scholarly journals Posaconazole MIC Distributions for Aspergillus fumigatus Species Complex by Four Methods: Impact of cyp51A Mutations on Estimation of Epidemiological Cutoff Values

2018 ◽  
Vol 62 (4) ◽  
pp. e01916-17 ◽  
Author(s):  
A. Espinel-Ingroff ◽  
J. Turnidge ◽  
A. Alastruey-Izquierdo ◽  
E. Dannaoui ◽  
G. Garcia-Effron ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Species Complex ◽  
Susceptibility Testing ◽  
Analytical Techniques ◽  
Wild Type ◽  
Testing Methods ◽  
Content Type ◽  
Mutational Status ◽  
Type Population ◽  
Almost All

ABSTRACT Estimating epidemiological cutoff endpoints (ECVs/ECOFFS) may be hindered by the overlap of MICs for mutant and nonmutant strains (strains harboring or not harboring mutations, respectively). Posaconazole MIC distributions for the Aspergillus fumigatus species complex were collected from 26 laboratories (in Australia, Canada, Europe, India, South and North America, and Taiwan) and published studies. Distributions that fulfilled CLSI criteria were pooled and ECVs were estimated. The sensitivity of three ECV analytical techniques (the ECOFFinder, normalized resistance interpretation [NRI], derivatization methods) to the inclusion of MICs for mutants was examined for three susceptibility testing methods (the CLSI, EUCAST, and Etest methods). The totals of posaconazole MICs for nonmutant isolates (isolates with no known cyp51A mutations) and mutant A. fumigatus isolates were as follows: by the CLSI method, 2,223 and 274, respectively; by the EUCAST method, 556 and 52, respectively; and by Etest, 1,365 and 29, respectively. MICs for 381 isolates with unknown mutational status were also evaluated with the Sensititre YeastOne system (SYO). We observed an overlap in posaconazole MICs among nonmutants and cyp51A mutants. At the commonly chosen percentage of the modeled wild-type population (97.5%), almost all ECVs remained the same when the MICs for nonmutant and mutant distributions were merged: ECOFFinder ECVs, 0.5 μg/ml for the CLSI method and 0.25 μg/ml for the EUCAST method and Etest; NRI ECVs, 0.5 μg/ml for all three methods. However, the ECOFFinder ECV for 95% of the nonmutant population by the CLSI method was 0.25 μg/ml. The tentative ECOFFinder ECV with SYO was 0.06 μg/ml (data from 3/8 laboratories). Derivatization ECVs with or without mutant inclusion were either 0.25 μg/ml (CLSI, EUCAST, Etest) or 0.06 μg/ml (SYO). It appears that ECV analytical techniques may not be vulnerable to overlap between presumptive wild-type isolates and cyp51A mutants when up to 11.6% of the estimated wild-type population includes mutants.

scholarly journals Method-Dependent Epidemiological Cutoff Values for Detection of Triazole Resistance in Candida and Aspergillus Species for the Sensititre YeastOne Colorimetric Broth and Etest Agar Diffusion Methods

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
A. Espinel-Ingroff ◽  
J. Turnidge ◽  
A. Alastruey-Izquierdo ◽  
F. Botterel ◽  
E. Canton ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Species Complex ◽  
Susceptibility Testing ◽  
Yeast Species ◽  
Sensu Stricto ◽  
Wild Type ◽  
Pichia Kudriavzevii ◽  
Cutoff Value ◽  
Content Type ◽  
Clavispora Lusitaniae

