Abstract
Cryptococcosis is primarily caused by Cryptococcus neoformans and Cryptococcus gattii. These two pathogenic species each divide into four distinct molecular genotypes. In this study, we examined whether genotype influenced susceptibility to antifungal drugs used to treat cryptococcosis using the broth microdilution method described by the Clinical and Laboratory Standards Institute. C. gattii isolates belonging to molecular genotype VGII had significantly higher MIC values for flucytosine and all azole antifungal agents tested, particularly fluconazole, than isolates of other C. gattii genotypes. In an extended analysis of fluconazole susceptibility, VGII isolates from the north and west of Australia required higher drug levels for inhibition than those from Vancouver Island, Canada. Within C. neoformans, genotype VNII had significantly lower geometric mean MICs for fluconazole than genotype VNI. These results indicate that cryptococcal species, molecular genotype, and region of origin may be important when deciding treatment options for cryptococcosis.
Cryptococcosis, caused by the encapsulated yeasts Cryptococcus neoformans and Cryptococcus gattii, is a fungal disease of humans and animals (10). C. neoformans is a worldwide, usually opportunistic pathogen that typically infects immunosuppressed patients, including those with HIV/AIDS (38, 41). In contrast, C. gattii is a primary pathogen that affects immunocompetent people and has caused significant outbreaks in animals in Australia and in humans and animals in Canada (18, 40).
The two pathogenic Cryptococcus species divide into eight major molecular types: VNI to VNIV for C. neoformans and VGI to VGIV for C. gattii. There is increasing evidence that these molecular types may represent cryptic species (4, 36), with differences found in important traits, including virulence, geographic range, epidemiology, and population genetics (3, 8). VNI and VGI molecular types are widespread and cause most of the disease attributed to C. neoformans and C. gattii, respectively. C. neoformans VNII to VNIV and C. gattii VGIII and VGIV are less common, and VGIII, VGIV, and VNIV appear to be geographically restricted (15, 21). C. gattii VGII, until recently considered to be rare, has received increasing attention due to its link with a large, ongoing outbreak of cryptococcosis that originated on Vancouver Island, Canada, and has now extended into the Pacific Northwest of the United States (5, 7, 21). VGII also predominates in some parts of Australia and South America and has likely been endemic in these regions for a substantial period (31, 45).
Clinical data suggest that the response to antifungal therapy is slower in C. gattii infection than in C. neoformans infection (41, 42) and that more prolonged treatment may be required (13). This is corroborated by various in vitro studies that have found that C. gattii may be less susceptible than C. neoformans to antifungal agents, in particular, fluconazole (13, 17, 44); however, other studies report no significant differences in susceptibility (9, 43). A possible reason for these contradictory findings is that most studies have not considered genetic or geographic differences within the two Cryptococcus species. Iqbal et al. (19) found significant differences in susceptibility between VGII subgenotypes VGIIa, VGIIb, and VGIIc that occur in the Pacific Northwest of Canada and the United States, suggesting that genotype and origin may influence MICs to antifungal agents.
The aim of the present study was to determine whether the most common genotypes of C. gattii and C. neoformans differ in their in vitro susceptibility to common antifungal agents. Reduced susceptibility to fluconazole was evident in C. gattii VGII, and this was further explored in an extended set of isolates from Australia and Vancouver Island, Canada, where C. gattii VGII accounts for a substantial proportion of infection.
MATERIALS AND METHODS
Fungal strains and antifungal agents.
This study was conducted in two parts: (i) an initial assessment of susceptibility to five commonly used antifungal agents—amphotericin B (AMB; Sigma-Aldrich), voriconazole (VRC; Pfizer Australia, Ryde, New South Wales), itraconazole (ITC; Sigma-Aldrich), fluconazole (FLC; Sigma-Aldrich), and flucytosine (5-fluorocytosine [5FC]; Sigma-Aldrich)—was conducted on 52 isolates representing the major genotypes of C. gattii and a selection of C. neoformans strains (see Table S1 in the supplemental material), and (ii) an extended analysis of susceptibility to FLC was undertaken in an additional 58 C. gattii VGII and 16 C. neoformans isolates (see Table S2 in the supplemental material), giving a total of 126 isolates that included 103 C. gattii (19 VGI, 72 VGII, and 12 VGIII) and 23 C. neoformans (11 VNI, 10 VNII, and 2 VNIV) strains. C. gattii strains were from clinical, veterinary, and environmental sources and were predominantly from Australia and Canada, with some strains from other regions, including Papua New Guinea, India, Portugal, France, French Guyana, Brazil, Senegal, Aruba, Columbia, Asia, Mexico, and the United States of America. C. neoformans strains were largely from infected animals from Australia that had a very low likelihood of previous exposure to azole antifungal agents. The molecular type of isolates was determined by DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis, according to Meyer et al. (32). Quality control type strains of Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included in each antifungal susceptibility assay (35).
