Frequency and evolution of Azole resistance in Aspergillus fumigatus associated with treatment failure.
ABSTRACT: Azoles are the mainstay of oral therapy for aspergillosis. Azole resistance in Aspergillus has been reported infrequently. The first resistant isolate was detected in 1999 in Manchester, UK. In a clinical collection of 519 A. fumigatus isolates, the frequency of itraconazole resistance was 5%, a significant increase since 2004 (p<0.001). Of the 34 itraconazole-resistant isolates we studied, 65% (22) were cross-resistant to voriconazole and 74% (25) were cross-resistant to posaconazole. Thirteen of 14 evaluable patients in our study had prior azole exposure; 8 infections failed therapy (progressed), and 5 failed to improve (remained stable). Eighteen amino acid alterations were found in the target enzyme, Cyp51A, 4 of which were novel. A population genetic analysis of microsatellites showed the existence of resistant mutants that evolved from originally susceptible strains, different cyp51A mutations in the same strain, and microalterations in microsatellite repeat number. Azole resistance in A. fumigatus is an emerging problem and may develop during azole therapy.
Project description:The rapid and global emergence of azole resistance in the human pathogen Aspergillus fumigatus has drawn attention. Thus, a thorough understanding of its mechanisms of drug resistance requires extensive exploration. In this study, we found that the loss of the putative calcium-dependent protein-encoding gene algA causes an increased frequency of azole-resistant A. fumigatus isolates. In contrast to previously identified azole-resistant isolates related to cyp51A mutations, only one isolate carries a point mutation in cyp51A (F219L mutation) among 105 independent stable azole-resistant isolates. Through next-generation sequencing (NGS), we successfully identified a new mutation (R243Q substitution) conferring azole resistance in the putative A. fumigatus farnesyltransferase Cox10 (AfCox10) (AFUB_065450). High-performance liquid chromatography (HPLC) analysis verified that the decreased absorption of itraconazole in related Afcox10 mutants is the primary reason for itraconazole resistance. Moreover, a complementation experiment by reengineering the mutation in a parental wild-type background strain demonstrated that both the F219L and R243Q mutations contribute to itraconazole resistance in an algA-independent manner. These data collectively suggest that the loss of algA results in an increased frequency of azole-resistant isolates with a non-cyp51A mutation. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus and that algA defects make it possible to isolate drug-resistant alleles. In addition, our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to identify the contribution of single nucleotide polymorphism (SNP) diversity to drug resistance.
Project description: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.
Project description:ASP2397 is a new compound with a novel and as-yet-unknown target different from that of licensed antifungal agents. It has activity against Aspergillus and Candida glabrata. We compared its in vitro activity against wild-type and azole-resistant A. fumigatus and A. terreus isolates with that of amphotericin B, itraconazole, posaconazole, and voriconazole. Thirty-four isolates, including 4 wild-type A. fumigatus isolates, 24 A. fumigatus isolates with alterations in CYP51A TR/L98H (5 isolates), M220 (9 isolates), G54 (9 isolates), and HapE (1 isolate), and A. terreus isolates (2 wild-type isolates and 1 isolate with an M217I CYP51A alteration), were analyzed. EUCAST E.Def 9.2 and CLSI M38-A2 MIC susceptibility testing was performed. ASP2397 MIC50 values (in milligrams per liter, with MIC ranges in parentheses) determined by EUCAST and CLSI were 0.5 (0.25 to 1) and 0.25 (0.06 to 0.25) against A. fumigatus CYP51A wild-type isolates and were similarly 0.5 (0.125 to >4) and 0.125 (0.06 to >4) against azole-resistant A. fumigatus isolates, respectively. These values were comparable to those for amphotericin B, which were 0.25 (0.125 to 0.5) and 0.25 (0.125 to 0.25) against wild-type isolates and 0.25 (0.125 to 1) and 0.25 (0.125 to 1) against isolates with azole resistance mechanisms, respectively. In contrast, MICs for the azole compounds were elevated and highest for itraconazole: >4 (1 to >4) and 4 (0.5 to >4) against isolates with azole resistance mechanisms compared to 0.125 (0.125 to 0.25) and 0.125 (0.06 to 0.25) against wild-type isolates, respectively. ASP2397 was active against A. terreus CYP51A wild-type isolates (MIC 0.5 to 1), whereas MICs of both azole and ASP2397 were elevated for the mutant isolate. ASP2397 displayed in vitro activity against A. fumigatus and A. terreus isolates which was independent of the presence or absence of azole target gene resistance mutations in A. fumigatus. The findings are promising at a time when azole-resistant A. fumigatus is emerging globally.
