Correlation of Genotype and In Vitro Susceptibilities of Cryptococcus gattii Strains from the Pacific Northwest of the United States

Abstract

Cryptococcus gattii emerged in North America in 1999 as a human and veterinary pathogen on Vancouver Island, British Columbia. The emergent subtype, VGIIa, and the closely related subtype VGIIb can now be found in the United States in Washington, Oregon, and California. We performed multilocus sequence typing and antifungal susceptibility testing on 43 isolates of C. gattii from human patients in Oregon, Washington, California, and Idaho. In contrast to Vancouver Island, VGIIa was the most frequent but not the predominant subtype in the northwest United States. Antifungal susceptibility testing showed statistically significant differences in MICs between the subtypes. This is the first study to apply antifungal susceptibility testing to C. gattii isolates from the Pacific Northwest and the first to make direct comparisons between subtypes.

Cryptococcus species are basidiomycetous fungi, some of which are capable of causing invasive infection in both humans and animals. The major pathogens within the genus Cryptococcus are Cryptococcus neoformans and Cryptococcus gattii (2). C. neoformans and C. gattii differ significantly with regard to their geographical distributions and their ecological niches. While C. neoformans has a global distribution and is primarily associated with soil contaminated by bat or bird guano (19), C. gattii was until recently thought to be largely confined to tropical and subtropical regions, such as Australia and New Zealand, where it has been found primarily in association with Eucalyptus trees (11). C. gattii has also been isolated from clinical specimens from numerous other parts of the world, including Mexico, parts of Latin America, Europe, southern California, Hawaii, India, and Africa (15, 19, 25, 30).

In human immunodeficiency virus (HIV)-positive patients, the two species share similar clinical aspects; however, the diseases may differ significantly in otherwise healthy patients. C. neoformans primarily causes meningoencephalitis, while C. gattii has a propensity to cause cryptococcomas, focal lesions in the central nervous system (CNS), and significant neurological sequelae (5, 25).

In 1999, C. gattii began infecting animals and humans living in or traveling from Vancouver Island (VI), British Columbia, a temperate climate very different from that which was considered the traditional ecological niche of C. gattii (10, 14, 18). The majority of cases in this outbreak were caused by C. gattii of the clonal subtype VGIIa, with a minority of isolates caused by the clonal subtype VGIIb (12, 17, 18), but other subtypes have also been isolated during this ongoing outbreak (17).

In 2005, investigators documented the presence of C. gattii subtype VGIIa isolates in environmental samples taken from parts of the Pacific northwestern United States (16). Since that time, both human and animal cryptococcoses have been recognized in Washington and Oregon (4, 21, 29). Many of these cases have been caused by subtype VGIIa, but VGIIb and VGIIc isolates also play a role in this emergence (4). Because the VI outbreak major strain (VGIIa) is a unique subtype seen in no other part of the world (18), there has been substantial interest in subtyping all strains of C. gattii isolated from the Pacific Northwest to monitor whether other unique strains were emerging or whether the region where VGIIa could be found was expanding.

Through the collaboration of a public health working group, 38 recent and 5 archived isolates of C. gattii from the Pacific Northwest states of California, Washington, Oregon, and Idaho have been collected, genotyped, and tested for susceptibility to six antifungal drugs.

MATERIALS AND METHODS

Isolates.

A total of 43 isolates of Cryptococcus gattii were analyzed at the Centers for Disease Control and Prevention. Public health institutions from the states of California, Oregon, Washington, and Idaho submitted 38 human and animal isolates from recent cases, including 28 isolates from the state of Oregon (17 human, 11 veterinary), four isolates from California (one human, three veterinary), five veterinary isolates from Washington, and one human isolate from Idaho. The CDC also had five archived C. gattii isolates, including two human isolates from Oregon from 2005, two human isolates from California from 1992 (3), and one veterinary isolate from California from 2001 (23). All isolates were confirmed as C. gattii using canavanine-glycine-bromthymol blue (CGB) medium (20). A single isolate that grew poorly and did not develop the characteristic cobalt blue color on CGB medium was confirmed as C. gattii by molecular typing, as described below.

