Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Garcia-Effron, G. Articles by Perlin, D. S. Search for Related Content PubMed PubMed Citation Articles by Garcia-Effron, G. Articles by Perlin, D. S.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, April 2008, p. 1200-1206, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02330-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid Detection of Triazole Antifungal Resistance in Aspergillus fumigatus

Guillermo Garcia-Effron,¹ Amanda Dilger,¹ Laura Alcazar-Fuoli,² Steven Park,¹ Emilia Mellado,² and David S. Perlin^1*

Public Health Research Institute, International Center for Public Health, UMDNJ-New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103-3535,¹ Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo Km2, Majadahonda, Madrid 28220, Spain²

Received 4 December 2007/ Returned for modification 6 January 2008/ Accepted 22 January 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Triazole resistance in Aspergillus fumigatus is an uncommon but rising phenomenon. Susceptibility testing is rarely performed and can take 48 h or longer, which is an impediment to effective therapy. Molecular diagnostic probing of well-defined resistance mechanisms, which serve as surrogate markers, provides an alternative approach to rapidly (within hours) and efficiently identify resistant strains. The mechanisms of triazole resistance in A. fumigatus are limited to amino acid substitutions in the drug target Cyp51A and include amino acid substitutions at the positions Gly 54, Gly 138, Met 220, and Leu 98, coupled with a tandem repetition in the gene promoter. We report the development of a real-time PCR assay utilizing molecular beacons to assess triazole resistance markers in A. fumigatus. When combined in a multiplex platform, the assay provides a comprehensive evaluation of drug resistance in A. fumigatus.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Numerous advances in transplant medicine over the past decade have enabled patients to live longer with reduced mortality. However, infectious complications, such as invasive pulmonary aspergillosis (IA) continue to be a major cause of morbidity and mortality in such patients, as well as those with a variety of hematological malignancies. Aspergillus fumigatus accounts for most of these infections and is the most important airborne-pathogenic fungus (24, 25). IA remains difficult to diagnose, at an early stage, despite advances in imaging technology and the development of antigen-based serological diagnostics (15, 22). Hence, IA is still associated with a high mortality rate that ranges from 30 to 90% (4, 7, 20, 21, 30, 43).

The treatment of IA typically involves primary therapy with an expanded-spectrum triazole drug, which targets the sterol 14- sterol demethylase, a key enzyme in the ergosterol pathway (12, 25). These drugs include itraconazole (ITC), voriconazole (VRC), and posaconazole (PSC), which all have good in vitro and in vivo activity against A. fumigatus (8, 14, 16, 19, 32, 35). Unlike Candida spp., primary resistance does not appear to be a problem, since most Aspergillus spp. are highly susceptible to these drugs (10). However, as the incidence of IA infections rise (25), there are an increasing number of reports of secondary acquired resistance in clinical strains of A. fumigatus strains (3, 6, 8, 9, 13, 18, 23, 28, 29, 31). It was recently reported in The Netherlands that, since 2002, a significant number of clinical A. fumigatus isolates with multiple resistance to VRC, ITC, ravuconazole (RVC), and PSC have been detected (44).

Triazole resistance mechanisms in Aspergillus, like Candida, can involve both overexpression of drug efflux transporters (ABC- and MFS-type) and modification of the target site 14- sterol demethylase, encoded by cyp51 (9, 23, 33, 39). In A. fumigatus there are two distinct but related 14- sterol demethylase (cyp51) proteins encoded by cyp51A and cyp51B (27), although only mutations in Cyp51A are important for clinical resistance. These mutations confer several different susceptibility profiles: (i) resistance to ITC and PSC associated with amino acid substitutions at Cyp51A glycine 54 (Gly 54) (9, 23, 33); (ii) a pattern of resistance to ITC and high VRC, RVC, and PSC MICs (strains exhibiting this susceptibility profile harbor amino acid substitutions at methionine 220 [Met 220]) (3, 28); (iii) a pattern of azole cross-resistance associated with higher cyp51A expression produced by a tandem repeat (TR) of a 34-bp sequence in the cyp51A gene promoter in combination with an amino acid substitution at Cyp51A leucine 98 (TR-L98H) (29, 44); and (iv) a triazole cross-resistance related to an amino acid change at Cyp51A glycine 138 to cysteine (G138C) (18).

