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Eukaryotic Cell, July 2008, p. 1075-1084, Vol. 7, No. 7
1535-9778/08/$08.00+0     doi:10.1128/EC.00062-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

Multilocus Sequence Typing of Pathogenic Candida Species{triangledown}

Frank C. Odds* and Mette D. Jacobsen

Aberdeen Fungal Group, Institute of Medical Sciences, Aberdeen, United Kingdom


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INTRODUCTION
 
Typing strains within a microbial species on the basis of DNA sequences at multiple loci has greatly advanced study of the epidemiology and evolutionary phylogenetics of many bacterial (9, 40, 60, 72) and fungal (26, 68) pathogens. Multilocus sequence typing (MLST) schemes have now been published for five of the opportunistically pathogenic Candida species: C. albicans (6, 7), C. dubliniensis (42), C. glabrata (19), C. krusei (30), and C. tropicalis (65). A sixth species, C. parapsilosis, shows too little sequence diversity to be typeable by MLST (64). This review discusses technical aspects and implications of findings from MLST studies of the genus Candida. There will inevitably be an emphasis on studies with C. albicans, since many more isolates of this species than of other Candida species have been subjected to MLST.

The ability to discriminate between microbial strain types has been, for very many years, a topic of great importance to epidemiologists. Strain typing facilitates tracing of sources and routes of transmission of infection outbreaks. However, a paradox exists at the heart of strain typing. The question most often posed by an epidemiologist is whether two microbial isolates are the same type, but the methodology used for typing is designed to reveal differences between strain types and is usually assessed statistically for its success in differentiation. Identity is inferred by implication when two isolates cannot be distinguished; however, increasing technological finesse means that two isolates classed as identical types in one system may be shown to differ in another. One possible resolution to the paradox of proving strain identity would be to compare whole-genome sequences for two isolates, something that is already a technical possibility for selected isolates and which seems likely to become a more common event in the near future. However, because sequence differences may arise rapidly in highly variable regions of the genome, evidence for strain identity may never achieve its theoretical perfection.

MLST, while a long way from providing complete genome sequences, generates evidence for similarities and differences between isolates from sequences determined, typically, for six to eight independent chromosomal genes. The MLST approach, originally devised by Maiden and colleagues (41), offers a number of advantages over most earlier phenotypic and genotypic typing methods. Sequencing data offer a higher level of reproducibility and minimal subjectivity in analysis compared with other technologies, and the sequences can be stored in Internet databases, offering an unprecedented degree of portability and accessibility to all interested users. (MLST databases can be accessed at two main host sites, http://pubmlst.org/ and http://www.mlst.net/.) Moreover, MLST is based on the same conceptual premise as multilocus enzyme electrophoresis, allelic variation, which means that MLST provides information relevant to population genetic studies as well as to epidemiology. The field of Candida epidemiology has already been substantially explored by a range of technologies. MLST data confirm the many previous studies in the field and offer an increment of refinement in the phylogenetic detail with which epidemiology can be explored experimentally.

Strain typing as a routine clinical laboratory procedure is more familiar to bacteriologists than to mycologists, which is perhaps why, with the exception of Aspergillus fumigatus (2), MLST data only for Candida species and Cryptococcus neoformans have been made available on the central MLST databases. In fact, the MLST approach to strain delineation was used with Coccidioides immitis (31) at least as early as the year when bacteriologists first set up their Web-based MLST systems (41). MLST has been applied to epidemiological and phylogenetic research with other fungal opportunist pathogens, including Cryptococcus gattii (8, 26), Fusarium solani (74), and Batrachochytrium dendrobatidis (43), but the authors of these publications have not utilized the central MLST Web repository for their data, which makes it less easy for others to add or reanalyze the data. The opposite applies to MLST for Cryptococcus neoformans var. grubii (35), the latest fungal addition to the central MLST database (http://www.mlst.net/).


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HISTORY AND BASIS OF CANDIDA MLST SCHEMES
 
Although some investigators had previously used genetic single-nucleotide polymorphisms (SNPs) to differentiate C. albicans strains (16, 39), the earliest publication describing a Candida MLST scheme similar in style and essence to bacterial MLST for clinical use was by Bougnoux and colleagues for C. albicans in 2002 (6). A C. albicans MLST system that included some different genes was published the following year from our laboratory (67), and collaboration between the two groups led to the establishment of a consensus MLST gene set that provided optimum differentiation for C. albicans isolates (7). MLST for C. glabrata (19), C. tropicalis (65), C. krusei (30), and C. dubliniensis (42) appeared over the following years. MLST for the phenotypically identical species C. guilliermondii and C. fermentati has been described (32); however, that study was aimed more at species differentiation than at strain typing. No Web database has been created for sequences of C. guilliermondii or C. fermentati.

Genes chosen to be sequenced for bacterial MLST traditionally encoded "housekeeping" functions, i.e., metabolic activities that are subject to stabilizing selection, because diversifying selection may obscure relationships among isolates (40). The SNPs for most of the genes used for MLST with Candida species generate ratios (dN/dS) of nonsynonymous to synonymous amino acid changes in their encoded peptides of less than 1.0, the criterion for stabilizing selection. However, we agree with Taylor and Fisher (68) that the choice of MLST loci needs to provide as much sequence diversity as possible; our scheme for C. krusei (30) includes loci with dN/dS ratios of >1 for precisely this reason.

The choice of genes with potential value for MLST can be made easily when full genomic information is available to help locate potential SNPs. In the absence of genomic information, the approach involves some hit-and-miss testing of a set of putatively distinct isolates by sequencing genes for which information is available, sometimes with the use of degenerate primers designed against conserved regions of likely housekeeping genes. This, for example, was the approach we used to devise the gene set for C. krusei MLST (30). The eight-gene set for typing C. dubliniensis was drawn directly from the set used to type C. albicans but with redesigned primers and, for two genes, with extended lengths of DNA sequenced (42).

In practice, to type an isolate by MLST, DNA is extracted; six (for C. glabrata, C. krusei, and C. tropicalis), seven (for C. albicans), or eight (for C. dubliniensis) PCRs are set up with the appropriate primer sets; and the PCR products are purified and used for bidirectional sequencing reactions (normally done with automated capillary sequencing equipment). The sequencing chromatograms are checked visually and scrutinized for heterozygous SNPs (C. albicans, C. dubliniensis, C. krusei, and C. tropicalis). Each sequence is compared with the reference database for the species. If it matches an existing sequence, it is assigned that sequence's allele number (C. glabrata) or (diploid) genotype number (C. albicans, C. dubliniensis, C. krusei, and C. tropicalis). If it is a new sequence, details are sent to the Web database curator, who assigns the sequence a new allele/genotype number. The set of six to eight allele/genotype numbers defines a strain type, numbered once again by reference to the database or by addition to it. The end product of the process is therefore the sequence type (ST) for haploid organisms or a diploid ST (DST) for diploid organisms. If two isolates have different STs or DSTs, this is evidence that the strain types are different, although the STs/DSTs themselves provide no information on the extent of SNP differences between two sequences. As already explained, if two isolates have the same ST or DST, this is evidence that the strains are indistinguishable by MLST but not necessarily that they are identical strains. Table 1 provides an overview of the current status of MLST data for five Candida spp.


