Itraconazole-resistant Strains Research Articles

Relationships between Secreted Aspartyl Proteinase 2 and General Control Nonderepressible 4 gene in the Candida albicans resistant to itraconazole under planktonic and biofilm conditions.

This study aimed to explore the roles of SAP2 and GCN4 in itraconazole (ITR) resistance of C. albicans under different conditions, and their correlations. A total of 20 clinical strains of C. albicans, including 10 ITR resistant strains and 10 sensitive strains, were used. Then, SAP2 sequencing and GCN4 sequencing were performed, and the biofilm formation ability of different C. albicans strains was determined. Finally, real-time quantitative PCR was used to measure the expression of SAP2 and GCN4 in C. albicans under planktonic and biofilm conditions, as well as their correlation was also analyzed. No missense mutations and three synonymous mutation sites, including T276A, G543A, and A675C, were found in SAP2 sequencing. GCN4 sequencing showed one missense mutation site (A106T (T36S)) and six synonymous mutation sites (A147C, C426T, T513C, T576A, G624A and C732T). The biofilm formation ability of drug-resistant C. albicans strains was significantly higher than that of sensitive strains (P < 0.05). Additionally, SAP2 and GCN4 were up-regulated in the ITR-resistant strains, and were both significantly higher in C. albicans under biofilm condition. The mRNA expression levels of SAP2 and GCN4 had significantly positive correlation. The higher expression levels of SAP2 and GCN4 were observed in the ITR-resistant strains of C. albicans under planktonic and biofilm conditions, as well as there was a positive correlation between SAP2 and GCN4 mRNA expression.

Can the essential oil of rosemary (Rosmarinus officinalis Linn.) protect rats infected with itraconazole-resistant Sporothrix brasiliensis from fungal spread?

IntroductionItraconazole is the first-choice option to treat human and animal sporotrichosis. However, the emergence of itraconazole-resistant strains has encouraged research on new active antifungals. Among them, the essential oil of rosemary (Rosmarinus officinalis Linn., Lamiaceae) has shown antifungal activity in vitro. ObjectiveAssessing, for the first time, the effectiveness of rosemary essential oil in vivo in experimental cutaneous sporotrichosis, as well as its chemical composition and action mode. MethodsItraconazole-resistant Sporothrix brasiliensis was inoculated in the left foot pad of 30 Wistar rats, which were randomized (n=10) for treatment with saline solution (control, CONT), itraconazole (ITRA, 10 mg/kg) and rosemary oil (ROSM, 250 mg/kg) for 30 days at an oral dose of 1 mL, daily. Clinical evolution, histopathology and fungal burden were investigated. GC-MS was used for chemical analysis; sorbitol protection and ergosterol effect were used to evaluate the action mechanism of rosemary oil. ResultsROSM was the only group evolving to skin lesion remission, lack of edema and exudate, and mild-to-absent yeast cells. Rosemary oil delayed fungal spreading and protected systemic organs, mainly liver and spleen. The ROSM group presented lower fungal load than that observed for the CONT and ITRA groups (p<0.05). Antifungal action took place at complexation level after ergosterol application. Most compounds were 1,8-cineole/eucalyptol (47.91%), camphor (17.92%), and α-pinene (11.52%). ConclusionsThese findings have evidenced that rosemary oil is a promising antifungal to treat sporotrichosis, since it protects systemic organs from fungal spread.

Evaluation of virulence and drug resistance of Sporothrix globosa itraconazole-resistant strains in Galleria mellonella

Evaluation of virulence and drug resistance of Sporothrix globosa itraconazole-resistant strains in Galleria mellonella

In vitro and in vivo Efficacy of a Synergistic Combination of Itraconazole and Verapamil Against Aspergillus fumigatus.

