This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Perkhofer, S.
Right arrow Articles by Lass-Flörl, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Perkhofer, S.
Right arrow Articles by Lass-Flörl, C.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, February 2007, p. 791-793, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.01024-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Posaconazole Enhances the Activity of Amphotericin B against Aspergillus Hyphae In Vitro{triangledown}

Susanne Perkhofer,* Helene Lugger, Manfred P. Dierich, and Cornelia Lass-Flörl

Department of Hygiene, Microbiology and Social Medicine, Medical University of Innsbruck, Innsbruck, Austria

Received 16 August 2006/ Returned for modification 6 September 2006/ Accepted 8 November 2006


arrow
ABSTRACT
 
The MICs and fractional inhibitory concentrations of posaconazole (POS) and voriconazole (VRZ), alone and in combination with amphotericin B (AMB), for the conidia and hyphae of 100 Aspergillus isolates were evaluated. POS-AMB had more synergistic activity against hyphae (75% of isolates) than VRZ-AMB (37%) and significantly more synergistic activity against hyphae than against conidia (12%).


arrow
TEXT
 
Filamentous fungal pathogens are recognized as major and increasing sources of infection in immunocompromised hosts (6, 18). The most common species causing disease in patients is Aspergillus fumigatus (90%), followed by Aspergillus flavus, Aspergillus niger, and Aspergillus terreus (6, 8). In the meantime, besides amphotericin B (AMB), several other drugs are available as treatments for invasive aspergillosis, such as voriconazole (VRZ), posaconazole (POS), and caspofungin (1, 9, 23, 24). POS was recently approved for use for the treatment of patients with invasive infections that are refractory to other antifungal agents (17). The high rate of mortality from mold infections and the relatively limited efficacies of the current agents have produced a significant interest in the use of polyene- and azole-based combinations for these difficult-to-treat infections (3, 5, 11, 19). The present study evaluated the antifungal activity of either POS or VRZ, alone and in combination with AMB, against the conidia and hyphae of Aspergillus spp. in vitro.

We tested 25 clinical isolates each of A. fumigatus, A. terreus, A. flavus, and A. niger from patients with invasive aspergillosis. The MICs of AMB (Sigma Aldrich, Vienna, Austria), VRZ (kindly provided by Pfizer, Vienna, Austria), and POS (kindly provided by Schering-Plough Research Institute, Kenilworth, NJ) for Aspergillus spp. were tested according to the guidelines described in the CLSI (formerly the NCCLS) M38-A document (15). The MICs for hyphae were tested by the method of Lass-Flörl et al. (12). For all drugs the endpoints were read at 100% inhibition of conidia germination and hyphal growth after 48 h of incubation at 35°C. The endpoints read at 80% resulted in similar MIC data (not shown). Drug combinations were assessed by a checkerboard method. The synergy tests were evaluated by using the MIC endpoints of each drug. The fractional inhibitory concentration (FIC) of each drug for an individual isolate was calculated as the ratio of the concentration of the drug in combination that achieves the MIC endpoint to the MIC of the drug alone obtained by use of that endpoint. FIC index values were interpreted as follows: FIC ≤0.5, synergistic; FIC ≥1 to ≤4, indifferent; and FIC >4, antagonistic (3). Also, the metabolic activity of drug-treated hyphae was determined by their ability to reduce the tetrazolium compound 3-(4,5-dimethl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT), as described elsewhere (13). Duplicate testing was performed on separate days. The results of the in vitro tests were compared by the log-rank test. Significance was defined as a P value of <0.05.

The MIC ranges for conidia and hyphae of Aspergillus spp. are given in Table 1, and the interaction results are given in Table 2. The POS-AMB and VRZ-AMB interactions were synergistic to indifferent. The activity of the combination of POS and AMB was significantly more synergistic against hyphae than against conidia. Visual readings of growth inhibition were correlated with the colorimetric assessments of the metabolic activities of fungi. For hyphae, comparison of the visually determined endpoints with the results of the MTT method revealed that 84.7% of the visually determined MICs corresponded to a 95% or greater reduction in metabolic activity, as measured by determination of the optical density.