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  		TABLE 1. Overview of the contributions of sequenced gene fragments to MLST for five Candida speciesa


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ISSUES OF NOMENCLATURE
 
Every specialized research area tends to develop its own vocabulary, often hijacking existing terms with subtly different variations on the usual meaning. MLST is no exception, and the first matter of concern is the name MLST itself. "MLST" means, literally, no more than typing something by sequencing multiple loci. The term "MLST" was coined for subspecies strain typing, but multilocus sequence data can also be used to determine interspecies and intergeneric differences. However, such breadth of use can be misleading, and it is recommended that "MLST" is best reserved for use solely in its original context of strain typing.

Strain typing methodology, as the name implies, results in delineation of strain types: multilocus STs in the case of MLST. The term ST (40) can be applied to all organisms; however, as already indicated, the term DST is normally used for diploid species, such as C. albicans, C. dubliniensis, C. krusei, and C. tropicalis. More problematic is the appellation of the individual gene sequences that combine to create the STs or DSTs. For haploid organisms these can properly be referred to as alleles, but for diploid organisms the sequences have been named "genotypes" ever since the earliest publication on C. albicans MLST (6). This inevitably generates a complex nomenclature for the MLST operation, with terms such as "genotype" and "ST" being capable of creating ambiguous meanings when taken out of context.

Diploid species provide an advantage for MLST work, in that heterozygous SNPs add a third level of information to the strain differentiation process. More useful for phylogenetic analyses are "haplotypes," which are the exact biallelic combinations in a diploid strain. Haplotypes can be easily determined for a given gene fragment only when both alleles are identical or when they contain a single heterozygous SNP. With two or more heterozygous SNPs in a sequenced fragment, haplotype determination requires separate cloning and sequencing of each allele. However, with sufficient sequence data available, dedicated computer programs can estimate haplotype probabilities with apparently commendable accuracy.

The Candida literature has for several years employed the word "clade" to describe a cluster of genetically related isolates. In its strict phylogenetic sense, "clade" should refer to a common ancestral species and all its evolutionary progeny and is therefore a less appropriate term to define genetically related groups of strains without any robustly defined evolutionary lineage. However, the word "cluster" is already taken in the term "clonal cluster," which defines groups of strains delimited by software such as eBURST (see below), and can therefore cause ambiguity. In phylograms of analyzed MLST data, the branches at least resemble true phylogenetic clades. We will therefore join the rest of the Candida typing field in continuing informally to "take the liberty" (59) of defining major sets of genetically related isolates as clades.

A phenomenon well known for many years in C. albicans is evolutionary change represented by small alterations in the sequences of some genes. The phenomenon was first described in 1995 (36) and was called "microevolution." Although this name seems to be consistent with the phenomenon described, population biologists take "microevolution" to refer to small-scale changes in allele frequencies, and the term seems to have taken on many shades of meaning in recent years. We have therefore coined the term "microvariation" to describe sequence changes at the level of individual gene fragments (45), and "microvariation" will be used instead of "microevolution" in this review.


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TECHNICAL ISSUES
 
At its heart, MLST with a Candida species is no more than a matter of extracting DNA and sequencing gene fragments with manageable lengths of 400 to 600 bp. Our group initially used Pfu DNA polymerase in our sequencing work for its superior proofreading properties. We have subsequently reverted to less expensive, nonproofreading polymerases since it is exceedingly unlikely that an error will arise in an early cycle of the PCR fragment of ~500 bp used for sequencing. However, the need for sequencing to be as accurate as possible remains; for MLST both DNA strands are routinely sequenced to ensure that ambiguous base readings can be called as correctly as possible. Prospective evaluations of sequencing accuracy indicated a reproducibility of 99.72% for C. albicans MLST (63) and 100% for C. dubliniensis MLST (42).

One problem with sequencing a diploid genome concerns heterozygous SNPs. In many instances a result is easily interpreted as indicating heterozygosity when two peaks in a sequencing chromatogram overlap with equal heights approximately half the size of neighboring peaks. However, the majority of heterozygous SNPs appear in practice with overlapping peaks of less than perfectly equal heights. To avoid erroneous interpretation of double peaks as heterozygous SNPs when they may be no more than sequencing noise, we routinely take account of noise in neighboring positions and carefully compare the chromatograms for both strands sequenced. Our approach is illustrated in Fig. 1. For MLST with a diploid fungus, visual scrutiny of sequencing chromatograms is a highly labor-intensive aspect of the procedure. Inaccurate calls of heterozygosity at a polymorphic site undoubtedly occur and are probably the main source of irreproducibility in MLST data, even though such irreproducibility is demonstrably very low.


Figure 1
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  		FIG. 1. Sequencing chromatograms for bases 2 to 13 in the ACC1 fragment of C. albicans, indicating heterozygosity. For each isolate the forward and reverse chromatograms are shown. For isolates A and B, position 8 would be read as heterozygous AG, even though the A and G peaks coincide perfectly only in one of the four sets. For isolate C, the very small A peak at position 8 would be read as noise, by comparison with adjacent bases, and the result read as homozygous G for MLST purposes.


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DISCRIMINATORY POWER OF FUNGAL MLST
 
At the time of this writing (February 2008), the C. albicans MLST database shows over 1,100 DSTs for more than 1,500 isolates, the C. tropicalis database contains 202 DSTs for 270 isolates, and the C. krusei database shows 99 DSTs from 134 isolates. Our equivalent in-house databases contain more MLST test data, but these sets include strain and colony replicate tests and multiple isolates from single sources, which are not valid for upload to the Web database or for inclusion in phylogenetic analyses. With such artificial replicates of DST and allele numbers excluded, Fig. 2 illustrates how the rate of acquisition of new genotypes for each of the seven C. albicans MLST genes steadily decreases with increasing numbers of isolates sequenced, while the comparable curve for new DSTs responds far more slowly to the inevitable law of diminishing returns. The discriminatory power, D, of MLST, newly calculated from our current databases according to Hunter's formula (29), is 99.9% for C. albicans (n = 1,594), 99.8% for C. krusei (n = 134), and 99.9% for C. tropicalis (n = 270). For C. dubliniensis the equivalent published value is 90.9% (n = 50) (42). For C. glabrata no discriminatory power value has yet been quoted; it cannot be calculated here, as the database is currently incomplete.


Figure 2
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  		FIG. 2. Rates of acquisition of new genotypes and new DSTs for C. albicans MLST. (a) As the number of sequenced isolates increases, the rate of discovery of new genotypes falls off fairly rapidly (pink, VPS13; green, ZWF1b; brown, SYA1; black, AAT1a; blue, ADP1; red, PMIb; yellow, ACC1). (b) The rate of acquisition of new DSTs falls off much more slowly, reflecting the high discriminatory power of the method.


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WHAT DEFINES A CLADE OR CLUSTER?
 
Perhaps the most obvious thing to do with a set of strain typing data—it must be so, since almost every paper on microbial strain typing does this—is to apply a statistical cluster analysis that places strains of high similarity in groups and, typically, represents those groups with a dendrogram. However; for MLST, careful consideration has to be given to the choice of analytical approach. The problem is one of deciding what constitutes which level of difference with sequence data for multiple loci. Because evolutionary change can occur by mechanisms such as recombination as well as simple point mutation, it is possible that an extensive list of changes in SNPs at a single locus between two isolates in fact represents no more than a single event.