In vitro susceptibility testing for clinical isolates of A. fumigatus is time-consuming and expensive, and most clinical labs do not routinely perform such tests. However, for some institutions with patients at high risk for mold infections, such tests are warranted given the increasing number of high MIC isolates reported for A. fumigatus (11, 14, 16, 44). In an effort to improve the identification of triazole resistance in primary isolates of A. fumigatus, we describe a multiplex real-time PCR assay utilizing detection by allele-specific molecular beacons (MBs) to rapidly and accurately assess A. fumigatus cyp51A gene mutations conferring resistance to triazole drugs.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A. fumigatus strains. A total of 73 independent A. fumigatus strains were used through this work. A total of 49 A. fumigatus strains (43 clinical strains) were obtained from the Mycelial Collection of the Spanish National Center for Microbiology. Eleven strains belong to the Public Health Research Institute (PHRI) collection, eight of them were A. fumigatus clinical isolates (kindly provided by D. Denning, Manchester University, Manchester, United Kingdom) (18) and three laboratory-derived mutants (33). Moreover, 10 A. fumigatus with known cyp51A mutations (G54V, G54E, G54R, G54W, TR-L98H, G138C, M220V, M220K, M220T, and M220I) and 3 A. fumigatus wild-type (WT) strains (ATCC 13073, Af293, and AfR21) were used to validate the probes in this work. A. flavus ATCC 204304 and A. fumigatus ATCC 204305 were included among the isolates as reference strains for antifungal MIC testing.

Blinded study design. Each laboratory sequenced the cyp51A gene (coding sequence and promoter) and established the MICs for their strains, and DNA samples were prepared in the Servicio de Micología (Spain). A collection of 60 A. fumigatus genomic DNA samples was assembled and a blinded code number was assigned. The panel of A. fumigatus DNAs was returned to the PHRI Center, where the multiplex MB real-time PCR assay was performed. MB real-time results were sent to Spain to be evaluated with known genotypic indicators from DNA sequencing. Finally, all data was compiled in the PHRI Center.

DNA isolation. A. fumigatus strains were grown in either potato-dextrose agar or YPD (yeast extract [2%], Bacto peptone [4%], dextrose [4%]) broth medium at 37°C, and DNA extraction and purification were done as reported earlier (17, 27) or isolated with the Q-Biogene FastDNA kit (Irvine, CA) according to the manufacturer's instructions.

Antifungals and susceptibility testing. ITC (from Janssen Pharmaceutical, Madrid, Spain), VRC (from Pfizer S.A., Madrid, Spain), RVC (Bristol-Myers Squibb, Madrid, Spain), and PSC (Schering Plough, Madrid, Spain) were obtained as standard powder from their respective manufacturers. The individual MICs were determined by following the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) reference method (34), with modifications (5, 36, 38).

A. fumigatus cyp51A gene DNA sequencing. The full coding sequences and promoter (–500 from the start codon) of cyp51A were amplified and sequenced in both directions, as previously described (27). In Spain, DNA sequencing was performed by using a BigDye terminator cycle sequencing ready-reaction system (Applied Biosystems, Madrid, Spain) according to the manufacturer's instructions. Sequence analysis was performed on an ABI Prism 377 DNA sequencer (Applied Biosystems) using the facilities available at the Biopolymers Unit at Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. In the PHRI Center, DNA sequencing was performed with a CEQ dye terminator cycle sequencing QuickStart kit (Beckman Coulter, Fullerton, CA) according to the manufacturer's recommendations. Sequencing analyses were done with the CEQ 8000 genetic analysis system software (Beckman Coulter) and with the BioEdit sequence alignment editor (Ibis Therapeutics, Carlsbad, CA).