The incidence of aspergillosis continues to rise sharply, while the progress made in expanding the antifungal drug arsenal remains extremely slow, indicating an urgent need for new strategies. Previous studies have shown that the calcium signaling pathway, which is evolutionarily conserved in mammals and fungi, is involved in regulating the tolerance of azoles in fungi. In this study, we performed a preliminary screening among various combinations of different clinical calcium channel blockers and different antifungal drugs. We found that the combination of itraconazole and verapamil showed the best synergistic effect against Aspergillus fumigatus. Thereafter, using the checkboard assays we observed synergistic effects of the combination treatment against most of the A. fumigatus strains tested, including itraconazole-sensitive and itraconazole-resistant strains, with a fractional inhibitory concentration index (FICI) < 0.5. Furthermore, we showed that verapamil strongly decreased the cytosolic calcium transients following itraconazole stimulation by an aequorin-mediated method. Moreover, verapamil influenced the efflux of rhodamine 6G, an azole mimic substance. An ergosterol assay revealed that verapamil alone had no effect on ergosterol biosynthesis, but the combination of itraconazole and verapamil treatment decreased the ergosterol level. Further murine assays were performed using a luciferase-probed bioluminescence imaging method. Drug combination therapy reduced lung burden and improved survival rate. In conclusion, verapamil is a promising candidate to enhance the antifungal activity of itraconazole against A. fumigatus. In addition, our study suggests the effectiveness of an emerging approach based on bioluminescence imaging in monitoring the efficacy of drug combination therapy for invasive aspergillosis.

In vitro susceptibility of Cuban Aspergillus spp. strains of clinical and environmental origin

The behavior of antifungal susceptibility of Aspergillus spp. in Cuba remains unknown. The antifungals recommended to treat aspergillosis are amphotericin B, itraconazole, voriconazole and echinocandins. The influence of the environment may set off the emergence of drug-resistance in these microorganisms. To evaluate in vitro susceptibility of Aspergillus spp. strains to amphotericin B, itraconazole and voriconazol, and the relationship between susceptibility patterns and their origin. Minimum inhibitory concentrations of amphotericin B, itraconazole and voriconazole were determined for 60 Aspergillus spp. strains of clinical and environmental origin using the M38-A2 method of the Clinical and Laboratory Standards Institute. We found 21 amphotericin B resistant strains (mainly from clinical samples and hospital environments), as well as three itraconazole resistant strains (from non-hospital outdoor and indoor environments). No voriconazole resistance was found. No relationship was found between strain origin and susceptibility. Results suggest the possible existence of environmental factors or interactions with resistant genotypes which may give rise to resistant phenotypes in our country. This is the first report of in vitro Aspergillus spp. resistant strains in Cuba. These studies should be broadened and include molecular and phylogenetic analyses.

Molecular expression of itraconazole resistance in clinical isolates of Candida krusei

Objective To investigate the main molecular expressions responsible for itraconazole resistance in clinical isolates of Candida krusei (C.krusei). Methods The ERG11 gene in the 16 C. krusei clinical isolates was amplified by polymerase chain reaction (PCR). Point mutations were determined by nucleotide sequence and compared among itraconazole-resistant (R, n=5), itraconazole-susceptible dose dependent (SDD, n=8) and itraconazole-susceptible (S, n=3) C. krusei. Meanwhile, ERG11 and efflux transporter genes (ABC1 and ABC2) were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in 3 groups at the mRNA level. All variables were transformed to obey normal distribution and variance homogenity and compared by two sided t test between two groups. Results Seven point mutations in ERG11 gene of all the C. krusei clinical isolates were detected, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in all three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones (P=0.015 and P=0.002, respectively). ABC2 gene mRNA level in itraconazole-resistant group was significantly higher than the other two groups (P=0.016 and P<0.01, respectively), and the level of its expression in susceptible dose-dependent group was higher than dose susceptible group (P=0.007). ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in one C. krusei (CK10) strain resistant to both itraconazole and voriconazole were highest in all the itraconazole-resistant isolates. Conclusions There are ERG11 gene polymorphisms in clinical isolates of C. krusei. ERG11 gene mutations are not found to be involved in the development of itraconazole resistance in C. krusei. ERG11 and ABC2 upregulation might be responsible for the acquired itraconazole resistance of these clinical isolates. Key words: Candida krusei; Itraconazole; Drug resistance, multiple; Polymerase chain reaction; Reverse transcriptase polymerase chain reaction

