JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2006, p. 3647–3658 Vol. 44, No. 100095-1137/06/$08.00�0 doi:10.1128/JCM.00934-06Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Candida albicans Strain Maintenance, Replacement, and MicrovariationDemonstrated by Multilocus Sequence Typing

F. C. Odds,1* A. D. Davidson,1 M. D. Jacobsen,1 A. Tavanti,2 J. A. Whyte,1 C. C. Kibbler,3D. H. Ellis,4 M. C. J. Maiden,5 D. J. Shaw,1 and N. A. R. Gow1

Aberdeen Fungal Group, Institute of Medical Sciences, Aberdeen AB25 2ZD, United Kingdom1; Department of Biology, University ofPisa, Pisa, Italy2; Department of Medical Microbiology, Royal Free and University College Medical School, Royal Free Hospital,

London, United Kingdom3; Department of Microbiology and Immunology, Australian Candidaemia Study, Women’s andChildren’s Hospital, North Adelaide, SA, Australia4; and Peter Medawar Building for Pathogen Research and

Department of Zoology, University of Oxford, Oxford, United Kingdom5

Received 4 May 2006/Returned for modification 18 May 2006/Accepted 24 July 2006

We typed 165 Candida albicans isolates from 44 different sources by multilocus sequence typing (MLST) andABC typing of rRNA genes and determined their homozygosity or heterozygosity at the mating-type-like locus(MTL). The isolates represented pairs or larger sets from individual sources, which allowed the determinationof strain diversity within patients. A comparison of replicate sequence data determined a reproducibilitythreshold for regarding isolates as MLST indistinguishable. For 36 isolate sets, MLST and ABC typing showedindistinguishable or highly related strain types among isolates from different sites or from the same site atdifferent times from each patient. This observation included 11 sets with at least one isolate from a bloodculture and a nonsterile site from the same patient. For one patient, strain replacement was evidenced in theform of two sets of isolates from different hospital admissions where the strain types within each set were nearlyidentical but where the two sets differed both by MLST and ABC typing. MLST therefore confirms the existingview of C. albicans strain carriage. Microvariation, evidenced as small differences between MLST types,resulted in most instances from a loss of heterozygosity at one or more of the sequenced loci. Among isolatesets that showed major strain type differences, some isolates could be excluded as likely examples of handlingerrors during storage. However, for a minority of isolates, intermittent differences in ABC type for tightlyclustered MLST types and intermittent appearances of MTL homozygosity lead us to propose that some C.albicans isolates, or all isolates under yet-to-be-determined conditions, maintain a high level of genetic diversityby mechanisms such as recombination, gene conversion, or chromosomal ploidy change.

Candida albicans is a normal commensal of the human gutmicroflora that can cause invasive superficial and disseminatedinfections in immunologically susceptible hosts (11). A numberof different technical approaches to typing C. albicans isolateshave been developed since the early 1980s (78). For C. albi-cans, as for all microbial pathogens, a reproducible and dis-criminatory strain typing system is of benefit for clinical andepidemiological studies to provide information on sources, car-riage, and transmission of infection and on relations betweenstrain types and properties such as virulence and antimicrobialresistance. In many publications, multiple C. albicans isolatesfrom longitudinal surveys of patients or from surveillance cul-tures of different anatomical sites have been typed. Most workof this nature has confirmed a tendency towards the persis-tence of a unique strain in each human host, which was sug-gested even in the earliest studies based on phenotypic straintyping (45, 53). Minor variations in genetically determinedstrain types in surveys of multiple C. albicans isolates fromindividual patients have been described as “microevolution”(33, 35, 37, 64, 65, 76, 78) or genotypic shuffling (37, 69). (Thisis not the usual sense in which the term “microevolution” isused by population geneticists.) Evidence from strain typingwork confirms the view of C. albicans as a species that repro-

duces predominantly in a clonal manner but with a tendencyfor occasional genetic variation that may arise by mechanismssuch as recombination, gene replacement, or cryptic mating(22–24, 29, 30, 34, 38, 39, 42, 50, 66, 79, 80, 84, 98).

