scholarly journals Development of Novel PCR Assays To Detect Azole Resistance-Mediating Mutations of theAspergillus fumigatus cyp51AGene in Primary Clinical Samples from Neutropenic Patients

2012 ◽  
Vol 56 (7) ◽  
pp. 3905-3910 ◽  
Author(s):  
Birgit Spiess ◽  
Wolfgang Seifarth ◽  
Natalia Merker ◽  
Susan J. Howard ◽  
Mark Reinwald ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Positive Culture ◽  
Cross Reactivity ◽  
Resistant Isolate ◽  
Clinical Samples ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Pcr Assays

ABSTRACTThe increasing incidence of azole resistance inAspergillus fumigatuscausing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediatingcyp51Agene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of theAspergillus fumigatus cyp51Agene to establish PCR assays with consecutive DNA sequence analysis to detect and identify theA. fumigatus cyp51Atandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially dilutedA. fumigatusand human DNA,A. fumigatus cyp51Agene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg ofA. fumigatusDNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons forA. fumigatuswild-type DNA confirmed thecyp51Awild-type sequence, and PCR products from one azole-resistantA. fumigatusisolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.

scholarly journals Rapid Induction of Multiple Resistance Mechanisms in Aspergillus fumigatus during Azole Therapy: a Case Study and Review of the Literature

2011 ◽  
Vol 56 (1) ◽  
pp. 10-16 ◽  
Author(s):  
Simone M. T. Camps ◽  
Jan W. M. van der Linden ◽  
Yi Li ◽  
Ed J. Kuijper ◽  
Jaap T. van Dissel ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Resistance Mechanisms ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Multiple Resistance ◽  
Review Of The Literature ◽  
Wild Type ◽  
Content Type ◽  
Type Isolate ◽  
Rapid Induction

ABSTRACTNine consecutive isogenicAspergillus fumigatusisolates 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 thecyp51Agene 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.

scholarly journals In Vivo Efficacy of Liposomal Amphotericin B against Wild-Type and Azole-Resistant Aspergillus fumigatus Isolates in Two Different Immunosuppression Models of Invasive Aspergillosis

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Seyedmojtaba Seyedmousavi ◽  
Johan W. Mouton ◽  
Willem J. G. Melchers ◽  
Paul E. Verweij
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Amphotericin B ◽  
Liposomal Amphotericin ◽  
Treated Group ◽  
Liposomal Amphotericin B ◽  
Wild Type ◽  
Resistance Mutations ◽  
Content Type

ABSTRACT Using an immunocompetent murine model of invasive aspergillosis (IA), we previously reported that the efficacy of liposomal amphotericin B (L-AmB) (Ambisome) is not hampered by the presence of azole resistance mutations in Aspergillus fumigatus (S. Seyedmousavi, W. J. G. Melchers, J. W. Mouton, and P. E. Verweij, Antimicrob Agents Chemother 57:1866–1871, 2013, https://doi.org/10.1128/AAC.02226-12 ). We here investigated the role of immune suppression, i.e., neutropenia and steroid treatment, in L-AmB efficacy in mice infected with wild-type (WT) A. fumigatus and with azole-resistant A. fumigatus harboring a TR34/L98H mutation in the cyp-51A gene. Survival of treated animals at day 14 in both immunosuppressed models was significantly better than that of nontreated controls. A dose-response relationship was observed that was independent of the azole-resistant mechanism and the immunosuppression method used. In the neutropenic model, 100% survival was reached at an L-AmB dose of 16 mg/kg of body weight for the WT strain and the TR34/L98H isolate. In the steroid-treated group, 90.9% survival and 100% survival were achieved for the WT isolate and the TR34/L98H isolate with an L-AmB dose of 16 mg/kg, respectively. The 50% effective dose (ED50) was 1.40 mg/kg (95% confidence interval [CI], 0.66 to 3.00 mg/kg) for the WT isolate and 1.92 mg/kg (95% CI, 0.60 to 6.17 mg/kg) for the TR34/L98H isolate in the neutropenic model and was 2.40 mg/kg (95% CI, 1.93 to 2.97 mg/kg) for the WT isolate and 2.56 mg/kg (95% CI, 1.43 to 4.56 mg/kg) for the TR34/L98H isolate in the steroid-treated group. Overall, there were no significant differences between the two different immunosuppressed conditions in the efficacy of L-AmB against the wild-type and azole-resistant isolates (P > 0.9). However, the required L-AmB exposure was significantly higher than that seen in the immunocompetent model.

scholarly journals Multiplecyp51A-Based Mechanisms Identified in Azole-Resistant Isolates of Aspergillus fumigatus from China

2015 ◽  
Vol 59 (7) ◽  
pp. 4321-4325 ◽  
Author(s):  
Musang Liu ◽  
Rong Zeng ◽  
Lili Zhang ◽  
Dongmei Li ◽  
Guixia Lv ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Content Type ◽  
Resistant Strains

ABSTRACTSeventy-twoA. fumigatusclinical isolates from China were investigated for azole resistance based on mutations ofcyp51A. 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.

scholarly journals Is Azole Resistance in Aspergillus fumigatus a Problem in Spain?

