Susceptibility of Filamentous Fungi to Voriconazol
http://www.100md.com
微生物临床杂志 2005年第1期
Department of Microbiology, Faculty of Medicine, University of Cordoba, and Department of Microbiology, Hospital University Reina Sofía, Cordoba, Spain
ABSTRACT
The growing number of fungal infections, coupled with emerging resistance to classical antifungal agents, has led to the development of new agents, among them voriconazole. Susceptibility to voriconazole was tested by two microdilution techniques: the National Committee for Clinical Laboratory Standards reference method M38-A and a colorimetric method, Sensititre YeastOne. The study tested a total of 244 isolates: 223 Aspergillus (136 Aspergillus fumigatus, 37 A. niger, 26 A. terreus, 16 A. flavus, 7 A. nidulans, and 1 A. ustus), 14 Fusarium (8 Fusarium moniliformis, and 6 F. oxysporum), 6 Scedosporium apiospermum, and 1 Rhizomucor pusillus strain and four control strains (Candida parapsilosis ATCC 22019, C. krusei ATCC 6258, A. fumigatus ATCC 204305, and A. flavus ATCC 204304). For all tested species except one F. moniliforme strain and R. pusillus, the MIC, the MIC at which 50% of the isolates are inhibited (MIC50), and MIC90 ranges of 1 μg/ml were obtained for voriconazole, indicating excellent activity against these species. The high rate of agreement between the two methods used (97 to 99%) suggests that the Sensititre YeastOne colorimetric method may be a valuable tool for determining the susceptibility of filamentous fungi to voriconazole.
INTRODUCTION
In recent years, opportunist filamentous fungi have come to pose a serious threat to immunosuppressed patients. The incidence of Aspergillus infection has risen over the last 20 years to become the most frequent life-threatening invasive fungal infection (9, 27). Increased incidence appears to be linked to the growing use of immunosuppressive drugs for the treatment of autoimmune diseases, to the spread of AIDS, to the increasing use of chemotherapy against various tumors, and to the large number of transplants now performed (8, 25, 27).
Many emerging fungi pose problems of intrinsic resistance to classical antifungal agents; this is exacerbated by simultaneous acquired in vitro resistance, i.e., resistant clinical isolates belonging to previously susceptible species (37, 39). Increased resistance has led to the need for new antifungal agents.
These new agents, including voriconazole (5), offer enhanced clinical efficacy coupled with lower toxicity.
In 2002, the National Committee for Clinical Laboratory Standards (NCCLS) issued a protocol for testing the susceptibility of filamentous fungi, published as document M38-A (34). This standardization has enabled valid comparison of the results obtained in different susceptibility tests.
However, these standardized methods are complex and time-consuming, and the growing demand for tests of this sort has prompted a need for more accessible commercial methods. One such method is the Sensititre YeastOne colorimetric antifungal panel (13).
The present study compared two microdilution methods for testing the susceptibility of various species of filamentous fungi to voriconazole, the reference method (NCCLS M38-A) and Sensititre YeastOne, and compared results with those reported by other authors with the reference method.
MATERIALS AND METHODS
Recovery and identification of strains. A total of 244 strains were isolated from various clinical specimens as follows: 102 bronchoaspirates, 98 sputum samples, 14 ear exudate samples, 10 bronchoalveolar lavage samples, 6 biopsy samples, 3 nasal exudate samples, 3 cerebrospinal fluid samples, 2 pharyngeal exudate samples, 2 blood culture samples, 1 brain abscess sample, 1 granuloma sample, 1 conjunctival exudate sample, and 1 pulmonary valve supernatant sample.
The following species were isolated: 223 Aspergillus (136 Aspergillus fumigatus, 37 A. niger, 26 A. terreus, 16 A. flavus, 7 A. nidulans, and 1 A. ustus), 14 Fusarium (8 Fusarium moniliforme and 6 F. oxysporum), 6 Scedosporium apiospermum, and 1 Rhizomucor pusillus strain.
Strains were identified by simple microbiological methods, including gross inspection, and by microscopic examination after microculture and growth on different media (22).
Fungal isolates. All isolates were stored as suspensions in sterile distilled water at –70°C until the study was performed. Prior to testing, each isolate was subcultured on potato dextrose agar (34).
Antifungal agents. (i) NCCLS method:. Standard voriconazole antifungal powder was supplied by Pfizer, Inc., Central Research Division (Groton, Conn.). Dilutions were prepared according to NCCLS guidelines. Final voriconazole concentrations ranged from 0.03 to 16 μg/ml.
(ii) Sensititre YeastOne method. Disposable 96-well plates with incorporated Alamar Blue contained twofold serial dilutions of dried antifungal agents: amphotericin B (0.008 to 16 μg/ml), fluconazole (0.125 to 256 μg/ml), itraconazole (0.008 to 16 μg/ml), ketoconazole (0.008 to 16 μg/ml), flucytosine (0.03 to 64 μg/ml), and voriconazole (0.008 to 16 μg/ml.). Voriconazole occupied the seventh of the eight rows in each plate. The first well on the first row was used as a control (1A) (11, 29).
(iii) Culture media. RPMI 1640 was used both for the reference method M38-A and, with 1.5% added glucose, for the Sensititre YeastOne method (29).
Inoculum preparation. (i) NCCLS method. The inoculum was prepared according to M38-A guidelines (34).
(ii) Sensititre YeastOne method. Isolates were subcultured on potato dextrose agar and incubated at 30°C for 7 to 14 days. After colony growth, a sterile bacteriological loop was scraped gently over the surface growth to dislodge aerial mycelia (conidia and hyphal fragments);a loopful of culture was then suspended in sterile distilled water containing 1% Tween 80 (4). The optical density was adjusted to give a final inoculum concentration of 0.4 x 104 to 5 x 104 CFU/ml by McFarland standard.
Growth medium containing the inoculum suspension was added to plates containing antifungal agents at various concentrations, enabling dissolution of the final antifungal concentration and the pH indicator. Plates were sealed and incubated at 35°C for 48 to 72 h depending on the species.
Reading and interpretation of results: (i) NCCLS method. Readings were performed with the aid of an inverted mirror. As is customary for azole compounds such as voriconazole, the MIC was defined as the lowest concentration that produced a 100% reduction in fungal growth compared to that of the drug-free growth control (10, 16, 28).
