Abstract
In this study, the first such study in Greece, we used polyphasic identification combined with antifungal susceptibility study to analyze Aspergillus clinical isolates comprising 102 common and rare members of sections Fumigati, Flavi, Terrei, Nidulantes, Nigri, Circumdati, Versicolores, and Usti. High amphotericin B MICs (>2 μg/ml) were found for 17.6% of strains. Itraconazole, posaconazole, and voriconazole MICs of >4 μg/ml were shown in 1%, 5%, and 0% of the isolates, respectively. Anidulafungin, micafungin, and caspofungin minimum effective concentrations (MECs) of ≥2 μg/ml were correspondingly recorded for 4%, 9%, and 33%, respectively, of the strains.
TEXT
Current treatment guidelines for invasive aspergillosis recommend voriconazole as primary therapy assigning alternative therapeutic roles to amphotericin B, caspofungin, itraconazole, micafungin, and posaconazole (16, 27). Aspergillus species display variable antifungal susceptibility (4, 5, 24). Consequently, accurate identification of clinical Aspergillus isolates, supported by comparative sequence identification methods and ongoing Aspergillus taxonomy revision (5, 18, 23), sponsor meticulous associations between species and antifungal susceptibility.
We report antifungal susceptibilities of clinical Aspergillus isolates, collected during 1998 to 2009, using CLSI M38-A2 guidelines (7). All isolates were polyphasically identified, the first Aspergillus occurrence and susceptibility report from this part of Greece (Athens, Thessaloniki, and Larissa).
(Part of this study was presented at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 2009.)
Strains.
We studied 102 Aspergillus isolates from bronchial secretion, bronchoalveolar lavage, peritoneal fluid, lung, liver, skin, maxillary bone nail, and nasal biopsy specimens from 102 consecutive immunocompromised patients with confirmed aspergilloses who were admitted to tertiary health care centers located in the cities of Athens, Thessaloniki, and Larissa in Greece. The isolates were stored at −80°C in the Hellenic Collection of Pathogenic Fungi UOA929 (a member of the World Data Centre for Microorganisms) until tested.
Polyphasic identification.
Morphology was studied in malt extract and Czapeck agars, at 25°C followed by physiological studies (22, 23). Isolates were identified by ribosomal internal transcribed spacer (ITS) sequencing as described previously (28). Beta-tubulin was sequenced when ITS sequences were inadequate for definitive identification (11). Sequences were compared (http://www.ncbi.nlm.nih.gov/BLAST) and deposited in GenBank (EU714322 and see below). Identification was concluded when there was ≥99% sequence homology. The ITS and beta-tubulin sequences were each aligned (ClustalX) (25), and phylogenetic relations were derived via the neighbor-joining method and the MEGA v.4 software (14). GenBank homolog sequences were used as reference (10, 23, 29).
Antifungal susceptibility testing.
Pure amphotericin B (AMB), itraconazole (ITZ), posaconazole (POS), voriconazole (VOR), anidulafungin (AND), caspofungin (CAS), and micafungin (MCF) compounds were tested at concentrations from 0.032 to 64 μg/ml. The CLSI M38-A2 guideline was employed for recording MICs for amphotericin B and the azoles and minimum effective concentrations (MECs) for the candins (7). Testing was repeated at three independent occasions, using different batches of stock drug solutions. Reference strains, per the CLSI M38-A2 guideline, were used at each independent trial.
Statistical analysis.
Repeated-measure analysis of variance (ANOVA) (2) was employed on transformed data followed by post hoc pairwise comparisons with Bonferroni's corrections for the four most common Aspergillus isolates A. fumigatus, A. flavus, A. terreus, and Emericella nidulans.