2013 ◽  
Vol 57 (6) ◽  
pp. 2815-2820 ◽  
Author(s):  
Pilar Escribano ◽  
Teresa Peláez ◽  
Patricia Muñoz ◽  
Emilio Bouza ◽  
Jesús Guinea
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Cryptic Species ◽  
Antifungal Susceptibility ◽  
Single Species ◽  
Azole Resistance ◽  
Tubulin Gene ◽  
Complex Species ◽  
Content Type ◽  
Β Tubulin

ABSTRACTAspergillus fumigatuscomplex comprisesA. fumigatusand other morphologically indistinguishable cryptic species. We retrospectively studied 362A. fumigatuscomplex isolates (353 samples) from 150 patients with proven or probable invasive aspergillosis or aspergilloma (2, 121, and 6 samples, respectively) admitted to the hospital from 1999 to 2011. Isolates were identified using the β-tubulin gene, and only 1 isolate per species found in each sample was selected. Antifungal susceptibility to azoles was determined using the CLSI M38-A2 procedure. Isolates were considered resistant if they showed an MIC above the breakpoints for itraconazole, voriconazole, or posaconazole (>2, >2, or >0.5 μg/ml). Most of the samples yielded only 1 species (A. fumigatus[n= 335],A. novofumigatus[n= 4],A. lentulus[n= 3],A. viridinutans[n= 1], andNeosartorya udagawae[n= 1]). The remaining samples yielded a combination of 2 species. Most of the patients were infected by a single species (A. fumigatus[n= 143] orA. lentulus[n= 2]). The remaining 5 patients were coinfected with multipleA. fumigatuscomplex species, althoughA. fumigatuswas always involved; 4 of the 5 patients were diagnosed in 2009 or later. Cryptic species were less susceptible thanA. fumigatus. The frequency of resistance amongA. fumigatuscomplex andA. fumigatusto itraconazole, voriconazole, and posaconazole was 2.5 and 0.3%, 3.1 and 0.3%, and 4.2 and 1.8%, respectively, in the per-isolate analysis and 1.3 and 0.7%, 2.6 and 0.7%, and 6 and 4% in the per-patient analysis. Only 1 of the 6A. fumigatusisolates in which thecyp51Agene was sequenced had a mutation at position G448. The proportion of patients infected by azole-resistantA. fumigatusisolates was low.

scholarly journals Azole Resistance in Aspergillus fumigatus Clinical Isolates from an Italian Culture Collection

2015 ◽  
Vol 60 (1) ◽  
pp. 682-685 ◽  
Author(s):  
Cristina Lazzarini ◽  
Maria Carmela Esposto ◽  
Anna Prigitano ◽  
Massimo Cogliati ◽  
Gabriella De Lorenzis ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Culture Collection ◽  
Resistance Rate ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Content Type ◽  
Italian Culture

ABSTRACTThe aims of the study were to investigate the prevalence of azole resistance amongAspergillus fumigatusclinical isolates. A total of 533 clinical isolates that had been collected between 1995 and 2006, from 441 patients, were screened. No resistance was detected in isolates collected between 1995 and 1997. Starting in 1998, the resistance rate was 6.9%; a total of 24 patients (6.25%) harbored a resistant isolate. The TR34/L98H substitution was found in 21 of 30 tested isolates.

scholarly journals Progressive Dispersion of Azole Resistance in Aspergillus fumigatus: Fatal Invasive Aspergillosis in a Patient with Acute Myeloid Leukemia Infected with an A. fumigatus Strain with a cyp51A TR46 Y121F M172I T289A Allele

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Susann Rößler ◽  
Oliver Bader ◽  
Friedrich Stölzel ◽  
Ulrich Sommer ◽  
Birgit Spiess ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Myeloid Leukemia ◽  
Treatment Options ◽  
Hematologic Malignancies ◽  
Azole Resistance ◽  
Hematopoietic Stem ◽  
Content Type ◽  
Acute Myeloid

ABSTRACT Patients with hematologic malignancies as well as allogeneic hematopoietic stem cell transplantation (HSCT) patients are at high risk for invasive aspergillosis. Here, we report a culture- and autopsy-proven fatal invasive aspergillosis in an allogeneic HSTC patient which he developed despite posaconazole prophylaxis. The agent was determined to be an azole-resistant Aspergillus fumigatus strain bearing the cyp51A mutation combination TR46 Y121F M172I T289A. At increasing frequency, the azole resistance of A. fumigatus is being reported globally, limiting treatment options and complicating regimens.

