Influence of hypoxia on antifungal susceptibility, sterol pattern and biomarker release of Aspergillus spp.

Ref ID: 19376

Author:

U. Binder,1 E. Maurer,1 C. Mueller,2 F. Bracher2 and C. Lass-Floerl1

Author address:

1Medical University Innsbruck, Innsbruck, Austria and
2Department of Pharmacy, Ludwig Maximilians University
Munich, Germany

Full conference title:

6th Trends in Medical Mycology 2013

Date: 11 October 2014

Abstract:

Objectives Invasive aspergillosis (IA) is a major life-threatening dis-
ease in immunocompromised patients, with mortality rates from 40%
up to 90% in high-risk populations. The most common species caus-
ing aspergillosis is Aspergillus (A.) fumigatus, accounting for approxi-
mately 90% of infections. Depending on regional distinctions, A.
flavus and A. terreus are frequently reported. During infection, fungal
pathogens must adapt to microenvironmental stresses, including
hypoxia as well as high CO2 levels. Such oxystress conditions are
usually not taken into account in current in vitro models of infection,
the assessment of antifungal sensitivities or the release of biomarkers
used for diagnosis.
Methods Using Etest strips,the in vitro activity of amphotericin B
(amB), different azoles and echinocandins in hypoxic conditions (1%
O2, 5% CO2) to their activity in normoxic conditions against asper-
gilli. For evaluation of biomarker release, the amount of b-1,3 glucan
(BG) and galactomannan (GM) in Aspergillus supernatants was deter-
mined by commercially available detection kits (Platelia/Fungitell).
Changes in the sterol pattern or the amount of ergosterol was
evaluated by GC-MS.
Results On surface cultures, we found a reduction of the minimal
inhibitory concentration (MIC) for amB for all aspergilli in hypoxic
conditions. Similarly, a significant reduction in the MIC for all tested
azoles was demonstrated for A. terreus isolates, while for A. fumigatus
isolates differences were less pronounced. For echinocandins, little or
no change in the MEC (minimal effective concentration) was detected
between hypoxic and normoxic conditions for all aspergilli. Most
interestingly, A. terreus strains, that are resistant to amB in normox-
ia, exhibited sensitivity to amB in hypoxic conditions, defining a
breakpoint of > 2 lg/ml. Notably, for none of the strains tested, MIC/
MEC values increased in hypoxia. Our results so far indicate, that
there is no significant difference in the amount of ergosterol whether
mycelia is grown in hypoxia or normoxia.
The detection of circulating fungal antigens in serumfor Aspergillus
galactomannanor b-D-glucan has become an accepted diagnostic
strategy. However, sensitivity and specificity vary extremely and the
reasons are only partially clear; therefore, we are currently checking
whether hypoxia influences the physiological kinetics of GM and b-
glucan release.
Conclusion ECOFFs in hypoxia differ from those in normoxia for
antifungal drugs targeting ergosterol or its biosynthesis. Supplemen-
tation of test media with blood or ergosterol abrogated the reduction
of the MIC, but there is no direct link between ergosterol content and
increased susceptibility to amB and azoles, but further analysis of ste-
rol intermediates needs to be done in more detail.
P015
2005-2012 prevalence of triazole-resistant Aspergillus
fumigatus isolates in a cohort of patients with cystic
fibrosis from Haute-Normandie, France
L. Favennec,1 F. Verhaeghe,1 F. Morio,2 G. Gargala1 and
P. Lepape2
1University of Rouen, France and 2Laboratoire de Parasitologie-
Mycologie, EA1155 IICiMedCHU de Nantes, France
Objectives The aim of this work was to evaluate retrospectively the
prevalence of azole-resistance in Aspergillus fumigatus from a large
cohort of patients with cystic fibrosis..
Methods Aspergillus fumigatus isolates obtained from 334 sputa and
bronchial aspirations of 97 cystic fibrosis (CF) patients (mean
age = 22 years, range 4-58) attending the Rouen University Hospi-
tal from December 2005 to June 2012 were cryopreserved. After
thawing, 326 isolates (93 patients) were grown and plated on Sab-
ouraud’s agar medium containing 4 mg/ml itraconazole (ITZ). Mini-
mal Inhibitory Concentrations (MICs) to itraconazole were
determined according to the EUCAST standardized methodology. Spe-
cies identification of A. fumigatus isolates was obtained by sequencing
of the b-tubulin gene. Sequencing analysis of the CYP51A gene and
its promoter region was performed.
Results 6/326 (1.84%) isolates identified as Aspergillus fumigatus
species obtained from 6/93 (6.4%) patients were resistant to itraco-
nazole (MICs >2 mg/ml) (Table). Three out of 6 isolates (3 patients)
obtained since 2011 displayed the TR34/L98H mutation that suggest
acquisition through environmental exposure of de novo azole-resis-
tant isolate in these patients. Mutations at residue 248 have been
previously described although their involvment in azole resistance
remains to be established. Strikingly, a non-CYP51 mediated azole
resistance mechanism was evidenced for two patients (Pt1 and 5) as
one isolate had the wild-type sequence whereas the remaining exhib-
ited mutations previously reported in azole-susceptible isolates
(M172V, E427K).
Table Characteristics of the six itraconazole-resistant Aspergillus fu-
migatus isolates obtained for patients with cystic fibrosis
Conclusion In the present study, we report a 6.4% prevalence of itr-
aconazole-resistant Aspergillus fumigatus mostly driven by TR34/L98H
In CF patients from the Haute-Normandie. This large cattle breeding
area also showed some agricultural treatments based on azole fungi-
cides as prothioconazole, fluzilazole, epoxiconazole and tebuconazole.
Finally these results underline the need for routine azole susceptibil-
ity testing. Additional studies are ongoing to determine the molecular
mechanisms leading to azole resistance in some of these isolates.

Abstract Number: NULL

Conference Year: 2013

Link to conference website: NULL

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