Growth media and agar.
RPMI 1640 medium (Sigma-Aldrich, Castle Hill, New South Wales, Australia) supplemented with 0.03% (wt/vol) l-glutamine and 2% (wt/vol) d-glucose and buffered to a pH of 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma-Aldrich) was used for Candida controls, while yeast nitrogen base (YNB; Becton, Dickinson and Company) medium supplemented with 0.5% d-glucose and buffered to a pH of 7.0 with 0.165 M MOPS was used for Cryptococcus isolates. Sabouraud dextrose agar (SAB; 10 g/liter peptone, 40 g/liter glucose, and 15 g/liter agar) was used for subculturing all isolates prior to antifungal testing.
In vitro antifungal susceptibility testing.
The broth microdilution method described in the Clinical and Laboratory Standards Institute (CLSI) document M27-A2 (35), with a few minor modifications for Cryptococcus, was used to determine the susceptibility of strains in this study. Briefly, Cryptococcus isolates were subcultured onto SAB agar and incubated at 30°C for 48 h prior to the test. Cells were standardized to a cell density of a 0.5 McFarland standard (1 × 106 to 5 × 106 cells/ml) by spectrophotometric absorbance at 530 nm. The cell suspension was diluted 1:100 with YNB medium to achieve a final concentration of 5.0 × 103 to 2.5 × 104 cells/ml. Final concentrations of the antifungal agents ranged from 0.0625 to 32 mg/liter for 5FC, 0.125 to 64 mg/liter for FLC, and 0.0156 to 8 mg/liter for VRC, ITC, and AMB. For each test run, a drug-free, positive growth control and a cell-free, negative control were included. Plates were incubated at 35°C and read at 72 h. The MIC endpoint (35) was defined for AMB as the lowest concentration of drug that inhibited 100% of growth, and for the other drugs as the lowest concentration of drug that inhibited 50% of growth compared to the positive control. The inoculum strengths and purities of the diluted cell suspensions were checked by colony counts on SAB plates incubated for 48 to 72 h. All tests were performed in duplicate for each antifungal agent in a single experiment, and two separate experiments were carried out.
Statistical analysis.
The results obtained from the tests were analyzed using PASW Statistics 17 (SPSS Inc., version 17). MIC data were compared to detect differences among genotypes by one-way analysis of variance (AVONA) (when comparing three or more groups of results) or the independent sample t test (when comparing two groups of results). Homogeneity of variances was determined with the Levene statistic. When equal variances were obtained, the least significant difference (LSD) test was applied to determine significance among the molecular groups within the ANOVA. When equal variances were not obtained, the Tamhane test was used. Correlation was determined using Spearman's rho by using the online calculator available at http://www.wessa.net/rwasp_spearman.wasp. A P value of less than 0.05 was considered significant.
RESULTS
In vitro MIC data for 52 Cryptococcus isolates to antifungal agents.