MB and PCR and target primer design. The A. fumigatus gene cyp51A sequence with the GenBank accession number AF338659 was used for MB and primer design. All MBs and PCR primers used in the study are listed in Table 1. MBs covering the loci Gly 54, Leu 98, Gly 138, and Met 220 of the cyp51A coding sequence and the 34-bp TR in the cyp51A promoter (–288 and –322 from the start codon) were designed with Beacon Designer software (version 2.12; PREMIER Biosoft International, Palo Alto, CA). MBs were labeled with the following fluorophores (5' ends): 5-carboxyfluorescein (FAM), 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (HEX), CAL Flour Red 610 (CFR), and Quasar 670 (Q670). The 3' ends were labeled with benzoic acid succinimidyl ester (DABCYL) for FAM and HEX 5'-labeled MBs and with Black Hole Quencher (BHQ) when the 5' labeling was done with CFR or Q670. All of the MBs were purchased from Biosearch Technologies, Inc. (Novato, CA). PCR primers were designed by using the oligonucleotide design tool of the IDT SciTools (Integrated DNA Technologies, Coralville, IA) and were purchased from Integrated DNA Technologies.

View this table: [in this window] [in a new window]

TABLE 1. Oligonucleotide primers and molecular beacon sequences used in this study

MB thermal denaturation profiling. The oligonucleotides used as MB targets are listed in Table 1. Target sequences were designed to be complementary to a specific MB. Target-MB hybridization profiling was performed with the Stratagene Mx3005P multiplex quantitative PCR system (Stratagene, La Jolla, CA) using the "MB melting curve" option in the software for data monitoring and analysis. Each hybridization reaction mixture contained 1x Core PCR buffer (Stratagene), 4 mM MgCl₂, 100 pmol of individual target oligonucleotides, and 5 pmol of MBs. The test reaction mixtures were subjected to heating at 95°C for 1 min and a subsequent cooling down in 72 10-s steps with a temperature gradient of –0.5°C/step. Fluorescence outputs were measured at the end of each step. The melting temperatures values were obtained by the Mx3005P software using the fluorescence output first derivative [–Rn'(T)]. Each MB and corresponding primer set were validated in real-time assays using DNA from specific A. fumigatus reference strains at an annealing temperature of 55°C. In all cases, the PCR detection of the mutant or WT allele correlated with the sequences obtained for the reference DNAs.

Real-time PCR. Real-time PCR experiments were performed on a Stratagene Mx3005P multiplex quantitative PCR system using the "quantitative PCR (multiple standards)" setting. Eurogentec qPCR MasterMix-No ROX (Eurogentec North America, Inc., San Diego, CA) was used for all reactions. Each real-time PCR was carried out in a 50-µl reaction volume containing 25 µl of 2x Eurogentec qPCR MasterMix, 20 pmol of each MB, 25 pmol of each corresponding primer, and 1 ng of A. fumigatus genomic DNA. In multiplex PCR experiments, the MB and primer concentrations were changed to 10 pmol for G138-MB and G54-MB, 20 pmol for L98-MB and M220-MB, 12.5 pmol for the G138-F and G138-R primers, and 25 pmol for the G54-F, G54-R, L98-F, L98-R, M220-F, and M220-R primers (Table 1). Amplifications were performed according to the following protocol: 1 cycle of 10 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C. The annealing temperature was changed to 58°C in multiplex (MB panel 1) and duplex (MB panels 2A, 2B, and 2C) PCR experiments to improve the allele discrimination. The fluorescence measurement was done during the 30 s of the annealing step. The Mx3005P filters were set to recover fluorescence signals by four of its five channels using FAM (495 nm), HEX (535 nm), CFR (585 nm), and Q70 (670 nm) filters.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Multiplex real-time detection. A. fumigatus triazole clinical resistance has been strictly linked to cyp51A mutations (18, 26). Moreover, each mutation confers a particular triazole resistance pattern with the different triazole drugs. Therefore, specific mutations can be used as surrogate markers for phenotypic triazole resistance. A two-tier real-time PCR assay with MB detection was used to assess the triazole-resistant phenotypes of A. fumigatus strains. In the first tier, a dropout methodology was used to assess the presence of mutations at four resistance loci, Gly 54, Leu 98, Gly 138, and Met 220, covering all known triazole resistance mutations (Fig. 1). A multiplexed panel of MB probes was developed that recognized only WT sequences at each resistance loci. However, by taking advantage of the allele discriminating properties of MB (42), mutations occurring at a given locus prevent the beacons from opening and responding. A signal dropout signifies a mutation at a specific locus. The probability of a random noncoding mutation occurring at a given locus is extremely low, so failure to detect the WT signal strongly signifies a drug resistance mutation and an expected resistance phenotype (Fig. 2). In the first-tier real-time PCR assay, if all four probes fluoresce, then WT sequences were detected, and the strain was consider to be triazole susceptible.