Consider two C. albicans isolates with the same sequences for six of the seven MLST genes but with the seventh gene in one isolate containing, say, seven heterozygous SNPs while the other isolate has no heterozygous SNPs in that gene. This sort of situation is very common in the MLST database, and indeed loss of heterozygosity has been recognized for many years as a means by which C. albicans can generate microvariation and population diversity (15, 25, 48). At the level of the seven gene fragments sequenced for MLST, the difference between the two isolates is 1/7 (14.3%). At the level of nucleotide polymorphisms, the difference is 7 SNPs out of 185 total known SNPs across the seven genes sequenced (Table 1), or only 3.8%. Two isolates would need to differ in 26 of the 185 SNPs to appear as different as two isolates with 1/7 gene fragments different. We are unaware that analysis at either the locus or the SNP level is more "correct" in the context of MLST. In multilocus enzyme electrophoresis, differences would appear only at the equivalent of the allele level, whereas it can be argued that MLST allows greater finesse by consideration of numbers of SNP differences. The problem of comparing SNPs becomes further complicated by the diploid genomes of many Candida species. For clustering purposes, homozygous SNPs (e.g., CC versus TT) have been scored as twice the value of heterozygous differences (e.g., either CC or TT versus CT) (6, 44, 63). This reduces the calculated interisolate difference for the seven-heterozygous versus seven-homozygous SNP example to just 2%.

Analysis of strain differences based on whole-genotype or allele differences is relatively straightforward. The software called eBURST was written precisely for this purpose (23). It analyzes sets of genotype (allele) data and joins isolates that differ only in one of the whole set of genes sequenced in a "clonal cluster." The output from an eBURST analysis for an entire MLST data set is visualized in the form of a "snapshot" of (D)STs (Fig. 3) in which the structures of clonal clusters and the prevalence of "singletons" (isolates that do not belong to any cluster) can be reviewed. Although the default setting for an eBURST analysis is to generate clonal clusters in which (D)STs are linked to others that differ in a single allele, it is permissible, and sometimes more revealing, to use a more relaxed criterion for a cluster, such as differences in two alleles. The theory behind the eBURST software is that the putative founder of a clonal cluster is the most probable ancestor from which all other linked types have evolved. Recent research has shown, however, that while this premise appears to hold well for many organisms for which the snapshot shows few singletons, the snapshots for the three diploid Candida species, with their high prevalence of singletons (Fig. 3), are typical of populations with a high rate of recombination relative to mutation and for which eBURST does not reliably indicate ancestry (71). This finding explains why eBURST applied to a small population of multiple C. albicans isolates from the same set of patients entirely failed to correspond to likely ancestral relationships (45). For the three diploid Candida species, then, eBURST is a helpful clustering tool but otherwise does not contribute to phylogenetic analyses.


Figure 3
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  		FIG. 3. Computer printouts of eBURST snapshots for 1,564 isolates of C. albicans (a), 126 isolates of C. krusei (b), and 242 isolates of C. tropicalis (c). In each snapshot, lines join pairs of isolates that show sequence differences in only one of the panel of genes used for MLST. The positions of the clonal clusters and singletons and the lengths of lines are without relevance. For all three species, the snapshots indicate several clusters of related strains but also a high proportion of singletons, indicative of a high rate of recombination relative to mutation (70).

The most widely used approach for analyses of MLST data is to draw a dendrogram based on a statistical clustering algorithm. The unweighted-pair group method with arithmetic mean (UPGMA) has been widely used for many types of microbial strain typing analysis, and MLST is no exception. Applied to MLST data, this method analyzes data at the level of individual SNP differences. Its benefit, where MLST for Candida species is concerned, is that the major clades in the UPGMA dendrogram for C. albicans correlate extremely well with the clades previously defined by DNA fingerprinting with the moderately repetitive probe Ca3 (59). By including five isolates already assigned by Ca3 fingerprinting to each of the clades I, II, III, SA, and E, our group was able to show that four major branches in the MLST UPGMA dendrogram each included the corresponding isolates from the first four of the Ca3 fingerprinting clades (63). The MLST clades have therefore been designated 1 through 4 (63). The five isolates from Ca3 clade E did not fall into a single clade in the C. albicans MLST dendrogram; two were in clade 4 and three were in clade 11. The UPGMA-determined clades also correlate well with eBURST clonal clusters; no examples have been found in which clonal clusters cross UPGMA clade boundaries, although clades may contain more than a single clonal cluster.

It may reasonably be objected that the basis used for delineation of C. albicans clades, i.e., an arbitrary p distance cutoff, has no objective reality. (The cutoff of 0.04 used for C. albicans [44, 63] was chosen because it separates clades 2 and 4, which are well discriminated by other typing methods.) We acknowledge that a small minority of isolates can shift to different clades as the data set grows and also that very small clades may be assimilated into others. However, the assignments of isolates in the largest clade groupings appear to be robust and stable and have not altered as the size of the database has increased.

Bootstrap values for cluster nodes in the UPGMA dendrogram have proved unhelpful in delineating clade boundaries because they become very low in large data sets of (mainly) closely related strains, except for the smallest, tightest subclusters. We have attempted to use more robust phylogenetic clustering approaches for MLST data, such as maximum-parsimony and minimum-likelihood trees, but these methods require very long computational times or even fail, with some software packages, when applied to large numbers of isolates. Others using these methods have applied them only to small sets or subsets of isolates, typically fewer than 120 (8, 26, 31, 35). When larger isolate sets have been analyzed, e.g., the 218 F. solani isolates analyzed by Zhang et al. (74), only three sequenced loci were tested. We too have run maximum-parsimony analyses with the seven MLST gene loci run separately with small subsets of the C. albicans panel that represent the strain diversity found experimentally (44), but that approach involves our selecting isolates by their positions in the UPGMA dendrogram, thus defeating the object of establishing clades by objective criteria.


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BAYESIAN CLUSTERS, HAPLOTYPES, AND PHYLOGENETICS
 
An approach to definitions of strain groupings that has produced excellent, epidemiologically informative results with some bacterial MLST data (22) is analysis of alleles by software named Structure (21). The output of this package, which analyzes data in a Bayesian framework, is assignment of strains in a population to one of K groups based on the proportion of their genotype representing each of the K groups. The Structure package does not determine the value of K, which must be provided as an input, so in practice one runs several replicates for a range of values of K, determining the optimum value of K from a heuristic. The input data for Structure consists of haplotypes, which for C. albicans MLST sequences can be reconstructed with seemingly reliable precision by the program PHASE (61) or its more efficient successor fastPHASE (http://depts.washington.edu/ventures/UW_Technology/Express_Licenses/fastPHASE.php). For a C. albicans MLST data set that included one example of every known DST, we have run quintuplicates of Structure for values of K from 8 to 19, and the output indicates that division into 11 to 13 groups best defines the population under analysis (F. C. Odds, unpublished data). However, the assignments of individual isolates to groups differs from run to run in Structure. The positive outcome is that Structure analyses have consistently confirmed the assignment of isolates to the five largest UPGMA clades (1 to 4 and 11), with the exception of a very small number of DSTs. The Structure analysis also consistently shows good separation of the groups equivalent to clades 2 and 4, further justifying our basis for defining a p distance of 0.04 as a criterion for UPGMA clade separation.

