the centers for disease control and prevention ( cdc ; atlanta , ga , usa ) has been performing passive surveillance for c. gattii in the united states since 2009 .
isolates were collected as part of routine surveillance for c. gattii or submitted to the fungal reference laboratory at the cdc for identification .
we identified all isolates to the species level as described ( 8) and used the consensus protocol for multilocus sequence typing ( mlst ) ( 9 ) .
in the course of this surveillance , several isolates were received from states in the southeast ; genotypic analysis revealed that these isolates consisted of 2 molecular types , vgi and vgiii ( 8) .
of > 400 isolates received from throughout the united states , 42 were molecular type vgi .
mlst analysis of vgi isolates showed a cluster of isolates with a single mlst sequence type originating in the southeastern states ( figure 1 ) .
five isolates from georgia and 1 each from florida , tennessee , and michigan were indistinguishable by mlst ( sequence type 162 ) .
a second isolate from georgia differed from this group by 1 single - nucleotide polymorphism ( snp ) in the intergenic spacer gene locus .
maximum - parsimony tree of multilocus sequence typing analysis of vgi isolates of cryptococcus gattii from the southeastern united states . in the predominant clade ,
1 isolate was from michigan ; all remaining isolates were from the southeastern united states .
nearest neighbor isolates were included for comparison , and an environmental vgi isolate from australia was used as an outgroup .
scale bar indicates 1 single - nucleotide polymorphism . because the population of vgi isolates from the southeastern states ( vgi - se )
was only recently identified , it might be a newly emerging population similar to the independent emergences of vgiia and vgiic in the pnw ( 68 ) . to further characterize this cluster , we used whole - genome sequence typing ( wgst ) of isolates from this cluster , other closely related isolates , and unrelated control isolates ( table 1 ; figure 2 ) .
, san diego , ca , usa ) , used gatk unified genotyper version 2.4 ( broad institute , cambridge , ma , usa ) for snp detection , and constructed maximum - parsimony snp trees using paup * ( sinauer associates , inc . , sunderland , ma , usa ) , as previously described ( 6 ) .
maximum - parsimony tree of whole - genome sequence data of isolates of cryptococcus gattii from the southeastern united states .
nearest neighbor isolates were included for comparison , and an environmental vgi isolate from australia was used as an outgroup .
although the overall topology of the tree we generated remained almost identical to that seen by mlst , we were able to separate each of the isolates within the cluster using wgst ( figure 2 ) .
a total of 41,024 snps were within the vgi - se cluster , which had an average branch length of 4,558 snp differences between any 2 isolates .
the isolate from georgia that differed in mlst by 1 snp was placed well within the cluster by wgst . outside the cluster , the nearest neighbor , an isolate collected from a resident of oregon with a history of living near the southeastern united states in texas ( table 1 ) , differed by > 29,000 snps .
the other cluster of vgi isolates was separated from vgi - se by 49,992 snps .
previously , whole - genome sequencing was performed on isolates of c. gattii molecular type vgii exclusively from the pnw .
results showed that the vgii isolates consisted of 3 different highly clonal and recently emerged populations with only 107 , 132 , and 137 snps identified within the vgiia , vgiib , and vgiic populations , respectively ( 10 ) ( table 2 ) .
the average branch length between any 2 isolates was < 18 snps for the vgiia and vgiic populations and only slightly higher for the vgiib population . on the basis of snp diversity ,
the vgi - se subtype is substantially older than the clonal vgii populations in the pnw and has likely been in the united states for a much longer time . *
these isolates , accessed from the same national reference laboratory , were submitted without clinical history ; therefore , we can not discern whether they represent the diversity of 2 isolates from 1 patient over time or whether they are part of a local clonal population .
all of the patients for whom disease manifestation was known had meningitis ( table 1 ) .
this finding differs notably from c. gattii vgii patients in the pnw , among whom a substantial proportion ( 59% ) had a primary pulmonary infection ( 8) . the average age of the patients with isolates in the vgi - se cluster was 46 ( range 1870 ) years . for most patients ,
the source of infection was unknown , although 2 of the patients had been exposed to rotting wooden structures ( 1 demolished a wooden structure , 1 power - washed old houses ) .
our data indicate that the clonal diversity of the c. gattii vgi - se clade in the southeastern united states is an order of magnitude greater than that seen in isolates from the pnw .
hypothesized that the emergence in the pnw began in the mid-1990s in british columbia , canada , and spread to the united states in the early 2000s ( 12 ) .
comparison of the average number of snps detected among the 3 vgii clonal groups in the pnw with the average number of snps detected between the vgi - se isolates indicates that the vgi - se isolates have been in place substantially longer .
the limitation to this conclusion is that we do not know the rate of mutation for the vgi or vgii molecular types ; however , there is no reason to believe that they would be substantially different .
there may also be higher rate of recombination within the vgi - se clade related to opposite - sex or same - gender sexual recombination .
the isolates from this clade for which the mating type is known are all  , but that does not preclude the possibility that some mating type a isolates exist in the environment in the southeastern united states .
the first identified isolate of c. gattii in the united states was reported from west virginia in 1924 ( 11 ) , although the isolate was not recognized as c. gattii vgi until decades later ( 12 ) .
two other historical reports of c. gattii isolates from the southeastern united states exist : the first , from 1968 , describes clinical and environmental isolates from savannah , georgia ; the other , from 1982 , describes isolates from alabama , tennessee , and louisiana from patients with no travel history to a c. gattii  endemic region ( 13,14 ) .
although both of these reports use the previous name for c. gattii , c. neoformans serotypes b and c , their results clearly indicate that c. gattii has existed in the southeastern united states for > 40 years , as opposed to the recent emergence in the pnw during the past 2 decades .
a more recent report describes a c. gattii vgi isolate from an hiv - positive patient in north carolina ( 15 ) ; those authors surmised that the isolate was related to travel to san francisco , but that assumption may have to be revised .   no current molecular clock for c. gattii indicates the time required to generate the level of diversity seen in the vgi isolates
however , our findings lend credence to the hypothesis that c. gattii has circulated in the southeastern united states long enough to be considered endemic .
 cryptococcus gattii is a recognized pathogenic fungus along the pacific coast of the united states from california to washington . here 
 we report that c. gattii may also be endemic to the southeastern united states and has probably been present there longer than in the pacific northwest . 
The Study
Conclusions