Most longitudinal and/or multiple-site epidemiological workdemonstrating strain maintenance in C. albicans has been donewith superficial isolates, particularly with cultures from the oralcavities of human immunodeficiency virus (HIV)-infected pa-tients (2, 3, 6, 19, 31, 48, 51, 61, 71, 72, 90, 96), with vaginalisolates (1, 37, 49, 64, 65, 73, 76, 91), with other sources ofsuperficial isolates (4, 17, 74, 97), or with superficial surveil-lance cultures from hospitalized patients (60, 81, 89). Somesurveys of oral isolates have shown the same strain persistingfor months to years (4, 17). Occasionally, authors have sug-gested that multiple strain types coexist in samples from somepatients (27, 69, 83), but such findings are in the minority andmay reflect technical sensitivities and variabilities in the typingmethodologies used in some cases. The epidemiology of dis-seminated C. albicans infection has been studied much lessoften than that of superficial sites, but the results of surveysbased on karyotype electrophoretic patterns (62, 63, 93), ran-domly amplified PCR fragments (92), DNA fingerprinting witholigonucleotide probes (44, 67), and multilocus enzyme elec-trophoresis (12) consistently indicate that isolates from bloodare highly similar to, or indistinguishable from, isolates fromsuperficial sites in the same patient.

The overall picture of C. albicans colonization and infection

* Corresponding author. Mailing address: Aberdeen Fungal Group,Institute of Medical Sciences, Aberdeen AB25 2ZD, United Kingdom.Phone and fax: 44 01224 555828. E-mail: f.odds@abdn.ac.uk.

3647

is therefore mainly of clonal reproduction of strains, infectionby the spread of endogenous, colonizing strain types, but withsporadic changes at the level of sequences of individual DNAfragments, which we shall call microvariation rather than mi-croevolution. Microvariation refers to small but detectablechanges in DNA sequences among isolates obtained longitu-dinally or as separate clones from individual patients (35, 65).Although the original reports of microvariation suggested thatchanges arose from the reorganization of repetitive DNA se-quences in the genome, later work has consistently associatedthe loss of heterozygosity (LOH) as a common mechanism formicrovariation changes (16, 21, 70, 85, 88). LOH may resultfrom chromosome deletion or loss, recombination, and/orgene conversion events. Conjugational mating and nuclear fu-sion without subsequent meiosis can occur between C. albicanscells (24, 34, 42), and such mating is dependent on homozygousalleles, i.e., LOH, at the mating-type-like locus (MTL) onchromosome 5 (42, 79).

Multilocus sequence typing (MLST) is a highly discrimina-tory and portable approach to distinguishing strains within amicrobial species (43). DNA sequences from six or seven genefragments are compared to establish levels of similarity be-tween isolates and results, expressed with unique numbers forindividual sequences (genotypes) and for diploid sequencetypes (DSTs), unique combinations of the seven genotypes ineach isolate (43). Several statistical procedures have been de-vised for the analysis of MLST data, including eBURST (20),which compiles clusters of isolates that differ from each otherat only one of the sequenced loci. MLST schemes are nowavailable for several fungi, and the approach is well developedfor the typing of C. albicans strains (7–9, 68, 84, 86). The C.albicans data are stored on a central internet database (http://test1.mlst.net/) to allow free access to investigators. Resultsfrom MLST are comparable with those obtained by DNAfingerprinting with the moderately repetitive sequence Ca3,since both typing approaches assign the same sets of isolates tothe same clusters of highly related strains, with the exception ofDNA fingerprinting clade E, which is split between two MLSTclades (84).

In the course of our ongoing work to determine DSTs byMLST for the analysis of C. albicans population structures(84), we have now typed sets of multiple isolates of the fungusfrom 44 different sources, including instances of superficial andblood isolates from the same patient, isolates that have beenmaintained and passaged through different culture collections,and isolates that have been experimentally exposed to flucon-azole. We have determined the level of sequencing errors inreplicate experiments to establish criteria for regarding isolatesas indistinguishable. We have looked for evidence of micro-variation changes and LOH in these isolates and in isolatesrecovered from experimental infections. In addition to MLST,we have typed the isolates for the presence or absence of anintron in the internal transcribed spacer 1 (ITS1) region ofDNA sequences encoding rRNA (ABC typing) (46, 47) and forhomozygosity or heterozygosity at the MTL (86). These extradata augment the MLST results by providing extra typing in-formation for each isolate. Our findings confirm the prevailingviews of strain maintenance, predominant clonality, and occa-sional microvariation in C. albicans and that isolates fromblood cultures match those from nonsterile sites in the same

patient. We hypothesize that LOH may occur among some butnot all cells in the population colonizing or infecting a patientso that heterozygosity remains established in a proportion ofthe population. Our data also raise some caveats concerningthe maintenance and distribution of isolates within and be-tween laboratories.

