Antimicrobial Agents and Chemotherapy, August 2004, p . 3107-3111, Vol . 48, No . 8

In Vitro Activities of Ravuconazole and Four Other Antifungal Agents against Fluconazole-Resistant or -Susceptible Clinical Yeast Isolates

Manuel Cuenca-Estrella,^* Alicia Gomez-Lopez, Emilia Mellado, Guillermo Garcia-Effron, and Juan L . Rodriguez-Tudela

Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadajonda (Madrid), Spain

Received 23 March 2004/ Returned for modification 2 April 2004/ Accepted 18 April 2004

Ravuconazole has exhibited activity in vivo as well . It has been evaluated in several animal models of infection . Those studies have demonstrated that ravuconazole is efficient for the treatment of mucosal candidosis in mice (6), intra-abdominal abscesses due to C . albicans in rats (16), disseminated aspergillosis in guinea pigs (13), and systemic murine histoplasmosis (5) . Likewise, ravuconazole in combination with micafungin showed synergistic effects for the treatment of experimental pulmonary aspergillosis in neutropenic rabbits . The combination led to significant reductions in rates of mortality and fungal burdens compared with those achieved with monotherapies (22) .

Ravuconazole has been found to have very good pharmacokinetic and pharmacodynamic parameters (2) . Eight hours after oral administration of 10 mg/kg of body weight to female rats, the maximum concentration of drug in plasma was 1.68 µg/ml, and the concentrations in tissues were two times higher than the corresponding concentrations in blood (17) . A 2003 study (1) revealed that the critical pharmacodynamic parameter associated with ravuconazole treatment efficacy in a murine model of candidiasis was the area under the concentration-time curve/MIC ratio, with the ratio for ravuconazole being similar to that observed for fluconazole .

Taking into account these data, we have analyzed the activities of ravuconazole against a collection of 564 yeast clinical isolates with decreased susceptibilities in vitro to fluconazole (MICs 16 µg/ml) . Ravuconazole has exhibited activity in vitro against fluconazole-resistant isolates, but a limited number of strains were included in those studies and the majority of nonsusceptible isolates belonged to rare species or to species intrinsically resistant to fluconazole, such as Candida krusei . We have tested a large collection of isolates nonsusceptible to fluconazole, including the most clinically relevant yeast species (11, 23, 25) . In addition, we have compared the results of antifungal susceptibility testing with those obtained with a second collection of 1,232 clinical isolates that were susceptible to fluconazole (MICs < 16 µg/ml) .

Fungi. A collection of 1,796 isolates was included in the study; 1,232 strains were fluconazole susceptible and 466 were susceptible-dose dependent (S-DD; MICs = 16 to 32 µg/ml), and 198 were resistant to fluconazole in vitro (MICs > 64 µg/ml), according to the criteria of the NCCLS (20) . All strains were recovered from 71 hospitals over a period of 9 years, from 1995 to 2003 . The isolates were obtained from a variety of clinical sources, including a total of 935 strains from blood cultures and 352 from tissue biopsy specimens and other deep sites . A total of 509 isolates were recovered from superficial mucous membranes, skin, and nails . Each isolate was obtained from a different patient . The isolates were sent to the Mycology Reference Laboratory of the National Center for Microbiology of Spain for identification or susceptibility testing . The strains were classified into 31 distinct species (Table 1) . The isolates were identified by routine morphological and physiological tests: fermentation of and growth on carbon sources, growth on nitrogen sources, growth at various temperatures, and ability to hydrolyze urea (14) .

 
Antifungal susceptibility testing. Susceptibility testing strictly followed the recommendations proposed by the Antifungal Susceptibility Testing Subcommittee of the European Union Committee on Antibiotic Susceptibility Testing (EUCAST) for the testing of fermentative yeasts (discussion document 7.1) (26) . These recommendations are based on the NCCLS procedure described in document M27-A2 (20), but including some modifications in order to allow automation of the susceptibility testing method and to permit the incubation period to be shortened from 48 to 24 h (7) . Briefly, susceptibility testing included RPMI medium supplemented with 2% glucose as the assay medium, an inoculum size of 10⁵ CFU/ml, and flat-bottom trays; and the results were read with a spectrophotometer . In order to improve the growth of some species, particularly Cryptococcus and Trichosporon species, a minor modification was included (27) . That is, all microplates used to test those species were wrapped with a film sealer to prevent the medium from evaporating, attached to an electrically driven wheel inside the incubator, agitated at 350 rpm, and incubated at 30°C for 48 h . Candida parapsilosis ATCC 22019 and C . krusei ATCC 6258 were used as quality control strains .

