In Vitro Activity of Eight Antifungal Drugs Against a Global Collection of Genotyped Isolates of ...

13 Pages·2015·0.58 MB·English

AAC Accepted Manuscript Posted Online 3 August 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.01218-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 In Vitro Activity of Eight Antifungal Drugs Against a Global Collection of
2 Genotyped Isolates of Exserohilum
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4 Anuradha Chowdharya, Ferry Hagenb, Ilse Curfs-Breukerb, Hugo Madridc, G. Sybren de
5 Hoogc and Jacques F. Meisb,d*
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7 Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, a
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8 Delhi, Indiaa; Department of Medical Microbiology and Infectious Diseases, Canisius- d

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9 Wilhelmina Hospital, Nijmegen, The Netherlandsb; CBS-KNAW Fungal Biodiversity Centre, om

10 Utrecht, the Netherlandsc; Department of Medical Microbiology, Radboudumc, Nijmegen, ht
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11 The Netherlandsd //a
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13 Running title: Antifungal susceptibility testing of Exserohilum spp.
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15 Word count abstract: 69
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18 *Corresponding author: [email protected] 1
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20 This work was presented (poster M-1350) at the 53rd ICAAC 2013, Denver, CO. e
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21 Abstract
22 In vitro susceptibilities of 24 worldwide Exserohilum isolates belonging to 10 species from
23 human and environmental sources were determined for eight antifungal drugs. Strains were
24 characterized by ITS sequencing and Amplified Fragment Length Polymorphism
25 fingerprinting. Posaconazole had the lowest geometric mean MIC (0.16 µg/ml) followed by
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26 micafungin (0.21 µg/ml), amphotericin B (0.24 µg/ml), itraconazole (0.33 µg/ml), w
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27 voriconazole (0.8 µg/ml), caspofungin (1.05 µg/ml), isavuconazole (1.38 µg/ml) and lo
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28 fluconazole (15.6 µg/ml). e
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29 o
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30 Keywords: Exserohilum, antifungal susceptibility, isavuconazole, AFLP, ITS sequencing h
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47 The anamorphic genus Exserohilum (teleomorph Setosphaeria, family Pleosporaceae, order
48 Pleosporales) comprises approximately 35 species, which are common saprobic fungi on plant
49 debris worldwide (1). Until recently Exserohilum species were considered to be rare
50 opportunistic pathogens reported sporadically from cases of keratitis, cutaneous and
51 subcutaneous phaeohyphomycosis and invasive infections especially in immunosuppressed
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52 patients (2, 3). However, at the end of 2012 the meningitis outbreak in the United States due
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53 to Exserohilum displayed this fungus as an agent of severe threatening infections (4,5). n
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54 Although several fungi were implicated in the outbreak, the vast majority of infections were d
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55 caused by Exserohilium rostratum, which were traced to contaminated steroid injections as fr
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56 the source (4-8). Previously, E. rostratum, E. longirostratum and E. mcginnisii, had been
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57 reported as opportunistic human pathogens. However, molecular studies have demonstrated p
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58 that these species are conspecific, with E. rostratum being the accepted species (1). a
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59 m
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60 The pathobiology of this melanized mould including infections in the g
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61 immunocompromised host is not clearly understood. A recent report (9) suggested that n

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62 methylprednisolone-induced suppression of phagocytosis by polymorphonuclear leukocytes r
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63 was responsible for the rapid development of an E. rostratum outbreak in the USA. The 1
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64 therapeutic approach to combat this pathogen is not yet established, and therefore knowledge 0
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65 of its pathogenic potential and antifungal susceptibility is of paramount importance (8,10,11). b
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66 The large majority of patients in the recent outbreak were administered voriconazole with or u
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67 without amphotericin B; this was based on small case series, personal experience and the t
68 favourable pharmacokinetic profile of voriconazole with regard to cerebral infections (10).
69 Clinical outcome with voriconazole seemed to be satisfactory, although a recent case showed
70 failure after 4.5 months of voriconazole therapy with excellent trough levels (12) and
71 therefore its upfront use has been challenged (13, 14). In vitro antifungal susceptibility (AFS)
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72 data for Exserohilum species is scarce. Three studies reported AFS data on E. rostratum
73 (1,4,15). Given the fact that E. rostratum is a coincidental opportunist, there is no a priori
74 reason to believe that this will be the only Exserohilum species with this ability. Therefore,
75 we aimed to study the potential activity of isavuconazole, a new triazole recently approved by
76 FDA for treatment of invasive aspergillosis and mucormycosis, against E. rostratum (n=10)
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77 and 9 other species of Exserohilum (n=14) made available by the CBS-KNAW Fungal
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78 Biodiversity Centre (Utrecht, the Netherlands) and the Vallabhbhai Patel Chest Institute n
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79 (Delhi, India) (Table 1). They were analyzed by amplified fragment length polymorphism d
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80 (AFLP) genotyping (Fig. S1) as described previously (16,17) and subjected to sequencing of fr
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81 the ITS region (Fig. S2).The minimal inhibitory concentrations (MIC) of amphotericin B
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82 (Bristol Myers Squibb, Woerden, The Netherlands), fluconazole, voriconazole (Pfizer Central p
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83 Research, Sandwich, Kent, United Kingdom), itraconazole (Janssen Cilag, Tilburg, The a
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84 Netherlands), posaconazole (Merck, Whitehouse Station, NJ), isavuconazole (Basilea s
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85 Pharmaceutica, Basel, Switzerland), caspofungin (Merck) and micafungin (Astellas, Toyama, o
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86 Japan) were determined using microdilution method, in accordance with the guidelines of the o
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87 CLSI document M38-A2 (18). p
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88 Exserohilum rostratum isolates (n=8) clustered together in AFLP along with 2 isolates il 1
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89 that were previously identified by conventional methods as E. mcginnisii (CBS 325.87T, CBS 2
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90 120308), confirming their synonymy (Fig. S1)(1). Also, two isolates each of E. prolatum 9