ABSTRACT Although the Sensititre Yeast-One (SYO) and Etest methods are widely utilized, interpretive criteria are not available for triazole susceptibility testing of Candida or Aspergillus species. We collected fluconazole, itraconazole, posaconazole, and voriconazole SYO and Etest MICs from 39 laboratories representing all continents for (method/agent-dependent) 11,171 Candida albicans, 215 C. dubliniensis, 4,418 C. glabrata species complex, 157 C. guilliermondii (Meyerozyma guilliermondii), 676 C. krusei (Pichia kudriavzevii), 298 C. lusitaniae (Clavispora lusitaniae), 911 C. parapsilosis sensu stricto, 3,691 C. parapsilosis species complex, 36 C. metapsilosis, 110 C. orthopsilosis, 1,854 C. tropicalis, 244 Saccharomyces cerevisiae, 1,409 Aspergillus fumigatus, 389 A. flavus, 130 A. nidulans, 233 A. niger, and 302 A. terreus complex isolates. SYO/Etest MICs for 282 confirmed non-wild-type (non-WT) isolates were included: ERG11 (C. albicans), ERG11 and MRR1 (C. parapsilosis), cyp51A (A. fumigatus), and CDR2 and CDR1 overexpression (C. albicans and C. glabrata, respectively). Interlaboratory modal agreement was superior by SYO for yeast species and by the Etest for Aspergillus spp. Distributions fulfilling CLSI criteria for epidemiological cutoff value (ECV) definition were pooled, and we proposed SYO ECVs for S. cerevisiae and 9 yeast and 3 Aspergillus species and Etest ECVs for 5 yeast and 4 Aspergillus species. The posaconazole SYO ECV of 0.06 µg/ml for C. albicans and the Etest itraconazole ECV of 2 µg/ml for A. fumigatus were the best predictors of non-WT isolates. These findings support the need for method-dependent ECVs, as, overall, the SYO appears to perform better for susceptibility testing of yeast species and the Etest appears to perform better for susceptibility testing of Aspergillus spp. Further evaluations should be conducted with more Candida mutants.

scholarly journals Evaluation of MIC Strip Isavuconazole Test for Susceptibility Testing of Wild-Type and Non-Wild-Type Aspergillus fumigatus Isolates

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Paul Verweij ◽  
Henrik Vedel Nielsen
Keyword(s):  
Growth Inhibition ◽  
Aspergillus Fumigatus ◽  
Susceptibility Testing ◽  
Wild Type ◽  
Content Type

ABSTRACT We evaluated the MIC Strip Isavuconazole test against EUCAST E.Def 9.3 by using 40 wild-type and 39 CYP51A mutant Aspergillus fumigatus strains. The strip full inhibition endpoint (FIE) and 80% growth inhibition endpoint were determined by two independent readers, reader 1 (R1) and R2. The essential (within ±0, ±1, and ±2 twofold dilutions) and categorical agreements were best with the FIE (for R1/R2, 42%/41%, 75%/73%, and 90%/89% for essential agreement, and 91.1%/92.4% categorical agreement, with 6.3/8.9% very major errors and 0/1.3% major errors, respectively). The MIC Strip Isavuconazole test with the FIE appears to be useful.

scholarly journals First Detection of TR46/Y121F/T289A and TR34/L98H Alterations in Aspergillus fumigatus Isolates from Azole-Naive Patients in Denmark despite Negative Findings in the Environment

2014 ◽  
Vol 58 (9) ◽  
pp. 5096-5101 ◽  
Author(s):  
K. M. T. Astvad ◽  
R. H. Jensen ◽  
T. M. Hassan ◽  
E. G. Mathiasen ◽  
G. M. Thomsen ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Transplant Patient ◽  
Susceptibility Testing ◽  
Reference Method ◽  
Ecological Niches ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Negative Findings ◽  
Microbroth Dilution

