Print version ISSN 0074-0276
Mem. Inst. Oswaldo Cruz vol.107 no.3 Rio de Janeiro May 2012
Débora Alves Nunes MarioI, II, +; Laura Bedin DenardiI; Laíssa Arévalo BandeiraI; Milene Silva AntunesII; Janio Morais SanturioI; Luiz Carlos SeveroIII; Sydney Hartz AlvesI, II
IDepartamento de Microbiologia e Parasitologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil
IIPrograma de Pós-graduação em Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil
IIIComplexo Hospitalar Santa Casa de Misericórdia, Porto Alegre, RS, Brasil
The extensive use of azole antifungal agents has promoted the resistance of Candida spp to these drugs. Candida glabrata is a problematic yeast because it presents a high degree of primary or secondary resistance to fluconazole. In Brazil, C. glabrata has been less studied than other species. In this paper, we compared the activity of three major classes of antifungal agents (azoles, echinocandins and polyenes) against fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains. Cross-resistance between fluconazole and voriconazole was remarkable. Among the antifungal agents, the echinocandins were the most effective against FS and FR C. glabrata and micafungin showed the lowest minimal inhibitory concentrations.
Key words: Candida glabrata - echinocandins - antifungal drug resistance
In Europe and the United States of America (USA), Candida glabrata has emerged as the second most common cause of invasive candidiasis and an increasing number of reports show its importance in mucosal or bloodstream infections (Pfaller & Diekema 2007). Systemic infections due to C. glabrata are characterized by a high mortality rate and they are difficult to treat due to their intrinsically low susceptibility to azoles, particularly fluconazole (Pfaller et al. 2004). Numerous C. glabrata isolates have shown primary resistance to fluconazole, while others easily develop fluconazole resistance after exposure to the treatment (Bennett et al. 2004).
In Brazil, Colombo et al. (2006) evaluated the aetiology of candidaemia episodes in 11 Brazilian medical centres and showed that they occur in 2.49 cases for every 1,000 hospital admissions, which is two-15 times more frequently than those reported for centres in the Northern Hemisphere. However, based on percentages (4.9%), C. glabrata is apparently less important in Brazil (it holds 5th place among the Candida species), but it corresponds to 0.12 cases for every 1,000 hospital admissions, which is similar to Europe and the USA.
Echinocandins are antifungal agents licensed for the treatment of invasive candidiasis by the USA Food and Drug Administration (Brielmaier et al. 2008). These agents disrupt the cell walls of Candida species by inhibiting β-1.3-D-glucan synthase, which results in cell rupture and death (Messer et al. 2009). Echinocandins prevent the overlapping toxicities and drug interactions with mammalian cells observed between azoles and polyenes (Wiederhold & Lewis 2003).
In 2000, caspofungin was the first echinocandin to be marketed in Brazil. In 2009, anidulafungin was introduced in Brazil as a treatment for invasive candidiasis in non-neutropenic adult patients. Recently, in 2010, micafungin was approved by the National Agency of Sanitary Surveillance for use in Brazil and designated for the treatment of invasive candidiasis, prophylaxis in individuals undergoing haematopoietic stem cell transplant, Aspergillus infections and empiric therapy for febrile neutropenia in adults and children, as previously established in Europe and Japan. Micafungin has been studied in both the USA and Brazil; however, it is currently only approved in the USA for use in adults (Bormann & Morrison 2009).
The aim of this study was to evaluate the in vitro activity of anidulafungin, micafungin and caspofungin against fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata clinical strains that were isolated from candidaemia episodes in Brazil and compare them to amphotericin B and voriconazole.
Anidulafungin and voriconazole (Pfizer, Inc, New York, NY), amphotericin B and fluconazole (Sigma Chemical Co, St. Louis, MO), caspofungin (Merck Research Laboratories, Rahway, NJ) and micafungin (Fujisawa Pharmaceutical Co Ltd, Osaka, Japan) were obtained as standard powders and prepared according to the Clinical and Laboratory Standards Institute guidelines (CLSI 2008a, b).
We studied two groups of C. glabrata strains. The first group included clinical FS isolates (n = 36) obtained from blood cultures and from deep-site specimens or oropharyngeal exudates. The second group included FR (n = 36) derivatives obtained from susceptible isolates through an in vitro method of fluconazole resistance induction described by Fekete-Forgács et al. (1999). All of the yeasts were tested by the broth microdilution method using the CLSI M27-A3 and third informational supplement (M27-S3/2008) standardized reference method.
The Mann-Whitney U test was used to compare two independent samples to evaluate the different groups (susceptible strains vs. resistant strains).
All of the isolates that were subcultured with fluconazole according Fekete-Forgács method acquired resistance to fluconazole; the minimal inhibitory concentrations (MICs) to fluconazole in the FR group ranged from 64.0-256.0 µg/mL and the differences between the MICs of both groups were significant (p < 0.0001) (Table II).
Based on susceptibility parameters (MIC range, MIC50, MIC90 and geometric mean), the FS C. glabrata group was very susceptible to amphotericin B, showing a MIC90 of 0.125 µg/mL and a geometric mean of 0.093 µg/mL. For inhibition, the FR C. glabrata group required higher concentrations of amphotericin B, with a MIC90 of 2 µg/mL and a geometric mean of 0.824 µg/mL. In this group, the MIC range included nine strains with a MIC of 2 µg/mL. The Mann-Whitney U test showed significant differences between the susceptibility of FS vs. FR groups (p < 0.0001). C. glabrata shows a natural tendency to become resistant compared with other Candida species (Pfaller & Diekema 2004), which was confirmed in this study.