The initial analysis determined the MICs of a selection of 52 Cryptococcus isolates to 5FC, AMB, VRC, ITC, and FLC. The range of MICs, MIC50s, MIC90s, and geometric mean (GM) MICs are summarized in Table 1, with full details provided in Tables S1, S3, and S4 in the supplemental material. Within this group, none of the isolates had MIC values of ≥64 mg/liter, which is the level determined by CLSI where Candida species are considered resistant to antifungal agents (35) (note that breakpoints for Cryptococcus have yet to be defined). However, isolates of the VGII molecular type had generally higher GM MICs. VGII isolates had significantly higher GM MICs to VRC (P < 0.01) and FLC (P < 0.05) than the VGI, VGIII, and VN genotypes and had a significantly higher GM MIC to ITC than the VGIII and VN genotypes (P ≤ 0.011). There were no significant differences among genotypes in GM MIC for AMB (P ≥ 0.099). VGI had a lower GM MIC to 5FC than VGII, VGIII, and VN (P ≤ 0.024), and VGIII had a significantly lower GM MIC to ITC than VGI and VGII (P ≤ 0.005). MICs for isolates of a and α mating types were similar. When all isolates were grouped together regardless of genotype, no significant difference between the two Cryptococcus species against each antifungal agent tested was observed (P ≥ 0.184).
TABLE 1.
In vitro susceptibility of Cryptococcus to five antifungal agents
| Species (no. of isolates) | Compound | MIC (mg/liter)a |
|||
|---|---|---|---|---|---|
| Range | GM | 50% | 90% | ||
| C. gattii (45) | 5FC | 0.5-8.0 | 2.19 | 2.0 | 8.0 |
| AMB | 0.13-0.5 | 0.35 | 0.5 | 0.5 | |
| VRC | 0.03-0.5 | 0.11 | 0.13 | 0.25 | |
| ITC | 0.02-0.5 | 0.19 | 0.25 | 0.5 | |
| FLC | 1.0-8.0 | 3.54 | 4.0 | 8.0 | |
| VGI (19) | 5FC | 0.5-4.0 | 1.16 A | 1.0 | 2.0 |
| AMB | 0.25-0.5 | 0.36 | 0.5 | 0.5 | |
| VRC | 0.03-0.5 | 0.09 B | 0.13 | 0.25 | |
| ITC | 0.06-0.5 | 0.23 D | 0.25 | 0.5 | |
| FLC | 1.0-8.0 | 2.99 B | 2.0 | 8.0 | |
| VGII (14) | 5FC | 2.0-8.0 | 3.81 A | 4.0 | 8.0 |
| AMB | 0.25-0.5 | 0.39 | 0.5 | 0.5 | |
| VRC | 0.13-0.5 | 0.23 B | 0.25 | 0.25 | |
| ITC | 0.13-0.5 | 0.32 CDE | 0.25 | 0.5 | |
| FLC | 2.0-8.0 | 5.38 B | 4.0 | 8.0 | |
| VGIII (12) | 5FC | 1.0-8.0 | 3.18 A | 4.0 | 8.0 |
| AMB | 0.13-0.5 | 0.3 | 0.25 | 0.5 | |
| VRC | 0.03-0.13 | 0.06 B | 0.06 | 0.13 | |
| ITC | 0.02-0.25 | 0.08 CD | 0.13 | 0.25 | |
| FLC | 1.0-8.0 | 2.83 B | 2.0 | 8.0 | |
| C. neoformans (7) | 5FC | 2.0-4.0 | 2.97 A | 4.0 | 4.0 |
| AMB | 0.13-0.5 | 0.31 | 0.25 | 0.5 | |
| VRC | 0.03-0.25 | 0.06 B | 0.06 | 0.25 | |
| ITC | <0.02-0.25 | 0.11 CE | 0.25 | 0.25 | |
| FLC | 0.5-8.0 | 2.0 B | 4.0 | 8.0 | |
Letters A to E indicate significantly different comparisons (P < 0.05): A, VGII, VGIII, and C. neoformans > VGI; B, VGII > VGI, VGIII, and C. neoformans; C, VGII > VGIII and C. neoformans; D, VGI and VGII > VGIII; E, VGII > C. neoformans. See Table S4 in the supplemental material for a complete list of comparisons.
Susceptibility of Cryptococcus isolates to FLC.
Initial testing found FLC to be the least potent antifungal agent for all Cryptococcus strains, and this was particularly pronounced for C. gattii VGII isolates. To further investigate FLC susceptibility within C. gattii VGII and to extend the analysis to the genotypes within C. neoformans, 74 additional isolates were tested, including 58 C. gattii VGII isolates from various sources and geographic regions and eight isolates each of C. neoformans VNI and C. neoformans VNII from infected animals in Australia. The range of MICs, MIC50s, MIC90s, and GM MICs are summarized in Table 2 (also see Tables S1 to S4 in the supplemental material).