ABSTRACTAzole-resistantAspergillus fumigatusharboring the TR34/L98H or TR46/Y121F/T289A alterations is increasingly found in Europe and Asia. Here, we present the first clinical cases of TR46/Y121/T289A and three cases of TR34/L98H outside the cystic fibrosis (CF) population in Denmark and the results of environmental surveys. Four patients (2012 to 2014) with 11A. fumigatusand 4Rhizomucor pusillusisolates and 239 soil samples (spring 2010 and autumn 2013, respectively) with a total of 113A. fumigatusisolates were examined.Aspergillusisolates were screened for azole resistance using azole-containing agar. Confirmatory susceptibility testing was done using the EUCAST microbroth dilution EDEF 9.1 reference method. For relevantA. fumigatusisolates,CYP51Asequencing and microsatellite genotyping were performed. Three patients harbored TR34/L98H isolates. Two were azole naive at the time of acquisition and two were coinfected with wild-typeA. fumigatusorR. pusillusisolates, complicating and delaying diagnosis. The TR46/Y121F/T289A strain was isolated in 2014 from a lung transplant patient. Genotyping indicated that susceptible and resistantAspergillusisolates were unrelated and that no transmission between patients occurred. Azole resistance was not detected in any of the 113 soil isolates. TR34/L98H and TR46/Y121F/T289A alterations appear to be emerging in the clinical setting in Denmark and now involve azole-naive patients. Two recent soil-sampling surveys in Denmark were unable to indicate any increased prevalence of azole-resistantA. fumigatusin the environment. These findings further support the demand for real-time susceptibility testing of all clinically relevant isolates and for studies investigating the seasonal variation and ecological niches for azole-resistant environmentalA. fumigatus.

scholarly journals Pharmacodynamics of Itraconazole against Aspergillus fumigatus in anIn VitroModel of the Human Alveolus: Perspectives on the Treatment of Triazole-Resistant Infection and Utility of Airway Administration

2012 ◽  
Vol 56 (8) ◽  
pp. 4146-4153 ◽  
Author(s):  
Zaid Al-Nakeeb ◽  
Ajay Sudan ◽  
Adam R. Jeans ◽  
Lea Gregson ◽  
Joanne Goodwin ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Therapeutic Strategies ◽  
Wild Type ◽  
Content Type ◽  
Exposure Response ◽  
Prevention And Treatment ◽  
Resistant Infection ◽  
Resistant Strains ◽  
Type Strains

ABSTRACTItraconazole is used for the prevention and treatment of infections caused byAspergillus fumigatus. An understanding of the pharmacodynamics of itraconazole against wild-type and triazole-resistant strains provides a basis for innovative therapeutic strategies for treatment of infections. Anin vitromodel of the human alveolus was used to define the pharmacodynamics of itraconazole. Galactomannan was used as a biomarker. The effect of systemic and airway administration of itraconazole was assessed, as was a combination of itraconazole administered to the airway and systemically administered 5FC. Systemically administered itraconazole against the wild type induced a concentration-dependent decline in galactomannan in the alveolar and endothelial compartments. No exposure-response relationships were apparent for the L98H, M220T, or G138C mutant. The administration of itraconazole to the airway resulted in comparable exposure-response relationships to those observed with systemic therapy. This was achieved without detectable concentrations of drug within the endothelial compartment. The airway administration of itraconazole resulted in a definite but submaximal effect in the endothelial compartment against the L98H mutant. The administration of 5FC resulted in a concentration-dependent decline in galactomannan in both the alveolar and endothelial compartments. The combination of airway administration of itraconazole and systemically administered 5FC was additive. Systemic administration of itraconazole is ineffective against Cyp51 mutants. The airway administration of itraconazole is effective for the treatment of wild-type strains and appears to have some activity against the L98H mutants. Combination with other agents, such as 5FC, may enable the attainment of near-maximal antifungal activity.

scholarly journals Epidemiological cutoff values for a 96-well broth microdilution plate for high-throughput research antibiotic susceptibility testing of M. tuberculosis

2021 ◽  
Author(s):  
◽  
Philip W Fowler
Keyword(s):  
Genetic Variants ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Broth Microdilution ◽  
Wild Type ◽  
Genetic Sequencing ◽  
Indicator Tube ◽  
Cutoff Values ◽  
Type Population

