Received 12 May 2009/Returned for modification 29 July 2009/Accepted 9 August 2009

0095-1137/09/$08.00

⫹0 doi:10.1128/JCM.00940-09

Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Wild-Type MIC Distribution and Epidemiological Cutoff Values for

Aspergillus fumigatus and Three Triazoles as Determined by the

Clinical and Laboratory Standards Institute Broth

Microdilution Methods

䌤

M. A. Pfaller,

1,2

* D. J. Diekema,

1

M. A. Ghannoum,

3

J. H. Rex,

4

B. D. Alexander,

5

D. Andes,

6

S. D. Brown,

7

V. Chaturvedi,

8

A. Espinel-Ingroff,

9

C. L. Fowler,

10

E. M. Johnson,

11

C. C. Knapp,

12

M. R. Motyl,

13

L. Ostrosky-Zeichner,

14

D. J. Sheehan,

15

and T. J. Walsh

16

for the Clinical and Laboratory Standards

Institute Antifungal Testing Subcommittee

Roy J. and Lucille A. Carver College of Medicine

1

_{and College of Public Health,}

2

_{University of Iowa, Iowa City, Iowa 52242;}

Case Western Reserve University, Cleveland, Ohio 44106-5028

3

_{; AstraZeneca, Macclesfield, Cheshire, United Kingdom}

4

_;

Duke University Medical Center, Durham, North Carolina 27710

5

_{; University of Wisconsin, Madison, Wisconsin 53792}

6

_;

Clinical Microbiology Institute, Wilsonville, Oregon 97070

7

_{; New York State Department of Health,}

Albany, New York 12201-2002

8

_{; Medical College of Virginia, Richmond, Virginia 23298}

9

_{; BioMerieux, Inc.,}

Durham, North Carolina 27712

10

_{; HPA Centre for Infections, Kingsdown, Bristol, United Kingdom}

11

_;

Trek Diagnostic Systems, Cleveland, Ohio 44131

12

_{; Merck & Company, Inc., Rahway,}

New Jersey 07065-0900

13

_{; University of Texas Medical School at Houston, Houston,}

Texas 77030

14

_{; Pfizer, Inc., New York, NY 10017}

15

_{; and National Cancer Institute,}

Bethesda, Maryland 20892

16

Received 12 May 2009/Returned for modification 29 July 2009/Accepted 9 August 2009

Antifungal susceptibility testing of Aspergillus species has been standardized by both the Clinical and

Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility

Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatus with acquired

resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A (sterol demethylase)

gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the

EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions,

epidemio-logical cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical

isolates of A. fumigatus for which itraconazole MICs were

<2 ␮g/ml against posaconazole and voriconazole

using the CLSI BMD method. An ECV of

<1 ␮g/ml encompassed the WT population of A. fumigatus for

itraconazole and voriconazole, whereas an ECV of

<0.25 ␮g/ml was established for posaconazole. Our

results demonstrate that the WT distribution and ECVs for A. fumigatus and the mold-active triazoles were

the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which

itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between

itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between

itracon-azole and voriconitracon-azole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution

and ECVs for the azoles and A. fumigatus will be useful in resistance surveillance and is an important step

toward the development of clinical breakpoints.

Invasive aspergillosis (IA) is second only to candidiasis as

the most important invasive mycosis affecting humans (5, 10,

18, 37, 40). Although several hundred species of Aspergillus

have been described, relatively few are known to cause disease

in humans. Aspergillus fumigatus remains the most common

cause of IA, although the proportion of IA cases caused by this

species has fallen from

⬃90% of cases in the 1980s to between

50 and 60% of cases in the 1990s and into the 2000s (30, 36).

Antifungal susceptibility testing of Candida as an aid in

guiding clinical treatment has gained wide acceptance (3, 8, 16,

20, 22, 24, 39). The development and application of in vitro

susceptibility testing of Aspergillus spp. have lagged behind that

for Candida due to the difficulty in determining a clinical role

for this testing. Confounding the issue are numerous factors

that play either a positive or negative role in determining the

outcome of therapy (14, 26). Given the increasing number of

antifungal agents with systemic activity against Aspergillus spp.