Multilocus sequence typing.

All isolates were subtyped by multilocus sequence typing (MLST). The URA5, IGS1, and CAP59 gene fragments were amplified as described previously (22). Briefly, isolates were grown on yeast extract-peptone-dextrose (YPD) plus 0.5% NaCl, and DNA was prepared using an UltraClean DNA isolation kit as described by the manufacturer (MO BIO Laboratories, Carlsbad, CA). Gene fragments were amplified using the primers of Meyer et al. (22) and the same primers were used to sequence each fragment in both directions. Sequences were compared to published sequences in GenBank to assign an allele number and subtype for the IGS1 and CAP59 genes according to the supplementary allele table of Fraser et al. (12). Alleles for unpublished IGS1 and CAP59 sequences and alleles for the URA5 gene were assigned allele numbers by Wieland Meyer, University of Sydney, the current curator of the C. gattii MLST allele data set (W. Meyer, personal communication). The VGIIc alleles for CAP59 and IGS1 in this study were identical to the alleles first reported for VGIIc isolates by Byrnes and coworkers (4).

Susceptibility testing.

Antifungal susceptibility testing was performed on all isolates by broth microdilution with fluconazole, voriconazole, itraconazole, posaconazole, and flucytosine as outlined in the Clinical and Laboratory Standards Institute (CLSI) document M27-A3 (7) using RPMI broth and trays custom manufactured by TREK Diagnostics (Cleveland, OH). An Etest was used for amphotericin B susceptibility testing. All results were read visually after 72 h of incubation.

GenBank numbers.

The alleles in this study can be found under the following GenBank numbers: URA5_2 (AY973125), URA5_7 (AY973124), URA5_12 (AY973114), URA5_19 (AY973153), URA5_23 (GU250873), IGS1_1(DQ096311), IGS1_3 (DQ096313), IGS1_4 (DQ096314), IGS1_10 (DQ096319), IGS1_15 (DQ096324), IGS1_18 (DQ096327), CAP59_1 (DQ096432), CAP59_2 (DQ096433), CAP59_6 (EU937819), CAP59_16 (GU250874), CAP59_18 (GU250875), CAP59_29 (GU250876), and CAP59_30 (GU250877).

RESULTS

Passive surveillance for C. gattii in the Pacific Northwest.

Following the establishment of the C. gattii Public Health Working Group in 2008, public health institutions in Alaska, California, Idaho, Montana, Oregon, and Washington began passive surveillance for human and veterinary cases of C. gattii in their states. From this surveillance the CDC received 38 isolates from the states of Oregon, California, Washington, and Idaho. Isolates came from human cases as well as a number of different animals, including companion animals such as dogs and cats, as well as livestock, including llamas, alpacas, horses, and elk (Table 1).

TABLE 1.