AbstractDrug susceptibility testing of M. tuberculosis is rooted in a binary susceptible/resistant paradigm. There are considerable advantages in measuring the minimum inhibitory concentrations (MICs) of a panel of drugs for an isolate, including quantifying the magnitude of effect conferred by genetic variants and being able to identify isolates with elevated MICs that can still be treated with standard therapy. It is necessary, however, to measure the epidemiological cutoff values (ECOFF/ECVs) to permit comparison with qualitative data. Here we present ECOFF/ECVs for 13 anti-TB compounds, including bedaquiline and delamanid, derived from 20,637 clinical isolates collected by 14 laboratories based in 11 countries on five continents. Each isolate was incubated for 14 days on a dry 96-well broth microdilution plate and then read. Resistance to the majority of the drugs due to prior exposure is expected and the MIC distributions for many of the compounds are complex and therefore a phenotypically wild-type population could not be defined. Since a majority of samples also underwent genetic sequencing, we defined a genotypically wild-type population and measured the MIC of the 99th percentile by direct measurement and via fitting a Gaussian using interval regression. The proposed ECOFF/ECV values were then validated by comparing to the MIC distributions of high-confidence genetic variants that confer resistance and to qualitative drug susceptibility tests obtained via Mycobacterial Growth Indicator Tube and the Microscopic-Observation Drug-Susceptibility assay.

scholarly journals ThemtfATranscription Factor Gene Controls Morphogenesis, Gliotoxin Production, and Virulence in the Opportunistic Human Pathogen Aspergillus fumigatus

2014 ◽  
Vol 13 (6) ◽  
pp. 766-775 ◽  
Author(s):  
Timothy D. Smith ◽  
Ana M. Calvo
Keyword(s):  
Aspergillus Fumigatus ◽  
Protease Activity ◽  
Galleria Mellonella ◽  
Molecular Genetic ◽  
Colony Growth ◽  
Infection Model ◽  
Slight Reduction ◽  
Wild Type ◽  
Content Type ◽  
Factor Gene

ABSTRACTAspergillus fumigatusis the leading causative agent of invasive aspergillosis (IA). The number of cases is on the rise, with mortality rates as high as 90% among immunocompromised patients. Molecular genetic studies inA. fumigatuscould provide novel targets to potentially set the basis for antifungal therapies. In the current study, we investigated the role of the transcription factor genemtfAinA. fumigatus. Our results revealed thatmtfAplays a role in the growth and development of the fungus. Deletion or overexpression ofmtfAleads to a slight reduction in colony growth, as well as a reduction in conidiation levels, in the overexpression strain compared to the wild-type strain. Furthermore, production of the secondary metabolite gliotoxin increased whenmtfAwas overexpressed, coinciding with an increase in the transcription levels of the gliotoxin genesgliZandgliPwith respect to the wild type. In addition, our study showed thatmtfAis also necessary for normal protease activity inA. fumigatus; deletion ofmtfAresulted in a reduction of protease activity compared to wild-type levels. Importantly, the absence ofmtfAcaused a decrease in virulence in theGalleria mellonellainfection model, indicating thatmtfAis necessary forA. fumigatuswild-type pathogenesis.

scholarly journals EUCAST Determination of Olorofim (F901318) Susceptibility of Mold Species, Method Validation, and MICs

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Karin Meinike Jørgensen ◽  
Karen M. T. Astvad ◽  
Rasmus Krøger Hare ◽  
Maiken Cavling Arendrup
Keyword(s):  
Fusarium Solani ◽  
Susceptibility Testing ◽  
Wild Type ◽  
Preparation Methods ◽  
Content Type ◽  
Microtiter Plates ◽  
Upper Limits ◽  
Limited Variation