scholarly journals Azole Resistance of Environmental and Clinical Aspergillus fumigatus Isolates from Switzerland

2018 ◽  
Vol 62 (4) ◽  
Author(s):  
Arnaud Riat ◽  
Jérôme Plojoux ◽  
Katia Gindro ◽  
Jacques Schrenzel ◽  
Dominique Sanglard
Keyword(s):  
Aspergillus Fumigatus ◽  
Opportunistic Pathogen ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Clinical Settings ◽  
Content Type ◽  
Azole Antifungals ◽  
First Time

ABSTRACT Aspergillus fumigatus is a ubiquitous opportunistic pathogen. This fungus can acquire resistance to azole antifungals due to mutations in the azole target ( cyp51A ). Recently, cyp51A mutations typical for environmental azole resistance acquisition (for example, TR 34 /L98H) have been reported. These mutations can also be found in isolates recovered from patients. Environmental azole resistance acquisition has been reported on several continents. Here we describe, for the first time, the occurrence of azole-resistant A. fumigatus isolates of environmental origin in Switzerland with cyp51A mutations, and we show that these isolates can also be recovered from a few patients. While the TR 34 /L98H mutation was dominant, a single azole-resistant isolate exhibited a cyp51A mutation (G54R) that was reported only for clinical isolates. In conclusion, our study demonstrates that azole resistance with an environmental signature is present in environments and patients of Swiss origin and that mutations believed to be unique to clinical settings are now also observed in the environment.

scholarly journals Five-Year Survey (2014 to 2018) of Azole Resistance in Environmental Aspergillus fumigatus Isolates from China

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Duantao Cao ◽  
Ruilin Wu ◽  
Suxia Dong ◽  
Feiyan Wang ◽  
Chao Ju ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Soil Samples ◽  
Azole Resistance ◽  
Clinical Settings ◽  
Wild Type ◽  
Content Type

ABSTRACT A total of 191 soil samples from Hangzhou, China, were submitted to detect non-wild-type (non-WT) Aspergillus fumigatus and its associated mechanisms. There were 2 (4.7%), 13 (12.4%), and 31 (23.1%) isolates identified as non-WT in 2014, 2016, and 2018, respectively. The resistant mutations of TR34/L98H, TR46/Y121F/T289A, and TR34/L98H/S297T/F495I were found in 3, 5, and 5 non-WT isolates. The G448S mutation, previously only found in clinical settings, was detected in A. fumigatus from soil samples.

scholarly journals In VitroBiochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

2015 ◽  
Vol 59 (12) ◽  
pp. 7771-7778 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Josie E. Parker ◽  
Claire L. Price ◽  
W. David Nes ◽  
Steven L. Kelly ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Inhibitory Concentration ◽  
Point Mutations ◽  
Residual Activity ◽  
Azole Resistance ◽  
Wild Type ◽  
Biochemical Basis ◽  
Content Type ◽  
Resistant Phenotype

ABSTRACTThe incidence of triazole-resistantAspergillusinfections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinantAspergillus fumigatusCPR1 (AfCPR1), andEscherichia colimembrane suspensions containing recombinantA. fumigatusCYP51 proteins, allowingin vitroscreening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed thatA. fumigatusCYP51B (Af51B IC50, 0.50 μM) was 34-fold more susceptible to inhibition by fluconazole thanA. fumigatusCYP51A (Af51A IC50, 17 μM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 μM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 μM triazole, which could confer azole resistance inA. fumigatusstrains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance ofA. fumigatusstrains harboring G54W, L98H, and M220K Af51A point mutations.

scholarly journals Triazole Resistance Is Still Not Emerging in Aspergillus fumigatus Isolates Causing Invasive Aspergillosis in Brazilian Patients

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Clara E. Negri ◽  
Sarah S. Gonçalves ◽  
Ana Cristina P. Sousa ◽  
Maria Daniela Bergamasco ◽  
Marinês D. V. Martino ◽  
...  
Keyword(s):  
Global Health ◽  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Health Problem ◽  
Antifungal Susceptibility ◽  
Azole Resistance ◽  
Cutoff Value ◽  
Content Type ◽  
Global Health Problem

ABSTRACT Aspergillus fumigatus azole resistance has emerged as a global health problem. We evaluated the in vitro antifungal susceptibility of 221 clinical A. fumigatus isolates according to CLSI guidelines. Sixty-one isolates exhibiting MICs at the epidemiological cutoff value (ECV) for itraconazole or above the ECV for any triazole were checked for CYP51A mutations. No mutations were documented, even for the isolates (1.8%) with high voriconazole MICs, indicating that triazoles may be used safely to treat aspergillosis in Brazil.

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