(13, 15, 21, 38, 41, 42, 44, 48, 49), it is recognized that

antifun-gal susceptibility testing of these opportunistic pathogens may

be useful in guiding the selection of antifungal agents for the

treatment of IA (1, 34, 42, 44, 47). This is especially true for

itraconazole (11, 34) and the newer extended-spectrum

tria-zoles posaconazole and voriconazole (42, 44).

In vitro antifungal susceptibility testing of azoles versus

As-pergillus spp. has been standardized by both the CLSI (7) and

European Committee on Antimicrobial Susceptibility Testing

* Corresponding author. Mailing address: Medical Microbiology

Di-vision, C606 GH, Department of Pathology, University of Iowa

Col-lege of Medicine, Iowa City, IA 52242. Phone: (319) 345-8615. Fax:

(319) 356-4916. E-mail: [email protected].

(EUCAST) (27). Both methods use a 96-well broth

microdi-lution (BMD) format, RPMI 1640 broth medium, 48 h of

incubation, and a MIC endpoint of complete inhibition of

growth as determined by visual inspection. The EUCAST

method employs a higher inoculum concentration (2

⫻ 10

5

_to

5

⫻ 10

5

_{CFU/ml versus 0.4}

_{⫻ 10}

4

_{to 5}

_{⫻ 10}

4

_{CFU/ml) and}

additional glucose (2% versus 0.2% final concentration) in an

effort to improve fungal growth. Using these methods, A.

fu-migatus isolates exhibiting high MICs to azole drugs have been

described (2, 11, 19, 28, 29) and both methods have been

shown to reliably detect those strains with defined azole

resis-tance mechanisms (1, 4, 6, 17, 31–33, 42, 44).

Mechanisms of elevated azole MICs in A. fumigatus

discov-ered thus far involve the cyp51A (lanosterol demethylase) gene

and include mutations resulting in amino acid substitutions at

glycine 54 (G54) (12, 29, 35, 44) at G448 (50), and at

methi-onine 220 (M220) (31, 44) and an amino acid substitution of

leucine for histidine at position 98 (L98H) together with a

tandem repeat (TR) of a 34-bp sequence in the promoter of

the cyp51A gene (33). Cross-resistance to itraconazole and

posaconazole has been associated with the G54 substitution,

whereas cross-resistance to voriconazole and ravuconazole has

been associated with the G448 substitution (44, 50). Both the

M220 substitution and the L98H TR produce a

cross-resis-tance pattern to all four azoles (44).

Interpretive breakpoints based upon the correlation of in

vitro data with clinical outcome have not been established for

any Aspergillus-drug combination (14, 42, 44). In the absence of

the necessary clinical data, one practical approach to the use of

susceptibility testing data in detecting resistance or decreased

susceptibility has been to define the wild-type (WT)

distribu-tion of MICs for the relevant drug-organism combinadistribu-tions

(e.g., populations of organisms with no acquired resistance

mechanisms) and set epidemiological cutoff values (ECVs)

that would discriminate WT strains from those with acquired

resistance mechanisms (25, 44–46). The clinical relevance of

ECVs would still be uncertain, but ECVs could nonetheless

serve as the foundation for the laboratory detection of

ac-quired resistance (decreased susceptibility) and be used to

monitor resistance development (45).

Rodriguez-Tudela et al. (44) employed the EUCAST BMD

method to define the WT MIC distribution of four triazole

antifungal agents (itraconazole, posaconazole, ravuconazole,

and voriconazole) for A. fumigatus. They also used studies of

resistance mechanisms in A. fumigatus to demonstrate that

ECVs of

ⱕ1 ␮g/ml for itraconazole, ravuconazole, and

vori-conazole and

ⱕ0.25 ␮g/ml for posaconazole identified the WT

strains and provided separation of the WT population from

those strains with resistance mutations in the cyp51A gene (44).