ABSTRACT Olorofim is a novel antifungal agent with in vitro activity against Aspergillus and some other molds. Here, we addressed technical aspects for EUCAST olorofim testing and generated contemporary MIC data. EUCAST E.Def 9.3.1 testing was performed comparing two plate preparation methods (serial dilution in medium [serial plates] versus predilution in DMSO [ISO plates]), two lots of olorofim, visual (visual-MIC) versus spectrophotometer (spec-MIC) reading, and four polystyrene plates using 34 to 53 Aspergillus isolates from five genera. Subsequently, olorofim MICs were compared to itraconazole, voriconazole, posaconazole, and amphotericin B MICs for 298 clinical mold isolates (2016 to 2017). Wild-type upper limits (WT-UL) were determined following EUCAST principles for epidemiologic cutoff value (ECOFF) setting. Olorofim median MICs comparing serial plates and ISO plates were identical (25/36 [69%]) or one dilution apart (11/36 [31%]). Interperson agreement for visual-MICs was 92% to 94%/100% for ≤1/≤2 dilutions, respectively. The visual-MIC values across tested microtiter plates and olorofim lots revealed only discrete differences (≤1 dilution lower for treated plates). No single spec-MIC criterion was applicable to all species. Olorofim MICs were low against 275 Aspergillus species isolates (modal MIC, 0.06 mg/liter; MIC range, < 0.004 to 0.25 mg/liter) and three dermatophytes (MICs 0.03 to 0.06 mg/liter). MICs against Fusarium were diverse, with full inhibition of F. proliferatum (MIC, 0.016), 50% growth inhibition of Fusarium solani at 1 to 2 mg/liter, and no inhibition of F. dimerum. Olorofim displayed potent in vitro activity against most mold isolates and was associated with limited variation in EUCAST susceptibility testing.

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 Substitutions at Position 105 in SHV Family β-Lactamases Decrease Catalytic Efficiency and Cause Inhibitor Resistance

2012 ◽  
Vol 56 (11) ◽  
pp. 5678-5686 ◽  
Author(s):  
Mei Li ◽  
Benjamin C. Conklin ◽  
Magdalena A. Taracila ◽  
Rebecca A. Hutton ◽  
Marion J. Skalweit
Keyword(s):  
Clavulanic Acid ◽  
Susceptibility Testing ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
Oxyanion Hole ◽  
Content Type ◽  
Class A ◽  
Inhibitor Resistance ◽  
Structural Probes ◽  
Variant Residue

ABSTRACTAmbler position 105 in class A β-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine β-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed thatEscherichia coliDH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all β-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, ≤2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increasedKm. Clavulanic acid and penemKivalues were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.

scholarly journals Implications of the EUCAST Trailing Phenomenon inCandida tropicalisfor theIn VivoSusceptibility in Invertebrate and Murine Models

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
K. M. T. Astvad ◽  
D. Sanglard ◽  
E. Delarze ◽  
R. K. Hare ◽  
M. C. Arendrup
Keyword(s):  
Susceptibility Testing ◽  
Galleria Mellonella ◽  
Growth Control ◽  
Resistant Isolate ◽  
Murine Models ◽  
Wild Type ◽  
Content Type ◽  
Gradual Loss

ABSTRACTCandida tropicalisisolates often display reduced but persistent growth (trailing) over a broad fluconazole concentration range during EUCAST susceptibility testing. Whereas weak trailing (<25% of the positive growth control) is common and found not to impair fluconazole efficacy, we investigated if more pronounced trailing impacted treatment efficacy. Fluconazole efficacy against two weakly (≤25% growth), two moderately (26% to 50% growth), and one heavily (>70% growth) trailing resistant isolate and one resistant (100% growth) isolate were investigatedin vitroandin vivo(in aGalleria mellonellasurvival model and two nonlethal murine models).CDR1expression levels andERG11sequences were characterized. The survival in fluconazole-treatedG. mellonellawas inversely correlated with the degree of trailing (71% to 9% survival in treatment groups). In mice, resistant and heavily trailing isolates responded poorly to fluconazole treatment.CDR1expression was significantly higher in trailing and resistant isolates than in wild-type isolates (1.4-fold to 10-fold higher). All isolates exhibitedERG11wild-type alleles. Heavily trailing isolates were less responsive to fluconazole in allin vivomodels, indicating an impact on fluconazole efficacy.CDR1upregulation may have contributed to the observed differences. Moderately trailing isolates responded less well to fluconazole in larvae only. This confirms clinical data suggesting fluconazole is effective against infections with such isolates in less severely ill patients and supports the current 50% growth endpoint for susceptibility testing. However, it is still unclear if the gradual loss of efficacy observed for moderately trailing isolates in the larva model may be a reason for concern in selected vulnerable patient populations.

Sign in / Sign up

Export Citation Format

Share Document