Project description:We investigated the antifungal susceptibilities and the cyp51 mutant strains among Aspergillus fumigatus clinical isolates obtained from 10 university hospitals in Korea. Of the 84 isolates examined, two itraconazole-resistant isolates were found with no amino acid substitution in the cyp51A/cyp51B genes. However, 19 (23.2%) azole-susceptible isolates harbored amino acid substitutions: Nine isolates harbored one to five mutations in cyp51A with high polymorphism, and 11 isolates exhibited the same Q42L mutation in cyp51B. Overall, a low azole resistance rate and high frequency of cyp51A/cyp51B amino acid substitutions were observed in the azole-susceptible A. fumigatus isolates in Korea.
Project description:The past decade has seen an increase in aspergillosis in humans and animals due to Aspergillus viridinutans species complex members. Azole resistance is common to these infections, carrying a poor prognosis. cyp51A gene mutations are the main cause of acquired azole resistance in Aspergillus fumigatus This study aimed to determine if the azole-resistant phenotype in A. viridinutans complex members is associated with cyp51A mutations or extrolite profiles. The cyp51A gene of clinical and environmental isolates was amplified using novel primers, antifungal susceptibility was tested using the Clinical and Laboratory Standards Institute methodology, and extrolite profiling was performed using agar plug extraction. Very high azole MICs were detected in 84% of the isolates (31/37). The MICs of the newer antifungals luliconazole and olorofim (F901318) were low for all isolates. cyp51A sequences revealed 113 nonsynonymous mutations compared to the sequence of wild-type A. fumigatus M172A/V and D255G, previously associated with A. fumigatus azole resistance, were common among all isolates but were not correlated with azole MICs. Two environmental isolates with nonsusceptibility to itraconazole and high MICs of voriconazole and isavuconazole harbored G138C, previously associated with azole-resistant A. fumigatus Some novel mutations were identified only among isolates with high azole MICs. However, cyp51A homology modeling did not cause a significant protein structure change for these mutations. There was no correlation between extrolite patterns and susceptibility. For A. viridinutans complex isolates, cyp51A mutations and the extrolites that they produced were not major causes of antifungal resistance. Luliconazole and olorofim show promise for treating azole-resistant infections caused by these cryptic species.
Project description:Nine consecutive isogenic Aspergillus fumigatus isolates cultured from a patient with aspergilloma were investigated for azole resistance. The first cultured isolate showed a wild-type phenotype, but four azole-resistant phenotypes were observed in the subsequent eight isolates. Four mutations were found in the cyp51A gene of these isolates, leading to the substitutions A9T, G54E, P216L, and F219I. Only G54 substitutions were previously proved to be associated with azole resistance. Using a Cyp51A homology model and recombination experiments in which the mutations were introduced into a susceptible isolate, we show that the substitutions at codons P216 and F219 were both associated with resistance to itraconazole and posaconazole. A9T was also present in the wild-type isolate and thus considered a Cyp51A polymorphism. Isolates harboring F219I evolved further into a pan-azole-resistant phenotype, indicating an additional acquisition of a non-Cyp51A-mediated resistance mechanism. Review of the literature showed that in patients who develop azole resistance during therapy, multiple resistance mechanisms commonly emerge. Furthermore, the median time between the last cultured wild-type isolate and the first azole-resistant isolate was 4 months (range, 3 weeks to 23 months), indicating a rapid induction of resistance.