Definition of the WT distribution and ECVs for azoles and A.

fumigatus is important in order to have a clear understanding

of which MICs can be considered microbiologically susceptible

(25, 44, 46). Furthermore, these studies showed that the MIC

phenotype obtained by means of a standardized methodology

was sufficient to identify nonsusceptible strains of A. fumigatus

and their pattern of cross-resistance without the need for

se-quence analysis of cyp51A to detect the underlying mutation

(44).

Similar studies using CLSI BMD to test itraconazole,

posaconazole, ravuconazole, and voriconazole against 553

lates tested against itraconazole, posaconazole, and

voricon-azole by the CLSI method to provide further documentation of

the WT MIC distribution and ECVs for the azoles and A.

fumigatus.

MATERIALS AND METHODS

Organisms.Between January 2005 and December 2007, 637 unique patient isolates of A. fumigatus were obtained from more than 60 different medical centers worldwide. All isolates were submitted to the Molecular Epidemiology and Fungus Testing Laboratory (MEFTL) at the University of Iowa College of Medicine as part of a global antifungal surveillance program. Each participating center was instructed to submit fungal isolates from sterile sites or from respi-ratory samples when they were determined to be significant pathogens by local criteria. The isolates were obtained from a variety of sources, including sputum, bronchoscopy, and tissue biopsy specimens. All isolates were identified by stan-dard microscopic morphology and were stored as spore suspensions in sterile distilled water at room temperature until used in the study. Before testing, each isolate was subcultured at least twice on potato dextrose agar (Remel, Lenexa, KS) to ensure viability and purity. As a screen for cryptic species within the A.

fumigatus complex (e.g., A. lentulus), all A. fumigatus isolates for which the MIC

of any azole wasⱖ2 ␮g/ml were tested for growth at 50°C. All isolates screened grew at 50°C, confirming that they were likely to be A. fumigatus.

Antifungal susceptibility testing.Itraconazole (Janssen), posaconazole (Scher-ing-Plough), and voriconazole (Pfizer) were all obtained as reagent-grade pow-ders from their respective manufacturers. The BMD method was performed according to the CLSI M38-A2 standard (7). Trays containing a 0.1-ml aliquot of the appropriate drug solution (2⫻ the final drug concentration) in each well were sealed and stored at⫺70°C until used in the study. The stock conidial suspension (106_{spores/ml) was diluted to a final inoculum concentration of 0.4⫻ 10}4_{to 5⫻}

104

CFU/ml and dispensed into the microdilution wells. The final concentrations of drugs in the wells ranged from 0.007 to 8␮g/ml. The inoculated microdilution trays were incubated at 35°C and read at 48 h. The MIC endpoint for the azoles was defined as the lowest concentration that produced complete inhibition of growth.

In addition, 43 isolates of A. fumigatus which had been submitted to the MEFTL as part of global antifungal surveillance projects since January 2000 and for which itraconazole MICs wereⱖ2 ␮g/ml (2 to ⬎8 ␮g/ml) were used to assess cross-resistance patterns.

Quality control.Quality control was ensured by testing the following strains recommended in CLSI standard M38-A2 (7): Candida parapsilosis ATCC 22019,

Candida krusei ATCC 6258, and Aspergillus flavus ATCC 204304.

Definitions.The definitions of WT and ECVs were those outlined previously by Kahlmeter et al. (25), Turnidge and Paterson (46), and Rodriguez-Tudela et al. (44). A WT organism is defined as a strain which does not harbor any acquired resistance to the particular antimicrobial agent (azole) being examined (46). In this particular instance, a WT MIC distribution for the azoles and A. fumigatus was ensured by omitting those isolates for which an itraconazole MIC of 4␮g/ml or greater was obtained (44).

The ECV for each azole was obtained by considering the WT MIC distribu-tion, the modal MIC for each distribudistribu-tion, and the inherent variability of the test (usually⫾1 log2dilution). In general, the ECV should encompass at least 95%

of isolates in the WT distribution (43). Organisms with acquired resistance mechanisms may be identified as those with a MIC higher than the highest MIC of the WT (⬎ECV) (25, 44).