The antifungal agents used in the study were as follows: amphotericin B (Sigma Aldrich Quimica S.A., Madrid, Spain), flucytosine (Sigma Aldrich Quimica), fluconazole (Pfizer S.A, Madrid, Spain), itraconazole (Janssen S.A., Madrid, Spain), voriconazole (Pfizer S.A.), and ravuconazole (Bristol-Myers Squibb, Princeton, N.J.) . The MICs were determined at 24 and 48 h . MICs were obtained by measuring the absorbance at 530 nm with an MRXII reader (Dynatech, Cultek, Madrid, Spain) . For amphotericin B the MIC endpoints were defined as the lowest drug concentrations resulting a reduction in growth of 90% or more compared with that of the control growth . For flucytosine and the azole drugs the MIC endpoint was defined as 50% inhibition .

Analysis of results. Differences in proportions were determined by Fisher's exact test or ² analysis . The significance of the differences in MICs was determined by Student's t test (unpaired, unequal variance) . In order to approximate a normal distribution, the MICs were transformed to log₂ values to establish differences in susceptibilities between species . Both on-scale and off-scale results were included in the analysis . The off-scale MICs were converted to the next concentration up or down . The relationship between the distributions of the MICs of the antifungal agents was calculated by use of the Pearson correlation coefficient, which was expressed over a maximum value of 1 . A P value <0.01 was considered significant . Statistical analysis was done with the Statistical Package for the Social Sciences (version 12.0; SPSS S.L, Madrid, Spain) .

Table 1 displays the geometric mean (GM) MICs and the ranges of MICs of the antifungal agents tested, grouped by species and fluconazole susceptibility . The fluconazole-resistant group included fluconazole-resistant and S-DD isolates, according to the criteria of the NCCLS . Overall, ravuconazole exhibited potent activity in vitro, with GM MICs of 0.05 µg/ml . This value was somewhat better than those of amphotericin B, itraconazole, and voriconazole, which had GM MICs of 0.14, 0.07, and 0.06 µg/ml, respectively .

By analysis of the results by fluconazole susceptibility, ravuconazole showed a strong effect in vitro against fluconazole-resistant, S-DD, and susceptible isolates . Its activity was similar to that of amphotericin B and slightly better than those of itraconazole and voriconazole against resistant and S-DD isolates . Ravuconazole was highly active in vitro against fluconazole-susceptible isolates . However, a significant difference in the ravuconazole MICs was observed between those for fluconazole-susceptible strains and those for S-DD and resistant isolates . The ravuconazole MICs for both fluconazole-resistant and S-DD organisms were consistently higher that those for susceptible strains (GM MICs, 0.48 µg/ml for resistant isolates and 0.25 µg/ml for S-DD organisms, compared with a GM MIC of 0.02 µg/ml for susceptible strains [P < 0.01]) . Likewise, similar significant differences were observed between the itraconazole and voriconazole MICs for the two groups of isolates . Differences with statistical relevance were not found when the results were analyzed by the site of isolation .

Notably, the MICs of amphotericin B and flucytosine for fluconazole-resistant and S-DD isolates were significantly higher as well (P < 0.01), indicating that resistance to fluconazole could lead to rises in the MICs of antifungal agents other than azole compounds . Calculation of correlation coefficients showed that the MICs of amphotericin B, flucytosine, itraconazole, voriconazole, and ravuconazole correlated significantly and proportionally with the MICs of fluconazole . As might be expected, correlations between the MICs of fluconazole and those of itraconazole, voriconazole, and ravuconazole were particularly significant, with correlation coefficients of 0.85, 0.86, and 0.82 (P < 0.01), respectively .