PHASE-reconstructed C. albicans haplotypes were analyzed for possible recombination events detectable by comparison of all pairs of SNPs in each haplotype. These occurred often enough within the species that its propagation cannot be regarded as exclusively clonal (44). This finding accords with the high prevalence of singleton strains in eBURST snapshots (71). In a related analysis the data for seven genes sequenced for C. albicans MLST showed a lack of congruence suggestive of separate evolutionary histories for each gene (44). The phylogenetic data therefore clearly point to significant levels of recombination, and even possibly sexual matings, in the evolutionary history of C. albicans.

For C. dubliniensis, where so far only 50 isolates have been typed, the data indicated a purely clonal mode of reproduction (42). One notable feature of the first C. dubliniensis MLST study is the relative lack of SNP diversity in this species compared with C. albicans (42). From Table 1 it can be seen that more than 6% of the bases sequenced for C. albicans are SNPs, compared with fewer than 1% for C. dubliniensis. Even when only the first 86 isolates of C. albicans had been typed, 2.9% of the bases sequenced were SNPs. It is not possible similarly to combine and compare MLST data for other Candida species, since they are based on sequencing of different genes. However, for C. parapsilosis, the near-total absence of SNPs was interpreted as indicting a relatively recently evolved species (64); perhaps the low proportion of SNPs among the C. dubliniensis genes sequenced for MLST also points to an evolutionary origin more recent than that for C. albicans. However, on the evidence from comparisons of nuclear DNA sequences (17) and from whole-genome phylogenies (24), it seems more likely that both species arose from a common ancestor. The fact that the positions of the MLST SNPs in the same genes from the two species are mostly different (42) also argues for a common ancestor rather than sequential origin. Like C. albicans and C. dubliniensis, C. guilliermondii and C. fermentati are two very closely related species. Isolates of the two species could be easily differentiated by sequencing five genes, although minimal intraspecies variation was seen, possibly because the genes sequenced were likely to be highly conserved (32).


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EPIDEMIOLOGICAL FINDINGS FROM MLST
 
The basic features of the epidemiology of Candida infections have been known for many years from a variety of DNA typing approaches. Most individuals carry and/or are infected by a single strain type, though mixed types are found in some instances; strains undergo microvariation within their host, often involving changes in zygosity of diploid allele pairs; and strains show strong, though imperfect, geographical associations (57, 59). MLST studies have confirmed and, in some instances, amplified the detail of our understanding of the epidemiology of Candida species as commensals and pathogens.

The principal observation common to MLST surveys of C. albicans and C. glabrata is the tendency for isolates from individual geographical areas to cocluster within different clades. This leads to notably high or low representation of particular geographical sources within some clades, which is a predictable phenomenon, since strains given time to evolve in separate geographical compartments will inevitably develop locally associated traits. However, because most of the pathogenic Candida species are carried as human commensals, movements of human populations, particularly those that have occurred during the most recent centuries, are likely to have contributed to admixture of strain populations across the planet. Thus, among 103 isolates of C. glabrata, those from Europe were significantly underrepresented in MLST group III while isolates from Japan were significantly overrepresented in MLST group IV (19).

Among C. albicans clades, clade 2 contains a very high proportion of isolates from the United Kingdom, while clade 11 is enriched with isolates from continental Europe. Isolates from Pacific Rim countries dominate clades 14 and 17 (44). Only clade 1 isolates show a fully worldwide distribution, although even among clade 1 strains, subclusters with geographical specificity can be seen. This is depicted in Fig. 4, where the isolates comprising C. albicans clade 1 are seen to contain two eBURST clonal clusters, one with only 6% of isolates from Asian countries and the other with 78% of Asian isolates. A subset analysis of the C. albicans MLST data based only on isolates from Japan, England/Wales, and North America confirmed clonal clusters with geographical specificity, emphasizing in particular the high prevalence of Japanese isolates in clades other than 1 to 4 (62). It is entirely possible that the perception of clades 1 to 4 as the "major" ones is an artifactual consequence of the high numerical predominance of strains of non-Asian origin in C. albicans typing surveys.


Figure 4
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  		FIG. 4. Enlarged view of the clustering of 462 isolates in C. albicans clade 1 as defined by a UPGMA dendrogram drawn for 1,433 isolates currently in our database. Two eBURST clonal clusters that constitute the majority of clade 1 isolates are indicated; the second cluster is heavily enriched with isolates from southeast Asia.

Geographical associations of strain groups were not seen by MLST of C. dubliniensis, C. krusei, and C. tropicalis, but this may partly reflect the comparatively low numbers of isolates typed so far or, in some cases, the relative geographical homogeneity of the isolate set tested (30, 42, 65).

Other associations of isolate properties with MLST clades include examples of resistance to antifungal agents. The majority of C. albicans isolates with resistance to flucytosine have been found in Ca3/MLST clade 1 and, so far, all clade 1 isolates resistant to flucytosine have had a mutation (R101C) in FUR1, i.e., a common mechanism of resistance within the clade, while flucytosine-resistant isolates from other clades have not shown this mutation (18, 63). Similarly, a higher number of isolates resistant to amphotericin B was found in Ca3 clade SA, which corresponds to MLST clade 4 (4). In C. dubliniensis, a clade originally defined by DNA fingerprinting and confirmed by MLST was enriched with flucytosine-resistant isolates (1, 42).

Clade-associated properties extend to other features unrelated directly to the genes sequenced for MLST and therefore indicating evolutionary codivergence in a species with predominantly clonal propagation. Significant differences between clades have been found for alleles of tandem repeat sequences in the genes ALS3, ALS5, and ALS6, which encode cell wall adhesins (47, 75). Recent work in our laboratory extends this list of clade-associated tandem repeat alleles to ALS1 and ALS7 and also other genes encoding glycosylphosphatidylinositol-anchored proteins, including HYR1 and HYR3 (D. M. MacCallum et al., unpublished data).

Analysis of a European subset of C. albicans isolates (the subset was chosen to minimize geographical influence on clade differences) showed a significantly higher proportion of isolates from superficial infections and commensal carriage compared with blood isolates in clade 1 compared with the four other largest clades (44). This raises the intriguing proposition that the apparent numerical dominance and pan-global distribution of clade 1 C. albicans may be a consequence of slightly higher fitness to colonize mucosal surfaces. Schmid and colleagues have found a "general-purpose" set of isolates, defined by Ca3 typing, whose members have a greater propensity than other types to cause infections (27, 53). This set of isolates seems to correlate largely with clade 1. A small statistical advantage for clade 1 isolates as colonizers or infecting strains is compatible with their numerical dominance among the whole population.

Two small but well differentiated sets of C. albicans isolates merit special mention. The first (clade 13 [44]) comprises almost entirely isolates that were originally described as a new species, C. africana (69). The main characteristics of these isolates were their predominantly (but not exclusively) African origin and their genital, typically vaginal, site of isolation. Among the 16 isolates presently included in clade 13, all from different patients and from 10 different countries, only three DSTs are represented (14 isolates are DST 182), 14 isolates are from genitalia (one other comes from sputum and one from blood culture), and the most dissimilar isolate in the clade, which is still within a p distance of 0.02, comes from Japan (reference 44 and unpublished data). The second small strain set of interest comprises only four isolates, all of them DST 1031 and all originally identified as C. stellatoidea type 1, i.e., strains unable to assimilate sucrose, including CBS1905, the C. stellatoidea type strain (29a). It is perhaps worth noting that the "C. stellatoidea" isolates join with five singleton isolates and with 13 isolates designated clade 16 to form a set of isolates more dissimilar than any others from the "mainstream" clades of C. albicans. They have extremely diverse geographical origins.


