Minimum Inhibitory Concentration Of Itraconazole Research Articles

In vitro Antifungal Susceptibility Testing of Clinical and Environmental Fusarium Isolates in Iran

Background: Members of the genus Fusarium are common soil saprophytes and important plant pathogens which also cause a wide spectrum of diseases ranging from onychomycosis to the life - threatening systemic infections. The antifungal susceptibility patterns of Fusarium isolates varies in different species. Objectives: This study was undertaken to investigate the antifungal susceptibility pattern of environmental and clinical Fusarium isolates to conventionally - used azole antifungal drugs in Iran. Methods: A total of 36 Fusarium isolates (16 clinical and 20 environmental) were included in this study. All environmental isolates were obtained from the culture collection of medical mycology laboratory of the School of Public Health at Tehran University of Medical Sciences, Tehran, Iran. Clinical isolates were obtained from patients with onychomycosis and were identified by PCR - sequencing of a fragment of translation elongation factor 1 alpha gene. All clinical and environmental isolates were tested for their in vitro susceptibility to itraconazole (ITC) and voriconazole (VRC) according to the CLSI M38 - A2 standard. Statistical analysis of data was performed using SPSS version 21. Results: The majority of clinical isolates were identified as F. proliferatum (N = 6) followed by F. oxysporum (N = 4), F. solani (N = 3), F. verticillioides (N = 1), F. acutatum (N = 1), and F. thapsinum (N = 1). The lowest minimum inhibitory concentration (MIC) values of ITC was observed for environmental F. verticillioides isolates (N = 5, GM = 13.93 µg/mL). For VRC, the lowest MICs were recorded for environmental F. verticillioides isolates (N = 5, GM = 2.3 µg/mL), and the highest MICs were observed for clinical F. solani isolates (N = 3, GM = 10.08 µg/mL). ITC was inactive against all clinical and the majority of environmental Fusarium isolates (MICs ≥ 16 µg/mL). Significant differences were observed between MICs of ITC and VRC against environmental F. proliferatum and F. verticillioides isolates (P < 0.001 for both). Similarly, the susceptibility of clinical F. proliferatum, F. oxysporum and F. solani isolates to VRC was significantly different (P = 0.023). Conclusions: The resistance pattern in Fusarium isolates is species specific and, therefore, identification at the species level is important for choosing the proper antifungal treatment.

Screening of antifungal susceptibility in cave-dwelling aspergilli and report of an amphotericin B-resistant Aspergillus flavus

Caves are stable environments that favour the development of several microorganisms. The aspergilli represent a large number of species isolated from caves including strains capable of causing serious invasive opportunistic infections in humans. Considering that caves may harbour resistant strains to many antibiotics, investigation on the response of opportunistic aspergilli, isolated from pristine and tourist caves to antifungal agents and the mechanisms involved in resistance might be clinically relevant. A total of 32 strains of the species Aspergillus candidus, A. flavus, A. fumigatus, A. niger, A. tamarii, and A. terreus were isolated from caves in the iron quadrangle in Brazil. The strains were tested for their susceptibility to amphotericin B (AMB), itraconazole, voriconazole and terbinafine. One strain was analysed for the mechanism involved in the AMB-resistance, i.e., ergosterol content, lipid peroxidation and enzymatic activity of the antioxidant system. Terbinafine minimum inhibitory concentrations (MICs) ranged between 0.003 and 1.0 µg/mL; voriconazole MICs ranged between 2.0 and >16.0 µg/mL; itraconazole MICs ranged between 0.25 and 8.0 µg/mL and amphotericin B MICs ranged between 0.03 and 4.0 µg/mL. The AMB-resistant strain of A. flavus was detected with MIC value of 4 µg/mL. Resistance to AMB relied on higher ergosterol levels and increased enzymatic activity of the peroxidase and superoxide-dismutase, with lower lipid peroxidation. These results enhance the knowledge of natural antifungal resistance in the subterranean ecosystem, and broaden the knowledge about the subterranean microbiota.

Cryptococcus neoformans and C. gattii isolates from both HIV-infected and uninfected patients: antifungal susceptibility and outcome of cryptococcal disease.