ERG11 and ABC2 Overexpression but Not ERG11 Gene Mutations Are Involved in the Development of Itraconazole Resistance in Candida Krusei

Background: Recent years, the incidence and mortality of fungal infection has been on the rise in the patients with hematologic malignancies. This is mainly associated with antifungal resistance and the restricted number of available antifungal drugs. Candida species is one of the most prevalent pathogens in these immunodeficient patients. However, the study of azole resistance mechanisms of Candida has focused on C.albicans, C.glabrata, C.tropicalis. And few studies talked about resistance mechanisms of C.krusei, especially resistant to itraconazole. It was reported that the mutation or overexpression of 14¦Á-demethylases (encoded by ERG11) and upregulation of efflux transporters (encoded by ABC1 and ABC2) may be involved in azole resistance of C.krusei. Here, The purpose of the present study is to preliminarily explore the main molecular mechanisms responsible for Candida krusei clinical isolates to itraconazole, and may provide new sight into fungal infection therapy.Methods: The 14¦Á-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction (PCR), and their nucleotide sequences were determined to detect point mutations. Meanwhile, ERG11 and efflux transporters (ABC1 and ABC2) genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for their expression in itraconazole-resistant (R), itraconazole-susceptible dose dependent (SDD) and itraconazole- susceptible (S) C.krusei at the mRNA level.Results: We found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in the three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole- susceptible ones (P=0.015 and P=0.002 respectively). ABC2 gene mRNA levels in itraconazole-resistant group was significantly higher than the other two groups, and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole (P=0.007 in SDD compared with S, P=0.016 in SDD with R, and P<0.001 in S with R respectively). While ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in a C.krusei (CK10) resistant to both itraconazole and voriconazole were expressed highest in all the itraconazole-resistant isolates. The relative mRNA levels of gene ABC2 and ERG11 can be found in Fig.1 and Fig.2 respectively.Conclusions: There are ERG11 gene polymorphisms in clinical isolates of C.krusei. ERG11 gene mutations were not found to be involved in the development of itraconazole resistance in C.krusei. ERG11 and ABC2 overexpression might be responsible for the acquired itraconazole resistance of these clinical isolates. Therefore, combination of azole and selective efflux transporter inhibitors may help reverse azole resistance and enhance antifungal effect. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Overexpression of Both ERG11 and ABC2 Genes Might Be Responsible for Itraconazole Resistance in Clinical Isolates of Candida krusei.

ObjectiveTo study the main molecular mechanisms responsible for itraconazole resistance in clinical isolates of Candida krusei.MethodsThe 14α-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction (PCR), and their nucleotide sequences were determined to detect point mutations. Meanwhile, ERG11 and efflux transporters (ABC1 and ABC2) genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for their expression in itraconazole-resistant (R), itraconazole-susceptible dose dependent (SDD) and itraconazole-susceptible (S) C.krusei at the mRNA level.ResultsWe found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in the three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones (P = 0.015 and P = 0.002 respectively). ABC2 gene mRNA levels in itraconazole-resistant group was significantly higher than the other two groups, and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole (P = 0.007 in SDD compared with S, P = 0.016 in SDD with R, and P<0.001 in S with R respectively). While ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in a C.krusei (CK10) resistant to both itraconazole and voriconazole were expressed highest in all the itraconazole-resistant isolates.ConclusionsThere are ERG11 gene polymorphisms in clinical isolates of C.krusei. ERG11 gene mutations may not be involved in the development of itraconazole resistance in C.krusei. ERG11 and ABC2 overexpression might be responsible for the acquired itraconazole resistance of these clinical isolates.