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

  		TABLE 1. MIC results for the various Aspergillus spp. tested


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

  		TABLE 2. FIC results for the various Aspergillus spp. tested

Our MIC data for POS and VRZ for conidial suspensions of Aspergillus spp. were comparable to those presented in previously published reports (3, 7, 9, 14, 22). The in vitro activities of POS in combination with AMB against conidia of Aspergillus ranged from indifferent (88% of isolates) to synergistic (12% of isolates). The activity of the VRZ-AMB combination was more equally distributed between indifferent and synergistic (57% and 43%, respectively). The results of a number of in vivo and in vitro studies with the POS-AMB and VRZ-AMB combinations have been published, and all studies showed a lack of antagonism (5, 14, 19, 21). These data and those from our study suggest evidence of a possible additive and/or synergistic effect. However, antifungal combination therapies are still controversial (16, 19). Some studies have suggested that azole antifungal agents would antagonize the effects of AMB (16, 20). Also, in vitro combination tests must evaluate complex events that are difficult to assess (19). Using the same combinations, different authors have observed a spectrum from antagonism to synergism, depending on the methodology and analysis used (16, 19). However, the antagonism of the POS-AMB and VRZ-AMB combinations has not been reported so far.

The onset of invasive Aspergillus infection is associated with the appearance of hyphae (10). Consequently, an agent must be active against the hyphal form in order to be clinically effective. POS and VRZ exerted strong activities against the hyphae of Aspergillus spp.; and the POS-AMB combination yielded excellent results, as the FIC indices were synergistic for 75% of isolates. These FIC indices were significantly (P < 0.05) higher than those for synergistic activity against the conidia (12%). The underlying mechanism for this effect is unknown. Differences in the sterol compositions, the fungal cell membrane transporters, and the cell wall compositions of hyphae and conidia could account for this finding (2). One possible explanation for the synergy could be that polyene (AMB) binding to the fungus destabilizes the membrane and facilitates the entry of the azole (POS or VRZ). Our results suggest that a combination of AMB with POS might be effective against infections due to Aspergillus spp., as shown by Najvar et al. (14).

Clinical studies confirm that POS has a favorable safety profile during treatment of seriously ill patients with invasive fungal infections (3, 9, 17). POS appears to be well tolerated (9, 17), making this substance a promising candidate for use for the prevention and treatment of fungal infections in immunocompromised patients. The lack of antagonism in vitro and in vivo (3, 4, 14, 21) suggests that POS-AMB and VRZ-AMB may be used as combination therapies for the treatment of fungal infections.

In conclusion, POS and VRZ exhibited excellent in vitro activities against the hyphae of Aspergillus spp., and the combination of POS and AMB was significantly more active against hyphae than against conidia (P < 0.05). Further synergy tests with this drug combination are warranted, and the impact of the combination on patient outcomes needs to be further investigated.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Hygiene, Microbiology and Social Medicine, Medical University Innsbruck, Fritz-Pregl Str. 3, A-6020 Innsbruck, Austria. Phone: 43 512 9003 70729. Fax: 43 512 9003 73700. E-mail: Susanne.Perkhofer{at}i-med.ac.at. Back