Sensititre YeastOne method. Plates were incubated until a change in color from blue (indicative of no growth) to red (indicative of growth) was observed in the growth control well. MICs were determined after 48 to 72 h of incubation. Growth was evident as a change in the Alamar Blue growth indicator from blue to pink; this change facilitates clearer identification of cutoff points than the turbidimetric method, thus reducing the trailing effect characteristic of azole antifungal agents that hinders the interpretation of results when dilution techniques are used (13).
For other antifungal agents, the MIC is defined as the lowest drug concentration at which there is no change of color, i.e., the first blue well. For azoles, however, the change from pink to blue is not always complete, and there may be an intermediate purple color (due to the trailing effect) indicative of partial growth inhibition; in such cases, the MIC was defined as the lowest drug concentration resulting in a purple color (14).
The turbidity of wells was not taken into account when reading MICs. These data were used to calculate the MIC50 and the MIC90 (the antifungal MICs required to inhibit 50 and 90% of isolates, respectively) for genera and species of which more than five strains were tested.
(i) Quality controls. Each test included a reference strain in order to detect any alteration in the antifungal agent should the MIC obtained fail to fall within the reference range.
Four fungal strains with known susceptibility to voriconazole were used as quality controls: Candida parapsilosis ATCC 22019, C. krusei ATCC 6258, Aspergillus fumigatus ATCC 204305, and A. flavus ATCC 204304 (1, 34).
RESULTS
The most frequently encountered genus was Aspergillus, which accounted for 223 of the 244 strains tested, i.e., 91.39% of total strains. The species most often identified was A. fumigatus, with 136 strains (55.73% of all strains). The highest percentage of clinical samples was from bronchoaspirates, which accounted for 102 of the 244 (41.80%) strains tested.
Using both methods, voriconazole MICs for the reference strains tested, C. krusei ATCC 6258, C. parapsilosis ATCC 22019, A. fumigatus 204305, and A. flavus ATCC 204304 were within the range specified in NCCLS documents M27-A2 and M38-A del NCCLS.
All strains produced detectable growth (pink color in the growth control well) after 48 and the longest time to grow was 72 h of incubation. The strains taking the longest time to grow were A. fumigatus (five strains), A. niger (two strains), most Fusarium strains (eight strains), and S. apiospermum (five strains).
Susceptibility to voriconazole, as tested by the Sensititre YeastOne method and the reference method, is shown in Table 1. The maximum MIC range for the various genera was 1 μg/ml, with the exception of a single strain of F. moniliforme, which recorded MICs of 4 μg/ml using Sensititre YeastOne and of 2 μg/ml using the NCCLS reference method. An MIC of 2 μg/ml was found with both methods for the only R. pusillus strain tested and for F. moniliforme with an MIC of 2 μg/ml by one method and an MIC of 4 μg/ml by the other, suggesting that they were both the least susceptible to voriconazole. TheMIC50 and MIC90 values obtained by using the two microdilution methods for the remaining genera also differed by 1 μg/ml.
Differences in MICs of no more than two dilutions between the two methods were used to calculate the percent agreement. Where differences exceeded two dilutions, there was considered to be no agreement (24).
For 86 of 136 (63%) A. fumigatus isolates the voriconazole MICs were determined to be the same by both methods, for 32 of 136 (24%) isolates the voriconazole MICs differed by one dilution, and for 16 of 136 (12%) isolates the voriconazole MICs differed by two dilutions. For 28 of 37 (76%) A. niger isolates the voriconazole MIC was determined to be the same by both methods, 7 of 37 (19%) isolates differed in voriconazole MIC by one dilution, and 1 of 37 (3%) isolates differed in voriconazole MIC by two dilutions. For 21 of 26 (81%) A. terreus isolates the voriconazole MIC was the same as determined by both methods, 3 of 26 (12%) isolates differed in voriconazole MIC by one dilution, and 1 of 26 (4%) isolates differed in voriconazole MIC by two dilutions. For 14 of 16 (88%) A. flavus isolates the voriconazole MIC was the same as determined by both methods, 1 of 16 (6%) isolates differed in voriconazole MIC by one dilution, and 1 of 16 (6%) isolates differed in voriconazole MIC by two dilutions.
The highest rate of agreement between the two methods (99%) was found for A. fumigatus, A. nidulans, A. ustus, and R. pusillus, although the number of strains tested for the latter two species was not significant. Strain-by-strain comparison of the results obtained by the two methods disclosed 97 to 99% agreement for all species tested.
Breakdown of these results by species enabled examination of the differences between the two methods: for A. fumigatus (n = 136) the same voriconazole MIC was recorded for 86 strains, a difference of one dilution was found for 32 strains, and of two dilutions for 16 strains; for 2 additional strains, there was no agreement between microdilution methods. For A. niger, the same voriconazole MIC was recorded for 28 of the 37 strains tested, a difference of one dilution was found for 7 strains and of two dilutions for 1 strain; there was no agreement (>2-dilution difference) for only one strain. The full results for all strains are shown in Table 2.
DISCUSSION
These results were compared to the susceptibility findings of other authors with voriconazole. The voriconazole MIC90 obtained here for A. flavus and A. niger agreed with that 0.5 μg/ml reported by Murphy et al. (33) and differed by 1 or 2 dilutions from the 1 μg/ml obtained by Espinel-Ingroff (15) and from the values recorded by Oakley (35), who report a MIC90 of 1 μg/ml for A. niger and of 2 μg/ml for A. flavus. The MIC90 values obtained here also matched those reported for A. flavus by Johnson et al. (20).
Our results for A. fumigatus agreed with those of both Espinel-Ingroff (15) and Oakley (35) but differed by one or two dilutions from those reported by Johnson (20) and Murphy (33), respectively. The highest MIC obtained by Dannaoui et al. (7) for A. fumigatus was 0.5 μg/ml, i.e., a difference of one dilution with respect to the present results.
Emerging invasive Scedosporium and Fusarium opportunistic infections are thought to be among the most aggressive and treatment-refractory infections (18). Since they are often resistant to both amphotericin B and the azole group, they are associated with high mortality rates (2); hence, the need for a drug with activity against both fungi.
In the present study, S. apiospermum displayed in vitro susceptibility to voriconazole (MIC = 0.5 to 1 μg/ml) (6, 12, 23, 39). Other authors report similar or even greater activity. Johnson et al. (20) found a voriconazole MIC90 of 0.5 μg/ml, with an MIC range of 0.06 to 0.25 μg/ml for the 10 strains tested; Espinel-Ingroff (15) reported a range of 0.06 to 1 μg/ml for 15 strains tested, with a voriconazole MIC90 similar to that recorded by Johnson et al. (20).