ABSTRACTOne of the factors causing treatment failure in cryptococcosis is the resistance of Cryptococcus spp. to antifungal drugs, which has motivated the susceptibility assessment of isolates from patients with cryptococcosis, different clinical conditions and infections outcomes. Clinical isolates of Cryptococcus spp. from three different groups of patients were studied in the present investigation: 19 HIV-positive patients with relapsing and/or refractory meningitis (Group 1), 30 HIV-positive patients who experienced a single and limited episode of cryptococcosis (Group 2), and 19 HIV-negative patients with cryptococcosis (Group 3). Eighty C. neoformans var. grubii isolates and 7 C. gattii isolates were studied. The minimum inhibitory concentration (MIC) of amphotericin B, azole drugs and flucytosine was determined for Cryptococcus spp. by broth microdilution test and E-test. The MIC50 and MIC90 were 0.25 and 0.50 µg/mL for amphotericin B, 4.0 and 8.0 µg /mL for fluconazole, 0.06 and 0.25 µg/mL for itraconazole, 0.25 and 0.50 µg/mL for voriconazole, and 8.0 and 16.0 µg/mL for flucytosine, respectively. Amphotericin B and itraconazole showed higher MICs for C. neoformans var. grubii and C. gattii, respectively. The MICs of fluconazole and itraconazole obtained with the E-test were higher than those obtained with broth microdilution. Isolates from non-HIV coinfected were less sensitive to the azoles. There was no difference in the susceptibility of C. neoformans var. grubii isolates from patients with a favorable or unfavorable outcome or along the episodes of relapsing and/or refractory meningitis.

Clinico-mycological study of dermatophytic infections and their sensitivity to antifungal drugs in a tertiary care center.

Worldwide, dermatophytic infections are running a chronic course either due to ineffective treatment or emerging drug resistance. In the past three decades, there has been an increase in incidence and non-responsiveness to conventional antifungals, which suggests that there is a need of antifungal sensitivity testing. This study was aimed at identifying clinico-mycological pattern of dermatophytic infections in patients attending thedermatology outpatient department of a tertiary care hospital, and to obtain the sensitivity pattern of isolates against six commonly used oral antifungals (fluconazole, terbinafine, itraconazole, ketoconazole, griseofulvin and voriconazole). Patients with suspected dermatophytoses attending the outpatient department of Sir Sunderlal Hospital, Varanasi, were enrolled in the study. A detailed history, clinical examination and sample collection for mycological examinations was done. In vitro antifungal sensitivity testing was done on species isolated from culture as per the Clinical and Laboratory Standard Institute M38-A standards, with broth microdilution method. There were 256 patients recruited in the study, with a male: female ratio of 3:1. The most commonly affected age group was 20-40 years (52.4%). Tinea corporis et cruris was the most common type observed (27.2%). Potassium hydroxide positivity was seen in 211 samples (79.6%) and culture positivity was found in 139 samples (52.4%). The most common species identified was Trichophyton mentagrophytes (75.9%). Sensitivity testing was done on fifty isolates of T. mentagrophytes. Minimum inhibitory concentrations of itraconazole, ketoconazole, terbinafine and voriconazole were comparable, while griseofulvin showed the highest minimum inhibitory concentration. Itraconazole was found to be the most effective drug, followed by ketoconazole, terbinafine and fluconazole. Griseofulvin was the least effective drug among the tested antifungals. This is a hospital-based study, and may not reflect the true pattern in the community. Sensitivity pattern of only one species T. mentagrophytes was carried out. Inadequate and irregular use of antifungal drugs has led to the emergence of resistant strains, which cause poor treatment outcomes. Thus, it is very important to test for antifungal sensitivity to check for resistance to antifungals.

Molecular and antifungal susceptibility study on trichosporonemia and emergence of Trichosporon mycotoxinivorans as a bloodstream pathogen.

A total of 21 Trichosporon spp. isolates from blood over a period of 5 years (January 2009 to December 2013) were included in the study. The most common underlying diseases found were pancreatitis (33.3%) and cancer (33.3%). Trichosporon asahii (80.9%) was the commonest species followed by Trichosporon mycotoxinivorans (14.2%) and Trichosporon faecale (4.7%). On IGS1 region sequencing the most predominant T. asahii type in our region was genotype 1 (16/17 isolates; 94.1%) and one isolate belonged to genotype 4. Following the interpretative breakpoints for Candida albicans according to CLSI guidelines amphotericin B minimum inhibitory concentrations (MICs) were ≤1 μg/ml for 38% of isolates. Fluconazole MICs were ≤4 μg/ml for 33.3% of the isolates. Itraconazole MICs were ≤0.5 μg/ml for 52.3% of the isolates. However, the MICs to posaconazole and voriconazole were ≤0.5 μg/ml for all the isolates. The MICs to caspofungin and micafungin were ≤0.5 μg/ml for only 0.09% of the isolates. This study reemphasizes that IGS1 sequencing is the most reliable technique for accurate identification of Trichosporon spp. and also to identify the newer species like T. mycotoxinivorans, which still remains rare. Surveillance of antifungal susceptibility patterns can provide the local drug resistance data to the clinicians which can further aid better management of patients.