Regarding analysis per species, ravuconazole exhibited in vitro activity against the majority of species tested . However, the ravuconazole MIC was 1 µg/ml for 115 of 1,796 isolates (6.4%) . Thirteen of those 115 strains belonged to the fluconazole-susceptible group, 73 were S-DD isolates, and 29 were fluconazole-resistant strains . The ravuconazole MIC was 1 µg/ml for 9 of 92 (9%) fluconazole-resistant and S-DD C . albicans isolates . In this sense, ravuconazole MICs were 1 µg/ml for 12 of 26 (46%) fluconazole-resistant and S-DD Candida tropicalis isolates, 25 of 64 (39%) C . glabrata isolates, 16 of 97 (16%) C . neoformans var . neoformans strains, 8 of 16 (50%) C . neoformans var . gattii isolates, 8 of 24 (33%) Rhodotorula mucilaginosa isolates, and 10 of 16 (62%) fluconazole-resistant and S-DD strains of Trichosporon spp . The ravuconazole MIC was as high as 4 µg/ml for 25 of 564 fluconazole-resistant and S-DD (4.4%) isolates, that is, seven C . tropicalis isolates, four C . glabrata isolates, seven Trichosporon spp., two Debaryomyces hansenii isolates, two Candida haemulonii isolates, two R . mucilaginosa isolates, and one Cryptococcus laurentii isolate .

The MICs for the two quality control strains were consistently within 2 or 3 twofold dilutions . These values agreed with those depicted in discussion document 7.1 of EUCAST and NCCLS document M27-A2 (3, 20, 26) .

The results of this work confirm data reported previously (11, 23) . A study conducted in 2002 with 239 fluconazole-resistant isolates and 463 S-DD isolates indicated that ravuconazole was more active than fluconazole and itraconazole against Candida spp . in vitro . Although the MICs of ravuconazole for C . albicans and C . glabrata isolates that were resistant to fluconazole and itraconazole were also found to be elevated, those isolates that were resistant to fluconazole alone and those for which fluconazole MICs were 16 to 32 µg/ml were susceptible to 1 µg of ravuconazole per ml (25) . In this sense, our study shows that ravuconazole has inhibitory effects in vitro for the majority of fluconazole-resistant and S-DD strains, being as active as amphotericin B . In addition, the ravuconazole MICs were lower than those of itraconazole and voriconazole . By taking into account these data, ravuconazole could be an alternative for the treatment of infections caused by fluconazole-resistant isolates and it could be considered an extended-spectrum triazole . However, the ravuconazole MICs for fluconazole-resistant and S-DD strains were significantly and proportionally higher than the MICs for fluconazole-susceptible isolates (P < 0.01), and ravuconazole MICs were 1 µg/ml for a percentage of both fluconazole-resistant and S-DD organisms, indicating that ravuconazole MICs could also be high for some isolates for which fluconazole MICs are 16 to 32 µg/ml . By analysis of these results by species, with regard to Candida spp., ravuconazole MICs were 1 µg/ml for 46% of fluconazole-resistant and S-DD C . tropicalis isolates and 39% of fluconazole-resistant and S-DD C . glabrata isolates . These proportions were less than 10% for C . albicans, C . parapsilosis, and C . krusei . Ravuconazole MICs were 1 µg/ml for 21% of fluconazole-resistant and S-DD C . neoformans isolates . Fluconazole-resistant and S-DD isolates belonging to other species exhibited resistance to ravuconazole in vitro as well, but in general, the number of resistant strains was lower . Ravuconazole MICs were as high as 4 µg/ml for some clinical isolates of some species with less medical relevance, such as Trichosporon spp .

The MICs of amphotericin B and flucytosine were significantly higher for fluconazole-resistant isolates than for fluconazole-susceptible strains . We should exercise caution in interpreting these results, but statistical calculations were significant (P < 0.01) . Correlation coefficients between the distributions of MICs of fluconazole and those of both amphotericin B and flucytosine were also statistically significant, with values of 0.44 and 0.49, respectively . The increases in amphotericin B MICs were particularly marked for some species, such as C . tropicalis, C . parapsilosis, Candida guilliermondii, D . hansenii, and Geotrichum capitatum . These results indicate that further studies on drug resistance mechanisms are needed, but such studies were beyond the scope of this work .

In conclusion, ravuconazole was active in vitro against the majority of fluconazole-resistant and S-DD isolates . However, the ravuconazole MIC was 1 µg/ml for a total of 102 of 562 (18%) fluconazole-resistant and S-DD isolates . A significant number of these strains were C . tropicalis, C . glabrata, and C . neoformans . Ravuconazole represents an alternative for the treatment of infections due to fluconazole-resistant strains, but correct characterization of these isolates and susceptibility testing could be therapeutically significant in view of the possibility of cross-resistance .

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