As is the case with yeast-like organisms, there are reports of potential cross-resistance between voriconazole and other azoles. However, no significant resistance was observed here. Other authors (32, 38) also report that an increase in the MIC for itraconazole tends to be matched by an increase in that of voriconazole.
The 10 Fusarium strains tested displayed greater susceptibility to voriconazole (MICs of 0.25 to 4 μg/ml as determined by Sensititre and the reference method, respectively) than that reported by other authors, including Johnson et al. (20) and Espinel-Ingroff (15), who obtained MICs of between 2 and 8 μg/ml.
The results obtained here confirm the in vitro efficacy of voriconazole as an alternative treatment for invasive infection by filamentous fungi, particularly by Aspergillus species resistant to other antifungal agents (32).
Voriconazole is currently an excellent option for the treatment of fungal infections; it offers perhaps the broadest spectrum of activity, greater even than that of amphotericin B, with the additional benefit of oral and parenteral administration (21, 36).
The clinical value of the MIC as a predictor of the resistance or susceptibility of filamentous fungi to voriconazole has yet to be established in clinical studies. However, results from animal models and clinical trials suggest that voriconazole is effective in treating Aspergillus and Scedosporium infections (9, 17, 33); some trials report a good correlation between voriconazole MICs and clinical results (17).
Herbrecht et al. (19) found a better overall response to voriconazole than to amphotericin B in immunocompromised patients with invasive aspergillosis infection. Voriconazole was better tolerated than amphotericin B, with fewer adverse effects and a lower rate of abandonment. Survival rates were also higher with voriconazole.
No studies have yet compared susceptibility of filamentous fungi as measured by Sensititre YeastOne versus the reference method. However, comparisons of both methods with other antifungal agents such as amphotericin B and itraconazole have yielded agreement rates of 77 to 93.4% for itraconazole and 66 to 90.2% for amphotericin B (30, 31).
Yamaguchi et al. (40) studied the susceptibility of Aspergillus species to a range of antifungal agents, including voriconazole, by using the reference method and a colorimetric method, obtaining 94% agreement.
The efficacy of voriconazole against the species tested, as measured by using Sensititre YeastOne, was very similar to that reported by other authors (3, 15, 20, 24, 26, 33) by using the NCCLS reference method M38-A, thus underlining the value of the Sensititre method for determining the susceptibility of filamentous fungi to voriconazole.
In summary, the high agreement rate (97 to 99%) between the two methods for all species tested confirms that Sensititre YeastOne is an efficient and effective alternative to the reference method for determining the susceptibility of filamentous fungi to voriconazole in clinical microbiology laboratories.
ACKNOWLEDGMENTS
We thank Josefa Gonzáles Lopez for technical assistance.
REFERENCES
Barry, A. L., M. A. Pfaller, S. D. Brown, A. Espinel-Ingroff, M. A. Ghannoum, C. Knapp, R. P. Rennie, J. H. Rex, and M. G. Rinaldi. 2000. Quality control limits for broth microdilution susceptibility test of ten antifungal agents. J. Clin. Microbiol. 38:3457-3459.
Boutati, E. I., and E. J. Anaissie. 1997. Fusarium, a significant emerging pathogen in patients with hematologic malignancy: ten years' experience at a cancer center and implications for management. Blood 90:999-1008.
Carrillo Muoz, A. J., G. Quindos, M. Ruesga, S. Brio, O. del Valle, V. Rodriguez, J. M. Hernández-Molina, E. Canton, J. Pemán, and P. Santos. 2001. Actividad del itraconazol frente a aislamientos clínicos de Aspergillus spp. y Fusarium spp. determinada por el metodo M-38-P del NCCLS. Rev. Esp. Quimioterap. 14:281-285.
Cermeo-Vivas, J. R., J. M. Torres-Rodriguez. 1998. Metodo espectrofotometrico en la preparacion del inoculo de hongos dematiáceos. Rev. Iberoam. Micol. 15:155-157.
Chandrasekar, P. H., and E. Manavathu. 2001. Voriconazole: a second-generation triazole. Drugs Today 37:135-148.
Cuenca-Estrella, M., B. Ruiz-Díez, J. V. Martínez-Suárez, A. Monzon, and J. L. Rodríguez-Tudela. 1999. Comparative in vitro activity of voriconazole (UK-109.496) and six other antifungal agents against clinical isolates of Scedosporium prolificans and Scedosporium apiospermum. J. Antimicrob. Chemother. 43:149-151.
Dannaoui, E., J. Meletiadis, A. M. Totorano, F. Symoens, N. Nolard, M. A. Viviani, M. A. Piens, B. Lebeau, P. E. Verweij, R. Grillot, et al. 2004. Susceptibility testing of sequential isolates of Aspergillus fumigatus recovered from treated patients. J. Med. Microbiol. 53:129-134.
Denning, D. W. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781-803.
Denning, D. W., P. Ribaud, N. Milpied, D. Caillot, R. Herbrecht, E. Thiel, A. Haas, M. Ruhnke, and H. Lode. 2002. The efficacy and safety of voriconazole in the treatment of acute invasive aspergillosis. Clin. Infect. Dis. 34:563-571.
Espinel-Ingroff, A., C. W. Kish, Jr., T. M. Kerkering, R. A. Fromtling, K. Bartizal, J. N. Galgiani, K. Villareal, M. A. Pfaller, T. Gerarden, M. G. Rinaldi, and A. Fothergill. 1992. Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests. J. Clin. Microbiol. 30:3138-3145.
Espinel-Ingroff, A., J. L. Rodríguez-Tudela, and J. V. Martínez-Suárez. 1995. Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards Reference Macrodilution Method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans. J. Clin. Microbiol. 33:3154-3158.
Espinel-Ingroff, A. 1998. In vitro activity of the new triazole voriconazole (UK-109,496) against opportunistic filamentous and dimorphic fungi and common and emerging yeast pathogens. J. Clin. Microbiol. 36:198-202.
Espinel-Ingroff, A., M. Pfaller, S. A. Messer, C. C. Knapp, S. Killian, H. A. Norris, and M. A. Ghaunoum. 1999. Multicenter comparison of the Sensititre yeast one colorimetric antifungal panel with the National Committee for clinical laboratory standard M27-A reference method for testing clinical isolates of common and emerging Candida spp., Cryptococcus spp., and other yeast-like organisms. J. Clin. Microbiol. 37:591-595.