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91 (CBS 128058, CBS 128059, no type strain) and E. antillanum (CBS 412.93T) clustered with g
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92 E. rostratum; E. antillanum can thus be regarded as a further synonym of E. rostratum. s
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93 Notably, in the ITS phylogenetic tree also E. antillanum could not be discriminated from E.
94 rostratum (Fig. S2). Exserohilum rostratum isolates exhibited somewhat variable banding
95 patterns, suggesting genotypic diversity in this complex. Two isolates of E. pedicellatum,
96 none of which was a type strain, clustered together (Fig. S1). Further, two isolates for each of
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97 the species E. gedarefense (CBS 297.80T) and E. neoregeliae (CBS 132832T) and a solitary
98 isolate of E. fusiforme (CBS 132709T) and E. protrudens (CBS 132710T) all type strains
99 represented four different species. Overall, ITS sequencing could not discriminate E.
100 rostratum, E. gedarefense, E. antillanum, and E. mcginnisii. Also, similar but not identical
101 AFLP profiles were observed for E. holmii (no type strain) and E. curvatum (CBS 505.90T)
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102 representing two different species, which was in conformity with ITS phylogeny.
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103 MIC ranges, MIC50/ MIC90 and geometric mean MICs of all isolates are presented in Table 2 nlo
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104 along with the three previously published studies for comparison (1,4,15). Table 1 d
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105 summarizes the origin and source of the isolates. They originated from USA (n=3), Australia fr
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106 (n=2), Canada (n=6), Cuba (n=1), India (n=2), Japan (n=2), Sudan (n=2), Turkey (n=1), and
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107 Venezuela (n=1). Of these, 10 were environmental and 10 clinical, whereas 4 were of p
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108 unknown origin and source. Posaconazole, itraconazole and amphotericin B had the lowest a
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109 MIC90s across all Exserohilum isolates whereas fluconazole had no activity. The MIC range sm
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110 of 10 E. rostratum isolates (inclusive E. mcginnisii) for isavuconazole spread over 2-3 two- o
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111 fold dilutions (1-8 µg/ml) and were similar to posaconazole (0.06-0.5 µg/ml), voriconazole o
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112 (0.5-2 µg/ml), itraconazole (0.125-0.5 µg/ml), caspofungin (0.5-4 µg/ml), amphotericin B p
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113 (0.06-1 µg/ml) and micafungin (0.008- 8 µg/ml). The geometric mean MIC of isavuconazole il 1
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114 for E. rostratum was 3 μg/ml compared to 1.15 μg/ml for voriconazole and 0.16 μg/ml for 2
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115 posaconazole. Notably, GM MICs of isavuconazole for all Exserohilum spp. (1.38 μg/ml) was 9