RESULTS AND DISCUSSION

The WT MIC distributions for A. fumigatus and the three

triazoles are shown in Fig. 1. These distributions are essentially

the same as those described by Rodriguez-Tudela et al. (44)

using the EUCAST BMD method. By excluding those isolates

for which the itraconazole MICs were

ⱖ4 ␮g/ml, we have

ensured that the remaining isolates comprising these MIC

dis-tributions are free of acquired azole resistance mutations (44).

The modal MICs for itraconazole, posaconazole, and

voricon-azole were, respectively, 0.25

␮g/ml (41.3% of all values), 0.03

␮g/ml (43.2% of all values), and 0.25 ␮g/ml (53.5% of all

values) (Table 1 and Fig. 1). The modal MIC

⫾ 1 twofold

dilution comprised 90.3% of the strains for itraconazole,

88.9% of the strains for posaconazole, and 94.0% of the strains

for voriconazole. The limitations of the twofold dilution

scheme used in the CLSI BMD method are especially apparent

for itraconazole, where the true mode lies between 0.12 and

0.25

␮g/ml. Similarly, the MIC distributions for both

posacon-azole and voriconposacon-azole show a prominent shoulder at the next

higher MIC immediately adjacent to the designated modal

MIC (encompassing 29.7% of the posaconazole MICs and

38.6% of the voriconazole MICs) (Fig. 1). Given these rather

broad central tendencies, visual inspection of the MIC

distri-butions supports the ECVs assigned by Rodriguez-Tudela et

al. (44):

ⱕ1 ␮g/ml for itraconazole and voriconazole and ⱕ0.25

␮g/ml for posaconazole (Table 1). These cutoffs comprise

99.8% of itraconazole and posaconazole MICs and 99.2% of

voriconazole MICs.

Given these findings, isolates for which the itraconazole

MICs are 2

␮g/ml or greater may be considered as outliers with

an increased probability of harboring an azole resistance

mech-anism (44). Table 2 shows the results of cross-resistance studies

using a collection of 43 isolates of A. fumigatus for which

itraconazole MICs fell outside of the ECV: range of 2 to

⬎8

␮g/ml. A high degree of cross-resistance is apparent between

itraconazole and posaconazole but not between itraconazole

and voriconazole. The posaconazole MIC distribution for

these isolates is clearly different from the WT distribution,

whereas that for voriconazole is essentially the same as the WT

distribution (Tables 1 and 2). Although the mechanisms of

resistance were not determined in this study, the

itraconazole-posaconazole cross-resistance phenotype observed here

sug-gests a G54 substitution in cyp51A (44). It has been postulated,

based on molecular modeling studies, that a G54 substitution

confers resistance to posaconazole and itraconazole by

per-TABLE 1. MIC distribution and ECVs for azoles and A. fumigatus

Antifungal agent

Result from:

This study Study by Rodriguez-Tudela et al. (44)

No. of isolates tested

MIC (␮g/ml) _{No. of isolates}

tested

MIC (␮g/ml)

Range Mode ECV (%)a _{Range Mode ECV (%)}a

Itraconazole

637

0.015–2

0.25

1 (99.8)

393

0.06–2

0.25

1 (99.7)

Posaconazole

637

0.06–1

0.03

0.25 (99.8)

393

0.015–2

0.06

0.25 (98.6)

Voriconazole

637

0.12–4

0.25

1 (99.2)

393

0.06–2

0.5

1 (97.8)

turbing the binding of the long side chain in the hydrophobic

channel of the enzyme (50). Substitutions at G54 would be

predicted to have less effect on the binding of voriconazole,

which lacks a long side chain. The MICs for three of the

isolates were higher than the ECVs for all three azoles,

sug-gesting either an M220 substitution or the L98H TR.