Espinel-Ingroff, A., D. W. Warnock, J. A. Vazquez, and B. A. Arthington-Skaggs. 2000. In vitro antifungal susceptibility methods and clinical implications of antifungal resistance. Med. Mycol. 38:293-304.
Espinel-Ingroff, A. 2001. In vitro fungicidal activities of voriconazole, itraconazole, and amphotericin B against opportunistic moniliaceous and dematiaceous fungi. J. Clin. Microbiol. 39:954-958.
Espinel-Ingroff, A., M. Barlett, V. Chaturvedi, M. Ghannoum, K. C. Hazen, M. A. Pfaller, M. Rinaldi, T. J. Walsh. 2001. Optimal susceptibility testing conditions for detection of azole resistance in Aspergillus spp.: NCCLS collaborative evaluation. National Committee for Clinical Laboratory Standards. Antimicrob. Agents Chemother. 45:1828-1835.
Fortun, J., P. Martín-Davila, M. A. Sánchez, V. Pintado, M. E. Alvarez, A. Sánchez-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.
Groll, A. H., and T. J. Walsh. 2001. Uncommon opportunistic fungi: new nosocomial threats. Clin. Microbiol. Infect. 7(Suppl. 2):8-24.
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, B. de Pauw, et al. 2002. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N. Engl. J. Med. 347:408-415.
Johnson, E. M., A. Szekely, and D. W. Warnock. 1998. In vitro activity of voriconazole, itraconazole and amphotericin B against filamentous fungi. J. Antimicrob. Chemother. 42:741-745.
Johnson, L. B., and C. A. Kauffman. 2003. Voriconazole a new triazole antifungal agent. Clin. Infect. Dis. 36:630-637.
Klich, M. A. 2002. Identification of common Aspergillus species. Cemtraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
Lazarus, H. M., J. L. Blumer, S. Yanovich, H. Schlamm, and A. Romero. 2002. Safety and pharmacokinetics of oral voriconazole in partients at risk of fungal infections: a dose escalation study. J. Clin. Pharmacol. 42:395-402.
Li, R. K., C. M. Elie, G. E. Clayton, and M. A. Ciblak. 2000. Comparison of a new colorimetric assay with the NCCLS broth microdilution method (M-27A) for antifungal drug MIC determination. J. Clin. Microbiol. 38:2334-2338.
Lin, S. J., J. Schranz, and S. M. Teutsch. 2001. Aspergillosis case-fatality rate: systematic review of the literature. Clin. Infect. Dis. 32:358-366.
Linares, M. J., G. Charriel, F. Solís, and M. Casal. 2004. Comparison of two microdilution methods for testing susceptibility of Candida spp. to voriconazole. J. Clin. Microbiol. 42:899-902.
Manuel, R. J., and C. C. Kibbler. 1998. The epidemiology and prevention of invasive aspergillosis. J. Hosp. Infect. 39:95-109.
Marco, F., M. A. Pfaller, S. Messer, and R. N. Jones. 1998. In vitro activities of voriconazole (UK-109.496) and four other antifungal agents against 394 clinical isolates of Candida spp. Antimicrob. Agents Chemother. 42:161-163.
Martín-Mazuelos, E., E. Canton Lacasa, and A. Espinel-Ingroff. 2001. Otros metodos para el estudio de la sensibilidad a los antifúngicos, p. 16-1-16-9. In J. Pemán, E. Martín-Mazuelos, and M. C. Rubio Calvo (ed.), Guía Práctica de identificacion y diagnostico en Micología Clínica, 1st ed. Revista Iberoamericana de Micología, Bilbao, Spain.
Martín-Mazuelos, E., J. Pemán, A. Valverde, M. Chaves, M. C. Serrano, and E. Canton. 2003. Comparison of the Sensititre YeastOne colorimetric antifungal panel and Etest with the NCCLS M38-A method to determine the activity of amphotericin B and itraconazole against clinical isolates of Aspergillus spp. Antimicrob. Chemother. 52:365-370.
Meletiadis, J., J. W. Mouton, J. F. Meis, B. A. Bouman, and P. E. Verweij. 2002. Comparison of the Etest and the Sensititre colorimetric methods with the NCCLS proposed standard for antifungal susceptibility testing of Aspergillus species. J. Clin. Microbiol. 40:2876-2885.
Mosquera, J., and D. W. Denning. 2002. Azole cross-resistance in Aspergillus fumigatus. Antimicrob. Agents Chemother. 46:556-557.
Murphy, M., E. M. Bernard, T. Ishimaru, and D. Armstrong. 1997. Activity of voriconazole (UK-109,496) against clinical isolates of species and its effectiveness in a experimental model of invasive pulmonary aspergillosis. Antimicrob. Agents Chemother. 41:696-698.
National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of conidium forming filamentous fungi. Proposed standard M38-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Oakley, K. L., C. B. Moore, and D. W. Denning. 1998. In vitro activity of voriconazole against Aspergillus spp. and comparison with itraconazole and amphotericin B. J. Antimicrob. Chemother. 42:91-94.
Pahisa, A. 2003. Introduccion. Monográfico: "Voriconazol." Enf. Infec. Microbiol. Clin. 2:1-2.
Perfect, J. R., and W. A. Schell. 1996. The new fungal opportunists are coming. Clin. Infect. Dis. 22:S112-S118.
Pfaller, M. A., S. A. Messer, R. J. Hollis, and R. N. Jones. 2002. Antifungal activities of posaconazole, ravuconazole, and voriconazole compared to those of itraonazole and amphotericin B against 239 clinical isolates of Aspergillus spp. and other filamentous fungi: report from SENTRY Antimicrobial Surveillance Program 2000. Antimicrob. Agents Chemother. 46:1032-1037.
Pfaller, M. A., D. J. Diekema, S. A. Messer, L. Boyken, R. J. Hollis, R. N. Jones, et al. 2003. In vitro activities of voriconazole, posaconazole, and four licensed systemic antifungal agents against Candida species infrequently isolated from blood. J. Clin. Microbiol. 41:78-83.
