Paradoxical Effect of Echinocandins across Candida Species In Vitro: Evidence for Echinocandin-Specific and Candida Species-Related Differences

Abstract

Paradoxical growth of some Candida isolates occurs at concentrations above the MIC for echinocandins. In 60 Candida bloodstream isolates from cancer patients (20 C. albicans isolates and 10 isolates each of C. parapsilosis, C. tropicalis, C. krusei, and C. glabrata), paradoxical growth was more frequent with caspofungin than micafungin or anidulafungin, was unrelated to MIC, and was strikingly absent in C. glabrata isolates.

The echinocandins are a new class of antifungal agents that target the fungal cell wall by inhibiting 1,3-β-d-glucan synthetase (5). Caspofungin and the recently approved echinocandins micafungin and anidulafungin have broad-spectrum fungicidal activity against Candida species and have been shown to be active for the treatment of invasive candidiasis (3, 5, 7, 11).

Of concern, however, is the paradoxical growth of some Candida albicans isolates in vitro (12, 15) and in vivo (4) observed at caspofungin concentrations that are high yet theoretically achievable in humans. This phenomenon has similarities to the paradoxical, or “eagle,” effect observed for other cell wall-active antimicrobial agents, such as penicillins. Although the molecular mechanisms responsible for the paradoxical effect of echinocandins remain largely unknown, it has been postulated that stress response pathways, such as the cell wall integrity and calcineurin pathways (14, 16), play a critical role. Hence, a compensatory increase in cell wall chitin synthesis was recently shown in a single C. albicans isolate exhibiting paradoxical growth (14). Notably, the paradoxical effect has been reported only for caspofungin; the frequency of the paradoxical effect has not been systematically evaluated across different echinocandins and Candida species. Because echinocandins differ in chemical structure (5), we hypothesized that there are echinocandin-specific and Candida species-related differences in the frequency of the paradoxical effect in vitro.

To test our hypothesis, we obtained caspofungin (institutional pharmacy), anidulafungin (institutional pharmacy), and micafungin (Fujisawa Healthcare, Inc., Deerfield, IL) in powder forms, prepared drug stock solutions in distilled water (1.28 mg/ml), and stored the solutions at −80°C until use. We determined the MIC of each antifungal agent for 60 bloodstream isolates of Candida (20 C. albicans, 10 C. parapsilosis, 10 C. tropicalis, 10 C. krusei, and 10 C. glabrata isolates) collected from cancer patients at The University of Texas M. D. Anderson Cancer Center (January 2003 to December 2005). We performed susceptibility testing according to Clinical and Laboratory Standards Institute-approved document M27-A2 in standard RPMI 1640 medium (9) in microtitration plates (Corning, New York, NY) containing serial twofold dilutions of each echinocandin and a final inoculum of 1 × 10³ to 5 × 10³ yeast cells/ml of each isolate; yeast cell counts were determined by using a hemocytometer. The MIC of each antifungal agent was determined visually 48 h after a shaking incubation at 35°C as the lowest concentration that resulted in prominent (>80%) decrease in turbidity of all drugs (9). We defined the paradoxical effect as a progressive increase in turbidity (growth) occurring after at least two drug dilutions above the MIC and tested a range of concentrations of all drugs (0.03 to 256 μg/ml) (12). We performed all experiments in triplicate on different days using the C. parapsilosis strain ATCC 20199 as a reference standard. For statistical comparisons, we used the Mann-Whitney U test where appropriate. Statistical analyses were performed with Graph-Pad Prism software, version 4.0 (San Diego, CA). We considered P values of less than 0.05 to be statistically significant.

Among all the isolates tested, C. parapsilosis exhibited the highest MICs to all the echinocandins tested (Table 1). The prevalence of the paradoxical effect for caspofungin was 90%, 60%, 40%, and 10% in C. parapsilosis, C. albicans, C. tropicalis, and C. krusei isolates, respectively (Table 1). For micafungin, the paradoxical effect was observed only in C. tropicalis (70%) and C. krusei (60%) isolates. Similarly, for anidulafungin, paradoxical growth occurred only in C. albicans (40%) and C. tropicalis (20%) isolates. Notably, paradoxical effect was absent in C. glabrata isolates for all three drugs tested (P < 0.001 compared with the frequency of the paradoxical effect in all other Candida species) (Table 1).

TABLE 1.

