1 Polyphasic identification and susceptibility to seven antifungals of 102 1 Aspergillus isolates

17 Pages·2011·0.32 MB·English

AAC Accepts, published online ahead of print on 28 March 2011
Antimicrob. Agents Chemother. doi:10.1128/AAC.01491-10
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Polyphasic identification and susceptibility to seven antifungals of 102
2 Aspergillus isolates recovered from immunocompromised hosts in Greece.
3
4 Michael Arabatzis,1* Manousos Kambouris,2 Miltiades Kyprianou,1 Aikaterini
5 Chrysaki,3 Maria Foustoukou,4 Maria Kanellopoulou,5 Lydia Kondyli,6 Georgia
6 Kouppari,7 Chrysa Koutsia-Karouzou,8 Evangelia Lebessi,4 Anastasia Pangalis,3
D
7 Efthimia Petinaki,9 Ageliki Stathi,3 Eleftheria Trikka-Graphakos,10 Erriketi Vartzioti,6 o
w
n
8 Aliki Vogiatzi,11 Timoleon-Achilleas Vyzantiadis,12 Loukia Zerva,13 Aristea Velegraki1 lo
a
d
e
9 d

f
r
10 1Mycology Laboratory, Department of Microbiology, Medical School, National and om

h
11 Kapodistrian University of Athens, Athens, Greece tt
p
:
/
12 2Department of Medical Laboratories, Faculty of Health and Caring Professions, /a
a
c
.
13 Technological Educational Institute of Athens, Athens, Greece a
s
m
14 3Microbiology Laboratory, Aghia Sophia Children’s Hospital, Athens, Greece .o
r
g
/
15 4Microbiology Laboratory, Aglaia Kyriakou Children’s Hospital, Athens, Greece o
n

A
16 5Microbiology Laboratory, Sismanoglion General Hospital of Athens, Athens, Greece p
r
il
17 6Microbiology Laboratory, Red Cross General Hospital, Athens, Greece 2
,

2
0
18 7Microbiology Laboratory, A. Fleming General Hospital, Athens, Greece 1
9

b
19 8Microbiology Laboratory, Asclepieion Voula General Hospital, Athens, Greece y

g
u
20 9Department of Microbiology, Medical School, University of Thessaly, Larissa, e
s
t
21 Greece
22 10Microbiology Laboratory, Thriassio General Hospital of Eleusis, Magoula, Attika,
23 Greece
24 11Microbiology Laboratory, Penteli Pediatric Hospital, Penteli, Greece
1
25 12Laboratory for Systemic Mycoses, First Department of Microbiology, Medical
26 School, Aristotle University of Thessaloniki, Thessaloniki, Greece
27 13Clinical Microbiology Laboratory, Attikon University General Hospital, Medical
28 School, National and Kapodistrian University of Athens, Athens, Greece
29
30 SHORT TITLE: Aspergillus identification and susceptibilities
D
31 o
w
n
32 *Corresponding author. lo
a
d
e
33 Mycology Laboratory d

f
r
o
34 Department of Microbiology m

h
35 Medical School, National and Kapodistrian University of Athens tt
p
:
/
/
36 Mikras Asias 75, Goudi 11527, Athens, Greece a
a
c
.
37 Phone: [phone] a
s
m
.
38 Fax: [phone] o
r
g
/

39 E-mail: [email protected] o
n

A
40 p
r
il
2
41
,

2
0
42 1
9

b
43 y

g
u
44 e
s
t
45
46
47
48
49
2
50 Abstract (Abstract word count = 74)
51 Polyphasic identification combined with antifungal susceptibility study of Aspergillus
52 spp. clinical isolates comprising 102 common and rare members of Sections Fumigati,
53 Flavi, Terrei, Nidulantes, Nigri, Circumdati, Versicolores and Usti is the first in
54 Greece.
55 High amphotericin B MIC (>2µg/ml) exhibited 17.6% of strains. Itraconazole,
D
56 posaconazole and voriconazole MIC>4µg/ml demonstrated 1%, 5% and 0% of the o
w
n
57 isolates respectively. Anidulafungin, micafungin and caspofungin MECs≥2 µg/ml lo
a
d
e
58 were correspondingly recorded for 4%, 9% and 33% of the strains. d