View larger version (7K): [in this window] [in a new window]

FIG. 1. Schematic representation of the A. fumigatus cyp51A locus mutations associated with triazole resistance. The antifungal drugs in the boxed areas include ITR and PSC. The suffix "-R" indicates resistance.

View larger version (32K): [in this window] [in a new window]

FIG. 2. Multiplex assay formats: MB panel conformation with all possible results and interpretations. (A) Primary panel. Each quadrant in the circles represents an allele-specific MB showing fluorescence (+) or an absence of fluorescence (–). The different fluorophores in each MB are represented by circles as followa: white, Q670; striped, CFR; black, FAM; and gray, HEX. BHQ and DABCYL quenchers are represented as small black circles. (B) Secondary panels. Each half circle represents an allele-specific MB. S and R means susceptible and resistant, respectively. *, Mutations other than G138C have never been seen in A. fumigatus. Thus, an expected phenotype is indicated.

For some loci, such as Met 220, all known mutations confer ITC resistance and elevated MICs for all other triazole drugs (28). Thus, a dropout signal for this locus strongly indicates resistance. The situation is a bit more complex at the Gly 54 locus. Mutations producing either G54V, G54E, G54R, or G54W amino acid substitutions confer resistance to ITC. However, a G54W substitution also confers cross-resistance to PSC (9, 23, 46). To address this more refined level of resistance phenotype, a second-tier MB panel was developed that recognizes the specific G54W mutation (Fig. 2B). In this way, it is possible to distinguish between mutations in the primary panel that confer only ITC resistance from those that also confer resistance to PSC. Similarly, a variety of possible amino acid substitutions at Gly 138 could confer triazole drug resistance. However, only a G138C substitution is known to confer cross-resistance to triazole drugs (18). A secondary panel was also used to positively identify this resistance mutation. Finally, a secondary panel was required to assess a L98H modification, since this mutation alone was insufficient to yield a change in susceptibility. However, when coupled with a 34-bp TR in the promoter of cyp51A, the dual mutant displays cross-resistance to multiple triazole drugs (except PSC) (29, 44) (Fig. 2B). The TR alone is also insufficient to confer a resistance phenotype. In all of the secondary probe panels, positive controls recognizing Gly 54 or Gly 138, as appropriate, were incorporated into the multiplexed assay.

Validation of the drug resistance panels. The utility of the two steps multiplex real-time PCR assay was assessed using a blinded collection of 60 A. fumigatus isolates comprising triazole-susceptible (n = 31) and triazole-resistant clinical and laboratory mutants (n = 29) strains (Table 2). The multiplex assay using the primary panel revealed only the presence of WT or mutant cyp51A alleles, while the secondary duplex panels uncovered the therapeutic significance of the particular mutations. Of 60 total strains tested, 35 isolates (58.3%) revealed mutant cyp51A alleles, which corresponded 100% to genotypes conferring the triazole resistance phenotype determined by DNA sequencing (Table 2). The mutant genotypes indicated that these strains were likely to be triazole resistant.

View this table: [in this window] [in a new window]

TABLE 2. Comparison of results from real-time MB panels 1 and 2 with DNA sequencing and in vitro susceptibilities of the A. fumigatus samples included in this study