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TRANSMISSION, MAINTENANCE, AND MICROVARIATION OF C. ALBICANS STRAIN POPULATIONS
 
Historically, all of the many approaches to C. albicans strain differentiation have shown that most individuals are colonized at multiple anatomical sites with a single strain type (57). However, minor genetic changes characterized as microvariation ("microevolution") or "substrain shuffling" have shown that C. albicans strains are probably able to adapt with time to new microenvironments by selection of subpopulations with presumed fitness advantages (3, 11, 34, 36, 37, 49-51, 55). The microvariation phenomenon has been demonstrated by passaging C. albicans strains in vitro (12-14).

MLST has similarly confirmed consistent intraindividual strain types, microvariation, and occasional strain replacement (5, 10, 45). C. albicans strain types were known to be transmissible between sex partners (54), and MLST has extended the range of transmission to include families (5).

Loss of genetic heterozygosity as a mechanism underpinning microvariation in C. albicans (15, 25, 52, 66, 70) is inevitably picked up also by MLST (5, 45). However, one striking feature of MLST analyses of successive isolates from the same patient is the finding of loss of heterozygosity at one or more sequenced loci, followed by regain of heterozygosity (45). While loss of heterozygosity can be readily accounted for by phenomena now well demonstrated in C. albicans, i.e., chromosomal loss and reduplication or mitotic recombination (25, 33, 56, 58, 70, 73), the regain of heterozygosity suggests that populations colonizing or infecting an individual may contain mixtures of heterozygous and homozygous variants (45). C. albicans colonies with different morphologies plated from a single sample were shown to have identical or similar Ca3 fingerprinting patterns (28). We recently typed by MLST randomly picked, separate C. albicans colonies from primary isolation plates and found that the rates of zygosity variation (and even of strain replacement) within a sample are similar to those seen between samples when single clones are typed from sequential isolations of the fungus from a single patient (Jacobsen et al., submitted). All these findings combine to suggest that populations of C. albicans even within the same site in an individual patient may be mixtures of strain types.

Finally, an MLST study of C. albicans isolates from a variety of animals has confirmed and extended earlier work, based on Ca3 fingerprinting (20), showing that strains colonizing or infecting animals are related to those found in human samples but with clear evidence of significant differences, suggestive of a separate evolution in the different hosts (Jacobsen et al., submitted).


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MLST OF CANDIDA SPECIES: SOME CONCLUSIONS
 
It is clear that MLST has added a substantial contribution to our detailed understanding of the epidemiology, transmission, and phylogenetics of the main pathogenic Candida species. Future investigations of mechanisms of pathogenesis of Candida infection need to include strains representative at least of the major clades of each species. To what extent continuation of typing studies based on MLST is justified is less clear. Findings from many genotyping methods have shown the same basic features of Candida epidemiology.

It is possible to view the constant addition of new isolates to the now-large C. albicans database as a biological resource, but in reality the steady increase in numbers of strains typed has ultimately contributed more to the statistical robustness of conclusions drawn than to the creation of new conclusions. The databases for the other typeable Candida species are much smaller than that for C. albicans, so these will benefit from further growth. Isolates of C. albicans that are indistinguishable by MLST can be differentiated by other approaches such as microsatellite typing (Jacobsen and Odds, unpublished data), and SNP arrays for typing Candida strains based on MLST sequences have already emerged (38) in the same way as they have for other organisms, from bacteria to mammals. The present MLST scheme may need this type of further adaptation to be of clinical utility, for example, to identify putative outbreaks of C. albicans infection within an institution. In a survey of candidemia in which all C. albicans isolates were typed by MLST, indistinguishable DSTs were found for some bloodstream isolates from hospitals in different cities (46). This is unlikely to be evidence for interhospital strain transmission; it probably is just a reminder of the difficulty mentioned at the start of this review in proving that isolates are the same.


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ACKNOWLEDGMENTS
 
The authors' research on MLST of Candida species has been variously supported with grants from the Wellcome Trust; Euresfun; Merck, Sharp & Dohme, Ltd.; and Pfizer UK.

We owe a particular debt of gratitude to (in alphabetical order) Jude Bain, Amanda Davidson, Arianna Tavanti, and Julie Whyte, who also undertook all the tedious work of sequencing, and to Duncan Shaw, whose facility with genetics and statistics has contributed hugely to our analyses of MLST data. Our colleagues Marie-Elisabeth Bougnoux, Christophe d'Enfert, and Shu-Ying Li, in Paris and Taiwan, have all been true pioneers, generating masses of data and shrewd observations in the Candida MLST world. Finally, thanks are due to Neil Gow and Martin Maiden, whose encouragement prompted our laboratory's research into typing fungi by multilocus sequencing.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Sciences, Aberdeen AB25 2ZD, United Kingdom. Phone and fax: 44 1224 555828. E-mail: f.odds{at}abdn.ac.uk Back