Clinical Aspergillus isolates in our region belonged to eight sections (Table 1), encompassing the rare species A. aculeatus, A. calidoustus, A. fumigatiaffinis, A. sclerotiorum, A. tubingensis, Emericella variecolor, Emericella unguis, and Neosartorya hiratsukae. Currently, ITS sequencing is recommended for Aspergillus molecular identification to the subgenus/section level and beta-tubulin sequencing to the species level (5, 6). We used ITS sequencing for all isolates and beta-tubulin only where ITS was inadequate for species identification (A. aculeatus, A. calidoustus, A. fumigatiaffinis, A. niger, A. tubingensis, E. nidulans, E. variecolor, and N. hiratsukae). This strategy evolved from (i) the general clinical laboratory use of ITS as the main target for identification of fungi, (ii) the adequacy of ITS sequencing for identifying common A. fumigatus, A. flavus, and A. terreus clinical isolates, and (iii) the ad hoc use of minimum targets. Besides, beta-tubulin may not be an orthologous gene in all Aspergillus sections (18). Therefore, other protein-coding genes, either cadmodulin or rodlet A, may be finally selected as preferred single target for Aspergillus identification (18, 23).
Table 1.
Polyphasic identification, MICs and MECs, and distributions for 50% and 90% of 102 tested Aspergillus isolatesa
| Aspergillus section (no. of isolates) | Aspergillus or Emericella species (no. of isolates)b | Antifungal agentc | MIC or MECd (μg/ml) range | GM MIC or MECd (μg/ml) (95% CI)e | MIC50/MIC90 (μg/ml) | No. of strains inhibited by the following MIC or MECd(μg/ml) value: |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.032 | 0.064 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | ||||||
| Fumigati (42) | A. fumigatiaffinis (3) | AMB | 1–4 | 0.04 | 1 | 2 | ||||||||||
| ITZ | 0.5 | 0.5 | 3 | |||||||||||||
| POS | 0.064–0.5 | 0.2 | 1 | 1 | 1 | |||||||||||
| VOR | 0.25–1 | 0.5 | 1 | 1 | 1 | |||||||||||
| AND | 0.5–1 | 0.63 | 2 | 1 | ||||||||||||
| CAS | 0.125–0.5 | 0.31 | 1 | 2 | ||||||||||||
| MCF | 0.5 | 3 | ||||||||||||||
| A. fumigatus (37) | AMB | 0.016–16 | 0.28 (0.19–0.41) | 0.25/1 | 2 | 2 | 9 | 11 | 8 | 3 | 1 | 1 | ||||
| ITZ | 0.032–32 | 0.52 (0.35–0.77) | 0.5/2 | 2 | 3 | 5 | 15 | 7 | 4 | 1 | ||||||
| POS | 0.032–32 | 0.34 | 0.25/1 | 2 | 1 | 9 | 13 | 6 | 2 | 1 | 3 | |||||
| VOR | 0.064–1 | 0.28 | 0.25/0.5 | 2 | 5 | 19 | 7 | 4 | ||||||||
| AND | 0.032–2 | 0.16 | 0.125/0.5 | 10 | 1 | 9 | 6 | 7 | 2 | 2 | ||||||