MATERIALS AND METHODS

C. albicans isolates. Details of the 165 isolates studied are shown in Table 1. Inour laboratory, all the isolates were stored on beads in 50% glycerol at �80°C.For experimental use, a bead was placed onto a plate of CHROMagar Candidamedium (M-Tech Diagnostics, Warrington, United Kingdom) and incubated at37°C for 48 h. All the isolates formed green colonies on this medium, verifyingtheir prior identification as C. albicans.

The isolates came from 44 separate sources and are grouped in numbered setsaccording to source in Table 1. Most of the isolates were pairs or triplet isolatesfrom a human subject or, in one instance, an animal. The subjects includedhealthy volunteers, patients with symptomatic vaginitis, patients undergoing che-motherapy for hematological malignancy, and patients with proven candidemia.Many of the isolates came from previously published studies, as indicated inTable 1. The oldest isolates in the panel were originally cultured in the 1970s.Many of the isolates have been maintained in the first author’s collection offungal strains, which was initiated in Leeds, United Kingdom, in 1973 andtransported to Leicester, United Kingdom, and Beerse, Belgium, and which isnow in Aberdeen, United Kingdom. With each move, all isolates in the collectionwere subcultured and restocked at least once (52, 57). The histories of theseisolates are indicated in Table 1.

Some sets of isolates merit special comment. In set 1, isolate RV4688 was astrain used in experiments by J. Van Cutsem in Beerse, Belgium, that hadbecome spontaneously avirulent (58). It was originally isolated in Zaire by R.Vanbreuseghem. Records at the culture collection of the Scientific Institute ofPublic Health (SIPH) (formerly the Institute of Hygiene and Epidemiology) inBrussels, Belgium, show that isolate IHEM3742 was received in 1988, havingpassed from R. Vanbreuseghem to J. Van Cutsem to the American Type CultureCollection (as strain ATCC 28516). IHEM16346 is not listed in the public SIPHcatalog but came directly from the original Zaire isolate from R. Vanbreuseg-hem, according to SIPH collection manager N. Nolard (personal communica-tion). 81/005 was the number given to an isolate that was received by the firstauthor from J. Van Cutsem in 1981. From their histories, these isolates should beindistinguishable by MLST. The isolates in set 15 share a similar complex history.An oral isolate from an AIDS patient in Leicester, United Kingdom, in 1988 wassent to J. Schmid at the University of Iowa and designated OD8826. A subcultureof the isolate was taken by J. Schmid to Massey University in New Zealand, andthe isolate was supplied from New Zealand for the present study. Two other oralisolates from the same patient in 1988 were removed to Beerse, Belgium, andwere then added to the SIPH collection and were supplied as isolatesIHEM20491 and IHEM20493 for the present study. The isolates in set 18, alloriginally from an AIDS patient in London, United Kingdom, had also beensubcultured and relocated in different culture collections before inclusion in thepresent study. Set 23 comprises examples of isolates derived from T118 that hadbeen passaged for a measured number of generations in the presence of flucon-azole (14, 16). For each isolate, the two digits after the letter “D” indicate thesubclone number from T118, and the three final digits indicate the number ofgenerations. Finally, set 44 comprises the isolates still available from a previousstudy in which examples of the widely used C. albicans strain 3153 were submit-ted from several laboratories for comparison by an earlier strain typing meth-odology (41).

Strain typing. The isolates were tested by MLST as previously described (9,84). The ABC type (A, B, or C) and MTL types (a/�, a/a, or �/�) were deter-mined by PCR as previously described (86). Similarities between sequence datawere analyzed in terms of p distance with MEGA version 2.1 (28), and resultswith 500 bootstrap replications are depicted as a dendrogram by the unweighted-pair group method with arithmetic averages (UPGMA). The analyses were basedon concatenated data from all known polymorphic sites, duplicated to allow thediscrimination of homozygous and heterozygous differences (84). The sequenceswere also analyzed to determine clonal clusters by eBURST, version 2 (20).Clusters were defined as groups of isolates with six of the seven genotypesequences being identical.