The results of this study confirm those of Rodriguez-Tudela

et al. (44) and demonstrate that the WT MIC distribution and

ECVs for A. fumigatus and the mold-active triazoles are

essentially the same when determined by either the CLSI or

EUCAST BMD method. Although azole resistance is rare among

clinical isolates of A. fumigatus, it does appear to be increasing

and thus warrants continued surveillance (2, 6, 9, 23, 47). We

agree with Rodriguez-Tudela et al. (44) that isolates of A.

fumigatus that are susceptible to itraconazole thus far have not

displayed resistance to other azole antifungal agents, and this

agent will continue to prove useful in tracking the emergence

of azole resistance and cross-resistance profiles of A. fumigatus.

Although ECVs do not take the place of clinical breakpoints,

they will be very useful in resistance surveillance and serve as

an important step in the establishment of clinical breakpoints.

ACKNOWLEDGMENTS

We thank Caitlin Howard for excellent secretarial support.

This study was supported in part by research and educational grants

from Pfizer Pharmaceuticals and the Schering-Plough Research

Insti-tute.

REFERENCES

1. Alcazar-Fuoli, L., E. Mellado, A. Alastruey-Izquierdo, M. Cuenca-Estrella,

and J. Rodriguez-Tudela.2008. Aspergillus section Fumigati: antifungal sus-ceptibility patterns and sequence-based identification. Antimicrob. Agents Chemother. 52:1244–1251.

2. Arendrup, M. C., S. Perkhofer, S. J. Howard, G. Garcia-Effron, A.

Vishu-kumar, D. Perlin, and C. Lass-Flo¨rl.2008. Establishing in vitro-in vivo correlations for Aspergillus fumigatus: the challenge of azoles versus echino-candins. Antimicrob. Agents Chemother. 52:3504–3511.

3. Baddley, J. W., M. Patel, M. James, et al. 2004. Utility of real-time antifungal susceptibility testing for fluconazole in the treatment of candidemia. Diagn. Microbiol. Infect. Dis. 50:119–124.

4. Balashov, S. V., R. Gardiner, S. Park, and D. S. Perlin. 2005. Rapid, high-throughput, multiplex, real-time PCR for identification of mutations in the

cyp51A gene of Aspergillus fumigatus that confer resistance to itraconazole.

J. Clin. Microbiol. 43:214–222.

5. Barnes, P. D., and K. A. Marr. 2006. Aspergillosis: spectrum of disease, diagnosis, and treatment. Infect. Dis. Clin. N. Am. 20:545–561.

6. Chen, J., H. Li, R. Li, D. Bu, and Z. Wan. 2005. Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus serially iso-lated with a patient with lung aspergilloma. J. Antimicrob. Chemother.

55:31–37.

7. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi: approved standard, 2nd ed. M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA.

8. Collins, C. D., G. A. Eschenauer, S. L. Salo, and D. W. Newton. 2007. To test or not to test: a cost minimization analysis of susceptibility testing for

pa-tients with documented Candida glabrata fungemias. J. Clin. Microbiol. 45: 1884–1888.

9. Dannaoui, E., D. Garcia-Hermoso, J. M. Naccache, I. Meneau, D. Sanglard,

C. Bouges-Michel, D. Valeyre, and O. Lortholary.2006. Use of voriconazole in a patient with aspergilloma cased by an itraconazole-resistant strain of

Aspergillus fumigatus. J. Med. Microbiol. 55:1457–1459.

10. Dasbach, E. J., G. M. Davies, and S. M. Teutsch. 2000. Burden of aspergil-losis-related hospitalizations in the United States. Clin. Infect. Dis. 31:1524– 1528.

11. Denning, D. W., K. Venkataswarlu, K. L. Oakley, M. J. Anderson, N. J.

Manning, D. A. Stevens, D. W. Warnock, and S. L. Kelly.1997. Itraconazole resistance in Aspergillus fumigatus. Antimicrob. Agents Chemother. 41:1364– 1368.