| CAS | 0.032–32 | 0.74 | 0.25–32 | 1 | 1 | 5 | 13 | 7 | 1 | 4 | 5 | |||||
| MCF | 0.032–1 | 0.26 (0.20–0.35) | 0.5/0.5 | 2 | 1 | 10 | 4 | 19 | 1 | |||||||
| N. hiratsukae (2) | AMB | 1 | 1 | 2 | ||||||||||||
| ITZ | 0.125–2 | 0.5 | 1 | 1 | ||||||||||||
| POS | 0.25 | 0.25 | 2 | |||||||||||||
| VOR | 0.032–1 | 0.18 | 1 | 1 | ||||||||||||
| AND | 0.125–0.25 | 0.18 | 1 | 1 | ||||||||||||
| CAS | 0.5 | 0.5 | 2 | |||||||||||||
| MCF | 2 | 2 | 2 | |||||||||||||
| Flavi (23) | A. flavus (23) | AMB | 0.016–16 | 0.94 (0.44–2.03) | 1/16 | 2 | 2 | 7 | 5 | 2 | 2 | 3 | ||||
| ITZ | 0.064–1 | 0.31 (0.23–0.41) | 0.5/0.5 | 1 | 4 | 6 | 11 | 1 | ||||||||
| POS | 0.032–1 | 0.26 | 0.25/0.5 | 1 | 1 | 5 | 7 | 7 | 2 | |||||||
| VOR | 0.125–1 | 0.28 | 0.25/0.5 | 6 | 8 | 8 | 1 | |||||||||
| AND | 0.032–2 | 0.28 | 0.25/1 | 4 | 1 | 1 | 5 | 7 | 4 | 1 | ||||||
| CAS | 0.032–32 | 1.20 | 0.5/32 | 2 | 1 | 3 | 7 | 2 | 2 | 1 | 5 | |||||
| MCF | 0.032–32 | 0.62 (0.32–1.19) | 0.5/2 | 2 | 1 | 3 | 6 | 6 | 3 | 1 | 1 | |||||
| Terrei (12) | A. terreus (12) | AMB | 0.25–4 | 1.41 (0.75–2.67) | 2/4 | 1 | 3 | 1 | 3 | 4 | ||||||
| ITZ | 0.064–1 | 0.22 (0.14–0.35) | 0.25/0.5 | 1 | 3 | 6 | 1 | 1 | ||||||||
| POS | 0.032–4 | 0.34 | 0.5/1 | 2 | 2 | 5 | 2 | 1 | ||||||||
| VOR | 0.064–4 | 0.38 | 0.5/1 | 1 | 3 | 1 | 4 | 2 | 1 | |||||||
| AND | 0.032–0.5 | 0.28 | 0.5/0.5 | 2 | 2 | 8 | ||||||||||
| CAS | 0.032–0.5 | 4.01 | 4–32 | 1 | 2 | 1 | 1 | 1 | 1 | 5 | ||||||
| MCF | 0.125–1 | 0.42 (0.29–0.62) | 0.5/1 | 1 | 3 | 6 | 2 | |||||||||
| Nidulantes (11) | E. nidulans (9) | AMB | 0.25–4 | 1.47 (0.72–2.99) | 2/4 | 1 | 1 | 1 | 4 | 2 | ||||||
| ITZ | 0.125–2 | 0.29 (0.15–0.58) | 0.25/0.5 | 3 | 3 | 2 | 1 | |||||||||
| POS | 0.032–4 | 0.25 | 0.5/2 | 2 | 1 | 2 | 1 | 1 | 1 | 1 | ||||||
| VOR | 0.032–4 | 0.19 | 0.25/1 | 3 | 1 | 2 | 1 | 1 | 1 | |||||||
| AND | 0.125–0.5 | 0.37 | 0.5/0.5 | 1 | 2 | 6 | ||||||||||
| CAS | 0.25–32 | 1.08 | 0.5/32 | 2 | 5 | 2 | ||||||||||
| MCF | 0.5 | 0.5 (0.50–0.50) | 0.5/0.5 | 9 | ||||||||||||
| E. unguis (1) | AMB | 0.125 | 1 | |||||||||||||
| ITZ | 0.25 | 1 | ||||||||||||||
| POS | 2 | 1 | ||||||||||||||
| VOR | 0.125 | 1 | ||||||||||||||
| AND | 0.125 | 1 | ||||||||||||||
| CAS | 0.25 | 1 | ||||||||||||||
| MCF | 0.5 | 1 | ||||||||||||||
| E. variecolor (1) | AMB | 2 | 1 | |||||||||||||
| ITZ | 0.25 | 1 | ||||||||||||||
| POS | 0.5 | 1 | ||||||||||||||
| VOR | 1 | 1 | ||||||||||||||