C. gattii isolates in this study with state of origin, host, and genotype

Isolate no.	State	Host	Isolation date	Genotype	URA5 allele	IGS1 allele	CAP59 allele
B7394	Washington	Cat	2008	VGIIb	2	10	2
B7395	Washington	Dog	2008	VGIIa	7	4	1
B8239	Washington	Cat	2009	VGIIa	7	4	1
B8240	Washington	Cat	2009	VGIIa	7	4	1
B8260	Washington	Cat	2009	VGIII	19	18	29
B7390	Idaho	Human	2008	VGIIc	2	15	30
B7422	Oregon	Cat	2009	VGIIa	7	4	1
B7432	Oregon	Human	2009	VGIIc	2	15	6
B7434	Oregon	Human	2008	VGIIc	2	15	6
B7435	Oregon	Elk	2009	VGIIb	2	10	2
B7428	Oregon	Human	2008	VGIIb	2	10	2
B7430	Oregon	Cat	2008	VGIIa	7	4	1
B7463	Oregon	Dog	2009	VGIIa	7	4	1
B7464	Oregon	Dog	2008	VGIIa	7	4	1
B7465	Oregon	Camelid	2009	VGIIa	7	4	1
B7466	Oregon	Cat	2008	VGIIc	2	15	6
B7467	Oregon	Porpoise	2009	VGIIa	7	4	1
B7488	Oregon	Human	2009	VGI	12	3	16
B7489	Oregon	Human	2009	VGIIa	7	4	1
B7490	Oregon	Human	2009	VGIIa	7	4	1
B7491	Oregon	Human	2009	VGIIc	2	15	6
B7641	Oregon	Cat	2009	VGIIc	2	15	6
B7493	Oregon	Ovine	2009	VGIIc	2	15	6
B7735	Oregon	Human	2009	VGIIb	2	10	2
B7736	Oregon	Human	2009	VGIIa	7	4	1
B7737	Oregon	Human	2009	VGIIc	2	15	6
B7765	Oregon	Dog	2009	VGIIc	2	15	6
B8214	Oregon	Human	2009	VGIIc	2	15	6
B8216	Oregon	Human	2009	VGIIb	2	10	2
B8217	Oregon	Human	2009	VGIIa	7	4	1
B8209	Oregon	Human	2009	VGIIa	7	4	1
B8211	Oregon	Human	2009	VGIIb	2	10	2
B8210	Oregon	Human	2009	VGIIc	2	15	6
B8212	Oregon	Human	2009	VGIII	19	1	18
B6863	Oregon	Human	2005	VGIIc	2	15	6
B6864	Oregon	Human	2005	VGIIa	7	4	1
B7415	California	Alpaca	2008	VGIII	19	1	18
B7414	California	Horse	2008	VGIIb	2	10	2
B7495	California	Human	2009	VGIII	23	1	1
B7436	California	Alpaca	2009	VGIIa	7	4	1
B6253	California	Dolphin	2001	VGI	12	3	16
B8262	California	Human	1992	VGIII	19	1	18
B8263	California	Human	1992	VGI	12	3	16

Subtyping of C. gattii isolates.

Because the Vancouver Island major outbreak strain VGIIa was a unique subtype seen in no other part of the world (18), there has been substantial interest in subtyping all strains of C. gattii isolated from the Pacific Northwest to monitor the potential for emergence of other unique strains and/or expansion of the region where VGIIa could be found. To this end, we typed all of the submitted isolates by MLST (22). Our results showed that 16 of the isolates were the major VI outbreak strain VGIIa, 7 of the isolates were the minor VI outbreak strain VGIIb, 12 of the isolates were the unique Oregon strain VGIIc (4), and 8 of the isolates were either subtype VGI or VGIII (Table 1).

Antifungal susceptibility testing.

The MICs for each isolate against four azole drugs (fluconazole, voriconazole, itraconazole, and posaconazole), flucytosine, and amphotericin B are shown in Table 2. All isolates had relatively low MICs with flucytosine and amphotericin B. There was a wide range of MICs for all four of the azole drugs. The range for voriconazole was 0.008 to 1 μg/ml, for fluconazole, 0.5 to 32 μg/ml, and for itraconazole, 0.06 to 2 μg/ml (Table 3). The range for posaconazole was somewhat smaller, 0.12 to 1 μg/ml.

TABLE 2.

Susceptibility profiles for all isolates against the most commonly used antifungal drugs