However, triazole resistance was confirmed in only 29 of the 35 A. fumigatus cyp51A mutants (Table 2). The remaining six isolates showed either a T364A mutation conferring the L98H amino acid change (n = 3) or a TR in the promoter (n = 3) (Table 2), but these mutations alone were insufficient to confer drug resistance. Thus, the 29 strains identified by molecular testing were found to have a resistant phenotype, which correlated 100% with drug susceptibility testing. When the second tier panel results were compared directly to DNA sequencing data, three azole-susceptible mutants harboring cyp51A promoter alterations failed to be detected by the primary MB multiplex assays. The reason for this false-negative result reflected the design of the primary panel, which was intended to detect resistance mutations within the coding region of cyp51A. In fact, all three strains harboring promoter mutations were fully susceptible to azoles because these mutations alone are insufficient to confer resistance.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Early and rapid detection of Aspergillus infections with an accurate assessment of potential drug resistance is essential for effective clinical management of patients with invasive disease (7). In clinical samples, triazole resistance rates vary between 2 and 6.6% (13, 26, 45). At present, in vitro azole susceptibility testing according to the Clinical and Laboratory Standards Institute protocol takes at least 48 h (34, 37), and most testing is performed in reference laboratories, which can cause additional delays in reporting. This lag often results in the adoption of empirical treatment, which may be ineffective if the infecting strain is drug resistant. To circumvent this delay, we have developed a comprehensive multiplexed PCR assay to assess rapidly triazole resistance. The assay takes advantage of allele discriminating MB probes, which have previously been used to detect ITC and caspofungin resistance (1, 2, 41). To be effective for clinical applications, the new assay was designed in a two-tier format to capture a broad range of mutations known to confer triazole resistance in clinical isolates of A. fumigatus (Fig. 1) (3, 18, 28, 29). The first tier consists of a probe panel that effectively distinguishes triazole-susceptible from triazole-resistant A. fumigatus (Fig. 2A). The second tier panel provides possible therapeutic options depending on the mutation detected (Fig. 2B). In this sense, the design is methodologically similar to the design first described by our group (1) to determine the exact mutation in the codon Gly 54 of the cyp51A gene of A. fumigatus. However, the present approach is far more comprehensive, since a wider array of mutations encoding triazole-resistance are assessed, and the assay differentiates between possible different triazole drug resistance patterns. The utility of the assay was confirmed in a blinded study of 60 strains comparing the two-tier panel results with sequencing and MICs. The assay displayed a high-level of detection of triazole resistance associated with cyp51A alterations (Table 2). These promising results suggest that this platform is suitable for use in clinical laboratories. In principle, the platform can be automated and the sensitivity of the PCR system should enable directly detect from respiratory fluids (e.g., sputum and bronchoalveolar lavage), which could reduce the resistance detection time less than 3 to 4 h, including the DNA isolation. As a potential methodological limitation, it could be argued that this platform does not detect triazole resistance unassociated with cyp51A gene alterations (e.g., drug pumps), and it does not detect new triazole resistance mutations that might be present in the A. fumigatus cyp51A gene. However, such mechanisms have rarely been described in clinical isolates, although they have been reported in laboratory mutants (33, 40). Nevertheless, the dynamic platform can accommodate new resistance mutations as they arise. In conclusion, a rapid and simple multiplexed PCR assay is described for assessing genetic alterations in the cyp51A gene associated with triazole resistance in A. fumigatus. The assay provides a comprehensive identification of triazole resistance in clinical isolates of A. fumigatus, and it distinguishes between resistance to various expanded-spectrum triazole drugs.

 
This study was supported by National Institutes of Health grants AI066561 and AI069397 to D.S.P. and by a National Project grant from the Instituto de Salud Carlos III (MPY1175/06) and the Ministerio de Educacion y Ciencia (SAF2005-06541) to E.M.

 
* Corresponding author. Mailing address: Public Health Research Institute, International Center for Public Health, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-3535. Phone: (973) 854-3200. Fax: (973) 854-3101. E-mail: perlinds{at}umdnj.edu