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116 lower than that of E. rostratum (3.03 μg/ml). In the present study isavuconazole had the g
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117 lowest in vitro activity among azoles, which was in conformity with the data of the USA s
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118 meningitis outbreak E. rostratum strains (4). Further the E. rostratum outbreak strains
119 exhibited equivalent susceptibility against itraconazole, posaconazole, amphotericin B, and to
120 a lesser extent voriconazole (4). Notably, in the present study for all Exserohilum species GM
121 MEC of caspofungin (1.18 μg/ml) was 3 fold higher than micafungin (0.36 μg/ml). Further,
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122 in contrast to previous findings with Candida all the isolates tested had reproducible MECs
123 for caspofungin when performed on two occasions.
124 Regarding the published reviews of the therapeutic outcome of cases of sinusitis and
125 cutaneous infections with Exserohilum, successful outcomes with amphotericin B and more
126 recently with itraconazole and voriconazole has been on reported (2,11,19). The expert panel
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127 for the USA outbreak recommended voriconazole for therapy of meningitis in patients with
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128 less-severe disease and the lipid formulation of amphotericin B for those with severe n
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129 extraneural disease or refractory infections (10). The present study showed low amphotericin d
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130 B MICs against E. rostratum while voriconazole had geometric mean MICs of >1 μg/ml. In fr
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131 contrast, Da Cunha et al. (1) reported low geometric mean MICs of voriconazole (0.1μg/ml)
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132 for 34 E. rostratum isolates originating from the USA. The different susceptibility to p
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133 voriconazole in the present study could be attributed to the geographically variable strains a
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134 tested in this study. Recently, using whole genome analysis, significant genomic variability s
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135 has been reported among E. rostratum strains unrelated to the outbreak strains (20). o
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136 Although clinical relevance of MIC data for Exserohilum have not been established we o
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137 conclude that amphotericin B, itraconazole and posaconazole are in vitro the most active p
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138 drugs exhibiting MICs below 1 μg/ml, with both isavuconazole and voriconazole showing il 1
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139 MICs below achievable serum trough levels. 2
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141 Potential Conflict of Interest g
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142 J.F.M received grants from Astellas, Basilea and Merck. He has been a consultant to Astellas, s
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143 Basilea and Merck and received speaker’s fees from Merck and Gilead.
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147 Acknowledgement
148 This work was partially funded by Astellas, USA. AC was supported by Indian Council of
149 Medical Research, New Delhi, India (Ref no. 5/3/3/26/200-ECD-I) and HM was funded by
150 Comisión Nacional de Investigación Científica y Tecnológica (CONICYT), Fondo Nacional
151 de Desarrollo Científico y Tecnológico (FONDECYT), Chile, Project 11140562.
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153 References n
lo
a
154 1. da Cunha KC, Sutton DA, Gené J, Capilla J, Cano J, Guarro J. 2012. Molecular d
e
d
155 identification and in vitro response to antifungal drugs of clinical isolates of Exserohilum. fr
o
m
156 Antimicrob Agents Chemother. 56:4951-4954.
h
t
t
157 2. Katragkou A, Pana ZD, Perlin DS, Kontoyiannis DP, Walsh TJ, Roilides E. 2014. p
:
/
/
a
158 Exserohilum infections: review of 48 cases before the 2012 United States outbreak. Med a
c
.
a
159 Mycol. 52: 376-386. s
m
.
160 3. Chowdhary A, Perfect JR, de Hoog GS. 2015. Black molds and melanised yeasts o
r
g
/
161 pathogenic to humans. p. 517-537. In: Casadevall A, Mitchell AP, Berman J, Kwon- o
n

A
162 Chung KJ, Perfect JR, Heitman J. (eds.), Human Fungal pathogens.Cold Spring Harbor p
r
163 Laboratory Press, Cold Spring Harbor, New York, USA. il 1
1
,

164 4. Lockhart SR, Pham CD, Gade L, Iqbal N, Scheel CM, Cleveland AA, Whitney AM, 2
0
1
165 Noble-Wang J, Chiller TM, Park BJ, Litvintseva AP, Brandt ME. 2013. Preliminary 9

b
y
166 laboratory report of fungal infections associated with contaminated methylprednisolone g
u
e
167 injections. J Clin Microbiol. 51: 2654-2661. s
t
168 5. Smith RM, Schaefer MK, Kainer MA, Wise M, Finks J, Duwve J, Fontaine E, Chu
169 A, Carothers B, Reilly A, Fiedler J, Wiese AD, Feaster C, Gibson L, Griese S,
170 Purfield A, Cleveland AA, Benedict K, Harris JR, Brandt ME, Blau D, Jernigan J,
7
171 Weber JT, Park BJ. 2013. Fungal infections associated with contaminated
172 methylprednisolone injections - preliminary report. N Engl J Med. 369: 1598-1609.
173 6. Ritter JM, Muehlenbachs A, Blau DM, Paddock CD, Shieh WJ, Drew CP, Batten
174 BC, Bartlett JH, Metcalfe MG, Pham CD, Lockhart SR, Patel M, Liu L, Jones TL,
175 Greer PW, Montague JL, White E, Rollin DC, Seales C, Stewart D, Deming MV,
D
176 Brandt ME, Zaki SR. 2013. Exserohilum infections associated with contaminated steroid
o
w
177 injections: A clinicopathologic review of 40 cases. Am J Pathol. 183: 881-892. n
lo
a
178 7. Smith RM, Derado G, Wise M, Harris JR, Chiller T, Meltzer MI, Park BJ. 2015. d
e
d
179 Estimated deaths and illnesses averted during fungal meningitis outbreak associated with fr
o
m
180 contaminated steroid injections, United States, 2012–2013. Emerg Infect Dis. 21:933-940.
h
t
t
181 8. Kontoyiannis DP, Perlin DS, Roilides E, Walsh TJ. 2013. What can we learn and what p
:
/
/
a
182 do we need to know amidst the iatrogenic outbreak of Exserohilum rostratum meningitis? a
c
.
a
183 Clin Infect Dis. 57:853-859. s
m
.
184 9. Simitsopoulou M, Walsh TJ, Kyrpitzi D, Petraitis V, Kontoyiannis DP, Perlin DS, o
r
g
/
185 Roilides E. 2015. Methylprednisolone impairs conidial phagocytosis but does not o
n