Antifungal Resistance and Virulence Among Candida spp. from Captive Amazonian manatees and West Indian Manatees: Potential Impacts on Animal and Environmental Health.

This work aimed at evaluating the antifungal susceptibility and production of virulence factors by Candida spp. isolated from sirenians in Brazil. The isolates (n=105) were recovered from the natural cavities of Amazonian and West Indian manatees and were tested for the susceptibility to amphotericin B, itraconazole, and fluconazole and for the production of phospholipases, proteases, and biofilm. The minimum inhibitory concentrations (MICs) for amphotericin B ranged from 0.03 to 1µg/mL, and no resistant isolates were detected. Itraconazole and fluconazole MICs ranged from 0.03 to 16µg/mL and from 0.125 to 64µg/mL, respectively, and 35.2% (37/105) of the isolates were resistant to at least one of these azole drugs. Concerning the production of virulence factors, phospholipase activity was observed in 67.6% (71/105) of the isolates, while protease activity and biofilm production were detected in 50.5% (53/105) and 32.4% (34/105) of the isolates, respectively. Since the natural cavities of manatees are colonized by resistant and virulent strains of Candida spp., these animals can act as sources of resistance and virulence genes for the environment, conspecifics and other animal species, demonstrating the potential environmental impacts associated with their release back into their natural habitat.

Invitro resistance of Aspergillus fumigatus to azole farm fungicide.

Azole resistance in Aspergillus fumigatus is mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which encodes the target of azole fungicides. Moreover, overexpression of CYP51B or multidrug resistance (MDR) gene is supposedly related to the mechanism of azole resistance in A.fumigatus. In this study, we tried to induce resistance to tetraconazole, an azole fungicide, in strains of A.fumigatus from a farm and then investigated mutation and expression of their CYP51A, CYP51B, and multidrug resistance (MDR) genes. Three tetraconazole resistant strains were induced and their minimum inhibitory concentration (MIC) for tetraconazole was 145mg/L. However, the MICs of itraconazole (ITZ), posaconazole (POS), and voriconazole (VRZ) obtained by an E-test of the three tetraconazole resistant strains were 0.064-0.19mg/L for ITZ, 0.023-0.32mg/L for POS, and 0.047-0.064mg/L for VRZ. No gene mutations were detected in the CYP 51A sequence amplified in these strains. RT-PCR of cyp51A and cyp51B indicated that the tetraconazole resistant strains more highly expressed these genes than the susceptible strain in tetraconazole containing medium.

Cyp51A gene silencing using RNA interference in azole‐resistant Aspergillus fumigatus

An increasing number of reports have described the emergence of acquired resistance of Aspergillus fumigatus to azole compounds. The primary mechanism of resistance in clinical isolates is the mutation of the azole drug target enzyme, which is encoded by the cyp51A gene. The aim of this study was to evaluate the impact of silencing the cyp51A gene in azole-resistant A. fumigatus isolates. A 21-nucleotide small-interfering RNA (siRNA) was designed based on the cDNA sequence of the A. fumigatus cyp51A gene. After silencing the cyp51A gene in germinated conidia (15, 20, 25 and 50 nM), azole-resistant A. fumigatus was cultured on broth media and gene expression was analysed by measuring the cyp51A mRNA level using RT-PCR assay. Hyphae were successfully transfected by siRNA and expression of the cyp51A gene was significantly reduced by siRNA at the concentration of 50 nM (P ≤ 0.05). In addition, at this siRNA concentration, the minimum inhibitory concentration of itraconazole for the treated cells was decreased, compared with that for untreated control cells, from 16 to 4 μg/ml.

Determination of minimum inhibitory concentrations of itraconazole, terbinafine and ketoconazole against dermatophyte species by broth microdilution method