{triangledown} Published ahead of print on 2 May 2008. Back


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REFERENCES
 
    1
  1. Al Mosaid, A., D. J. Sullivan, I. Polacheck, F. A. Shaheen, O. Soliman, S. Al Hedaithy, S. Al Thawad, M. Kabadaya, and D. C. Coleman. 2005. Novel 5-flucytosine-resistant clade of Candida dubliniensis from Saudi Arabia and Egypt identified by Cd25 fingerprinting. J. Clin. Microbiol. 43:4026-4036.[Abstract/Free Full Text]
    2. 2
  2. Bain, J. M., A. Tavanti, A. D. Davidson, M. D. Jacobsen, D. Shaw, N. A. R. Gow, and F. C. Odds. 2007. Multilocus sequence typing of the pathogenic fungus Aspergillus fumigatus. J. Clin. Microbiol. 45:1469-1477.[Abstract/Free Full Text]
    3. 3
  3. Ball, L. M., M. A. Bes, B. Theelen, T. Boekhout, R. M. Egeler, and E. J. Kuijper. 2004. Significance of amplified fragment length polymorphism in identification and epidemiological examination of Candida species colonization in children undergoing allogeneic stem cell transplantation. J. Clin. Microbiol. 42:1673-1679.[Abstract/Free Full Text]
    4. 4
  4. Blignaut, E., J. Molepoa, C. Pujol, D. R. Soll, and M. A. Pfaller. 2005. Clade-related amphotericin B resistance among South African Candida albicans isolates. Diagn. Microbiol. Infect. Dis. 53:29-31.[CrossRef][Medline]
    5. 5
  5. Bougnoux, M.-E., D. Diogo, N. Francois, B. Sendid, S. Veirmeire, J. F. Colombel, C. Bouchier, H. Van Kruiningen, C. d'Enfert, and D. Poulain. 2006. Multilocus sequence typing reveals intrafamilial transmission and microevolutions of Candida albicans isolates from the human digestive tract. J. Clin. Microbiol. 44:1810-1820.[Abstract/Free Full Text]
    6. 6
  6. Bougnoux, M.-E., S. Morand, and C. d'Enfert. 2002. Usefulness of multilocus sequence typing for characterization of clinical isolates of Candida albicans. J. Clin. Microbiol. 40:1290-1297.[Abstract/Free Full Text]
    7. 7
  7. Bougnoux, M. E., A. Tavanti, C. Bouchier, N. A. R. Gow, A. Magnier, A. D. Davidson, M. C. J. Maiden, C. d'Enfert, and F. C. Odds. 2003. Collaborative consensus for optimized multilocus sequence typing of Candida albicans. J. Clin. Microbiol. 41:5265-5266.[Abstract/Free Full Text]
    8. 8
  8. Bovers, M., M. R. Diaz, F. Hagen, L. Spanjaard, B. Duim, C. E. Visser, H. L. Hoogveld, J. Scharringa, I. M. Hoepelman, J. W. Fell, and T. Boekhout. 2007. Identification of genotypically diverse Cryptococcus neoformans and Cryptococcus gattii isolates by Luminex xMAP technology. J. Clin. Microbiol. 45:1874-1883.[Abstract/Free Full Text]
    9. 9
  9. Chan, M. S., M. C. J. Maiden, and B. G. Spratt. 2001. Database-driven multi locus sequence typing (MLST) of bacterial pathogens. Bioinformatics 17:1077-1083.[Abstract/Free Full Text]
    10. 10
  10. Chen, K. W., Y. C. Chen, H. J. Lo, F. C. Odds, T. H. Wang, C. Y. Lin, and S. Y. Li. 2006. Multilocus sequence typing for analyses of clonality of Candida albicans strains in Taiwan. J. Clin. Microbiol. 44:2172-2178.[Abstract/Free Full Text]
    11. 11
  11. Chong, P. P., Y. L. Lee, B. C. Tan, and K. P. Ng. 2003. Genetic relatedness of Candida strains isolated from women with vaginal candidiasis in Malaysia. J. Med. Microbiol. 52:657-666.[Abstract/Free Full Text]
    12. 12
  12. Cowen, L. E., J. B. Anderson, and L. M. Kohn. 2002. Evolution of drug resistance in Candida albicans. Annu. Rev. Microbiol. 56:139-165.[CrossRef][Medline]
    13. 13
  13. Cowen, L. E., L. M. Kohn, and J. B. Anderson. 2001. Divergence in fitness and evolution of drug resistance in experimental populations of Candida albicans. J. Bacteriol. 183:2971-2978.[Abstract/Free Full Text]
    14. 14
  14. Cowen, L. E., A. Nantel, M. S. Whiteway, D. Y. Thomas, D. C. Tessier, L. M. Kohn, and J. B. Anderson. 2002. Population genomics of drug resistance in Candida albicans. Proc. Natl. Acad. Sci. USA 99:9284-9289.[Abstract/Free Full Text]
    15. 15
  15. Cowen, L. E., D. Sanglard, D. Calabrese, C. Sirjusingh, J. B. Anderson, and L. M. Kohn. 2000. Evolution of drug resistance in experimental populations of Candida albicans. J. Bacteriol. 182:1515-1522.[Abstract/Free Full Text]
    16. 16
  16. Cowen, L. E., C. Sirjusingh, R. C. Summerbell, S. Walmsley, S. Richardson, L. M. Kohn, and J. B. Anderson. 1999. Multilocus genotypes and DNA fingerprints do not predict variation in azole resistance among clinical isolates of Candida albicans. Antimicrob. Agents Chemother. 43:2930-2938.[Abstract/Free Full Text]
    17. 17
  17. Diezmann, S., C. J. Cox, G. Schonian, R. J. Vilgalys, and T. G. Mitchell. 2004. Phylogeny and evolution of medical species of Candida and related taxa: a multigenic analysis. J. Clin. Microbiol. 42:5624-5635.[Abstract/Free Full Text]
    18. 18
  18. Dodgson, A. R., K. J. Dodgson, C. Pujol, M. A. Pfaller, and D. R. Soll. 2004. Clade-specific flucytosine resistance is due to a single nucleotide change in the FUR1 gene of Candida albicans. Antimicrob. Agents Chemother. 48:2223-2227.[Abstract/Free Full Text]
    19. 19
  19. Dodgson, A. R., C. Pujol, D. W. Denning, D. R. Soll, and A. J. Fox. 2003. Multilocus sequence typing of Candida glabrata reveals geographically enriched clades. J. Clin. Microbiol. 41:5709-5717.[Abstract/Free Full Text]
    20. 20
  20. Edelmann, A., M. Kruger, and J. Schmid. 2005. Genetic relationship between human and animal isolates of Candida albicans. J. Clin. Microbiol. 43:6164-6166.[Abstract/Free Full Text]
    21. 21
  21. Falush, D., M. Stephens, and J. K. Pritchard. 2003. Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164:1567-1587.[Abstract/Free Full Text]
    22. 22
  22. Falush, D., T. Wirth, B. Linz, J. K. Pritchard, M. Stephens, M. Kidd, M. J. Blaser, D. Y. Graham, S. Vacher, G. I. Perez-Perez, Y. Yamaoka, F. Mégraud, K. Otto, U. Reichard, E. Katzowitsch, X. Wang, M. Achtman, and S. Suerbaum. 2003. Traces of human migrations in Helicobacter pylori populations. Science 299:1582-1585.[Abstract/Free Full Text]
    23. 23
  23. Feil, E. J., B. C. Li, D. M. Aanensen, W. P. Hanage, and B. G. Spratt. 2004. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J. Bacteriol. 186:1518-1530.[Abstract/Free Full Text]
    24. 24
  24. Fitzpatrick, D. A., M. E. Logue, J. E. Stajich, and G. Butler. 2006. A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis. BMC Evol. Biol. 6:99.[CrossRef][Medline]
    25. 25
  25. Forche, A., G. May, and P. T. Magee. 2005. Demonstration of loss of heterozygosity by single-nucleotide polymorphism microarray analysis and alterations in strain morphology in Candida albicans strains during infection. Eukaryot. Cell 4:156-165.[Abstract/Free Full Text]