12. Diaz-Guerra, T. M., E. Mellado, M. Cuenca-Estrella, and J. L.

Rodriguez-Tudela.2003. A point mutation in the 14␣-sterol demethylase gene cyp51A contributes to itraconazole resistance in Aspergillus fumigatus. Antimicrob. Agents Chemother. 47:1120–1124.

13. Diekema, D. J., S. A. Messer, R. J. Hollis, R. N. Jones, and M. A. Pfaller. 2003. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi. J. Clin. Microbiol. 41:3623–3626.

14. Espinel-Ingroff, A. 2003. In vitro antifungal activities of anidulafungin and micafungin, licensed agents and the investigational triazole posaconazole as determined by NCCLS methods for 12,052 fungal isolates: review of the literature. Rev. Iberoam. Micol. 20:121–136.

15. Espinel-Ingroff, A., M. Bartlett, V. Chaturvedi, M. Ghannoum, K. C.

Hazen, M. A. Pfaller, M. Rinaldi, and T. J. Walsh.2001. Optimal sus-ceptibility testing conditions for detection of azole resistance in

Aspergil-lus spp.: NCCLS collaborative evaluation. Antimicrob. Agents

Che-mother. 45:1828–1835.

16. Forrest, G. 2006. Role of antifungal susceptibility testing in patient manage-ment. Curr. Opin. Infect. Dis. 19:538–543.

17. Garcia-Effron, G., E. Mellado, A. Gomez-Lopez, L. Alcazar-Fuoli, M.

Cuenca-Estrella, and J. L. Rodriguez-Tudela.2005. Differences in inter-actions between azole drugs related to modifications in the 14-␣ sterol demethylase gene (cyp51A) of Aspergillus fumigatus. Antimicrob. Agents Chemother. 49:2119–2121.

18. Garcia-Vidal, C., A. Upton, K. A. Kirby, and K. A. Marr. 2008. Epidemiology of invasive mold infections in allogeneic stem cell transplant recipients: biological risk factors for infection according to time after transplantation. Clin. Infect. Dis. 47:1041–1050.

19. Guinea, J., S. Recio, T. Pela´ez, M. Torres-Narbona, and E. Bouza.2008. Clinical isolates of Aspergillus species remain fully susceptible to voricon-azole in the post-voriconvoricon-azole era. Antimicrob. Agents Chemother. 52:3444– 3446.

20. Hadley, S., J. A. Martinez, L. McDermott, et al. 2002. Real-time antifungal susceptibility screening aids management of invasive yeast infections in im-munocompromised patients. J. Antimicrob. Chemother. 49:415–419. 21. Herbrecht, R., D. W. Denning, T. F. Patterson, J. E. Bennett, R. E. Green,

J. W. Oestmann, W. V. Kern, K. A. Marr, P. Ribaud, O. Lortholary, R. Sylvester, R. H. Rubin, J. R. Wingard, P. Stark, C. Durand, D. Caillot, E. Thiel, P. H. Chandrasekar, M. R. Hodges, H. T. Schlamm, P. F. Troke, and B. de Pauw.2002. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N. Engl. J. Med. 347:408–415.

22. Hospenthal, D. R., C. K. Murray, and M. G. Rinaldi. 2004. The role of antifungal susceptibility testing in the therapy of candidiasis. Diagn. Micro-biol. Infect. Dis. 48:153–160.

23. Howard, S. J., I. Webster, C. B. Moore, R. E. Gardiner, S. Park, D. S. Perlin,

and D. W. Denning.2006. Multi-azole resistance in Aspergillus fumigatus. Int. J. Antimicrob. Agents 28:450–453.

24. Johnson, E. M. 2008. Issues in antifungal susceptibility testing. J. Antimi-crob. Chemother. 61(Suppl. 1):i13–i18.

25. Kahlmeter, G., D. F. J. Brown, F. W. Goldstein, A. P. MacGowen, J. W.

Mouten, A. Osterlund, A. Rodloff, M. Steinbakk, P. Urbaskova, and A. Vatopoulos.2003. European harmonization of MIC breakpoints for antimi-crobial susceptibility testing of bacteria. J. Antimicrob. Chemother. 52:145– 148.