Isolate	Subtype	MIC (μg/ml)a
		Vori	Fluc	Itra	5-FC	Posa	AmpB
B6253	VGI	0.06	0.5	0.06	0.5	0.25	0.047
B7488	VGI	0.06	4	1	0.5	1	0.19
B8263	VGI	0.06	2	0.25	1	0.25	0.19
B7415	VGIII	0.06	4	1	0.5	0.5	0.125
B7495	VGIII	0.03	2	0.12	1	0.25	0.064
B8212	VGIII	0.12	4	0.5	4	0.5	0.125
B8260	VGIII	0.12	4	0.12	2	0.5	0.032
B8262	VGIII	0.06	2	0.12	2	0.12	0.047
B6864	VGIIa	0.12	4	0.25	4	0.25	0.19
B7395	VGIIa	0.06	8	0.5	4	0.5	0.19
B7422	VGIIa	0.03	8	1	2	1	0.25
B7430	VGIIa	0.06	2	1	2	0.5	0.125
B7436	VGIIa	0.12	8	0.25	4	0.12	0.19
B7463	VGIIa	0.12	4	0.12	2	0.12	0.19
B7464	VGIIa	0.06	4	0.25	2	0.12	0.19
B7465	VGIIa	0.12	8	0.5	4	0.5	0.19
B7467	VGIIa	0.12	4	0.12	2	0.25	0.19
B7489	VGIIa	0.06	8	0.5	2	0.5	0.19
B7490	VGIIa	0.06	8	0.5	2	0.25	0.19
B7736	VGIIa	0.12	4	0.25	4	0.25	0.19
B8209	VGIIa	0.12	8	0.25	8	0.5	0.125
B8217	VGIIa	0.06	4	0.12	4	0.25	0.125
B8239	VGIIa	0.12	4	0.25	4	0.25	0.19
B8240	VGIIa	0.12	4	0.25	4	0.25	0.125
B7394	VGIIb	0.12	16	2	2	1	0.064
B7414	VGIIb	0.06	16	1	1	1	0.125
B7428	VGIIb	0.12	4	0.5	2	0.25	0.19
B7435	VGIIb	0.12	4	1	2	0.5	0.25
B7735	VGIIb	0.008	16	0.5	2	0.5	0.25
B8211	VGIIb	0.5	32	0.5	8	0.5	0.125
B8216	VGIIb	0.12	4	0.25	4	0.25	0.125
B7390	VGIIc	0.06	16	0.12	2	0.12	0.19
B6863	VGIIc	0.25	4	0.12	2	0.5	0.125
B7432	VGIIc	0.25	16	0.5	2	1	0.19
B7434	VGIIc	0.5	8	0.5	2	0.5	0.19
B7466	VGIIc	0.25	16	1	2	0.5	0.19
B7491	VGIIc	0.5	16	1	1	1	0.19
B7493	VGIIc	0.25	16	0.25	4	0.5	0.094
B7641	VGIIc	0.008	8	0.25	4	0.5	0.094
B7737	VGIIc	1	32	2	8	1	0.19
B7765	VGIIc	0.12	16	0.5	8	1	0.19
B8210	VGIIc	0.25	16	0.5	4	0.5	0.19
B8214	VGIIc	0.12	8	0.5	4	0.5	0.19

^aVori, voriconazole; Fluc, fluconazole; Itra, itraconazole; 5-FC, flucytosine; Posa, posaconazole; AmpB, amphotericin B.

TABLE 3.

Compiled MIC data for all 43 C. gattii isolates

Antifungal agent	MIC range (μg/ml)	MIC50 (μg/ml)	MIC90 (μg/ml)
Fluconazole	0.5-32	8	16
Voriconazole	0.008-1	0.12	0.25
Itraconazole	0.06-2	0.5	1
Posaconazole	0.12-1	0.5	1
Flucytosine	0.5-8	2	4
Amphotericin B	0.032-0.25	0.19	0.19

The geometric mean MICs for all isolates of each subtype are given in Table 4. The geometric mean MICs vary widely by subtype, with VGIIc isolates generally having the highest values and VGI/VGIII isolates generally having the lowest values. There is a statistically significant difference in MICs between VGI/VGIII isolates and VGIIc isolates for four of the six antifungal drugs tested (Table 5). There was a statistically significant difference in MICs between VGIIa and VGIIc isolates for fluconazole, voriconazole, and posaconazole. Itraconazole was the only drug tested for which there were no statistically significant differences in MICs among subtypes.