Published ahead of print on 30 January 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. 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.[Abstract/Free Full Text]
2
2. Balashov, S. V., S. Park, and D. S. Perlin. 2006. Assessing resistance to the echinocandin antifungal drug caspofungin in Candida albicans by profiling mutations in FKS1. Antimicrob. Agents Chemother. 50:2058-2063.[Abstract/Free Full Text]
3
3. 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 isolated from a patient with lung aspergilloma. J. Antimicrob. Chemother. 55:31-37.[Abstract/Free Full Text]
4
4. Clark, T. A., and R. A. Hajjeh. 2002. Recent trends in the epidemiology of invasive mycoses. Curr. Opin. Infect. Dis. 15:569-574.[Medline]
5
5. Cuenca-Estrella, M., T. M. Diaz-Guerra, E. Mellado, and J. L. Rodriguez-Tudela. 2001. Influence of glucose supplementation and inoculum size on growth kinetics and antifungal susceptibility testing of Candida spp. J. Clin. Microbiol. 39:525-532.[Abstract/Free Full Text]
6
6. Dannaoui, E., E. Borel, M. F. Monier, M. A. Piens, S. Picot, and F. Persat. 2001. Acquired itraconazole resistance in Aspergillus fumigatus. J. Antimicrob. Chemother. 47:333-340.[Abstract/Free Full Text]
7
7. Denning, D. W. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781-803.[Medline]
8
8. Denning, D. W., S. A. Radford, K. L. Oakley, L. Hall, E. M. Johnson, and D. W. Warnock. 1997. Correlation between in-vitro susceptibility testing to itraconazole and in-vivo outcome of Aspergillus fumigatus infection. J. Antimicrob. Chemother. 40:401-414.[Abstract/Free Full Text]
9
9. Diaz-Guerra, T. M., E. Mellado, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela. 2003. A point mutation in the 14alpha-sterol demethylase gene cyp51A contributes to itraconazole resistance in Aspergillus fumigatus. Antimicrob. Agents Chemother. 47:1120-1124.[Abstract/Free Full Text]
10
10. 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. 337.[Abstract/Free Full Text]
11
11. Fortun, J., P. Martin-Davila, M. A. Sanchez, V. Pintado, M. E. Alvarez, A. Sanchez-Sousa, and S. Moreno. 2003. Voriconazole in the treatment of invasive mold infections in transplant recipients. Eur. J. Clin. Microbiol. Infect. Dis. 22:408-413.[CrossRef][Medline]
12
12. Ghannoum, M. A., and L. B. Rice. 1999. Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin. Microbiol. Rev. 12:501-517.[Abstract/Free Full Text]
13
13. Gomez-Lopez, A., G. Garcia-Effron, E. Mellado, A. Monzon, J. L. Rodriguez-Tudela, and M. Cuenca-Estrella. 2003. In vitro activities of three licensed antifungal agents against Spanish clinical isolates of Aspergillus spp. Antimicrob. Agents Chemother. 47:3085-3088.[Abstract/Free Full Text]
14
14. Gothard, P., and T. R. Rogers. 2004. Voriconazole for serious fungal infections. Int. J. Clin. Pract. 58:74-80.[Medline]
15
15. Greene, R. 2005. The radiological spectrum of pulmonary aspergillosis. Med. Mycol. 43(Suppl. 1):S147-S154.[CrossRef][Medline]
16
16. Herbrecht, R., D. W. Denning, T. F. Patterson, J. E. Bennett, R. E. Greene, 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.[Abstract/Free Full Text]
17
17. Holden, D. W. 1994. DNA miniprep method for Aspergillus fumigatus (and other filamentous fungi), p. 3-9. In B. Maresca and G. S. Kobayashi (ed.), Molecular biology of pathogenic fungi: a laboratory manual. Telos Press, New York, NY.
18
18. 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.[CrossRef][Medline]
19
19. Imai, J. K., G. Singh, K. V. Clemons, and D. A. Stevens. 2004. Efficacy of posaconazole in a murine model of central nervous system aspergillosis. Antimicrob. Agents Chemother. 48:4063-4066.[Abstract/Free Full Text]
20
20. Kontoyiannis, D. P., and G. P. Bodey. 2002. Invasive aspergillosis in 2002: an update. Eur. J. Clin. Microbiol. Infect. Dis. 21:161-172.[CrossRef][Medline]