26. Kanafani, Z. A., and J. R. Perfect. 2008. Resistance to antifungal agents: mechanisms and clinical impact. Clin. Infect. Dis. 46:120–128.

27. Lass, F., M. Cuenca-Estrella, D. W. Denning, and J. L. Rodriguez-Tudela. 2006. Antifungal susceptibility testing in Aspergillus spp. according to EUCAST methodology. Med. Mycol. 44:325.

28. Manavathu, E. K., J. A. Vazquez, and P. H. Chandrasekar. 1999. Reduced susceptibility in laboratory-selected mutants of Aspergillus fumigatus to itra-conazole due to decreased intracellular accumulation of the antifungal agent. Int. J. Antimicrob. Agents 12:213–219.

29. Mann, P. A., R. M. Parmegiani, S.-Q. Wei, C. A. Mendrick, X. Li, D.

Loebenberg, B. DiDomenico, R. S. Hare, S. S. Walker, and P. M. McNicholas.

2003. Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cyto-chrome P450 14␣-demethylase. Antimicrob. Agents Chemother. 47:577–581. 30. Marr, K. A., R. A. Carter, B. Boechkh, P. Martin, and L. Corey. 2002.

TABLE 2. Cross-resistance between itraconazole, posaconazole,

and voriconazole among 43 isolates of A. fumigatus with

decreased susceptibility to itraconazole

Antifungal agent MIC (␮g/ml)a % of MICsⱕ ECV Range Mode 50% 90%

Itraconazole

2–

⬎8

2

2

⬎8

0

Posaconazole

0.006–2

0.5

0.5

1

46.5

Voriconazole

0.12–4

0.25

0.25

1

93.0

a_{50% and 90%, MIC}

Invasive aspergillosis in allogeneic stem cell transplant recipients: change in epidemiology and risk factors. Blood 100:4358–4366.

31. Mellado, E., G. Garcia-Effron, L. Alcazar-Fuoli, M. Cuenca-Estrella, and

J. L. Rodriguez-Tudela.2004. Substitutions at methionine 220 in the

14␣-sterol demethylase (Cyp51A) of Aspergillus fumigatus are responsible for resistance in vitro to azole antifungal drugs. Antimicrob. Agents Chemother.

48:2747–2750.

32. Mellado, E., G. Garcia-Effron, M. J. Buitrago, L. Alcazar-Fuoli, M.

Cuenca-Estrella, and J. L. Rodriguez-Tudela.2005. Targeted gene disruption of the 14-␣ sterol demethylase (cyp51A) in Aspergillus fumigatus and its role in azole drug susceptibility. Antimicrob. Agents Chemother. 49:2536–2538. 33. Mellado, E., G. Garcia-Effron, L. Alca´zar-Fuoli, W. J. G. Melchers, P. E.

Verweij, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela.2007. A new

Aspergillus fumigatus resistance mechanism conferring in vitro

cross-resis-tance to azole antifungals involves a combination of cyp51A alterations. Antimicrob. Agents Chemother. 51:1897–1904.

34. Mosquera, J., and D. W. Denning. 2002. Azole cross-resistance in Aspergillus

fumigatus. Antimicrob. Agents Chemother. 46:556–557.

35. Nascimento, A. M., G. H. Goldman, S. Park, S. A. Marras, G. Delmas, U.

Oza, K. Lolans, M. N. Dudley, P. A. Mann, and D. S. Perlin.2003. Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resistance to itraconazole. Antimicrob. Agents Chemother. 47:1719–1726. 36. Neofytos, D., D. Horn, E. Anaissie, et al. 2009. Epidemiology and outcome of

invasive fungal infections in adult hematopoietic stem cell transplant recip-ients: analysis of multicenter PATH Alliance Registry. Clin. Infect. Dis.

48:265–273.

37. Patterson, T. F., W. R. Kirkpatrick, M. White, et al. 2000. Invasive aspergil-losis: disease spectrum, treatment practices, and outcomes. Medicine 79: 250–260.