f
r
o
59 m

h
60 tt
p
:
/
/
61 a
a
c
.
62 a
s
m
.
63 o
r
g
/

64 o
n

A
65 p
r
il
2
66
,

2
0
67 1
9

b
68 y

g
u
69 e
s
t
70
71
72
73
74
3
75 (Text word count = 1207)
76 Current treatment guidelines for invasive aspergillosis recommend voriconazole as
77 primary therapy assigning alternative therapeutic roles to amphotericin B,
78 caspofungin, itraconazole, micafungin, and posaconazole (16, 27). Aspergillus species
79 display variable antifungal susceptibility (4, 5, 24). Consequently, accurate
80 identification of clinical Aspergillus isolates, supported by comparative sequence
D
81 identification methods and ongoing Aspergillus taxonomy revision (5, 18, 23), o
w
n
82 sponsor meticulous associations between species and antifungal susceptibility. lo
a
d
e
83 We report CLSI M38-A2-attuned (7) antifungal susceptibilities of clinical Aspergillus d

f
r
o
84 isolates, collected during 1998-2009. All isolates were polyphasically identified, m

h
85 embodying the first Aspergillus spp. occurrence and susceptibility report from our tt
p
:
/
/
86 geographical region. a
a
c
.
87 Strains We studied 102 Aspergillus isolates from bronchial secretions, broncho- a
s
m
.
88 alveolar lavage, peritoneal fluid, lung, liver, skin, maxillary bone nail and nasal o
r
g
/

89 biopsies from 102 consecutive immunocompromised patients with confirmed o
n

A
90 aspergilloses who were admitted to tertiary health care centres located in the cities of p
r
il
2
91 Athens, Thessaloniki and Larissa. The isolates were stored at -80°C in the Hellenic
,

2
0
92 Collection of Pathogenic Fungi UOA9291 until tested. 1
9

b
93 Polyphasic identification Morphology was studied in malt extract and Czapeck y

g
u
94 agars, at 25 °C followed by physiological studies (22, 23). Isolates were identified by e
s
t
95 ribosomal Internal Transcribed Spacer (ITS) sequencing as described previously (28).
96 Beta-tubulin was sequenced where ITS sequences were inadequate for definitive
97 identification (11). Sequences were compared (http://www.ncbi.nlm.nih.gov/BLAST)
98 and deposited to GenBank (EU714322, FJ878624-717, GQ376120-43, GQ461899-

1 Member of the World Data Centre for Microorganisms.
4
99 912). Identification was concluded when there was >99% sequence homology. The
100 ITS and beta-tubulin sequences were each aligned (CLUSTAL-X) (23) and
101 phylogenetic relations were derived via the Neighbour-Joining method and the
102 MEGA v.4 software (14). GenBank homolog sequences were used as reference (10,
103 23, 29).
104 Antifungal susceptibility testing Pure amphotericin B (AMB), itraconazole (ITZ),
D
105 posaconazole (POS), voriconazole (VOR), anidulafungin (AND), caspofungin (CAS), o
w
n
106 and micafungin (MCF) compounds were tested at concentrations 0.032 to 64 µg/ml. lo
a
d
e
107 The CLSI M38-A2 was employed for recording minimum inhibitory concentrations d

f
r
o
108 (MIC) for amphotericin B and the azoles and minimum effective concentrations m

h
109 (MEC) for the candins (7). Testing was repeated at three independent occasions, using tt
p
:
/
/
110 different batches of stock drug solutions. Reference strains, as per M38-A2 a
a
c
.
111 guidelines, were used at each independent trial. a
s
m
.
112 Statistical analysis Repeated measures ANOVA (2) was employed on transformed o
r
g
/

113 data followed by post-hoc pairwise comparisons with Bonferroni corrections for the o
n

A
114 four more common isolates A. fumigatus, A. flavus, A. terreus and E. nidulans. p
r
il
2
115
,