21
21. Latge, J. P. 1999. Aspergillus fumigatus and aspergillosis. Clin. Microbiol. Rev. 12:310-350.[Abstract/Free Full Text]
22
22. Maertens, J. A., R. Klont, C. Masson, K. Theunissen, W. Meersseman, K. Lagrou, C. Heinen, B. Crepin, J. Van Eldere, M. Tabouret, J. P. Donnelly, and P. E. Verweij. 2007. Optimization of the cutoff value for the Aspergillus double-sandwich enzyme immunoassay. Clin. Infect. Dis. 44:1329-1336.[CrossRef][Medline]
23
23. 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 cytochrome P450 14-demethylase. Antimicrob. Agents Chemother. 47:577-581.[Abstract/Free Full Text]
24
24. Marr, K. A., R. A. Carter, F. Crippa, A. Wald, and L. Corey. 2002. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin. Infect. Dis. 34:909-917.[CrossRef][Medline]
25
25. Maschmeyer, G., A. Haas, and O. A. Cornely. 2007. Invasive aspergillosis: epidemiology, diagnosis, and management in immunocompromised patients. Drugs 67:1567-1601.[CrossRef][Medline]
26
26. Mellado, E., L. Alcazar-Fuoli, G. Garcia-Effron, A. Alastruey-Izquierdo, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela. 2006. New resistance mechanisms to azole drugs in Aspergillus fumigatus and emergence of antifungal drugs-resistant A. fumigatus atypical strains. Med. Mycol. 44(Suppl.):367-371.[CrossRef]
27
27. Mellado, E., T. M. Diaz-Guerra, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela. 2001. Identification of two different 14-alpha sterol demethylase-related genes (cyp51A and cyp51B) in Aspergillus fumigatus and other Aspergillus species. J. Clin. Microbiol. 39:2431-2438.[Abstract/Free Full Text]
28
28. 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.[Abstract/Free Full Text]
29
29. Mellado, E., G. Garcia-Effron, L. Alcazar-Fuoli, W. J. Melchers, P. E. Verweij, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela. 2007. A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations. Antimicrob. Agents Chemother. 51:1897-1904.[Abstract/Free Full Text]
30
30. Mihu, C. N., E. King, O. Yossepovitch, Y. Taur, A. Jakubowski, E. Pamer, and G. A. Papanicolaou. Risk factors and attributable mortality of late aspergillosis after T-cell-depleted hematopoietic stem cell transplantation. Transpl. Infect. Dis., in press.
31
31. Mosquera, J., and D. W. Denning. 2002. Azole cross-resistance in Aspergillus fumigatus. Antimicrob. Agents Chemother. 46:556-557.[Abstract/Free Full Text]
32
32. Munayyer, H. K., P. A. Mann, A. S. Chau, T. Yarosh-Tomaine, J. R. Greene, R. S. Hare, L. Heimark, R. E. Palermo, D. Loebenberg, and P. M. McNicholas. 2004. Posaconazole is a potent inhibitor of sterol 14-demethylation in yeasts and molds. Antimicrob. Agents Chemother. 48:3690-3696.[Abstract/Free Full Text]
33
33. 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.[Abstract/Free Full Text]
34
34. National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. Document M38-A. NCCLS, Wayne, PA.
35
35. Petraitiene, R., V. Petraitis, C. A. Lyman, A. H. Groll, D. Mickiene, J. Peter, J. Bacher, K. Roussillon, M. Hemmings, D. Armstrong, N. A. Avila, and T. J. Walsh. 2004. Efficacy, safety, and plasma pharmacokinetics of escalating dosages of intravenously administered ravuconazole lysine phosphoester for treatment of experimental pulmonary aspergillosis in persistently neutropenic rabbits. Antimicrob. Agents Chemother. 48:1188-1196.[Abstract/Free Full Text]
36
36. Petrikkou, E., J. L. Rodriguez-Tudela, M. Cuenca-Estrella, A. Gomez, A. Molleja, and E. Mellado. 2001. Inoculum standardization for antifungal susceptibility testing of filamentous fungi pathogenic for humans. J. Clin. Microbiol. 39:1345-1347.[Abstract/Free Full Text]
37