TABLE 4.

Comparison of the geometric mean MICs of isolates by subtype

Subtype	No. of isolates	Geometric mean MIC (μg/ml)a
		Fluc	Vori	Itra	Posa	5-FC	AmpB
VGI/VGIII	8	2.38	0.07	0.24	0.40	1.09	0.08
VGIIa	16	5.19	0.08	0.31	0.30	3.08	0.17
VGIIb	7	9.74	0.09	0.67	0.5	2.44	0.15
VGIIc	12	12.70	0.19	0.44	0.56	3.00	0.16
Overall	43	6.38	0.10	0.37	0.40	2.43	0.15

^aFluc, fluconazole; Vori, voriconazole; Itra, itraconazole; Posa, posaconazole; 5-FC, flucytosine; AmpB, amphotericin B.

TABLE 5.

Significant geometric mean MICs^a between C. gattii subtypes

Subtypes	Antifungal drug	P value
VGI/VGIII vs VGIIc	Voriconazole	0.007
VGIIa vs VGIIc	Voriconazole	0.02
VGI/VGIII vs VGIIc	Fluconazole	<0.001
VGIIa vs VGIIc	Fluconazole	0.001
VGI/VGIII vs VGIIa	Fluconazole	<0.001
VGI/VGIII vs VGIIb	Fluconazole	0.02
VGIIa vs VGIIc	Posaconazole	0.01
VGI/VGIII vs VGIIa	Flucytosine	0.002
VGI/VGIII vs VGIIc	Flucytosine	0.007
VGI/VGIII vs VGIIa	Amphotericin B	0.007
VGI/VGIII vs VGIIc	Amphotericin B	0.01

^aSignificance was determined using Student's t test.

DISCUSSION

We provide genotypic evidence that the strain dynamics of C. gattii isolates involved in the Pacific Northwest C. gattii emergence may be different from those seen on Vancouver Island, with VGIIb and VGIIc subtypes playing roles of equal significance to VGIIa types. We also show that human and animal infections caused by genotypes VGIIa, VGIIb, and VGIIc are occurring along the northern Pacific coast, now appear in California, and may have expanded eastward to Idaho. Because the described surveillance system is passive and not active, this study does not encompass the full scope of C. gattii disease in the Pacific Northwest United States, but it does provide a snapshot of human and animal cases that are occurring at this time.

There is no subtype bias in any one state in this study for either VGIIa or VGIIb, with each being found in Washington, Oregon, and California. This is the first report of the contemporary occurrence of genotypes VGIIa and VGIIb in California. As noted earlier by Byrnes and coworkers (4), we also found that the VGIIc subtype is unique to Oregon with the exception of the single isolate from Idaho. The lone case-patient from Idaho had a travel history to Oregon and Washington and might have acquired the infection in either state. However, it must be noted that the CAP59 allele from the Idaho isolate differs from all of the other VGIIc CAP59 alleles.

There were no significant differences in the distribution of subtypes between human and veterinary cases. Many of the veterinary cases had no history of travel, so it can be assumed that those cases arose from direct local environmental exposure. Because neither the strain types nor the MICs differed significantly between human and animal isolates, the animal cases may serve as a good sentinel for human cases in any particular area. Although we have no environmental isolates for comparison, earlier studies from both British Columbia and Washington state indicated that the genotypes of environmental isolates roughly correlated with the genotypes of patient isolates by the percentage of each genotype found (16, 21). It is interesting to note that no VGIII isolates were found during the environmental testing in British Columbia and Washington State (16, 21), and yet this type was not infrequent in either human or veterinary isolates in our study. Clearly, there is a need for more environmental testing in the Pacific Northwest United States.