Antifungal susceptibilities and identification of species of theSporothrix schenckiicomplex isolated in Brazil

Sporotrichosis is a subacute or chronic mycosis caused worldwide by the dimorphic species complex, Sporothrix schenckii. We studied 85 isolates recovered in Brazil to verify their identification and evaluate their in vitro antifungal susceptibility patterns. Based on phenotypic tests (microscopic features, ability to grow at 30°C and 37°C, colony diameters, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), the strains were identified as S. schenckii, S. brasiliensis and S. globosa, with a predominance of S. schenckii isolates. There was 37.7% disagreement between the phenotypic and genotypic identification methodologies. In general, terbinafine was the most active drug, followed by ketoconazole and itraconazole, and the less active fluconazole and voriconazole. Five isolates (one S. globosa and four S. schenckii) were found to be itraconazole-resistant strains but, in general, there were no differences in the in vitro antifungal susceptibility profiles among the Sporothrix species.

Cyp51A -Based Mechanisms of Aspergillus fumigatus Azole Drug Resistance Present in Clinical Samples from Germany

Since the mid-1990s, a steady increase in the occurrence of itraconazole-resistant Aspergillus fumigatus isolates has been observed in clinical contexts, leading to therapeutic failure in the treatment of aspergillosis. This increase has been predominantly linked to a single allele of the cyp51A gene, termed TR/L98H, which is thought to have arisen through the use of agricultural azoles. Here, we investigated the current epidemiology of triazole-resistant A. fumigatus and underlying cyp51A mutations in clinical samples in Germany. From a total of 527 samples, 17 (3.2%) showed elevated MIC0 values (the lowest concentrations with no visible growth) for at least one of the three substances (itraconazole, voriconazole, and posaconazole) tested. The highest prevalence of resistant isolates was observed in cystic fibrosis patients (5.2%). Among resistant isolates, the TR/L98H mutation in cyp51A was the most prevalent, but isolates with the G54W and M220I substitutions and the novel F219C substitution were also found. The isolate with the G54W substitution was highly resistant to both itraconazole and posaconazole, while all others showed high-level resistance only to itraconazole. For the remaining six isolates, no mutations in cyp51A were found, indicating the presence of other mechanisms. With the exception of the strains carrying the F219C and M220I substitutions, many itraconazole-resistant strains also showed cross-resistance to voriconazole and posaconazole with moderately increased MIC0 values. In conclusion, the prevalence of azole-resistant A. fumigatus in our clinical test set is lower than that previously reported for other countries. Although the TR/L98H mutation frequently occurs among triazole-resistant strains in Germany, it is not the only resistance mechanism present.

In Vitro Activity of a New Oral Glucan Synthase Inhibitor (MK-3118) Tested against Aspergillus spp. by CLSI and EUCAST Broth Microdilution Methods

MK-3118, a glucan synthase inhibitor derived from enfumafungin, and comparator agents were tested against 71 Aspergillus spp., including itraconazole-resistant strains (MIC, ≥ 4 μg/ml), using CLSI and EUCAST reference broth microdilution methods. The CLSI 90% minimum effective concentration (MEC(90))/MIC(90) values (μg/ml) for MK-3118, amphotericin B, and caspofungin, respectively, were as follows: 0.12, 2, and 0.03 for Aspergillus flavus species complex (SC); 0.25, 2, and 0.06 for Aspergillus fumigatus SC; 0.12, 2, and 0.06 for Aspergillus terreus SC; and 0.06, 1, and 0.03 for Aspergillus niger SC. Essential agreement between the values found by CLSI and EUCAST (± 2 log(2) dilution steps) was 94.3%. MK-3118 was determined to be a potent agent regardless of the in vitro method applied, with excellent activity against contemporary wild-type and itraconazole-resistant strains of Aspergillus spp.