A
186 attenuate hyphal damage by neutrophils against Exserohilum rostratum. Med Mycol. 53: p
r
187 189-193. il 1
1
,

188 10. Pappas PG, Kontoyiannis DP, Perfect JR, Chiller TM. 2013. Real-time treatment 2
0
1
189 guidelines: considerations during the Exserohilum rostratum outbreak in the United 9

b
y
190 States. Antimicrob Agents Chemother. 57: 1573-1576. g
u
e
191 11. Chowdhary A, Meis JF, Guarro J, de Hoog GS, Kathuria S, Arendrup MC, Arikan- s
t
192 Akdagli S, Akova M, Boekhout T, Caira M, Guinea J, Chakrabarti A, Dannaoui E,
193 van Diepeningen A, Freiberger T, Groll AH, Hope WW, Johnson E, Lackner M,
194 Lagrou K, Lanternier F, Lass-Flörl C, Lortholary O, Meletiadis J, Muñoz P, Pagano
195 L, Petrikkos G, Richardson MD, Roilides E, Skiada A, Tortorano AM, Ullmann AJ,
8
196 Verweij PE, Cornely OA, Cuenca-Estrella M. 2014. ESCMID and ECMM joint clinical
197 guidelines for the diagnosis and management of systemic phaeohyphomycosis: diseases
198 caused by black fungi. Clin Microbiol Infect. 20 (Suppl 3): 47-75.
199 12. Smith RM, Tipple M, Chaudry MN, Schaefer MK, Park BJ. 2013. Relapse of fungal
200 meningitis associated with contaminated methylprednisolone. N Engl J Med. 368: 2535-
D
201 2536.
o
w
202 13. Stevens DA. 2013. Reflections on the approach to treatment of a mycologic disaster. n
lo
a
203 Antimicrob Agents Chemother. 57: 1567-1572. d
e
d
204 14. Stevens DA. 2013. Mycologic catastrophe. Antimicrob Agents Chemother. 57: 2904. fr
o
m
205 15. Revankar SG, Moudgal V, Chandrasekar P, Sobel JD. 2014. In vitro studies of
h
t
t
206 Exserohilum rostratum with antifungal drugs and methylprednisolone. Antimicrob Agents p
:
/
/
a
207 Chemother. 58: 3564-3565. a
c
.
a
208 16. Kumar A, Babu R, Bijulal S, Abraham M, Sasidharan P, Kathuria S, Sharma C, s
m
.
209 Meis JF, Chowdhary A. 2014. Invasive mycosis due to multidrug resistant species of o
r
g
/
210 Blastobotrys in immunocompromised patients. J Clin Microbiol. 52: 4094-4099. o
n

A
211 17. Kathuria S, Sharma C, Singh PK, Agarwal P, Agarwal K, Hagen F, Meis JF, p
r
212 Chowdhary A. 2015. Molecular epidemiology and in-vitro antifungal susceptibility of il 1
1
,

213 Aspergillus terreus species complex isolates in Delhi, India: evidence of genetic diversity 2
0
1
214 by amplified fragment length polymorphism and microsatellite typing. PLoS ONE. 2015: 9

b
y
215 10(3):e0118997. doi: 10.1371/journal.pone.0118997. g
u
e
216 18. Clinical and Laboratory Standards Institute. 2008. Reference method for broth s
t
217 dilution antifungal susceptibility testing of filamentous fungi, 2nd ed. Approved standard.
218 CLSI document M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA.
219 19. Revankar SG, Sutton DA. 2010. Melanized fungi in human disease. Clin Microbiol Rev.
220 23: 884-928.
9
221 20. Litvintseva AP, Hurst S, Gade L, Frace MA, Hilsabeck R, Schupp JM, Gillece JD,
222 Roe C, Smith D, Keim P, Lockhart SR, Changayil S, Weil MR, MacCannell DR,
223 Brandt ME, Engelthaler DM. 2014. Whole-genome analysis of Exserohilum rostratum
224 from an outbreak of fungal meningitis and other infections. J Clin Microbiol. 52: 3216-
225 3222.
D
226
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