Purpose: Various antifungal agents both topical and systemic have been introduced into clinical practice for effectively treating dermatophytic conditions. Dermatophytosis is the infection of keratinised tissues caused by fungal species of genera Trichophyton, Epidermophyton and Microsporum, commonly known as dermatophytes affecting 20–25% of the world’s population. The present study aims at determining the susceptibility patterns of dermatophyte species recovered from superficial mycoses of human patients in Himachal Pradesh to antifungal agents; itraconazole, terbinafine and ketoconazole. The study also aims at determining the minimum inhibitory concentrations (MICs) of these agents following the recommended protocol of Clinical and Laboratory Standards Institute (CLSI) (M38-A2). Methodology: A total of 53 isolates of dermatophytes (T. mentagrophyte-34 in no., T. rubrum-18 and M. gypseum-1) recovered from the superficial mycoses were examined. Broth microdilution method M38-A2 approved protocol of CLSI (2008) for filamentous fungi was followed for determining the susceptibility of dermatophyte species. Results:T. mentagrophyte isolates were found more susceptible to both itraconazole and ketoconazole as compared to terbinafine (MIC50: 0.125 μg/ml for itraconazole, 0.0625 μg/ml for ketoconazole and 0.5 μg/ml for terbinafine). Three isolates of T. mentagrophytes (VBS-5, VBSo-3 and VBSo-73) and one isolate of T. rubrum (VBPo-9) had higher MIC values of itraconazole (1 μg/ml). Similarly, the higher MIC values of ketoconazole were observed in case of only three isolates of T. mentagrophyte (VBSo-30 = 2 μg/ml; VBSo-44, VBM-2 = 1 μg/ml). The comparative analysis of the three antifungal drugs based on t-test revealed that ‘itraconazole and terbinafine’ and ‘terbinafine and ketoconazole’ were found independent based on the P < 0.005 in case of T. mentagrophyte isolates. In case of T. rubrum, the similarity existed between MIC values of ‘itraconazole and ketoconazole’ and ‘terbinafine and ketoconazole’. Conclusion: The MIC values observed in the present study based on standard protocol M38-A2 of CLSI 2008 might serve as reference for further studies covering large number of isolates from different geographic regions of the state. Such studies might reflect on the acquisition of drug resistance among isolates of dermatophyte species based on MIC values.

In vitro activity of econazole in comparison with three common antifungal agents against clinical Candida strains isolated from superficial infections.

Background and Purpose: Candida species are the most common organisms involved in superficial fungal infections, worldwide. Although econazole is among the most frequently used topical formulations for the treatment of candidiasis, no information is available regarding the susceptibility profiles of Candida species in Iran.Materials and Methods: In vitro susceptibility of 100 clinical Candida isolates belonging to 6 species from superficial candidiasis of Iran towards to econazole was compared with three other common antifungal agents including itraconazole, fluconazole, and miconazole. Minimum inhibitory concentrations (MICs) values were analyzed according to the Clinical and Laboratory Standards Institute (CLSI) M38-A3 document. All isolates were previously identified to the species level, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on ITS region. Results:The MIC of econazole, itraconazole, miconazole, and fluconazole were within the range of 0.016-16, 0.032-16, 0.016-16, and 0.25-64 μg/ml, respectively. In general, econazole and miconazole were more active against Candida isolates, compared to the other two agents.Conclusion:The present study demonstrated that for Candida albicans isolates, miconazole and econazole had the best effect, but in non-albicans Candida species, itraconazole and miconazole displayed more activity than other antifungal agents.

In vitro susceptibility of filamentous fungi from mycotic keratitis to azole drugs

The in vitro antifungal activities of azole drugs viz., itraconazole, voriconazole, ketoconazole, econazole and clotrimazole were investigated in order to evaluate their efficacy against filamentous fungi isolated from mycotic keratitis. The specimen collection was carried out from fungal keratitis patients attending Aravind eye hospital and Post-graduate institute of ophthalmology, Coimbatore, India and was subsequently processed for the isolation of fungi. The dilutions of antifungal drugs were prepared in RPMI 1640 medium. Minimum inhibitory concentrations (MICs) were determined and MIC50 and MIC90 were calculated for each drug tested. A total of 60 fungal isolates were identified as Fusarium spp. (n=30), non-sporulating moulds (n=9), Aspergillus flavus (n=6), Bipolaris spp. (n=6), Exserohilum spp. (n=4), Curvularia spp. (n=3), Alternaria spp. (n=1) and Exophiala spp. (n=1). The MICs of ketoconazole, clotrimazole, voriconazole, econazole and itraconazole for all the fungal isolates ranged between 16 μg/mL and 0.03 μg/mL, 4 μg/mL and 0.015 μg/mL, 8 μg/mL and 0.015 μg/mL, 8 μg/mL and 0.015 μg/mL and 32 μg/mL and 0.06 μg/mL respectively. From the MIC50 and MIC90 values, it could be deciphered that in the present study, clotrimazole was more active against the test isolates at lower concentrations (0.12-5 μg/mL) when compared to other drugs tested. The results suggest that amongst the tested azole drugs, clotrimazole followed by voriconazole and econazole had lower MICs against moulds isolated from mycotic keratitis.