2
0
116 Clinical Aspergillus isolates in our region belonged to eight Sections (Table 1), 1
9

b
117 encompassing the rare species A. aculeatus, A. calidoustous, A. fumigatiaffinis, A. y

g
u
118 sclerotiorum, A. tubingensis, Emericella variecolor, E. unguis and Neosartorya e
s
t
119 hiratsukae. Currently, ITS sequencing is recommended for Aspergillus molecular
120 identification to subgenus/section level and beta-tubulin sequencing to species level
121 (5, 6). We used ITS sequencing for all isolates, and beta-tubulin only where ITS was
122 inadequate for species identification (A. aculeatus, A. calidoustus, A. fumigatiaffinis,
123 A. niger, A. tubingensis, E. nidulans, E. variecolor, N. hiratsukae). This strategy
5
124 evolved from (a) the general clinical laboratory use of ITS as the main target for
125 identification of fungi, (b) the adequacy of ITS sequencing for identifying common A.
126 fumigatus, A. flavus, and A. terreus clinical isolates, and (c) the ad hoc use of
127 minimum targets. Besides, beta-tubulin may not be an orthologous gene in all
128 Aspergillus Sections (18). Therefore, other protein-coding genes, either cadmodulin or
129 rodlet-A, may be finally selected as preferred single target for Aspergillus
D
130 identification (18, 23). o
w
n
131 lo
a
d
e
132 Regarding susceptibility to antifungals (Table 1), in total 18 strains (17.6%), d

f
r
o
133 comprising 5/23 A. flavus (21.7%), 4/37 A. fumigatus (10.8%), 3/4 A. niger (75%), m

h
134 4/12 A. terreus (33.3%) and 2/9 E. nidulans (22.2%) isolates, gave AMB MICs >2 tt
p
:
/
/
135 µg/ml. Such high AMB MICs are previously associated with treatment failure (16). a
a
c
.
136 Consequently, the relatively high MICs of AMB reported herein, among the highest a
s
m
.
137 reported (8), necessitates routine AMB susceptibility testing in our region. o
r
g
/
138 Additionally, ANOVA demonstrated (Table 2) that AMB MICs for A. fumigatus o
n

A
139 were notably lower (p<0.01) than those for A. flavus, A. terreus and E. nidulans. This p
r
il
2
140 aligns with previous reports where A. fumigatus is generally susceptible to AMB and
,

2
0
141 A. terreus resistant (24); whereas A. flavus usually registers as susceptible (8). 1
9

b
142 y

g
u
143 All isolates demonstrated VOR MIC≤4 µg/ml (Table 1). Consistently low VOR e
s
t
144 MICs, though similar to ones reported from Spain (12), differ from those reported
145 from USA where 4.1% of the tested Aspergillus isolates had MICs>4µg/ml (3);
146 possibly suggesting geographic heterogeneity in resistance trends. Among all tested
147 isolates, only one A. fumigatus had ITZ MIC>4 µg/ml (1%), despite previously
148 reported (13, 17) ITZ resistance (MICs ≥4 µg/ml). Herein, ITZ MICs were
6
149 significantly higher among Α. fumigatus in comparison to A. terreus (Table 2).
150 Previously reported Α. fumigatus ITZ resistance rates ranged from 2.1% to 7%, while
151 a rate of 2.7% was also detected in the present study. Lastly, five strains (4.9%) had
152 POS MIC>4 µg/ml including 1/2 A. calidoustus, 3/37 A. fumigatus (8.1%) and 1/4 A.
153 niger isolates. As is the case with other azoles, and in the absence of consensus
154 breakpoints, these somewhat increased rates of high POS MICs against Aspergillus
D
155 are weighed against the relevant host determinants, thus making unclear their clinical o
w
n
156 significance. lo
a
d
e
157 Recently, species dependent epidemiological cutoff values (ECVs) that may assist in d

f
r
o
158 azole resistance detection have been proposed for Aspergillus wild-type isolates (9, m

h
159 19, 26). Among the four most common tested species, the % of the non-wild type tt
p
:
/
/
160 isolates ITZ, POS and VOR (MIC>ECV) were respectively: A. fumigatus [13.5, 16, a
a
c
.
161 0], A. flavus [0, 8.6, 0], A. terreus [0, 25, 8.3], E. nidulans [0, 11, 11]. In accord to our a
s
m
.
162 findings, lower A. fumigatus susceptibility to posaconazole than to itraconazole has o
r
g
/
163 been recently recorded (21). This was attributed to either setting A. fumigatus o
n