Yamaguchi, H., K. Uchida, K. Nagino, and T. Matsunaga. 2002. Usefulness of a colorimetric method for testing antifungal drug susceptibilities of Aspergillus species to voriconazole. J. Infect. Chemother. 8:374-377.(Maria Jose Linares, Guada)
ABSTRACT
The growing number of fungal infections, coupled with emerging resistance to classical antifungal agents, has led to the development of new agents, among them voriconazole. Susceptibility to voriconazole was tested by two microdilution techniques: the National Committee for Clinical Laboratory Standards reference method M38-A and a colorimetric method, Sensititre YeastOne. The study tested a total of 244 isolates: 223 Aspergillus (136 Aspergillus fumigatus, 37 A. niger, 26 A. terreus, 16 A. flavus, 7 A. nidulans, and 1 A. ustus), 14 Fusarium (8 Fusarium moniliformis, and 6 F. oxysporum), 6 Scedosporium apiospermum, and 1 Rhizomucor pusillus strain and four control strains (Candida parapsilosis ATCC 22019, C. krusei ATCC 6258, A. fumigatus ATCC 204305, and A. flavus ATCC 204304). For all tested species except one F. moniliforme strain and R. pusillus, the MIC, the MIC at which 50% of the isolates are inhibited (MIC50), and MIC90 ranges of 1 μg/ml were obtained for voriconazole, indicating excellent activity against these species. The high rate of agreement between the two methods used (97 to 99%) suggests that the Sensititre YeastOne colorimetric method may be a valuable tool for determining the susceptibility of filamentous fungi to voriconazole.
INTRODUCTION
In recent years, opportunist filamentous fungi have come to pose a serious threat to immunosuppressed patients. The incidence of Aspergillus infection has risen over the last 20 years to become the most frequent life-threatening invasive fungal infection (9, 27). Increased incidence appears to be linked to the growing use of immunosuppressive drugs for the treatment of autoimmune diseases, to the spread of AIDS, to the increasing use of chemotherapy against various tumors, and to the large number of transplants now performed (8, 25, 27).
Many emerging fungi pose problems of intrinsic resistance to classical antifungal agents; this is exacerbated by simultaneous acquired in vitro resistance, i.e., resistant clinical isolates belonging to previously susceptible species (37, 39). Increased resistance has led to the need for new antifungal agents.
These new agents, including voriconazole (5), offer enhanced clinical efficacy coupled with lower toxicity.
In 2002, the National Committee for Clinical Laboratory Standards (NCCLS) issued a protocol for testing the susceptibility of filamentous fungi, published as document M38-A (34). This standardization has enabled valid comparison of the results obtained in different susceptibility tests.
However, these standardized methods are complex and time-consuming, and the growing demand for tests of this sort has prompted a need for more accessible commercial methods. One such method is the Sensititre YeastOne colorimetric antifungal panel (13).
The present study compared two microdilution methods for testing the susceptibility of various species of filamentous fungi to voriconazole, the reference method (NCCLS M38-A) and Sensititre YeastOne, and compared results with those reported by other authors with the reference method.
MATERIALS AND METHODS
Recovery and identification of strains. A total of 244 strains were isolated from various clinical specimens as follows: 102 bronchoaspirates, 98 sputum samples, 14 ear exudate samples, 10 bronchoalveolar lavage samples, 6 biopsy samples, 3 nasal exudate samples, 3 cerebrospinal fluid samples, 2 pharyngeal exudate samples, 2 blood culture samples, 1 brain abscess sample, 1 granuloma sample, 1 conjunctival exudate sample, and 1 pulmonary valve supernatant sample.
The following species were isolated: 223 Aspergillus (136 Aspergillus fumigatus, 37 A. niger, 26 A. terreus, 16 A. flavus, 7 A. nidulans, and 1 A. ustus), 14 Fusarium (8 Fusarium moniliforme and 6 F. oxysporum), 6 Scedosporium apiospermum, and 1 Rhizomucor pusillus strain.
Strains were identified by simple microbiological methods, including gross inspection, and by microscopic examination after microculture and growth on different media (22).
Fungal isolates. All isolates were stored as suspensions in sterile distilled water at –70°C until the study was performed. Prior to testing, each isolate was subcultured on potato dextrose agar (34).
Antifungal agents. (i) NCCLS method:. Standard voriconazole antifungal powder was supplied by Pfizer, Inc., Central Research Division (Groton, Conn.). Dilutions were prepared according to NCCLS guidelines. Final voriconazole concentrations ranged from 0.03 to 16 μg/ml.
(ii) Sensititre YeastOne method. Disposable 96-well plates with incorporated Alamar Blue contained twofold serial dilutions of dried antifungal agents: amphotericin B (0.008 to 16 μg/ml), fluconazole (0.125 to 256 μg/ml), itraconazole (0.008 to 16 μg/ml), ketoconazole (0.008 to 16 μg/ml), flucytosine (0.03 to 64 μg/ml), and voriconazole (0.008 to 16 μg/ml.). Voriconazole occupied the seventh of the eight rows in each plate. The first well on the first row was used as a control (1A) (11, 29).
(iii) Culture media. RPMI 1640 was used both for the reference method M38-A and, with 1.5% added glucose, for the Sensititre YeastOne method (29).
Inoculum preparation. (i) NCCLS method. The inoculum was prepared according to M38-A guidelines (34).
(ii) Sensititre YeastOne method. Isolates were subcultured on potato dextrose agar and incubated at 30°C for 7 to 14 days. After colony growth, a sterile bacteriological loop was scraped gently over the surface growth to dislodge aerial mycelia (conidia and hyphal fragments);a loopful of culture was then suspended in sterile distilled water containing 1% Tween 80 (4). The optical density was adjusted to give a final inoculum concentration of 0.4 x 104 to 5 x 104 CFU/ml by McFarland standard.
Growth medium containing the inoculum suspension was added to plates containing antifungal agents at various concentrations, enabling dissolution of the final antifungal concentration and the pH indicator. Plates were sealed and incubated at 35°C for 48 to 72 h depending on the species.
Reading and interpretation of results: (i) NCCLS method. Readings were performed with the aid of an inverted mirror. As is customary for azole compounds such as voriconazole, the MIC was defined as the lowest concentration that produced a 100% reduction in fungal growth compared to that of the drug-free growth control (10, 16, 28).