TABLE 2.
In vitro susceptibility of Cryptococcus genotypes to fluconazole in an extended number of strains
| Species or genotype (no. of isolates) | FLC MIC (mg/liter)a |
|||
|---|---|---|---|---|
| Range | GM | 50% | 90% | |
| C. gattii (103) | 1.0-32.0 | 4.96 A | 4.0 | 8.0 |
| VGI (19) | 1.0-8.0 | 2.99 B | 2.0 | 8.0 |
| VGII (72) | 2.0-32.0 | 6.23 B | 8.0 | 8.0 |
| VGIII (12) | 1.0-8.0 | 2.83 B | 2.0 | 8.0 |
| C. neoformans (23) | 0.25-8.0 | 1.94 AB | 4.0 | 8.0 |
| VNI (11) | 4.0-8.0 | 5.15 C | 4.0 | 8.0 |
| VNII (10) | 0.25-4.0 | 0.81 C | 0.5 | 2.0 |
| VNIV (2) | 0.5-1.0 | 0.75 C | 0.5 | 1.0 |
Letters A to C indicate significantly different comparisons (P < 0.05): A, C. gattii > C. neoformans; B, VGII > VGI, VGIII, and C. neoformans; C, VNI > VNII and VNIV. See Table S4 in the supplemental material for a complete list of comparisons.
Based on breakpoints for Candida species, none of the isolates in this collection are considered resistant to FLC (MIC ≥ 64 mg/liter); however, seven of the VGII isolates might be considered susceptible-dose dependent (8 mg/ liter < MIC < 64 mg/liter) (35). With the inclusion of more isolates, the difference in response between VGII and VGI and between VGII and C. neoformans (particularly VNII) became evident (P ≤ 0.011). C. neoformans genotype VNII isolates were inhibited by significantly lower levels of FLC than VNI isolates (P = 0.012). When all isolates were grouped together regardless of genotype, C. gattii had a significantly higher GM MIC for FLC than C. neoformans (P = 0.007), reflecting the influence of genotype composition of the Cryptococcus species.
MICs of C. gattii VGII to FLC based on source and geographic origin.
As reduced inhibition by FLC was observed in C. gattii VGII isolates, the 72 isolates of this genotype were further assessed considering their source and geographic region of origin. GM MICs are summarized in Table 3 (also see Tables S2, S5, and S6 in the supplemental material). Among these VGII isolates, the seven that were in the susceptible-dose-dependent range (based on Candida breakpoints) were all from Australia, including four from Western Australia and three from the Northern Territory. Overall, VGII isolates from Australia had significantly higher MICs for FLC than VGII isolates from Canada (P < 0.001). There were no significant differences among VGII populations sampled from the three different regions (New South Wales, Western Australia, and the Northern Territory) of Australia (P ≥ 0.220). However, only the VGII populations from Western Australia and the Northern Territory had significantly higher GM MICs for FLC than the VGII population from Vancouver Island, Canada (P = 0.004 and P < 0.001, respectively).
TABLE 3.
Comparison of fluconazole GM MICs among groups within C. gattii genotype VGII
| Comparison | Group (no. of isolates) | FLC GM MIC (mg/liter)a |
|---|---|---|
| Global | All (72) | 6.23 |
| Australia (50) | 7.46 A | |
| Other countries (8) | 6.17 | |
| Canada (14) | 3.28 A | |
| Regional | Western Australia, Australia (16) | 9.51 B |
| Northern Territory, Australia (28) | 6.73 C | |
| New South Wales, Australia (6) | 6.35 | |
| Vancouver Island, Canada (14) | 3.28 BC | |
| Source | Clinical (Australia) (11) | 9.08 F |
| Veterinary (Australia) (21) | 8.55 DE | |
| Environmental (Australia) (18) | 5.66 DFG | |
| Clinical (Canada) (1) | 4.0 | |
| Veterinary (Canada) (8) | 4.0 EH | |
| Environmental (Canada) (5) | 2.30 GH | |
| Clinical (global) (30) | 7.66 I | |
| Veterinary (global) (26) | 6.96 J | |
| Environmental (global) (16) | 4.82 IJ |
Letters A to J indicate significantly different comparisons: A, P < 0.001; B, P = 0.004; C, P < 0.001; D, P = 0.019; E, P = 0.021; F, P = 0.042; G, P < 0.001; H, P = 0.045; I, P = 0.034; J, P = 0.024.