Amphotericin B is generally recommended for primary therapy of severe infections by C. glabrata. However, it is becoming evident that the agent is not universally effective against C. glabrata and higher doses of amphotericin B may be required for efficient treatment (Kremery & Barnes 2002).
Susceptibility tests of the FS group to voriconazole showed a MIC90 of 1 µg/mL and a geometric mean of 0.389 µg/mL. On the other hand, the FR group was evidently resistant to this triazole, with a MIC90 of 16 µg/mL and a geometric mean of 6.229 µg/mL. The susceptibility between the two groups was significantly different (p < 0.0001).
The cross-resistance of C. glabrata to fluconazole and voriconazole was remarkable, showing a MIC50 of 8 µg/mL, a MIC90 of 16 µg/mL and a geometric mean of 6.229 µg/mL to voriconazole. Cross-resistance among azoles is a well-established phenomenon; therefore, fluconazole was proposed as a surrogate marker for voriconazole susceptibility, given the greater potency of voriconazole in comparison to fluconazole (Pfaller et al. 2007). Voriconazole, ravuconazole and posaconazole have shown efficacy against most FR Candida spp. These triazoles have also been significantly more effective against C. glabrata than fluconazole. Therefore, voriconazole has been suggested as an alternative therapy for C. glabrata strains that are resistant to fluconazole (Johnson & Kauffman 2003). However, when comparing MICs, the correlation between voriconazole and fluconazole cross-resistance is evident (Table II) (Bennett et al. 2004). Thus, we have confirmed that azoles are less effective against C. glabrata, as has been shown in previous studies (Pfaller & Diekema 2004).
The class of echinocandins showed the best activity against the FR group and the lowest MIC values were observed with micafungin. The FS group susceptibility profiles based on MIC90 were: micafungin (0.008 µg/mL), anidulafungin (0.031 µg/mL) and caspofungin (0.5 µg/mL). The MIC90 required by the FR group was similar to the FS group. Based on those parameters, the C. glabrata isolates of both groups were susceptible to echinocandins. In contrast, the statistical test comparing the MICs of the FS group vs. the MICs of the FR group indicated significant differences for caspofungin (p = 0.0023), anidulafungin (p < 0.0001) and micafungin (p < 0.0001).
We have demonstrated that echinocandins exhibited potent activity against all (FS and FR) C. glabrata strains. Micafungin showed the best antifungal activity. It has also been shown to be efficient against Candida biofilms and reduce the adherence of both azole-susceptible and azole-resistant C. albicans strains to epithelial cells (Borg-von Zepelin et al. 2002). Echinocandins may play an important role in therapy when azole resistance emerges. In this study, we found some disparity in the MIC values among the echinocandins. Anidulafungin and micafungin have demonstrated similar activities, but caspofungin required higher doses (MIC ± 4 dilutions) to inhibit strain growth (Table II). Ostrosky-Zeichner et al. (2003) found that micafungin was, on average, four times more potent than caspofungin, except when it was tested against Candida parapsilosis, which is consistent with our results. However, Perlin (2007) found that Candida spp FKS1 mutants expressing resistance to caspofungin were also cross-resistant to micafungin and anidulafungin. Pfeiffer et al. (2010) recently reported a case of C. glabrata breakthrough fungemia during micafungin treatment, with MIC values 4-8 µg/mL and FKS mutations. Such episodes emphasize the importance of monitoring azole and echinocandins resistance in C. glabrata.
Echinocandins are a class of antifungal agents with distinct mechanisms of action (Messer et al. 2009). However, the previous reports of multi-drug resistant Candida spp (Moudgal et al. 2005) involving azoles and amphotericin B or azoles and echinocandins suggest a connection between these two phenomena. Posteraro et al. (2006) reported a case of C. glabrata showing cross-resistance to multiple antifungal drugs in candidaemia treatment. Our results demonstrate that the MICs of echinocandins in the FS and the FR groups have increased slightly. Acquired resistance or reduced susceptibility to echinocandins has rarely been reported, with the majority of cases being associated with mutations in the FKS1 gene (Park et al. 2005).
We must emphasize that, based on susceptibility parameters (MIC50, MIC90 and MIC range) and established breakpoints, the FR C. glabrata group was susceptible to three echinocandins. Notably, however, when we applied a statistical test to compare the susceptibility between the two groups, the FR group was less susceptible than the FS group. Consequently, the continued application of antifungal susceptibility testing for the conventional and new antifungal agents is critical to detect the emergence of resistance in this important opportunistic fungal pathogen.
This study has clearly demonstrated the unparalleled spectrum and potency of echinocandin antifungal agents against a collection of C. glabrata with different susceptibility profiles. Micafungin, in particular, showed excellent effectiveness against the two groups of C. glabrata strains. Micafungin was the second drug in the echinocandin class to be approved in the USA and is efficient against Candida and Aspergillus spp. Overall, micafungin has proven to be safe and well tolerated and has few drug interactions. Future studies should include testing micafungin alone and as a component of combined antifungal therapy for invasive and refractory mould infections.
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Received 25 July 2011
Accepted 25 October 2012
Financial support: CAPES