Sensititre YeastOne method. Plates were incubated until a change in color from blue (indicative of no growth) to red (indicative of growth) was observed in the growth control well. MICs were determined after 48 to 72 h of incubation. Growth was evident as a change in the Alamar Blue growth indicator from blue to pink; this change facilitates clearer identification of cutoff points than the turbidimetric method, thus reducing the trailing effect characteristic of azole antifungal agents that hinders the interpretation of results when dilution techniques are used (13).
For other antifungal agents, the MIC is defined as the lowest drug concentration at which there is no change of color, i.e., the first blue well. For azoles, however, the change from pink to blue is not always complete, and there may be an intermediate purple color (due to the trailing effect) indicative of partial growth inhibition; in such cases, the MIC was defined as the lowest drug concentration resulting in a purple color (14).
The turbidity of wells was not taken into account when reading MICs. These data were used to calculate the MIC50 and the MIC90 (the antifungal MICs required to inhibit 50 and 90% of isolates, respectively) for genera and species of which more than five strains were tested.
(i) Quality controls. Each test included a reference strain in order to detect any alteration in the antifungal agent should the MIC obtained fail to fall within the reference range.
Four fungal strains with known susceptibility to voriconazole were used as quality controls: Candida parapsilosis ATCC 22019, C. krusei ATCC 6258, Aspergillus fumigatus ATCC 204305, and A. flavus ATCC 204304 (1, 34).
RESULTS
The most frequently encountered genus was Aspergillus, which accounted for 223 of the 244 strains tested, i.e., 91.39% of total strains. The species most often identified was A. fumigatus, with 136 strains (55.73% of all strains). The highest percentage of clinical samples was from bronchoaspirates, which accounted for 102 of the 244 (41.80%) strains tested.
Using both methods, voriconazole MICs for the reference strains tested, C. krusei ATCC 6258, C. parapsilosis ATCC 22019, A. fumigatus 204305, and A. flavus ATCC 204304 were within the range specified in NCCLS documents M27-A2 and M38-A del NCCLS.
All strains produced detectable growth (pink color in the growth control well) after 48 and the longest time to grow was 72 h of incubation. The strains taking the longest time to grow were A. fumigatus (five strains), A. niger (two strains), most Fusarium strains (eight strains), and S. apiospermum (five strains).
Susceptibility to voriconazole, as tested by the Sensititre YeastOne method and the reference method, is shown in Table 1. The maximum MIC range for the various genera was 1 μg/ml, with the exception of a single strain of F. moniliforme, which recorded MICs of 4 μg/ml using Sensititre YeastOne and of 2 μg/ml using the NCCLS reference method. An MIC of 2 μg/ml was found with both methods for the only R. pusillus strain tested and for F. moniliforme with an MIC of 2 μg/ml by one method and an MIC of 4 μg/ml by the other, suggesting that they were both the least susceptible to voriconazole. TheMIC50 and MIC90 values obtained by using the two microdilution methods for the remaining genera also differed by 1 μg/ml.
Differences in MICs of no more than two dilutions between the two methods were used to calculate the percent agreement. Where differences exceeded two dilutions, there was considered to be no agreement (24).
For 86 of 136 (63%) A. fumigatus isolates the voriconazole MICs were determined to be the same by both methods, for 32 of 136 (24%) isolates the voriconazole MICs differed by one dilution, and for 16 of 136 (12%) isolates the voriconazole MICs differed by two dilutions. For 28 of 37 (76%) A. niger isolates the voriconazole MIC was determined to be the same by both methods, 7 of 37 (19%) isolates differed in voriconazole MIC by one dilution, and 1 of 37 (3%) isolates differed in voriconazole MIC by two dilutions. For 21 of 26 (81%) A. terreus isolates the voriconazole MIC was the same as determined by both methods, 3 of 26 (12%) isolates differed in voriconazole MIC by one dilution, and 1 of 26 (4%) isolates differed in voriconazole MIC by two dilutions. For 14 of 16 (88%) A. flavus isolates the voriconazole MIC was the same as determined by both methods, 1 of 16 (6%) isolates differed in voriconazole MIC by one dilution, and 1 of 16 (6%) isolates differed in voriconazole MIC by two dilutions.
The highest rate of agreement between the two methods (99%) was found for A. fumigatus, A. nidulans, A. ustus, and R. pusillus, although the number of strains tested for the latter two species was not significant. Strain-by-strain comparison of the results obtained by the two methods disclosed 97 to 99% agreement for all species tested.
Breakdown of these results by species enabled examination of the differences between the two methods: for A. fumigatus (n = 136) the same voriconazole MIC was recorded for 86 strains, a difference of one dilution was found for 32 strains, and of two dilutions for 16 strains; for 2 additional strains, there was no agreement between microdilution methods. For A. niger, the same voriconazole MIC was recorded for 28 of the 37 strains tested, a difference of one dilution was found for 7 strains and of two dilutions for 1 strain; there was no agreement (>2-dilution difference) for only one strain. The full results for all strains are shown in Table 2.
DISCUSSION
These results were compared to the susceptibility findings of other authors with voriconazole. The voriconazole MIC90 obtained here for A. flavus and A. niger agreed with that 0.5 μg/ml reported by Murphy et al. (33) and differed by 1 or 2 dilutions from the 1 μg/ml obtained by Espinel-Ingroff (15) and from the values recorded by Oakley (35), who report a MIC90 of 1 μg/ml for A. niger and of 2 μg/ml for A. flavus. The MIC90 values obtained here also matched those reported for A. flavus by Johnson et al. (20).
Our results for A. fumigatus agreed with those of both Espinel-Ingroff (15) and Oakley (35) but differed by one or two dilutions from those reported by Johnson (20) and Murphy (33), respectively. The highest MIC obtained by Dannaoui et al. (7) for A. fumigatus was 0.5 μg/ml, i.e., a difference of one dilution with respect to the present results.
Emerging invasive Scedosporium and Fusarium opportunistic infections are thought to be among the most aggressive and treatment-refractory infections (18). Since they are often resistant to both amphotericin B and the azole group, they are associated with high mortality rates (2); hence, the need for a drug with activity against both fungi.
In the present study, S. apiospermum displayed in vitro susceptibility to voriconazole (MIC = 0.5 to 1 μg/ml) (6, 12, 23, 39). Other authors report similar or even greater activity. Johnson et al. (20) found a voriconazole MIC90 of 0.5 μg/ml, with an MIC range of 0.06 to 0.25 μg/ml for the 10 strains tested; Espinel-Ingroff (15) reported a range of 0.06 to 1 μg/ml for 15 strains tested, with a voriconazole MIC90 similar to that recorded by Johnson et al. (20).