38. Perfect, J. R., K. A. Marr, T. J. Walsh, R. N. Greenberg, B. Dupont, J. de la

Torrea-Cisneros, G. Just-Nubling, H. T. Schlamm, I. Lutsar, A. Espinel-Ingroff, and E. Johnson.2003. Voriconazole treatment for less common, emerging, or refractory fungal infections. Clin. Infect. Dis. 36:1122–1131. 39. Pfaller, M. A. 2008. New developments in the antifungal susceptibility testing

of Candida. Curr. Fungal Infect. Rep. 2:125–133.

40. Pfaller, M. A., and D. J. Diekema. 2007. Epidemiology of invasive candidi-asis: a persistent public health problem. Clin. Microbiol. Rev. 20:133–163. 41. Pfaller, M. A., and D. J. Diekema. 2007. Azole antifungal drug

cross-resis-tance: mechanisms, epidemiology, and clinical significance. J. Invasive Fun-gal Infect. 1:74–92.

42. Pfaller, M. A., S. A. Messer, L. Boyken, C. Rice, S. Tendolkar, R. J. Hollis,

and D. J. Diekema.2008. In vitro survey of triazole cross-resistance among more than 700 clinical isolates of Aspergillus species. J. Clin. Microbiol.

46:2568–2572.

43. Rodriguez-Tudela, J. L., J. P. Donnelly, M. A. Pfaller, E. Chryssantou, P.

Warn, D. W. Denning, A. Espinel-Ingroff, F. Barchiesi, and M. Cuenca-Estrella.2007. Statistical analysis of correlation between fluconazole MICs for Candida spp. assessed by standard methods set forth by the European Committee on Antimicrobial Susceptibility Testing (E.Dis. 7.1) and CLSI (M27-A2). J. Clin. Microbiol. 45:109–111.

44. Rodriguez-Tudela, J. L., L. Alcazar-Fuoli, E. Mellado, A.

Alastruey-Izqui-erdo, A. Monzon, and M. Cuenca-Estrella.2008. Epidemiological cut-offs and cross-resistance to azole drugs in Aspergillus fumigatus. Antimicrob. Agents Chemother. 52:2468–2472.

45. Turnidge, J., G. Kahlmeter, and G. Kronvall. 2006. Statistical characteriza-tion of bacterial wild-type MIC value distribucharacteriza-tions and the determinacharacteriza-tion of epidemiological cut-off values. Clin. Microbiol. Infect. 12:418–425. 46. Turnidge, J., and D. L. Paterson. 2007. Setting and revising antibacterial

susceptibility breakpoints. Clin. Microbiol. Rev. 20:391–408.

47. Verweij, P. E., E. Mellado, and W. J. Melchers. 2007. Multiple-triazole-resistant aspergillosis. N. Engl. J. Med. 356:1481–1483.

48. Walsh, T. J., I. Raad, T. F. Patterson, P. Chandrasekar, G. R. Donowitz, R.

Graybill, R. E. Greene, R. Hachem, S. Hadley, R. Herbrecht, A. Langston, A. Louie, P. Ribaud, B. H. Segal, D. A. Stevens, J. H. van Burik, C. S. White, G. Corcoran, J. Gogate, G. Krishna, L. Pedicone, C. Hardalo, and J. R. Perfect.

2007. Treatment of invasive aspergillosis with posaconazole in patients who are refractory to or intolerant of conventional therapy: an externally con-trolled trial. Clin. Infect. Dis. 44:2–12.

49. Walsh, T. J., E. J. Annaissie, D. W. Denning, et al. 2008. Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin. Infect. Dis. 46:327–360.

50. Xiao, L., V. Madison, A. S. Chau, D. Loebenberg, R. E. Palermo, and P. M.

McNicholas. 2004. Three-dimensional models of wild-type and mutated forms of cytochrome P450 14-␣ sterol demethylases from Aspergillus

fumiga-tus and Candida albicans provide insights into posaconazole binding.