Azole resistance and cyp51A mutation screening in Aspergillus fumigatus in Mexico

2019 ◽  
Vol 74 (7) ◽  
pp. 2047-2050 ◽  
Author(s):  
Maria F Gonzalez-Lara ◽  
Carla M Roman-Montes ◽  
Paulette Diaz-Lomeli ◽  
Andrea Rangel-Cordero ◽  
Maria O Valenzuela ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Screening Test ◽  
Mutation Screening ◽  
Tertiary Care ◽  
Dna Amplification ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Azole Resistance ◽  
Tertiary Care Centre

AbstractBackgroundFungicide exposure in the environment has driven the emergence of azole-resistant Aspergillus fumigatus worldwide. A screening test allows identification of resistant isolates.ObjectivesWe screened clinical samples for azole-resistant Aspergillus through azole-containing agar plates and identified mutations in the cyp51A gene of A. fumigatus.MethodsAspergillus isolates from clinical samples collected in a tertiary care centre from 2014 to 2017 were screened for azole resistance. Samples were subcultured in azole-containing agar plates. Isolates with a positive screening test were subject to DNA extraction, DNA amplification and sequencing of the cyp51A gene (coding and promoter regions). Clinical data were obtained from medical records.ResultsWe screened 43 Aspergillus isolates from 39 patients for azole resistance. Three isolates from three patients grew on azole-containing agar plates: two A. fumigatus and one Aspergillus flavus. PCR analysis and cyp51A sequencing identified the TR34/L98H mutation in both A. fumigatus isolates. The prevalence of cyp51A mutations among A. fumigatus was 8.3% (2/24). Both patients with TR34/L98H mutants were azole naive and presented with invasive aspergillosis; one had multiple myeloma and the other was a liver retransplant recipient. They suffered progressive disease and failed voriconazole therapy.ConclusionsTo the best of our knowledge, this is the first report of azole-resistant A. fumigatus with the TR34/L98H mutation in two azole-naive patients with refractory invasive aspergillosis in Mexico.

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 Prevalence and mechanisms of azole resistance in clinical isolates of Aspergillus section Fumigati species in a Canadian tertiary care centre, 2000 to 2013

2019 ◽  
Vol 75 (4) ◽  
pp. 849-858
Author(s):  
Maxime Parent-Michaud ◽  
Philippe J Dufresne ◽  
Eric Fournier ◽  
Benjamin Folch ◽  
Christine Martineau ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Tertiary Care ◽  
Geographic Area ◽  
Resistance Mechanisms ◽  
Sensu Stricto ◽  
Azole Resistance ◽  
Intrinsic Resistance ◽  
Tertiary Care Centre ◽  
Combined Use ◽  
Aspergillus Section

Abstract Objectives Azole resistance among Aspergillus fumigatus isolates is a growing concern worldwide. Induction of mutations during azole therapy, environment-acquired mutations caused by azole fungicides and intrinsic resistance of cryptic Fumigati species all contribute to the burden of resistance. However, there is a lack of data in Canada on this emerging threat. Methods To gain insights into the magnitude and mechanisms of resistance, a 14 year collection of Aspergillus section Fumigati comprising 999 isolates from 807 patients at a Montreal hospital was screened for azole resistance, and resistance mechanisms were investigated with the combined use of genome sequencing, 3D modelling and phenotypic efflux pump assays. Results Overall azole resistance was low (4/807 patients; 0.5%). A single azole-resistant A. fumigatus sensu stricto strain, isolated from a patient with pulmonary aspergillosis, displayed efflux-pump-mediated resistance. Three patients were colonized or infected with azole-resistant cryptic Fumigati species (one Aspergillus thermomutatus, one Aspergillus lentulus and one Aspergillus turcosus). Evidence is presented that azole resistance is efflux-pump-mediated in the A. turcosus isolate, but not in the A. lentulus and A. thermomutatus isolates. Conclusions Azole resistance is rare in our geographic area and currently driven by cryptic Fumigati species. Continued surveillance of emergence of resistance is warranted.

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 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.

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