This is the first analysis of antifungal susceptibility of C. gattii isolates from the ongoing emergence in the Pacific Northwest and the first time that MICs were compared among subtypes. While much research has been conducted on the antifungal susceptibilities of C. neoformans, very few studies have addressed the susceptibilities of C. gattii and none have compared the susceptibilities of specific subtypes. Only a few studies have used CLSI broth microdilution to report the susceptibilities of C. gattii (5, 6, 9, 24, 25, 27, 28), and none of these studies involved isolates from the Pacific Northwest emergence. As we found in our study, none of the earlier publications described elevated MICs to flucytosine or amphotericin B. Chen and coworkers (6) reported a higher geometric mean MIC of their 21 Taiwan isolates to amphotericin B than was found for the isolates in this study, but the mean was still within the range considered susceptible. Although we did not find any isolates with fluconazole MICs of >32 μg/ml, Trilles and coworkers reported fluconazole MICs of ≥64 μg/ml in their study of 57 C. gattii isolates from Brazil (28), although the proportion of their isolates with very high MICs was not given. Chen and coworkers also reported that 20% of the 18 C. gattii isolates from Australia that they tested had fluconazole MICs of ≥64 μg/ml (5). Our finding that 23.3% of the Pacific Northwest isolates have fluconazole MICs of 16 to 32 μg/ml is roughly double the 12.7% of isolates in this category reported from Spain (24) and reflects the findings of De Bedout and coworkers, who tested 15 C. gattii isolates from Colombia (9) and reported that 33% of their isolates had fluconazole MICs of 16 to 32 μg/ml. Voriconazole MICs in this study were relatively low, with the overall geometric mean MIC of 0.1 μg/ml and only a single isolate with an MIC of ≥1 μg/ml. Even so, our values are still higher than the geometric mean of 0.03 μg/ml reported for the isolates from Spain (24). Twenty-three percent of our isolates had itraconazole MICs that would be considered resistant for Candida species, but the clinical significance of these values for C. gattii is unknown.

The most interesting aspect of our study is that the MICs to fluconazole could be correlated with subtype. All of the VGI and VGIII isolates had comparatively low fluconazole MICs, while the majority of isolates with MICs of 16 to 32 μg/ml were of the subtype VGIIc. Clearly, there are differences between the subtypes, and these differences need to be further explored.

Unfortunately, what is not known is the relationship between elevated MICs and clinical outcome. While there have been no studies to date which correlate C. gattii MICs to clinical outcome, two recent studies of C. neoformans compared fluconazole MICs to clinical outcome (1, 8). The authors of these manuscripts reached opposite conclusions, with one study stating that among AIDS patients high fluconazole MICs may be predictive for patients who will not respond to fluconazole therapy (1), and the other study stating that among HIV-positive and -negative patients treated with fluconazole alone, there was no correlation between MICs and clinical success or failure (8). This is an area where more research is needed.

While fluconazole MICs of 16 and 32 μg/ml seem high compared to typical MICs for C. albicans and fluconazole, in the largest global study of C. neoformans susceptibility to date (1,811 human isolates) 44% of the isolates had an MIC to fluconazole of ≥8 μg/ml (26). Given this study and the studies cited above, it is clear that Cryptococcus species may have elevated MICs to fluconazole compared to values and breakpoints for Candida species. While we are not seeing MICs in the 64- to 256-μg/ml range, predictive of clinical failure for Candida species, VGIIb and VGIIc isolates from the Pacific Northwest have elevated MICs to fluconazole and comprise almost half of the collected isolates from the Pacific Northwest United States emergence. Unfortunately, very little is known about the clinical course and outcome for patients infected with these strains.

Treatment of C. gattii patients in the Pacific Northwest can be challenging, and these patients may have a difficult clinical course (31). In light of the difficulty in treating these patients and the 8.7% case fatality rate for these types of patients seen in British Columbia (13), it is prudent that physicians in the new areas of C. gattii emergence are aware that their patients may have an infection with C. gattii rather than with C. neoformans. It is also critical that we develop a better understanding of the correlation, if any, between MICs and clinical outcome. Public health surveillance for C. gattii infection, including tracking further spread of subtypes, may help us solve some of these problems.