| AND | 0.5 | 1 | ||||||||||||||
| CAS | 2 | 1 | ||||||||||||||
| MCF | 0.5 | 1 | ||||||||||||||
| Nigri (6) | A. aculeatus (1) | AMB | 0.125 | 1 | ||||||||||||
| ITZ | 0.125 | 1 | ||||||||||||||
| POS | 0.125 | 1 | ||||||||||||||
| VOR | 0.064 | 1 | ||||||||||||||
| AND | 0.064 | 1 | ||||||||||||||
| CAS | 0.5 | 1 | ||||||||||||||
| MCF | 1 | 1 | ||||||||||||||
| A. niger (4) | AMB | 0.125–16 | 2.83 | 1 | 2 | 1 | ||||||||||
| ITZ | 0.125–0.5 | 0.42 | 1 | 3 | ||||||||||||
| POS | 0.125–32 | 3.36 | 1 | 2 | 1 | |||||||||||
| VOR | 0.064–0.5 | 0.21 | 1 | 2 | 1 | |||||||||||
| AND | 0.064–0.5 | 0.21 | 1 | 2 | 1 | |||||||||||
| CAS | 0.032–0.5 | 0.25 | 1 | 3 | ||||||||||||
| MCF | 0.25–1 | 0.35 | 2 | 2 | ||||||||||||
| A. tubingensis (1) | AMB | 0.064 | 1 | |||||||||||||
| ITZ | 0.25 | 1 | ||||||||||||||
| POS | 0.064 | 1 | ||||||||||||||
| VOR | 0.125 | 1 | ||||||||||||||
| AND | 0.5 | 1 | ||||||||||||||
| CAS | 0.5 | 1 | ||||||||||||||
| MCF | 0.5 | 1 | ||||||||||||||
| Circumdati (3) | A. ochraceus (2) | AMB | 1 | 1 | 2 | |||||||||||
| ITZ | 0.5–1 | 0.71 | 1 | 1 | ||||||||||||
| POS | 1–2 | 1.41 | 1 | 1 | ||||||||||||
| VOR | 0.125–0.5 | 0.25 | 1 | 1 | ||||||||||||
| AND | 0.25–0.5 | 0.35 | 1 | 1 | ||||||||||||
| CAS | 2–32 | 8 | 1 | 1 | ||||||||||||
| MCF | 1 | 1 | 2 | |||||||||||||
| A. sclerotiorum (1) | AMB | 2 | 1 | |||||||||||||
| ITZ | 0.25 | 1 | ||||||||||||||
| POS | 2 | 1 | ||||||||||||||
| VOR | 1 | 1 | ||||||||||||||
| AND | 1 | 1 | ||||||||||||||
| CAS | 32 | 1 | ||||||||||||||
| MCF | 0.5 | 1 | ||||||||||||||
| Versicolores (3) | A. versicolor (3) | AMB | 1–2 | 1.26 | 2 | 1 | ||||||||||
| ITZ | 0.25–0.5 | 0.40 | 1 | 2 | ||||||||||||
| POS | 0.125–1 | 0.25 | 2 | 1 | ||||||||||||
| VOR | 0.25–0.5 | 0.40 | 1 | 2 | ||||||||||||
| AND | 0.064–0.25 | 0.16 | 1 | 2 | ||||||||||||
| CAS | 0.5–4 | 1.00 | 2 | 1 | ||||||||||||
| MCF | 0.5–2 | 0.79 | 2 | 1 | ||||||||||||
| Usti (2) | A. calidoustus (2) | AMB | 0.064–2 | 0.12 | 1 | 1 | ||||||||||
| ITZ | 0.25–0.5 | 0.40 | 1 | 1 | ||||||||||||
| POS | 0.5–32 | 4 | 1 | 1 | ||||||||||||
| VOR | 1–2 | 1.59 | 1 | 1 | ||||||||||||
| AND | 0.032–4 | 0.40 | 1 | 1 | ||||||||||||
| CAS | 0.5–32 | 6.35 | 1 | 1 | ||||||||||||
| MCF | 0.5–32 | 2 | 1 | 1 | ||||||||||||
Polyphasic identification, MIC and MEC ranges, geometric means and distributions for 50% (MIC50 and MEC50) and 90% (MIC90 and MEC90) MICs and MECs of 102 tested Aspergillus isolates are shown.