A
164 posaconazole ECV too low, or, alternatively, to the existence of a yet unidentified p
r
il
2
165 acquired resistance mechanism (21).
,

2
0
166 Echinocandins showed variable MECs, with AND exhibiting the lowest mean values 1
9

b
167 (Table 1). Only 4% of the isolates displayed AND MEC≥2 µg/ml; MCF following y

g
u
168 with 9%. In contrast to previous observations (20), 33% of our isolates had high CAS e
s
t
169 MECs, while lower anidulafungin and highest caspofungin MECs for nongerminated
170 conidia of the few Aspergillus strains tested before (1). Notably, CAS was on local
171 hospital formulatory since 2006, while AND and MCF were introduced in 2008.
172 Consequently, the great majority of tested isolates originated from hospitals where
173 mostly CAS was used. Also, isolates tested from 2008-2009 aspergilloses originated
7
174 from paediatric hospitals where anidulafungin and micafungin were not indicated for
175 use at the time. Nonetheless, echinocandin susceptibility appears patchy, depending
176 on the species/Sections (Table 1). This is also highlighted in a recent survey where
177 more than 99% of all isolates were inhibited by <0.06 µg/ml of these three agents
178 tested (20). Interestingly ANOVA, comparing echinocandin MECs for the four most
179 common species, determined that the only statistically significant difference (p<0.05)
D
180 was the higher A. flavus micafungin MECs, in comparison to those of A. fumigatus o
w
n
181 (Table 2). lo
a
d
e
182 d

f
r
o
183 In conclusion, the rare Aspergilus species A. aculeatus, A. calidoustous, A. m

h
184 fumigatiaffinis, A. sclerotiorum, A. tubingensis, E. variecolor, E. unguis, and N. tt
p
:
/
/
185 hiratsukae are aetiological agents of aspergilloses in Greece. Voriconazole and a
a
c
.
186 itraconazole showed the best overall in vitro performance against our isolates. a
s
m
.
187 Notwithstanding the professed susceptibility to these two agents, confirmed erratically o
r
g
/

188 high MICs or MECs recorded for all antifungals may warrant routine susceptibility o
n

A
189 testing of Aspergillus isolates from immunocompromised patients in our region and p
r
il
2
190 justify the underway ECV determinations of indigenous wild-type strains.
,

2
0
191 1
9

b
192 Part of this study has been presented in the 49th ICAAC, San Francisco, 2009. y

g
u
193 We thank Afroditi Milioni, Stavroula Kritikou, and Eirini Ilia for technical assistance. e
s
t
194 This work was supported by SARG K.A. 70/4/5905 and 70/3/6915, National and
195 Kapodistrian University of Athens, the Bodosakis Foundation and by an unrestricted
196 Pfizer research grant.
197
8
198 References
199 1. Antachopoulos, C., J. Meletiadis, T. Sein, E. Roilides, and T. J. Walsh.
200 2008. Comparative in vitro pharmacodynamics of caspofungin, micafungin,
201 and anidulafungin against germinated and nongerminated Aspergillus conidia.
202 Antimicrob. Agents Chemother. 52:321-328.
203 2. Arabatzis, M., M. Kyprianou, A. Velegraki, A. Makri, and A. Voyatzi.
D
o
204 2010. Microsporum canis antifungal susceptibilities: concerns regarding their w
n
lo
205 clinical predictability. Int. J. Antimicrob. Agents 36(4):385-386. a
d
e
206 3. Baddley, J. W., K. A. Marr, D. R. Andes, T. J. Walsh, C. A. Kauffman, D. d
f
r
o
m
207 P. Kontoyiannis, J. I. Ito, S. A. Balajee, P. G. Pappas, and S. A. Moser.