As is the case with yeast-like organisms, there are reports of potential cross-resistance between voriconazole and other azoles. However, no significant resistance was observed here. Other authors (32, 38) also report that an increase in the MIC for itraconazole tends to be matched by an increase in that of voriconazole.
The 10 Fusarium strains tested displayed greater susceptibility to voriconazole (MICs of 0.25 to 4 μg/ml as determined by Sensititre and the reference method, respectively) than that reported by other authors, including Johnson et al. (20) and Espinel-Ingroff (15), who obtained MICs of between 2 and 8 μg/ml.
The results obtained here confirm the in vitro efficacy of voriconazole as an alternative treatment for invasive infection by filamentous fungi, particularly by Aspergillus species resistant to other antifungal agents (32).
Voriconazole is currently an excellent option for the treatment of fungal infections; it offers perhaps the broadest spectrum of activity, greater even than that of amphotericin B, with the additional benefit of oral and parenteral administration (21, 36).
The clinical value of the MIC as a predictor of the resistance or susceptibility of filamentous fungi to voriconazole has yet to be established in clinical studies. However, results from animal models and clinical trials suggest that voriconazole is effective in treating Aspergillus and Scedosporium infections (9, 17, 33); some trials report a good correlation between voriconazole MICs and clinical results (17).
Herbrecht et al. (19) found a better overall response to voriconazole than to amphotericin B in immunocompromised patients with invasive aspergillosis infection. Voriconazole was better tolerated than amphotericin B, with fewer adverse effects and a lower rate of abandonment. Survival rates were also higher with voriconazole.
No studies have yet compared susceptibility of filamentous fungi as measured by Sensititre YeastOne versus the reference method. However, comparisons of both methods with other antifungal agents such as amphotericin B and itraconazole have yielded agreement rates of 77 to 93.4% for itraconazole and 66 to 90.2% for amphotericin B (30, 31).
Yamaguchi et al. (40) studied the susceptibility of Aspergillus species to a range of antifungal agents, including voriconazole, by using the reference method and a colorimetric method, obtaining 94% agreement.
The efficacy of voriconazole against the species tested, as measured by using Sensititre YeastOne, was very similar to that reported by other authors (3, 15, 20, 24, 26, 33) by using the NCCLS reference method M38-A, thus underlining the value of the Sensititre method for determining the susceptibility of filamentous fungi to voriconazole.
In summary, the high agreement rate (97 to 99%) between the two methods for all species tested confirms that Sensititre YeastOne is an efficient and effective alternative to the reference method for determining the susceptibility of filamentous fungi to voriconazole in clinical microbiology laboratories.
ACKNOWLEDGMENTS
We thank Josefa Gonzáles Lopez for technical assistance.
REFERENCES
Barry, A. L., M. A. Pfaller, S. D. Brown, A. Espinel-Ingroff, M. A. Ghannoum, C. Knapp, R. P. Rennie, J. H. Rex, and M. G. Rinaldi. 2000. Quality control limits for broth microdilution susceptibility test of ten antifungal agents. J. Clin. Microbiol. 38:3457-3459.
Boutati, E. I., and E. J. Anaissie. 1997. Fusarium, a significant emerging pathogen in patients with hematologic malignancy: ten years' experience at a cancer center and implications for management. Blood 90:999-1008.
Carrillo Muoz, A. J., G. Quindos, M. Ruesga, S. Brio, O. del Valle, V. Rodriguez, J. M. Hernández-Molina, E. Canton, J. Pemán, and P. Santos. 2001. Actividad del itraconazol frente a aislamientos clínicos de Aspergillus spp. y Fusarium spp. determinada por el metodo M-38-P del NCCLS. Rev. Esp. Quimioterap. 14:281-285.
Cermeo-Vivas, J. R., J. M. Torres-Rodriguez. 1998. Metodo espectrofotometrico en la preparacion del inoculo de hongos dematiáceos. Rev. Iberoam. Micol. 15:155-157.
Chandrasekar, P. H., and E. Manavathu. 2001. Voriconazole: a second-generation triazole. Drugs Today 37:135-148.
Cuenca-Estrella, M., B. Ruiz-Díez, J. V. Martínez-Suárez, A. Monzon, and J. L. Rodríguez-Tudela. 1999. Comparative in vitro activity of voriconazole (UK-109.496) and six other antifungal agents against clinical isolates of Scedosporium prolificans and Scedosporium apiospermum. J. Antimicrob. Chemother. 43:149-151.
Dannaoui, E., J. Meletiadis, A. M. Totorano, F. Symoens, N. Nolard, M. A. Viviani, M. A. Piens, B. Lebeau, P. E. Verweij, R. Grillot, et al. 2004. Susceptibility testing of sequential isolates of Aspergillus fumigatus recovered from treated patients. J. Med. Microbiol. 53:129-134.
Denning, D. W. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781-803.
Denning, D. W., P. Ribaud, N. Milpied, D. Caillot, R. Herbrecht, E. Thiel, A. Haas, M. Ruhnke, and H. Lode. 2002. The efficacy and safety of voriconazole in the treatment of acute invasive aspergillosis. Clin. Infect. Dis. 34:563-571.
Espinel-Ingroff, A., C. W. Kish, Jr., T. M. Kerkering, R. A. Fromtling, K. Bartizal, J. N. Galgiani, K. Villareal, M. A. Pfaller, T. Gerarden, M. G. Rinaldi, and A. Fothergill. 1992. Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests. J. Clin. Microbiol. 30:3138-3145.
Espinel-Ingroff, A., J. L. Rodríguez-Tudela, and J. V. Martínez-Suárez. 1995. Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards Reference Macrodilution Method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans. J. Clin. Microbiol. 33:3154-3158.
Espinel-Ingroff, A. 1998. In vitro activity of the new triazole voriconazole (UK-109,496) against opportunistic filamentous and dimorphic fungi and common and emerging yeast pathogens. J. Clin. Microbiol. 36:198-202.
Espinel-Ingroff, A., M. Pfaller, S. A. Messer, C. C. Knapp, S. Killian, H. A. Norris, and M. A. Ghaunoum. 1999. Multicenter comparison of the Sensititre yeast one colorimetric antifungal panel with the National Committee for clinical laboratory standard M27-A reference method for testing clinical isolates of common and emerging Candida spp., Cryptococcus spp., and other yeast-like organisms. J. Clin. Microbiol. 37:591-595.