Project description:Azole resistance in Aspergillus spp. has been increasingly reported worldwide. Acquired azole resistance is probably linked to environmental exposure to fungicides used in agriculture. We collected a total of 84 soil and leaf samples from eight farms in Southern Italy. Aspergillus isolates were tested for resistance to itraconazole, posaconazole, and voriconazole by the EUCAST method. Five out of 84 samples yielded A. fumigatus isolates: four of them were itraconazole-resistant and were identified as A. fumigatus sensu stricto, three of them were posaconazole-resistant, and two were also voriconazole-resistant. All three isolates harbored the TR34/L98H resistance mechanism, which was detected by DNA sequencing of the cyp51A gene. Fifteen out of 84 samples yielded Aspergillus spp. isolates and included 11 itraconazole-resistant isolates: Aspergillus section Nigri (9) and Aspergillus section Flavi (2). Our study reports for the first time the isolation of azole-resistant A. fumigatus harboring TR34/L98H mutation from the environment of Southern Italy. The present work provides a better understanding of the magnitude of the environmental spread of azole resistance in the context of a necessary effective surveillance program to improve the management of Aspergillus-related disease.
Project description:Seventy-two A. fumigatus clinical isolates from China were investigated for azole resistance based on mutations of cyp51A. We identified four azole-resistant strains, among which we found three strains highly resistant to itraconazole, two of which exhibit the TR34/L98H/S297T/F495I mutation, while one carries only the TR34/L98H mutation. To our knowledge, the latter has not been found previously in China. The fourth multiazole-resistant isolate (with only moderate itraconazole resistance) carries a new G432A mutation.
Project description:The opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in cytochromeP450 enzyme Cyp51. In this study, we indicated that in vitro azole situation results in emergence of azole-resistant mutations. There are previously identified azole-resistant cyp51A mutations (M220K, M220I, M220R, G54E and G54W mutations) and we successfully identified in this study two new mutations (N248K/V436A, Y433N substitution) conferring azole resistance among 18 independent stable azole-resistant isolates. The Galleria mellonella model of A. fumigatus infection experiment verified that Cyp51A mutations N248K/V436A and Y433N reduce efficacy of azole therapy. In addition, a predicted Cyp51A 3D structural model suggested that Y433N mutation causes the reduced affinities between drug target Cyp51A and azole antifungals. This study suggests that drug selection pressure make it possible to isolate unidentified cyp51A mutations conferring azole resistance in A. fumigatus.
Project description:Respiratory tract colonization by molds in patients with cystic fibrosis (CF) were analyzed, with particular focus on the frequency, genotype, and underlying mechanism of azole resistance among Aspergillus fumigatus isolates. Clinical and demographic data were also analyzed. A total of 3,336 respiratory samples from 287 CF patients were collected during two 6-month periods in 2007 and 2009. Azole resistance was detected using an itraconazole screening agar (4 mg/liter) and the EUCAST method. cyp51A gene sequencing and microsatellite genotyping were performed for isolates from patients harboring azole-resistant A. fumigatus. Aspergillus spp. were present in 145 patients (51%), of whom 63 (22%) were persistently colonized. Twelve patients (4%) harbored other molds. Persistently colonized patients were older, provided more samples, and more often had a chronic bacterial infection. Six of 133 patients (4.5%) harbored azole-nonsusceptible or -resistant A. fumigatus isolates, and five of those six patients had isolates with Cyp51A alterations (M220K, tandem repeat [TR]/L98H, TR/L98H-S297T-F495I, M220I-V101F, and Y431C). All six patients were previously exposed to azoles. Genotyping revealed (i) microevolution for A. fumigatus isolates received consecutively over the 2-year period, (ii) susceptible and resistant isolates (not involving TR/L98H isolates) with identical or very closely related genotypes (two patients), and (iii) two related susceptible isolates and a third unrelated resistant isolate with a unique genotype and the TR/L98H resistance combination (one patient). Aspergilli were frequently found in Danish CF patients, with 4.5% of the A. fumigatus isolates being azole nonsusceptible or resistant. Genotyping suggested selection of resistance in the patient as well as resistance being achieved in the environment.