The seven VGII isolates with MICs of ≥16 mg/liter included four veterinary isolates and three human isolates. On a global scale and in the Australia and Canadian populations, VGII isolates from veterinary and clinical sources had higher MICs for FLC than environmental VGII isolates (see Table S5 in the supplemental material). Australian veterinary and environmental VGII populations had significantly higher GM MICs for FLC than the corresponding veterinary and environmental populations from Canada (P = 0.021 and P < 0.001, respectively). There was no significant difference in GM MICs between veterinary and human VGII populations from Australia (P = 0.952).
DISCUSSION
Contradictory results have been reported in studies examining differences in antifungal susceptibility between C. neoformans and C. gattii. While a number of studies have found C. gattii to be less susceptible than C. neoformans to azole drugs, particularly FLC (13, 17, 44), others have reported no difference between the two species (9, 43). Data on susceptibility to AMB and 5FC are even more varied, with comparisons indicating that C. gattii is more resistant than (9), more susceptible than (17), or not different from (43, 44) C. neoformans. A possible reason for these findings is that none of the studies considered the effect of genotypic and geographic differences within the two Cryptococcus species. The differences in ecology, epidemiology, and virulence (3, 4, 8, 36) and the regional differences (15, 21) among cryptococcal genotypes are likely to reflect fundamental differences in their biology and physiology, which could affect their response to antifungal drugs. It is noteworthy that in the initial tests undertaken in this study, no significant difference between the two Cryptococcus species was observed, but this changed when additional isolates, which included a substantial number of the relatively resistant C. gattii genotype VGII and the relatively susceptible C. neoformans genotype VNII, were added to the collection (see Table S4 in the supplemental material). Overall, our study shows that genotype is an important factor in in vitro drug response and that relative resistance or susceptibility of the Cryptococcus species cannot be determined without taking this into account.
The key finding of the current study was the significantly higher MICs for azole drugs, particularly FLC, in C. gattii isolates of the molecular type VGII than in isolates of the other molecular types. This genotype is of increasing concern worldwide, as it has undergone rapid recent expansion in the Pacific Northwest of the United States and beyond (5, 7, 12, 21). These areas are dominated by particular clonal lineages within VGII that exhibit increased virulence compared to VGII isolates from Australia and other regions (6, 7, 15, 30). A recent study by Iqbal et al. (19) found that isolates belonging to subtype VGIIa, which dominates in Vancouver Island, Canada, were more susceptible to FLC and to other azole drugs than isolates in subtypes VGIIb (which is found in Australia and other parts of the world) (15) and VGIIc (which has recently emerged in Oregon) (5). Consistent with this, in the current study, Canadian isolates had significantly lower MICs to FLC than isolates from Australia. Most studies evaluating the correlation between in vitro susceptibility and the in vivo response to antifungal drugs have demonstrated that susceptibility testing is a reliable predictor of clinical response (22, 47) and that the clinical outcome may be poorer when higher concentrations of FLC are required (MIC ≥ 16 mg/liter) (1). Our data therefore predict a good outcome when treating Canadian C. gattii VGII isolates with azole drugs but suggest that some Australian isolates may be more refractory to treatment. Current treatment guidelines produced by the Infectious Diseases Society of America do not support routine susceptibility testing of Cryptococcus (37); however, the results presented here suggest that determining MICs should be considered in regions where isolates are documented to have reduced susceptibility.
A significant difference in response to FLC was noted between C. neoformans genotypes VNI and VNII (GM MICs of 5.15 mg/liter and 0.81 mg/liter, respectively; P = 0.012). Both genotypes belong to C. neoformans var. grubii, serotype A, which is responsible for most cryptococcosis worldwide. Globally, VNII is less common than VNI (14), but VNII isolates have been obtained from all of the inhabited continents (20). As with C. gattii, these findings emphasize the potential importance of genotype for predicting response to antifungal drugs and show how assumptions cannot be made based on species alone.