{triangledown} Published ahead of print on 20 November 2006. Back


arrow
REFERENCES
 
    1
  1. Bauters, T., F. Buyle, R. Peleman, and H. Robays. 2005. Antifungal drugs and rational use of antifungals in treating invasive aspergillosis: the role of the hospital pharmacist. Pharm. World Sci. 27:31-34.[CrossRef][Medline]
    2. 2
  2. Bernard, M., and J. P. Latgè. 2001. Aspergillus fumigatus cell wall: composition and biosynthesis. Med. Mycol. 39(Suppl. 1):9-17.[Medline]
    3. 3
  3. Cacciapuoti, A., M. Gurnani, J. Halpern, C. Norris, R. Patel, and D. Loebenberg. 2005. Interaction between posaconazole and amphotericin B in concomitant treatment against Candida albicans in vivo. Antimicrob. Agents Chemother. 49:638-642.[Abstract/Free Full Text]
    4. 4
  4. Cacciapuoti, A. D., D. Loebenberg, E. Corcoran, F. Menzel, E. L. Moss, C. Norris, M. Michalski, K. Raynor, J. Halpern, C. Mendrick, B. Arnold, B. Antonacci, R. Parmegiani, T. Yarosh-Tomaine, G. H. Miller, and R. S. Hare. 2000. In vitro and in vivo activities of SCH 56592 (posaconazole), a new triazole antifungal agent against Aspergillus and Candida. Antimicrob. Agents Chemother. 44:2017-2022.[Abstract/Free Full Text]
    5. 5
  5. Cuenca-Estrella, M., A. Gomez-Lopez, G. Garcia-Effron, L. Alcazar-Fuoli, E. Mellado, M. Buitrago, and J. L. Rodriguez-Tudela. 2005. Combined activity in vitro of caspofungin, amphotericin B, and azole agents against clinical isolates of Aspergillus fumigatus. Antimicrob. Agents Chemother. 49:1232-1235.[Abstract/Free Full Text]
    6. 6
  6. Denning, D. W. 1996. Therapeutic outcome in invasive aspergillosis. Clin. Infect. Dis. 23:608-615.[Medline]
    7. 7
  7. 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.[Abstract/Free Full Text]
    8. 8
  8. Garber, G. 2001. An overview of fungal infections. Drugs 61(Suppl. 1):1-12.[Medline]
    9. 9
  9. Groll, A., and J. T. Walsh. 2005. Posaconazole: clinical pharmacology and potential for management of fungal infections. Expert Rev. Anti-Infect. Ther. 4:467-487.
    10. 10
  10. Hogan, L. H., B. S. Klein, and S. M. Levitz. 1996. Virulence factors of medically important fungi. Clin. Microbiol. Rev. 4:469-488.
    11. 11
  11. Johnson, M. D., C. MacDougall, L. Ostrosky-Zeichner, J. R. Perfect, and J. H. Rex. 2004. Combination antifungal therapy. Antimicrob. Agents Chemother. 48:693-715.[Free Full Text]
    12. 12
  12. Lass-Flörl, C., M. Nagl, C. Speth, H. Ulmer, M. P. Dierich, and R. Würzner. 2001. Studies of the in vitro activities of voriconazole and itraconazole against Aspergillus hyphae using viability staining. Antimicrob. Agents Chemother. 45:124-128.[Abstract/Free Full Text]
    13. 13
  13. Levitz, S. M., and R. D. Diamond. 1985. A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT. J. Infect. Dis. 5:938-945.
    14. 14
  14. Najvar, L. K., A. Cacciapuoti, S. Hernandez, J. Halpern, R. Bocanegra, M. Gurnani, F. Menzel, D. Loebenberg, and J. R. Graybill. 2004. Activity of posaconazole combined with amphotericin B against Aspergillus flavus infection in mice: comparative studies in two laboratories. Antimicrob. Agents Chemother. 48:758-764.[Abstract/Free Full Text]
    15. 15
  15. National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard. Document M38-A. National Committee for Clinical Laboratory Standards, Wayne, PA.
    16. 16
  16. Polak, A. 1999. The past, present, and future of antimycotic combination therapy. Mycoses 42:355-370.[CrossRef][Medline]
    17. 17