In Vivo Emergence of Aspergillus terreus with Reduced Azole Susceptibility and a Cyp51a M217I Alteration

Azole resistance in Aspergillus terreus isolates was explored. Twenty related (MB) and 6 unrelated A. terreus isolates were included. CYP51A sequencing and RAPD genotyping was performed. Five MB isolates were itraconazole susceptible, whereas the minimum inhibitory concentrations (MICs) for 15 MB isolates were elevated (1-2 mg/L). Voriconazole and posaconazole MICs were 0.5-4 and 0.06-0.5 mg/L, respectively, for MB isolates but 0.25-0.5 and <0.03-0.06 mg/L, respectively, for controls. Sequencing identified a Cyp51Ap M217I alteration in all 15 isolates with elevated itraconazole MICs. Genotyping showed that 18 of 20 MB isolates were identical and unique, suggesting endogenous origin. In conclusion, itraconazole resistance in A. terreus was linked to an M217I Cyp51A alteration.

Molecular mechanisms of cross-resistance to azole antifungal agents in a clinical isolate of Aspergillus fumigatus: a preliminary study

Objective To investigate the molecular mechanisms of cross-resistance to azoles in a clinical isolate of Aspergillus fumigatus. Methods A. fumigatus was isolated from a patient with invasive aspergillosis.Clinical Laboratory Standard Institute M38-A2 broth microdilution method and E-test method were used to determine the minimum inhibitory concentrations (MICs) or minimum effective concentration (MEC) of itraconazole, voriconazole, amphotericin B, posaconazole and caspofungin for the A. fumigatus isolate. DNA was extracted from the isolate and subjected to the amplification of cyp51A gene encoding the target enzyme of azole antifungal agents followed by sequence analysis. Results The broth microdilution test showed that the MEC of caspofungin was 0.5 mg/L, and MICs of itraconazole, voriconazole and amphotericin B were ≥ 16 mg/L,8 mg/L and 1 mg/L, respectively, for this isolate; while E-test assay revealed that the MICs of caspofungin,itraconazole, voriconazole, amphotericin B and posaconazole were 0.047 mg/L, ≥32 mg/L,≥32 mg/L, 12 mg/L and ≥32 mg/L, respectively. Sequence analysis showed an insertion of a 34-bp tandem sequence in the promoter region of the cyp51A gene as well as a T364A point mutation causing the substitution of leucine 98 (L98H). In addition, there were some other mutations in the cyp51A gene of this isolate, such as A137T,G585A, C814A, G836C, T991C and A1350G, which could result in corresponding amino acid substitutions.Conclusions An A. fumigatus strain with cross-resistance to azole antifungal agents is isolated. There is an insertion of a 34-bp tandem sequence into the promoter region as well as a T364A point mutation in the cyp51A gene, which contribute to the cross resistance to azole antifungal agents including itraconazole, voriconazole,and posaconazole. In addition, other mutations causing amino acid substitutions have also been detected in the cyp51 A gene of this isolate. Key words: Aspergillus fumigatus; Azoles; Drug resistance, multiple, fungal; cyp51A

Lack of correlation of 24- vs. 48-h itraconazole minimum inhibitory concentrations with microbiological and survival outcomes in a guinea pig model of disseminated candidiasis

A 'trailing' effect has been commonly observed when azole antifungals are tested against Candida spp. Previous experience with fluconazole indicates that 24-h minimum inhibitory concentration (MIC) values are more compatible endpoints when compared with clinical outcomes. We evaluated the trailing effect of Candida isolates tested with itraconazole in a guinea pig model of systemic candidiasis. Survival and organ burden were only significantly affected by using a higher dose of itraconazole, irrespective of the MIC differences at 24 and 48 h. A fluconazole-resistant strain with susceptible dose-dependent MICs to itraconazole was successfully treated with high-dose itraconazole. Our data suggests that survival and microbiological response depend more on drug dosing than on the trailing phenotype of the isolates.

Onychomycosis caused by Fusarium proliferatum

Fusarium infections in humans are usually opportunistic, but the fungus sometimes infects healthy persons, causing keratomycosis or onychomycosis. Onychomycosis is usually caused by F. solani or F. oxysporum. We report the first two cases of onychomycosis caused by F. proliferatum, and discuss methods of diagnosis and effective treatment. Nail samples from the two patients were examined by direct microscopy, cultured, and identified morphologically and genetically as F. proliferatum. Both patients were treated successfully with oral itraconazole, even though the minimum inhibitory concentration of itraconazole was relatively high in Patient 1. This is the first report of F. proliferatum as an agent of onychomycosis. Itraconazole may be effective in the treatment of onychomycosis caused by F. proliferatum.