter (MFS7) that is important for fluconazole resistance. Here, we addressed the role of the ATP Binding Cassette (ABC) transporter CDR1 as additional mediator of azole resistance in C. lusitaniae. CDR1 expression in isolates with GOF MRR1 mutations was higher compared to wild types, which suggests that CDR1 is an additional (direct or indirect) target of MRR1. CDR1 deletion in the azole-resistant isolate P3 (V688G GOF) revealed that MICs of long-tailed azoles, itraconazole and posaconazole, were decreased compared to P3, which is consistent with the role of this ABC transporter in the efflux of these azoles. Fluconazole MIC was only decreased when CDR1 was deleted in the background of an mfs7Δ mutant from P3, which underpins the dominant role of MFS7 in the resistance of the short-tailed azole fluconazole. With R6G efflux readout as Cdr1 efflux capacity, our data showed that R6G efflux was increased in P3 compared to an azole-susceptible wild type parent, and diminished to background levels in mutant strains lacking CDR1. Milbemycin oxim A3, a known inhibitor of fungal ABC transporters, mimicked efflux phenotypes of cdr1Δ mutants. We therefore provided evidence that CDR1 is an additional mediator of azole resistance in C. lusitaniae, and that CDR1 regulation is dependent on MRR1 and associated GOF mutations.