If there were less than 2 strains tested, the geometric mean is not reported. If there were less than 5 strains tested, MIC50 and MIC90 values are not reported.
Antifungal agent abbreviations: AMB, amphotericin B; ITZ, itraconazole; POS, posaconazole; VOR, voriconazole; AND, anidulafungin; CAS, caspofungin; MCF, micafungin.
MICs are shown for amphotericin B, itraconazole, posaconazole, and voriconazole. Minimum effective concentrations (MECs) are shown for anidulafungin, caspofungin, and micafungin.
Abbreviations: GM, geometric mean; 95% CI, 95% confidence intervals corresponding to antifungals with differences determined by ANOVA.
Regarding susceptibility to antifungals (Table 1), in total 18 strains (17.6%), comprising 5 of 23 A. flavus isolates (21.7%), 4 of 37 A. fumigatus isolates (10.8%), 3 of 4 A. niger isolates (75%), 4 of 12 A. terreus isolates (33.3%), and 2 of 9 E. nidulans isolates (22.2%), gave AMB MICs of >2 μg/ml. Such high AMB MICs are previously associated with treatment failure (15). Consequently, the relatively high MICs of AMB reported in this study, among the highest reported (8), necessitate routine AMB susceptibility testing in our region. Additionally, ANOVA demonstrated that AMB MICs for A. fumigatus were notably lower (P < 0.01) than those for A. flavus, A. terreus, and E. nidulans (Table 2). This is consistent with previous reports where A. fumigatus is generally susceptible to AMB and A. terreus is resistant (24), whereas A. flavus usually registers as susceptible (8).
Table 2.
Statistically significant differences among four species for three antifungal agentsa
| Aspergillus species (no. of isolates) | Statistically significant difference for the MICs of isolates for the following antifungalb: |
||
|---|---|---|---|
| AMB | ITZ | MCF | |
| A. fumigatus (37) | A | A | A |
| A. flavus (23) | B | AB | B |
| A. terreus (12) | B | B | AB |
| E. nidulans (9) | B | AB | AB |
Statistically significant differences among A. flavus, A. fumigatus, A. terreus, and E. nidulans for the antifungals tested were found by ANOVA only for amphotericin B (AMB), itraconazole (ITZ), and micafungin (MCF). A. fumigatus appears significantly more resistant to amphotericin B than the other species, more resistant to itraconazole than A. terreus, and more resistant to micafungin than A. flavus.
Statistically significant differences for MICs by ANOVA are indicated by different letters. The presence of a different letter (A or B) indicates a significant difference. AB is not significantly different from A or B. For amphotericin B (AMB), ANOVA defined groups with degree of freedoms df3,77 = 8.0 and P < 0.01. For itraconazole (ITZ), ANOVA defined groups with df3,77 = 3.00 and P < 0.05. For micafungin (MCF), ANOVA defined groups with df3,77 = 3.55 and P < 0.05.