    26. 26
  26. Fraser, J. A., S. S. Giles, E. C. Wenink, S. G. Geunes-Boyer, J. R. Wright, S. Diezmann, A. Allen, J. E. Stajich, F. S. Dietrich, J. R. Perfect, and J. Heitman. 2005. Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak. Nature 437:1360-1364.[CrossRef][Medline]
    27. 27
  27. Giblin, L., A. Edelmann, N. X. Zhang, N. B. von Maltzahn, S. B. Cleland, P. A. Sullivan, and J. Schmid. 2001. A DNA polymorphism specific to Candida albicans strains exceptionally successful as human pathogens. Gene 272:157-164.[CrossRef][Medline]
    28. 28
  28. Hellstein, J., H. Vawterhugart, P. Fotos, J. Schmid, and D. Soll. 1993. Genetic similarity and phenotypic diversity of commensal and pathogenic strains of Candida albicans isolated from the oral cavity. J. Clin. Microbiol. 31:3190-3199.[Abstract/Free Full Text]
    29. 29
  29. Hunter, P. R. 1990. Reproducibility and indices of discriminatory power of microbial typing methods. J. Clin. Microbiol. 28:1903-1905.[Abstract/Free Full Text]
    30. 29
  30. Jacobsen, M. D., T. Boekhout, and F. C. Odds. 10 June 2008, posting date. Multilocus sequence typing confirms synonymy but highlights differences between Candida albicans and Candida stellatoidea. FEMS Yeast Res. doi:10.1111/j.1567-1364.2008.00392.x.
    31. 30
  31. Jacobsen, M. D., N. A. R. Gow, M. C. J. Maiden, D. J. Shaw, and F. C. Odds. 2007. Strain typing and determination of population structure of Candida krusei by multilocus sequence typing. J. Clin. Microbiol. 45:317-323.[Abstract/Free Full Text]
    32. 31
  32. Koufopanou, V., A. Burt, and J. W. Taylor. 1997. Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis. Proc. Natl. Acad. Sci. USA 94:5478-5482.[Abstract/Free Full Text]
    33. 32
  33. Lan, L., and J. P. Xu. 2006. Multiple gene genealogical analyses suggest divergence and recent clonal dispersal in the opportunistic human pathogen Candida guilliermondii. Microbiology 152:1539-1549.[Abstract/Free Full Text]
    34. 33
  34. Legrand, M., P. Lephart, A. Forche, F. M. C. Mueller, T. Walsh, P. T. Magee, and B. B. Magee. 2004. Homozygosity at the MTL locus in clinical strains of Candida albicans: karyotypic rearrangements and tetraploid formation. Mol. Microbiol. 52:1451-1462.[CrossRef][Medline]
    35. 34
  35. Lischewski, A., D. Harmsen, K. Wilms, G. Baier, U. Gunzer, H. Klinker, M. Wilhelm, A. Schwinn, and J. Hacker. 1999. Molecular epidemiology of Candida albicans isolates from AIDS and cancer patients using a novel standardized CARE-2 DNA fingerprinting technique. Mycoses 42:371-383.[CrossRef][Medline]
    36. 35
  36. Litvintseva, A. P., R. Thakur, R. Vilgalys, and T. G. Mitchell. 2006. Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana. Genetics 172:2223-2238.[Abstract/Free Full Text]
    37. 36
  37. Lockhart, S. R., J. J. Fritch, A. S. Meier, K. Schroppel, T. Srikantha, R. Galask, and D. R. Soll. 1995. Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing. J. Clin. Microbiol. 33:1501-1509.[Abstract]
    38. 37
  38. Lockhart, S. R., B. D. Reed, C. L. Pierson, and D. R. Soll. 1996. Most frequent scenario for recurrent Candida vaginitis is strain maintenance with substrain shuffling: demonstration by sequential DNA fingerprinting with probes Ca3, C1, and CARE2. J. Clin. Microbiol. 34:767-777.[Abstract]
    39. 38
  39. Lott, T. J., and R. T. Scarborough. 2008. Development of a MLST-based SNP microarray for Candida albicans. Fungal Genet. Biol. 45:803-811.[CrossRef][Medline]
    40. 39
  40. Luu, L. N., L. E. Cowen, C. Sirjusingh, L. M. Kohn, and J. B. Anderson. 2001. Multilocus genotyping indicates that the ability to invade the bloodstream is widespread among Candida albicans isolates. J. Clin. Microbiol. 39:1657-1660.[Abstract/Free Full Text]
    41. 40
  41. Maiden, M. C. J. 2006. Multilocus sequence typing of bacteria. Annu. Rev. Microbiol. 60:561-588.[CrossRef][Medline]
    42. 41
  42. Maiden, M. C. J., J. A. Bygraves, E. Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. J. Zhou, K. Zurth, D. A. Caugant, I. M. Feavers, M. Achtman, and B. G. Spratt. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA 95:3140-3145.[Abstract/Free Full Text]
    43. 42
  43. McManus, B. A., D. C. Coleman, G. Moran, E. Pinjon, D. Diogo, M. E. Bougnoux, S. Borecka-Melkusova, H. Bujdakova, P. Murphy, C. d'Enfert, and D. J. Sullivan. 2008. Multilocus sequence typing reveals that the population structure of Candida dubliniensis is significantly less divergent than that of Candida albicans. J. Clin. Microbiol. 46:652-664.[Abstract/Free Full Text]
    44. 43
  44. Morgan, J. A. T., V. T. Vredenburg, L. J. Rachowicz, R. A. Knapp, M. J. Stice, T. Tunstall, R. E. Bingham, J. M. Parker, J. E. Longcore, C. Moritz, C. J. Briggs, and J. W. Taylor. 2007. Population genetics of the frog-killing fungus Batrachochytrium dendrobatidis. Proc. Natl. Acad. Sci. USA 104:13845-13850.[Abstract/Free Full Text]
    45. 44
  45. Odds, F. C., M.-E. Bougnoux, D. J. Shaw, J. M. Bain, A. D. Davidson, D. Diogo, M. D. Jacobsen, M. Lecomte, S.-Y. Li, A. Tavanti, M. C. J. Maiden, N. A. R. Gow, and C. d'Enfert. 2007. Molecular phylogenetics of Candida albicans. Eukaryot. Cell 6:1041-1052.[Abstract/Free Full Text]
    46. 45
  46. Odds, F. C., A. D. Davidson, M. D. Jacobsen, A. Tavanti, J. A. Whyte, C. C. Kibbler, D. H. Ellis, M. C. J. Maiden, D. J. Shaw, and N. A. R. Gow. 2006. Candida albicans strain maintenance, replacement, and microvariation demonstrated by multilocus sequence typing. J. Clin. Microbiol. 44:3647-3658.[Abstract/Free Full Text]
    47. 46
  47. Odds, F. C., M. F. Hanson, A. D. Davidson, M. D. Jacobsen, P. Wright, J. A. Whyte, N. A. R. Gow, and B. L. Jones. 2007. One year prospective survey of Candida bloodstream infections in Scotland. J. Med. Microbiol. 56:1066-1075.[Abstract/Free Full Text]
    48. 47
  48. Oh, S. H., G. Cheng, J. A. Nuessen, R. Jajko, K. M. Yeater, X. M. Zhao, C. Pujol, D. R. Soll, and L. L. Hoyer. 2005. Functional specificity of Candida albicans Als3p proteins and clade specificity of ALS3 alleles discriminated by the number of copies of the tandem repeat sequence in the central domain. Microbiology 151:673-681.[Abstract/Free Full Text]
    49. 48
  49. Poulter, R. T. 1987. Natural auxotrophic heterozygosity in Candida albicans. Crit. Rev. Microbiol. 15:97-101.[Medline]
    50. 49
  50. Pujol, C., S. Joly, S. R. Lockhart, S. Noel, M. Tibayrenc, and D. R. Soll. 1997. Parity among the randomly amplified polymorphic DNA method, multilocus enzyme electrophoresis, and Southern blot hybridization with the moderately repetitive DNA probe Ca3 for fingerprinting Candida albicans. J. Clin. Microbiol. 35:2348-2358.[Abstract]