37. Rodriguez-Tudela, J. L., L. Alcazar-Fuoli, A. Alastruey-Izquierdo, A. Monzon, E. Mellado, and M. Cuenca-Estrella. 2007. Time of incubation for antifungal susceptibility testing of Aspergillus fumigatus: can MIC values be obtained at 24 hours? Antimicrob. Agents Chemother. 51:4502-4504.[Abstract/Free Full Text]
38
38. Rodriguez-Tudela, J. L., E. Chryssanthou, E. Petrikkou, J. Mosquera, D. W. Denning, and M. Cuenca-Estrella. 2003. Interlaboratory evaluation of hematocytometer method of inoculum preparation for testing antifungal susceptibilities of filamentous fungi. J. Clin. Microbiol. 41:5236-5237.[Abstract/Free Full Text]
39
39. Silva Ferreira, M. E., J. L. Capellaro, M. E. dos Reis, I. Malavazi, D. Perlin, S. Park, J. B. Anderson, A. L. Colombo, B. A. Arthington-Skaggs, M. H. Goldman, and G. H. Goldman. 2004. In vitro evolution of itraconazole resistance in Aspergillus fumigatus involves multiple mechanisms of resistance. Antimicrob. Agents Chemother. 48:4405-4413.[Abstract/Free Full Text]
40
40. Slaven, J. W., M. J. Anderson, D. Sanglard, G. K. Dixon, J. Bille, I. S. Roberts, and D. W. Denning. 2002. Increased expression of a novel Aspergillus fumigatus ABC transporter gene, atrF, in the presence of itraconazole in an itraconazole resistant clinical isolate. Fungal Genet. Biol. 36:199-206.[CrossRef][Medline]
41
41. Trama, J. P., E. Mordechai, and M. E. Adelson. 2005. Detection of Aspergillus fumigatus and a mutation that confers reduced susceptibility to itraconazole and posaconazole by real-time PCR and pyrosequencing. J. Clin. Microbiol. 43:906-908.[Abstract/Free Full Text]
42
42. Tyagi, S., D. P. Bratu, and F. R. Kramer. 1998. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16:49-53.[CrossRef][Medline]
43
43. Upton, A., K. A. Kirby, P. Carpenter, M. Boeckh, and K. A. Marr. 2007. Invasive aspergillosis following hematopoietic cell transplantation: outcomes and prognostic factors associated with mortality. Clin. Infect. Dis. 44:531-540.[CrossRef][Medline]
44
44. Verweij, P. E., E. Mellado, and W. J. Melchers. 2007. Multiple-triazole-resistant aspergillosis. N. Engl. J. Med. 356:1481-1483.[Free Full Text]
45
45. Verweij P. E., H. A. L. Van Der Lee, J. Kuijpers, E. Snelders, A. J. M. M. Rijs, and W. J. G. Melchers. 2007. Epidemiology of multiple-triazole-resistant Aspergillus fumigatus. Abstr. 47th Intersci. Conf. Antimicrob. Agents Chemother., abstr. M-2018.
46
46. 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 fumigatus and Candida albicans provide insights into posaconazole binding. Antimicrob. Agents Chemother. 48:568-574.[Abstract/Free Full Text]

---

Journal of Clinical Microbiology, April 2008, p. 1200-1206, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02330-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Araujo, R., Espinel-Ingroff, A. (2009). Comparison of Assessment of Oxygen Consumption, Etest, and CLSI M38-A2 Broth Microdilution Methods for Evaluation of the Susceptibility of Aspergillus fumigatus to Posaconazole. Antimicrob. Agents Chemother. 53: 4921-4923 [Abstract] [Full Text]  
• Cannon, R. D., Lamping, E., Holmes, A. R., Niimi, K., Baret, P. V., Keniya, M. V., Tanabe, K., Niimi, M., Goffeau, A., Monk, B. C. (2009). Efflux-Mediated Antifungal Drug Resistance. Clin. Microbiol. Rev. 22: 291-321 [Abstract] [Full Text]  
• Araujo, R., Coutinho, I., Espinel-Ingroff, A. (2008). Rapid method for testing the susceptibility of Aspergillus fumigatus to amphotericin B, itraconazole, voriconazole and posaconazole by assessment of oxygen consumption. J Antimicrob Chemother 62: 1277-1280 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Garcia-Effron, G. Articles by Perlin, D. S. Search for Related Content PubMed PubMed Citation Articles by Garcia-Effron, G. Articles by Perlin, D. S.