All isolates demonstrated VOR MICs of ≤4 μg/ml (Table 1). Consistently low VOR MICs, though similar to ones reported from Spain (12), differ from those reported from the United States where 4.1% of the tested Aspergillus isolates had MICs of >4 μg/ml (3), possibly suggesting geographic heterogeneity in resistance trends. Among all tested isolates, only one A. fumigatus isolate had an ITZ MIC of >4 μg/ml (1%), despite previously reported (13, 17) ITZ resistance (MICs ≥ 4 μg/ml). In this study, ITZ MICs were significantly higher among A. fumigatus isolates than among A. terreus isolates (Table 2). Previously reported A. fumigatus ITZ resistance rates ranged from 2.1% to 7%, while a rate of 2.7% was also detected in the present study. Last, five strains (4.9%) had POS MICs of >4 μg/ml, including 1 of 2 A. calidoustus isolates, 3 of 37 A. fumigatus (8.1%) isolates, and 1 of 4 A. niger isolates. As is the case with other azoles and in the absence of consensus breakpoints, these somewhat increased rates of high POS MICs against Aspergillus are weighed against the relevant host determinants, thus making unclear their clinical significance.
Recently, species-dependent epidemiological cutoff values (ECVs) that may assist in azole resistance detection have been proposed for Aspergillus wild-type isolates (9, 19, 26). Among the four most common tested species, the percentages of the non-wild-type isolates with MIC > ECV for ITZ, POS, and VOR, respectively, were as follows: 13.5, 16, and 0 for A. fumigatus; 0, 8.6, and 0 for A. flavus; 0, 25, and 8.3 for A. terreus; and 0, 11, and 11 for E. nidulans. In accord with our findings, lower A. fumigatus susceptibility to posaconazole than to itraconazole has recently been recorded (21). This was attributed to either setting A. fumigatus posaconazole ECV too low or, alternatively, to the existence of a yet unidentified acquired resistance mechanism (21).
Echinocandins showed variable MECs, with AND exhibiting the lowest mean values (Table 1). Only 4% of the isolates displayed AND MECs of ≥2 μg/ml, and 9% of the isolates displayed MCF MECs of ≥2 μg/ml. In contrast to previous observations (20), 33% of our isolates had high CAS MECs, while nongerminated conidia of the few Aspergillus strains tested before (1) had lower anidulafungin and highest caspofungin MECs. Notably, CAS has been in the local hospital pharmacies since 2006, while AND and MCF were introduced in 2008. Consequently, the great majority of tested isolates originated from hospitals where mostly CAS was used. Also, isolates tested from 2008 to 2009 that were from patients with aspergillosis originated from pediatric hospitals where anidulafungin and micafungin were not indicated for use at the time. Nonetheless, echinocandin susceptibility appears patchy, depending on the species/sections (Table 1). This is also highlighted in a recent survey where more than 99% of all isolates were inhibited by <0.06 μg/ml of these three agents tested (20). Interestingly, ANOVA analysis comparing echinocandin MECs for the four most common species determined that the only statistically significant difference (P < 0.05) was the higher A. flavus micafungin MECs, in comparison to those of A. fumigatus (Table 2).
In conclusion, the rare Aspergillus species A. aculeatus, A. calidoustus, A. fumigatiaffinis, A. sclerotiorum, A. tubingensis, E. variecolor, E. unguis, and N. hiratsukae are etiological agents of aspergilloses in Greece. Voriconazole and itraconazole showed the best overall in vitro performance against our isolates. Notwithstanding the professed susceptibility to these two agents, confirmed erratically high MICs or MECs recorded for all antifungals may warrant routine susceptibility testing of Aspergillus isolates from immunocompromised patients in this part of Greece (Athens, Thessaloniki, and Larissa) and justify the ECV determinations of indigenous wild-type strains that is under way.
Nucleotide sequence accession numbers.
The nucleotide sequences were deposited in GenBank under accession numbers FJ878624 to FJ878717, GQ376120 to GQ376143, and GQ461899 to GQ461912.
Acknowledgments
We thank Afroditi Milioni, Stavroula Kritikou, and Eirini Ilia for technical assistance.
This work was supported by SARG K.A. 70/4/5905 and 70/3/6915, the National and Kapodistrian University of Athens, the Antigoni Arseni Foundation, the Bodosakis Foundation, and by an unrestricted Pfizer research grant.
Footnotes
Published ahead of print on 28 March 2011.
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