    51. 50
  51. Pujol, C., S. Joly, B. Nolan, T. Srikantha, and D. R. Soll. 1999. Microevolutionary changes in Candida albicans identified by the complex Ca3 fingerprinting probe involve insertions and deletions of the full-length repetitive sequence RPS at specific genomic sites. Microbiology 145:2635-2646.[Abstract/Free Full Text]
    52. 51
  52. Samaranayake, Y. H., L. P. Samaranayake, R. S. Dassanayake, J. Y. Y. Yau, W. K. Tsang, B. P. K. Cheung, and K. W. S. Yeung. 2003. ‘Genotypic shuffling’ of sequential clones of Candida albicans in HIV-infected individuals with and without symptomatic oral candidiasis. J. Med. Microbiol. 52:349-359.[Abstract/Free Full Text]
    53. 52
  53. Sampaio, P., L. Gusmao, A. Correia, C. Alves, A. G. Rodrigues, C. Pina-Vaz, A. Amorim, and C. Pais. 2005. New microsatellite multiplex PCR for Candida albicans strain typing reveals microevolutionary changes. J. Clin. Microbiol. 43:3869-3876.[Abstract/Free Full Text]
    54. 53
  54. Schmid, J., S. Herd, P. R. Hunter, R. D. Cannon, M. S. M. Yasin, S. Samad, M. Carr, D. Parr, W. McKinney, M. Schousboe, B. Harris, R. Ikram, M. Harris, A. Restrepo, G. Hoyos, and K. P. Singh. 1999. Evidence for a general-purpose genotype in Candida albicans, highly prevalent in multiple geographical regions, patient types and types of infection. Microbiology 145:2405-2413.[Abstract/Free Full Text]
    55. 54
  55. Schmid, J., M. Rotman, B. Reed, C. Pierson, and D. Soll. 1993. Genetic similarity of Candida albicans strains from vaginitis patients and their partners. J. Clin. Microbiol. 31:39-46.[Abstract/Free Full Text]
    56. 55
  56. Schroppel, K., M. Rotman, R. Galask, K. Mac, and D. R. Soll. 1994. Evolution and replacement of Candida albicans strains during recurrent vaginitis demonstrated by DNA fingerprinting. J. Clin. Microbiol. 32:2646-2654.[Abstract/Free Full Text]
    57. 56
  57. Selmecki, A., S. Bergmann, and J. Berman. 2005. Comparative genome hybridization reveals widespread aneuploidy in Candida albicans laboratory strains. Mol. Microbiol. 55:1553-1565.[CrossRef][Medline]
    58. 57
  58. Soll, D. R. 2000. The ins and outs of DNA fingerprinting the infectious fungi. Clin. Microbiol. Rev. 13:332-370.[Abstract/Free Full Text]
    59. 58
  59. Soll, D. R. 2004. Mating-type locus homozygosis, phenotypic switching and mating: a unique sequence of dependencies in Candida albicans. Bioessays 26:10-20.[CrossRef][Medline]
    60. 59
  60. Soll, D. R., and C. Pujol. 2003. Candida albicans clades. FEMS Immunol. Med. Microbiol. 39:1-7.[CrossRef][Medline]
    61. 60
  61. Spratt, B. G., and M. C. J. Maiden. 1999. Bacterial population genetics, evolution and epidemiology. Philos. Trans. R. Soc. London B 354:701-710.[Abstract/Free Full Text]
    62. 61
  62. Stephens, M., and P. Donnelly. 2003. A comparison of Bayesian methods for haplotype reconstruction from population genotype data. Am. J. Hum. Genet. 73:1162-1169.[CrossRef][Medline]
    63. 62
  63. Takakura, S., S. Ichiyama, J. M. Bain, A. D. Davidson, M. D. Jacobsen, D. J. Shaw, N. A. R. Gow, and F. C. Odds. Comparison of Candida albicans strain types among isolates from three countries. Int. J. Med. Microbiol., in press.
    64. 63
  64. Tavanti, A., A. D. Davidson, M. J. Fordyce, N. A. R. Gow, M. C. J. Maiden, and F. C. Odds. 2005. Population structure and properties of Candida albicans, as determined by multilocus sequence typing. J. Clin. Microbiol. 43:5601-5613.[Abstract/Free Full Text]
    65. 64
  65. Tavanti, A., A. D. Davidson, N. A. R. Gow, M. C. J. Maiden, and F. C. Odds. 2005. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J. Clin. Microbiol. 43:284-292.[Abstract/Free Full Text]
    66. 65
  66. Tavanti, A., A. D. Davidson, E. M. Johnson, M. C. J. Maiden, D. J. Shaw, N. A. R. Gow, and F. C. Odds. 2005. Multilocus sequence typing for differentiation of strains of Candida tropicalis. J. Clin. Microbiol. 43:5593-5600.[Abstract/Free Full Text]
    67. 66
  67. Tavanti, A., N. A. R. Gow, M. C. J. Maiden, F. C. Odds, and D. J. Shaw. 2004. Genetic evidence for recombination in Candida albicans based on haplotype analysis. Fungal Genet. Biol. 41:553-562.[CrossRef][Medline]
    68. 67
  68. Tavanti, A., N. A. R. Gow, S. Senesi, M. C. J. Maiden, and F. C. Odds. 2003. Optimization and validation of multilocus sequence typing for Candida albicans. J. Clin. Microbiol. 41:3765-3776.[Abstract/Free Full Text]
    69. 68
  69. Taylor, J. W., and M. C. Fisher. 2003. Fungal multilocus sequence typing—it's not just for bacteria. Curr. Opin. Microbiol. 6:351-356.[CrossRef][Medline]
    70. 69
  70. Tietz, H. J., M. Hopp, A. Schmalreck, W. Sterry, and V. Czaika. 2001. Candida africana sp nov., a new human pathogen or a variant of Candida albicans?. Mycoses 44:437-445.[CrossRef][Medline]
    71. 70
  71. Tsang, P. W. K., B. Cao, P. Y. L. Siu, and J. Wang. 1999. Loss of heterozygosity, by mitotic gene conversion and crossing over, causes strain-specific adenine mutants in constitutive diploid Candida albicans. Microbiology 145:1623-1629.[Abstract/Free Full Text]
    72. 71
  72. Turner, K. M. E., W. P. Hanage, C. Fraser, T. R. Connor, and B. G. Spratt. 2006. Assessing the reliability of eBURST using simulated populations with known ancestry. BMC Microbiol. doi:10.1186/1471-2180-7-30.[CrossRef][Medline]
    73. 72
  73. Urwin, R., and M. C. J. Maiden. 2003. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol. 11:479-487.[CrossRef][Medline]
    74. 73
  74. Wu, W., C. Pujol, S. R. Lockhart, and D. R. Soll. 2005. Chromosome loss followed by duplication is the major mechanism of spontaneous mating-type locus homozygosis in Candida albicans. Genetics 169:1311-1327.[Abstract/Free Full Text]
    75. 74
  75. Zhang, N., K. O'Donnell, D. A. Sutton, A. Nalim, R. C. Summerbell, A. A. Padhye, and D. M. Geiser. 2006. Members of the Fusarium solani species complex that cause infections in both humans and plants are common in the environment. J. Clin. Microbiol. 44:2186-2190.[Abstract/Free Full Text]
    76. 75
  76. Zhao, X. M., S. H. Oh, R. Jajko, D. J. Diekerna, M. A. Pfaller, C. Pujol, D. R. Soll, and L. L. Hoyer. 2007. Analysis of ALS5 and ALS6 allelic variability in a geographically diverse collection of Candida albicans isolates. Fungal Genet. Biol. 44:1298-1309.[CrossRef][Medline]

Eukaryotic Cell, July 2008, p. 1075-1084, Vol. 7, No. 7
1535-9778/08/$08.00+0     doi:10.1128/EC.00062-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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