Paradoxical effect of caspofungin, micafungin, and anidulafungin against different Candida species^a

Candida species (no. of isolates)	MIC50/MIC90 (μg/ml)			Paradoxical efffect (%)			Medain paradoxical growth start point/endpoint (μg/ml)
	CAS	MICA	ANID	CAS	MICA	ANID	CAS	MICA	ANID
C. albicans (20)	0.125/0.25	0.25/0.25	0.5/1.0	60	0	40	8/32	NA	0.5/2.0
C. parapsilosis (10)	0.5/1.0	1.0/2.0	0.5/0.5	90	0	0	8/64	NA	NA
C. tropicalis (10)	1.0/1.0	0.06/0.06	0.5/1.0	40	70	20	16/48	12/48	0.125/1.0
C. krusei (10)b	0.06/1.0	0.5/0.5	0.25/0.25	10	60	0	8/32-64	0.125/0.5	NA
C. glabrata (10)	0.25/0.5	0.06/0.125	0.5/0.5	0	0	0	NA	NA	NA

^aThe range of concentrations tested was 0.03 to 256 μg/ml for all antifungals tested. CAS, caspofungin; MICA, micafungin; ANID, anidulafungin. NA, not applicable. The numbers shown in the table were obtained as described in the text.

^bIn two C. krusei isolates, paradoxical effect was observed at two different MICA concentrations (0.5 to 1.0 and 8 to 32).

The paradoxical effect was evident at median concentrations of 64× MIC, 96× MIC, and 16× MIC for caspofungin, micafungin, and anidulafungin, respectively. The median MIC of caspofungin was comparable between the Candida isolates that exhibited paradoxical growth and those in which the phenomenon was absent (Fig. 1). In contrast, an inverse relationship was noticed between the median MIC of micafungin and anidulafungin and the frequency of paradoxical growth in C. krusei and C. tropicalis isolates, respectively (Fig. 1). Hence, paradoxical growth was seen almost exclusively in C. krusei and C. tropicalis isolates that exhibited much lower MICs than the median MIC of the corresponding species to micafungin and anidulafungin, respectively (P < 0.0001; Fig. 1).

FIG. 1.

Association of caspofungin (CAS), micafungin (MICA), and anidulafungin (ANID) MICs with the presence of paradoxical effect. Note that the median MIC of caspofungin was comparable between the Candida isolates exhibiting paradoxical growth and those in which the phenomenon was absent. In contrast, paradoxical growth was seen almost exclusively in C. krusei and C. tropicalis isolates exhibiting significantly lower MICs than the median MIC of micafungin and anidulafungin for the corresponding species. *, MIC for C. krusei and C. tropicalis isolates with paradoxical effect compared with the corresponding isolates without paradoxical effect (P < 0.001). All other comparisons were not significant. +, presence of paradoxical effect; −, absence of paradoxical effect.

The paradoxical effect has been increasingly observed at high concentrations of caspofungin in Candida and Aspergillus isolates in vitro (1, 2, 4, 12-16) and in vivo (4, 17). It has been estimated that the phenomenon takes place in about 22% and 11% of isolates of C. albicans and non-C. albicans Candida species, respectively (12, 15). Notably, in previous studies the paradoxical effect was characteristically absent in all 51 clinical isolates of different Candida species tested against micafungin and anidulafungin (12).

Similar to previous observations (4, 12, 15), we found that the paradoxical effect occurs in vitro at a higher frequency and in a broader spectrum of Candida species with caspofungin than micafungin and anidulafungin. However, in contrast to previous studies, we also found that the paradoxical effect occurred in a Candida species-specific way for all echinocandins tested. Importantly, anecdotal case reports also suggest that there might be differences in echinocandin activity and mechanisms of in vivo resistance (8).

Notably, we did not observe any relationship between the MICs of caspofungin and the presence of paradoxical growth. Nonetheless, with respect to the other echinocandins, paradoxical effect was evidenced exclusively in isolates of C. krusei and C. tropicalis exhibiting low MICs to micafungin and anidulafungin, respectively. A similar phenomenon has also been observed in spontaneous Aspergillus fumigatus mutants that exhibit hypersensitivity to low concentrations of echinocandins, with restoration of nearly normal growth at relatively high concentrations, possibly due to the upregulation of cell wall proteins (6).

Of interest, paradoxical growth was strikingly absent in all C. glabrata isolates tested. In contrast, an extremely high prevalence (71%) of paradoxical growth in 24 C. dubliniensis isolates was recently reported, further emphasizing the concept that genetic differences across Candida species play an important role in the molecular mechanisms of this phenomenon (13).

A very complex picture regarding the mechanisms of the echinocandin eagle effect in regard to different echinocandins and Candida species emerges from our study. In addition, other factors might be important. For example, it has been shown that protein binding significantly decreases the frequency of the paradoxical effect in C. albicans isolates (10). Although the clinical significance of the echinocandin eagle effect remains elusive, deciphering the molecular pathways involved in this phenomenon could shed light on the molecular mechanisms of echinocandin resistance and sensitivity.

(This work was presented in part at the 16th Congress of the International Society for Human and Animal Mycology, Paris, France, 25 to 29 June 2006 [3a]).