  17. Raad, I. I., J. R. Graybill, A. B. Bustamante, O. A. Cornely, V. Gaona-Flores, C. Afif, D. R. Graham, R. N. Greenberg, S. Hadley, A. Langston, R. Negroni, J. R. Perfect, P. Pitisuttithum, A. Restrepo, G. Schiller, L. Pedicone, and A. J. Ullmann. 2006. Safety of long-term oral posaconazole use in the treatment of refractory invasive fungal infections. Clin. Infect. Dis. 42:1726-1734.[CrossRef][Medline]
    18. 18
  18. Richardson, M. D. 1998. Antifungal therapy in bone marrow failure. Br. J. Haematol. 100:619-628.[CrossRef][Medline]
    19. 19
  19. Steinbach, W., D. A. Stevens, and D. W. Denning. 2003. Combination and sequential therapy for invasive aspergillosis: review of published in vitro and in vivo interactions and 6281 clinical cases from 1966 to 2001. Clin. Infect. Dis. 37:S188-S224.
    20. 20
  20. Sugar, A. M., and X. P. Liu. 1998. Interactions of itraconazole with amphotericin B in the treatment of murine invasive candidiasis. J. Infect. Dis. 77:1660-1663.
    21. 21
  21. Sugar, A. M. 1991. Interactions of amphotericin B and SCH 39304 in the treatment of experimental murine candidiasis: lack of antagonism of a polyene-azole combination. Antimicrob. Agents Chemother. 35:1669-1671.[Abstract/Free Full Text]
    22. 22
  22. Torres, H. A., R. Hachem, R. F. Chemaly, D. P. Kontoyiannis, and I. Raad. 2005. Posaconazole: a broad-spectrum triazole antifungal. Lancet Infect. Dis. 12:775-785.
    23. 23
  23. Walsh, T. J., H. Teppler, G. R. Donowitz, J. A. Maertens, L. R. Baden, A. Dmoszynska, O. A. Cornely, M. R. Bourque, R. J. Lupinacci, C. A. Sable, and B. E. dePauw. 2004. Caspofungin versus liposomal amphotericin B for empirical antifungal therapy in patients with persistent fever and neutropenia. N. Engl. J. Med. 347:1391-1402.
    24. 24
  24. Walsh, T. J., P. Pappas, D. J. Winston, H. M. Lazarus, F. Petersen, J. Raffalli, S. Yanovich, P. Stiff, R. Greenberg, G. Donowitz, M. Schuster, A. Reboli, J. Wingard, C. Arndt, J. Reinhardt, S. Hadley, R. Finberg, M. Laverdiere, J. Perfect, G. Garber, G. Fioritoni, E. Anaissie, and J. Lee. 2002. Voriconazole compared with liposomal amphotericin B for empirical antifungal therapy in patients with neutropenia and persistent fever. N. Engl. J. Med. 346:225-234.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, February 2007, p. 791-793, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.01024-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Perkhofer, S., Lechner, V., Lass-Florl, C. (2009). In Vitro Activity of Isavuconazole against Aspergillus Species and Zygomycetes According to the Methodology of the European Committee on Antimicrobial Susceptibility Testing. Antimicrob. Agents Chemother. 53: 1645-1647 [Abstract] [Full Text]  
  • Perkhofer, S., Locher, M., Cuenca-Estrella, M., Ruchel, R., Wurzner, R., Dierich, M. P., Lass-Florl, C. (2008). Posaconazole Enhances the Activity of Amphotericin B against Hyphae of Zygomycetes In Vitro. Antimicrob. Agents Chemother. 52: 2636-2638 [Abstract] [Full Text]  
  • Perkhofer, S., Jost, D., Dierich, M. P., Lass-Florl, C. (2008). Susceptibility Testing of Anidulafungin and Voriconazole Alone and in Combination against Conidia and Hyphae of Aspergillus spp. under Hypoxic Conditions. Antimicrob. Agents Chemother. 52: 1873-1875 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Perkhofer, S.
Right arrow Articles by Lass-Flörl, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Perkhofer, S.
Right arrow Articles by Lass-Flörl, C.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»