Antifungal Susceptibilities of Sporothrix albicans, S. brasiliensis, and S. luriei of the S. schenckii Complex Identified in Brazil

We studied 40 strains of the species complex formerly classified as the single species Sporothrix schenckii to identify new species within this complex and evaluate their antifungal susceptibility profiles. Based on phenotypic tests (ability to grow at 37°C, colony diameters, and pigmentation of the colonies, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), here we report the identification of S. albicans, S. brasiliensis, S. luriei, and S. schenckii; two isolates of these species were detected as itraconazole-resistant strains.

Clinical Itraconazole-Resistant Strains of Aspergillus fumigatus, Isolated Serially from a Lung Aspergilloma Patient with Pulmonary Tuberculosis, can be Detected with Real-Time PCR Method

The invasive aspergillosis, which is commonly caused by Aspergillus fumigatus (A. fumigatus), has increased in recent years. Traditional methods for finding out antifungal resistant strains would take more than 2 weeks, which cannot satisfy the needs of rapid detection. In this study, a real-time PCR method for detection of the serial itraconazole-resistant strains of A. fumigatus isolated from a lung aspergilloma patient was developed. The results showed that the TacMAN-MGB probes, which were covered the loci Gly54, Leu98, Gly138, and Met220 of the enzyme CYP51A coded by the gene cyp51A, as well as the 34-bp tandem repeated sequence in the promoter region (-288 and -322 from the start codon) of this gene, could detect the serial itraconazole-resistant strains of A. fumigatus in our study. Besides, this method takes just 6 h to complete the whole detection.

New triazoles and echinocandins: mode of action, in vitro activity and mechanisms of resistance

Different types of mycoses, especially invasive mycoses caused by yeasts and molds, are a growing problem in healthcare. The most notable explanation for this increase is a rise in the number of immunocompromised patients owing to advances in transplantation, the emergence of AIDS and a rise in the number of invasive surgical procedures. Despite advances in medical practice, some therapeutic problems remain. In addition, intrinsic or acquired antifungal resistance may pose a serious problem to antifungal therapy. A new generation of triazole agents (voriconazole, posaconazole, isavuconazole, ravuconazole and albaconazole) and the recent class of the echinocandins (caspofungin, micafungin and anidulafungin) have become available, and represent an alternative to conventional antifungals for serious fungal infection management. Currently, only two of the recent triazole generation (voriconazole and posaconazole) and all three echinocandins are available for clinical use. More precisely, voriconazole and posaconazole are indicated for the treatment of invasive fungal infections and the echinocandins for the treatment of specific candidiasis. Voriconazole and posaconazole have a very broad spectrum of antifungal activity that includes Candida species, and filamentous and dimorphic fungi. Their activity extends to both fluconazole- and itraconazole-resistant strains of Candida. A major difference between posaconazole and voriconazole is that posaconazole has activity against Zygomycetes including Mucor spp., Rhizopus spp. and Cunninghamella spp., and voriconazole has no activity against this class of fungi. Ravuconazole, isavuconazole and albaconazole have shown very potent in vitro activity against species of Candida, Cryptococcus and Aspergillus, and they are currently in various stages of development. All three echinocandin agents, caspofungin, micafungin and anidulafungin, are similar in their spectrum of activity. Echinocandins do not possess in vitro activity against important basidiomycetes, including Cryptococcus, Rhodotorula and Trichosporon. This review attempts to deliver the most up-to-date knowledge on the mode of action and mechanisms of resistance to triazoles and echinocandins in fungal pathogens. In addition, the in vitro activity data available on triazoles and echinocandins are reported.