Espinel-Ingroff, A., D. W. Warnock, J. A. Vazquez, and B. A. Arthington-Skaggs. 2000. In vitro antifungal susceptibility methods and clinical implications of antifungal resistance. Med. Mycol. 38:293-304.
Espinel-Ingroff, A. 2001. In vitro fungicidal activities of voriconazole, itraconazole, and amphotericin B against opportunistic moniliaceous and dematiaceous fungi. J. Clin. Microbiol. 39:954-958.
Espinel-Ingroff, A., M. Barlett, V. Chaturvedi, M. Ghannoum, K. C. Hazen, M. A. Pfaller, M. Rinaldi, T. J. Walsh. 2001. Optimal susceptibility testing conditions for detection of azole resistance in Aspergillus spp.: NCCLS collaborative evaluation. National Committee for Clinical Laboratory Standards. Antimicrob. Agents Chemother. 45:1828-1835.
Fortun, J., P. Martín-Davila, M. A. Sánchez, V. Pintado, M. E. Alvarez, A. Sánchez-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.
Groll, A. H., and T. J. Walsh. 2001. Uncommon opportunistic fungi: new nosocomial threats. Clin. Microbiol. Infect. 7(Suppl. 2):8-24.
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, B. de Pauw, et al. 2002. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N. Engl. J. Med. 347:408-415.
Johnson, E. M., A. Szekely, and D. W. Warnock. 1998. In vitro activity of voriconazole, itraconazole and amphotericin B against filamentous fungi. J. Antimicrob. Chemother. 42:741-745.
Johnson, L. B., and C. A. Kauffman. 2003. Voriconazole a new triazole antifungal agent. Clin. Infect. Dis. 36:630-637.
Klich, M. A. 2002. Identification of common Aspergillus species. Cemtraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
Lazarus, H. M., J. L. Blumer, S. Yanovich, H. Schlamm, and A. Romero. 2002. Safety and pharmacokinetics of oral voriconazole in partients at risk of fungal infections: a dose escalation study. J. Clin. Pharmacol. 42:395-402.
Li, R. K., C. M. Elie, G. E. Clayton, and M. A. Ciblak. 2000. Comparison of a new colorimetric assay with the NCCLS broth microdilution method (M-27A) for antifungal drug MIC determination. J. Clin. Microbiol. 38:2334-2338.
Lin, S. J., J. Schranz, and S. M. Teutsch. 2001. Aspergillosis case-fatality rate: systematic review of the literature. Clin. Infect. Dis. 32:358-366.
Linares, M. J., G. Charriel, F. Solís, and M. Casal. 2004. Comparison of two microdilution methods for testing susceptibility of Candida spp. to voriconazole. J. Clin. Microbiol. 42:899-902.
Manuel, R. J., and C. C. Kibbler. 1998. The epidemiology and prevention of invasive aspergillosis. J. Hosp. Infect. 39:95-109.
Marco, F., M. A. Pfaller, S. Messer, and R. N. Jones. 1998. In vitro activities of voriconazole (UK-109.496) and four other antifungal agents against 394 clinical isolates of Candida spp. Antimicrob. Agents Chemother. 42:161-163.
Martín-Mazuelos, E., E. Canton Lacasa, and A. Espinel-Ingroff. 2001. Otros metodos para el estudio de la sensibilidad a los antifúngicos, p. 16-1-16-9. In J. Pemán, E. Martín-Mazuelos, and M. C. Rubio Calvo (ed.), Guía Práctica de identificacion y diagnostico en Micología Clínica, 1st ed. Revista Iberoamericana de Micología, Bilbao, Spain.
Martín-Mazuelos, E., J. Pemán, A. Valverde, M. Chaves, M. C. Serrano, and E. Canton. 2003. Comparison of the Sensititre YeastOne colorimetric antifungal panel and Etest with the NCCLS M38-A method to determine the activity of amphotericin B and itraconazole against clinical isolates of Aspergillus spp. Antimicrob. Chemother. 52:365-370.
Meletiadis, J., J. W. Mouton, J. F. Meis, B. A. Bouman, and P. E. Verweij. 2002. Comparison of the Etest and the Sensititre colorimetric methods with the NCCLS proposed standard for antifungal susceptibility testing of Aspergillus species. J. Clin. Microbiol. 40:2876-2885.
Mosquera, J., and D. W. Denning. 2002. Azole cross-resistance in Aspergillus fumigatus. Antimicrob. Agents Chemother. 46:556-557.
Murphy, M., E. M. Bernard, T. Ishimaru, and D. Armstrong. 1997. Activity of voriconazole (UK-109,496) against clinical isolates of species and its effectiveness in a experimental model of invasive pulmonary aspergillosis. Antimicrob. Agents Chemother. 41:696-698.
National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of conidium forming filamentous fungi. Proposed standard M38-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Oakley, K. L., C. B. Moore, and D. W. Denning. 1998. In vitro activity of voriconazole against Aspergillus spp. and comparison with itraconazole and amphotericin B. J. Antimicrob. Chemother. 42:91-94.
Pahisa, A. 2003. Introduccion. Monográfico: "Voriconazol." Enf. Infec. Microbiol. Clin. 2:1-2.
Perfect, J. R., and W. A. Schell. 1996. The new fungal opportunists are coming. Clin. Infect. Dis. 22:S112-S118.
Pfaller, M. A., S. A. Messer, R. J. Hollis, and R. N. Jones. 2002. Antifungal activities of posaconazole, ravuconazole, and voriconazole compared to those of itraonazole and amphotericin B against 239 clinical isolates of Aspergillus spp. and other filamentous fungi: report from SENTRY Antimicrobial Surveillance Program 2000. Antimicrob. Agents Chemother. 46:1032-1037.
Pfaller, M. A., D. J. Diekema, S. A. Messer, L. Boyken, R. J. Hollis, R. N. Jones, et al. 2003. In vitro activities of voriconazole, posaconazole, and four licensed systemic antifungal agents against Candida species infrequently isolated from blood. J. Clin. Microbiol. 41:78-83.
Yamaguchi, H., K. Uchida, K. Nagino, and T. Matsunaga. 2002. Usefulness of a colorimetric method for testing antifungal drug susceptibilities of Aspergillus species to voriconazole. J. Infect. Chemother. 8:374-377.(Maria Jose Linares, Guada)