h
t
t
208 2009. Patterns of susceptibility of Aspergillus isolates recovered from patients p
:
/
/
a
209 enrolled in the Transplant-Associated Infection Surveillance Network. J. Clin. a
c
.
a
210 Microbiol. 10:3271-3275. s
m
.
o
211 4. Balajee, S. A., J. L. Gribskov, E. Hanley, D. Nickle, and K. A. Marr. 2005. r
g
/

o
212 Aspergillus lentulus sp nov., a new sibling species of A. fumigatus. Eukaryot. n

A
p
213 Cell 4:625-632. r
il
2
214 5. Balajee, S. A., J. Houbraken, P. E. Verweij, S. B. Hong, T. Yaghuchi, J. ,
2
0
1
215 Varga, and R. A. Samson. 2007. Aspergillus species identification in the 9

b
y
216 clinical setting. Stud. Mycol. 59:39-46. g
u
e
217 6. Balajee, S. A., A. M. Borman, M. E. Brandt, J. Cano, M. Cuenca-Estrella, s
t
218 E. Dannaoui, J. Guarro, G. Haase, C. C. Kibbler, W. Meyer, K.
219 O’Donnell, C. A. Petti, J. L. Rodriguez-Tudela, D. Sutton, A. Velegraki,
220 and B. L. Wickes. 2009. Sequence-based identification of Aspergillus,
221 Fusarium, and Mucorales species in the clinical mycology laboratory: where
222 are we and where should we go from here? J. Clin. Microbiol. 4:877-884.
9
223 7. Clinical and Laboratory Standards Institute (CLSI). 2008. Reference
224 method for broth dilution antifungal susceptibility testing of filamentous
225 fungi; approved standard, 2nd ed. M38-A2. Clinical and Laboratory Standards
226 Institute, Wayne, PA.
227 8. Espinel-Ingroff, A., E. Johnson, H. Hockey and P. Troke. 2008. Activities
228 of voriconazole, itraconazole and amphotericin B in vitro against 590 moulds
D
229 from 323 patients in the voriconazole Phase III clinical studies. J. Antimicrob. o
w
n
230 Chemother. 61:616-620. lo
a
d
231 9. Espinel-Ingroff, A., D. J. Diekema, A. Fothergill, E. Johnson, T. Pelaez, ed

f
r
o
232 M. A. Pfaller, M. G. Rinaldi, E. Canton and J. Turnidge. 2010. Wild-type m

h
233 MIC distributions and epidemiological cutoff values for the triazoles and six tt
p
:
/
/
234 Aspergillus spp. for the CLSI broth microdilution method (M38-A2 a
a
c
.
235 document). J. Clin. Microbiol., in print. doi:10.1128/JCM.00536-10. a
s
m
236 10. Frisvad, J. C., J. M. Frank, J. A. M. P. Houbraken, A. F. A. Kuijpers, and .o
r
g
/
237 R. A. Samson. 2004. New ochratoxin A producing species of Aspergillus o
n

A
238 section Circumdati. Stud. Mycol. 50:23-43. p
r
il
239 11. Glass, N. L., and G. C. Donaldson. 1995. Development of primer sets 2
,

2
0
240 designed for use with the PCR to amplify conserved genes from filamentous 1
9

b
241 ascomycetes. Appl. Environ. Microbiol. 61:1323-1330. y

g
u
242 12. Guinea, J., S. Recio, T. Pelaez, M. Torres-Narbona, and E. Bouza. 2008. e
s
t
243 Clinical isolates of Aspergillus species remain fully susceptible to
244 voriconazole in the post-voriconazole era. Antimicrob. Agents Chemother. 52:
245 3444-3446.
246 13. Howard, S. J., D. Cerar, M. J. Anderson, A. Albarrag, M. C. Fisher, A. C.
247 Pasqualotto, M. Laverdiere, M. C. Arendrup, D. S. Perlin, and D. W.
10