Epidemiological Cutoffs and Cross-Resistance to Azole Drugs in Aspergillus fumigatus

Antifungal susceptibility testing of molds has been standardized in Europe and in the United States. Aspergillus fumigatus strains with resistance to azole drugs have recently been detected and the underlying molecular mechanisms of resistance characterized. Three hundred and ninety-three isolates, including 32 itraconazole-resistant strains, were used to define wild-type populations, epidemiological cutoffs, and cross-resistance between azole drugs. The epidemiological cutoff for itraconazole, voriconazole, and ravuconazole for the wild-type populations of A. fumigatus was < or =1 mg/liter. For posaconazole, the epidemiological cutoff was < or =0.25 mg/liter. Up till now, isolates susceptible to itraconazole have not yet displayed resistance to other azole drugs. Cross-resistance between azole drugs depends on specific mutations in cyp51A. Thus, a substitution of glycine in position 54 of Cyp51A confers cross-resistance between itraconazole and posaconazole. A substitution of methionine at position 220 or a duplication in tandem of a 34-bp fragment in the cyp51A promoter combined with a substitution of leucine at position 98 for histidine confers cross-resistance to all azole drugs tested. The results obtained in this study will help to develop clinical breakpoints for azole drugs and A. fumigatus.

Use of voriconazole in a patient with aspergilloma caused by an itraconazole-resistant strain of Aspergillus fumigatus

The case is reported of a patient with cavitary sarcoidosis complicated by an aspergilloma caused by an itraconazole-resistant strain of Aspergillus fumigatus, who was treated with voriconazole. The authors suggest that susceptibility testing of A. fumigatus strains is of value during long-term therapy with itraconazole, and that voriconazole may be a good option for treatment of patients infected with itraconazole-resistant strains of A. fumigatus.

Voriconazole

Background: Reports of resistance and intolerance to currently available antifungal agents are increasing. Voriconazole is a broad-spectrum azole antifungal agent structurally derived from fluconazole. It is indicated for the treatment of invasive aspergillosis and serious fungal infections caused by Scedosporium apiospermum and Fusarium species in patients who are unable to tolerate or are refractory to other antifungal therapy. Objective: This article reviews the pharmacologic and pharmacokinetic properties and clinical usefulness of voriconazole. Methods: Relevant information was identified through a search of MEDLINE (1966–December 2002), Iowa Drug Information Service (1966–December 2002), International Pharmaceutical Abstracts (1970–December 2002), and meeting abstracts of the Infectious Diseases Society of America (1996–2002) and the Interscience Conference on Antimicrobial Agents and Chemotherapy (1996–2002) using the terms voriconazole and UK-109,495. Results: In head-to-head comparative trials, voriconazole appeared to be as efficacious as amphotericin B for the treatment of invasive aspergillosis and the empiric treatment of fungal infections in patients with febrile neutropenia. In clinical studies, it was as efficacious as fluconazole for the treatment of oropharyngeal and esophageal candidiasis. The results of in vitro susceptibility studies and case reports suggested that voriconazole may be useful against fluconazole- and/or itraconazole-resistant strains of Candida. Although voriconazole may be associated with a lower incidence of serious systemic adverse effects compared with amphotericin B (13.4% vs 24.3% in 1 pivotal clinical study; P = NS), major adverse effects associated with voriconazole include visual abnormalities (∼30%), skin reactions (∼20%), and elevations in hepatic enzymes (≤20%). Voriconazole is available as oral and intravenous formulations. Pharmacokinetically, it has widespread distribution, including penetration into cerebral tissue. However, as 80% of voriconazole is hepatically eliminated, primarily via the cytochrome P450 (CYP) isozymes CYP2C19, CYP3A4, and CYP2C9, voriconazole has a high potential for drug interactions, and dose reduction is recommended in patients with mild to moderate hepatic dysfunction (Child-Pugh class A or B). Oral voriconazole may be preferred in patients with a creatinine clearance <50 mL/min due to the potential accumulation of the solubilizing excipient in the parenteral formulation of voriconazole. Conclusions: Voriconazole appears to be a useful alternative to conventional antifungal agents in cases of resistance or intolerance to initial therapy. However, dose adjustment is recommended in patients with hepatic dysfunction, as well as in those receiving medications that may interact with voriconazole via hepatic metabolism.