An important factor considered in the design of this study was the potential for prior exposure to antifungal agents to influence antifungal susceptibility. It is well known that antibiotic resistance can develop during exposure to sublethal levels of the agent (11, 27, 48), and FLC is used widely in prophylactic antifungal treatment in HIV/AIDS patients (24, 25, 29). Prior exposure to FLC was considered to be unlikely for C. gattii, which is a primary pathogen causing mostly sporadic disease in otherwise healthy people. In our comparisons of FLC susceptibility among C. neoformans genotypes, we targeted veterinary isolates where prior exposure to antifungal agents was very unlikely. However, we cannot completely rule out prior exposure to all azoles, as azole-based formulations are used in many countries, including Australia, to protect crops from pathogenic fungi and as wood preservatives (2, 16, 28). Studies have shown that some environmental fungi of clinical importance can develop cross-resistance to medical azoles following exposure to agricultural azoles (34, 39, 46). C. gattii and C. neoformans are environmental fungi, and contact with agricultural azoles may occur. The elevated FLC MICs in the Western Australia isolates are particularly interesting in this regard, as this is a region of extensive agriculture and forestry, and there is apparent widespread use of azole-based formulations on Western Australia cereals (http://www.agric.wa.gov.au/PC_93297.html). However, for the intergenotype comparisons, we included a range of isolates from various regions and sources in each genotype so that the influence of any particular environment or source would be minimized. It therefore seems likely that the differences seen among the C. gattii and C. neoformans genotypes are due to intrinsic differences that are specific to these genotypes. An interesting observation in the extended study of FLC susceptibility in the VGII genotype was the higher MICs for isolates from infected humans and animals compared to those obtained from the environment, suggesting a correlation between tolerance to FLC and virulence. Understanding what mediates these differences at the molecular level, and how they interact in cryptococcal infection, will be interesting areas of future study.
Among the three azoles tested, VRC showed the greatest potency and FLC the least, consistent with previous findings (26, 43). Importantly, although VGII isolates had higher MICs to all azoles than the other genotypes, they were still susceptible to VRC and ITC, indicating that these are suitable as alternative treatments to FLC. There was a high degree of correlation in the response to the different azole drugs (Spearman's rho for FLC versus VRC equals 0.77 [P < 0.0001] and for FLC versus ITC equals 0.67 [P < 0.0001]). Interestingly, the responses to FLC and 5FC were also correlated (Spearman's rho = 0.34 [P = 0.014]), which may suggest that generic factors, such as cell permeability or efflux pumps, underlie resistance to these drugs. However, susceptibility to AMB was not correlated with susceptibility to either FLC or 5FC (rho = 0.14 [P = 0.32] and rho = 0.08 [P = 0.56], respectively). AMB remains the most efficacious antifungal agent for the treatment of cryptococcosis, and the development of resistance is very rare (23, 33), which is supported by the current study.
This study documents the importance of genotype and geographic origin in an assessment of antifungal susceptibility within the C. neoformans species complex and concludes that there are distinct differences among the molecular types of C. neoformans and C. gattii in their in vitro response to antifungal agents. Geographic origin may further determine differences within C. gattii genotypes. Causative cryptococcal species, genotype, and geographic origin are thus important considerations when deciding treatment options for cryptococcosis.
Supplementary Material
Acknowledgments
We thank Mark Krockenberger and Nathan Saul (Faculty of Veterinary Science, University of Sydney, Sydney, Australia) for some of the strains used in this study. The help received from Alfred Widmer and Namfon Pantarat (Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead, Australia) is gratefully acknowledged.
This work was financially supported by a grant from the Australian National Health and Medical Research Council (grant number 571354) to D. Carter. R. Malik's position is funded by the Valentine Charlton Bequest administered by the Centre of Veterinary Education of the University of Sydney.
The work presented herein is part of the Ph.D. work of H. S. Chong. S. Chen is a member of the Antifungal Advisory Board of Gilead Sciences, Inc., and Pfizer Australia.
Footnotes
Published ahead of print on 15 September 2010.
Supplemental material for this article may be found at http://jcm.asm.org/.
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