When one or more isolates within a set of isolates from a common sourcediffered from the rest by MLST or ABC type, the sequencing chromatograms for

3648 ODDS ET AL. J. CLIN. MICROBIOL.

TABLE 1. Details of C. albicans isolates studied

Isolateset Isolate Yr

isolatedCountry

of origina Isolated fromb: Patient detail(s)c Storage historyd DST ABCtype

MTLtype Reference

1 IHEM16346 1970s Zaire Kidney? May be parent ofIHEM3742

Be2 258 C a/�

1 IHEM3742 1970s Zaire Kidney Unknown Be2 124 C a/�1 RV4688 1970s Kidney Originally

IHEM3742?Be1 87 C a/� 58

1 81/005 Subculture of RV4688received in 1981

Be1, UK2, Be1 480 C a/�

1 RV2LK Recovered fromexperimental mouseinfection with RV4688

269 C a/�

2 73/024 1973 UK Vagina Open-heart surgery UK1, UK2, Be1 145 A a/� 542 73/027 1973 UK Vagina Open-heart surgery UK1, UK2, Be1 485 A a/� 543 78/005 1978 UK Oropharynx Healthy volunteer UK2, Be1 96 A a/a3 78/010 1978 UK Oropharynx Healthy volunteer UK2, Be1 517 A �/�4 81/066 1980 UK Oropharynx Female, STD clinic UK2, Be1 396 A a/�4 81/114 1980 UK Vagina Female, STD clinic UK2, Be1 396 A a/�5 90/141 1980 Spain Folliculitis Heroin addict UK2, Be1 547 A a/� 565 90/142 1980 Spain Abscess Heroin addict UK2, Be1 482 A a/� 566 L206 1985 UK Feces Patient 11 UK2, Be1 255 B a/� 556 L249 1985 UK Feces Patient 11 UK2, Be1 255 B a/� 556 L258 1985 UK Oropharynx Patient 11 UK2, Be1 255 B a/� 556 L332 1985 UK Feces Patient 11 UK2, Be1 255 B a/� 556 L343 1985 UK Oropharynx Patient 11 UK2, Be1 80 B a/� 556 L475 1985 UK Feces Patient 11 UK2, Be1 255 B a/� 556 L485 1985 UK Feces Patient 11 UK2, Be1 256 B a/� 556 L527 1985 UK Feces Patient 11 UK2, Be1 257 B a/� 556 L531 1985 UK Feces Patient 11 UK2, Be1 255 B a/� 556 L548 1985 UK Oropharynx Patient 11 UK2, Be1 255 B a/� 556 L586 1985 UK Feces Patient 11 UK2, Be1 255 B a/� 556 L607 1985 UK Oropharynx Patient 11 UK2, Be1 255 B a/� 557 L344 1985 UK Oropharynx Patient 5, March 1985 UK2, Be1 273 C a/� 557 L345 1985 UK Sputum Patient 5, March 1985 UK2, Be1 273 C a/� 557 L367 1985 UK Oropharynx Patient 5, March 1985 UK2, Be1 272 C a/� 558 L479 1985 UK Feces Patient 5, May 1985 UK2, Be1 125 A a/� 558 L562 1985 UK Sputum Patient 5, May 1985 UK2, Be1 271 A a/� 558 L572 1985 UK Stomach Patient 5, May 1985 UK2, Be1 271 A a/� 558 L580 1985 UK Feces Patient 5, May 1985 UK2, Be1 270 A a/� 559 L217 1985 UK Oropharynx Patient 8 UK2, Be1 89 B a/� 559 L252 1985 UK Oropharynx Patient 8 UK2, Be1 89 B a/� 559 L261B 1985 UK Oropharynx Patient 8 UK2, Be1 267 B a/� 559 L283 1985 UK Oropharynx Patient 8 UK2, Be1 268 B a/� 559 L289 1985 UK Oropharynx Patient 8 UK2, Be1 89 B a/a 559 L441 1985 UK Oropharynx Patient 8 UK2, Be1 89 B a/� 549 L463 1985 UK Feces Patient 8 UK2, Be1 89 B a/� 5410 L121 1985 UK CSF UK2, Be1 141 B �/�10 L123 1985 UK Urine UK2, Be1 515 B a/�10 L124 1985 UK CSF UK2, Be1 542 B �/�11 L220 1985 UK Feces Patient 6 UK2, Be1 544 A a/� 5511 L262 1985 UK Oropharynx Patient 6 UK2, Be1 545 A a/� 5511 L353 1985 UK Oropharynx Patient 6 UK2, Be1 544 A a/� 5512 85/005 1985 UK Blood UK2, Be1 83 A a/a 1012 85/006 1985 UK Sputum UK2, Be1 541 A a/� 1013 L513 1986 UK Sputum UK2, Be1 546 B a/�13 L516 1986 UK Blood UK2, Be1 134 B a/�14 L517 1986 UK Anus Male, STD clinic UK2, Be1 216 A a/�14 L518 1986 UK Oropharynx Male, STD clinic UK2, Be1 216 A a/�15 OD8826 1988 UK Oropharynx UK2, Iowa, NZ 24 A a/� 7215 IHEM20491 1989 UK Oropharynx UK2, Be1, Be2 373 A a/� 7215 IHEM20493 1990 UK Oropharynx UK2, Be1, Be2 372 A a/� 7216 B59626 1991 Germany Oropharynx Be1 585 A a/�16 B59630 1991 Germany Oropharynx Be1 585 A a/�17 C19 1993 UK Sputum 429 B a/�17 C25 1993 UK Catheter tip 429 B a/�17 C26 1993 UK Blood 429 B a/�17 C27 1993 UK Catheter tip 429 B a/�18 IHEM20486 1993 UK Oropharynx AIDS patient UK2, Be1, Be2 309 B a/�

Continued on following page

VOL. 44, 2006 C. ALBICANS MICROVARIATION 3649

TABLE 1—Continued

Isolateset Isolate Yr

isolatedCountry

of origina Isolated fromb: Patient detail(s)c Storage historyd DST ABCtype

MTLtype Reference

18 IHEM20487 1993 UK Oropharynx AIDS patient UK2, Be1, Be2 389 B a/�18 IHEM20488 1993 UK Oropharynx AIDS patient UK2, Be1, Be2 302 B a/a18 J932570 1993 UK Oropharynx AIDS patient UK2, Be1 489 C a/�18 J932571 1993 UK Oropharynx AIDS patient UK2, Be1 379 B a/�18 J932575 1993 UK Oropharynx AIDS patient UK2, Be1 380 C a/�18 J941383 1994 UK Oropharynx AIDS patient UK2, Be1 378 A a/�19 J942148 1994 Germany Oropharynx AIDS patient Be1 381 A �/�19 J942149 1994 Germany Oropharynx AIDS patient Be1 382 A �/�20 TW2-76 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW2-80 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW2-81 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW2-82 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW2-83 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW2-84 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW2-85 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW2-86 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW3-55 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW8-43 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW8-44 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW8-45 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW8-46 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW8-47 1994 USA Oropharynx AIDS patient 79 A a/� 9520 TW12-79 1994 USA Oropharynx AIDS patient 254 A a/� 9520 TW12-99 1994 USA Oropharynx AIDS patient 79 A a/� 9521 S01 1995 Italy Oropharynx Liver transplant 88 C a/� 8721 S02 1995 Italy Oropharynx Liver transplant 264 C a/� 8721 S03 1995 Italy Oropharynx Liver transplant 88 C a/� 8721 S04 1995 Italy Oropharynx Liver transplant 264 C a/� 8721 S05 1995 Italy Sputum Liver transplant 265 C �/� 8721 S06 1995 Italy Sputum Liver transplant 88 C a/� 8721 S07 1995 Italy Sputum Liver transplant 88 C a/� 8721 S08 1995 Italy Sputum Liver transplant 266 C a/� 8721 S09 1995 Italy Sputum Liver transplant 265 C �/� 8721 S10 1995 Italy Bronchoalveolar lavage Liver transplant 88 C a/� 8722 T63 1995 Canada Oropharynx AIDS patient 222 B a/� 1522 T65 1995 Canada Oropharynx AIDS patient 222 B a/� 1522 T68 1995 Canada Oropharynx AIDS patient 223 C a/� 1523 T118 1995 Canada Oropharynx AIDS patient 151 A a/� 1523 D08-330 T118 population exposed

to fluconazole278 A a/� 16

23 D09-330 T118 population exposedto fluconazole

278 A a/� 16

23 D11-330 T118 population exposedto fluconazole

278 A a/� 16

23 D12-165 T118 population exposedto fluconazole

278 A a/� 16

23 D12-260 T118 population exposedto fluconazole

280 A a/� 16

23 D12-330 T118 population exposedto fluconazole

279 A a/� 16

24 J990102 1999 Belgium Vagina Symptomatic vaginitis Be1 45 B a/�24 J990103 1999 Belgium Cervix Symptomatic vaginitis Be1 490 B �/�25 AM2001/0019 2001 UK Sternal abscess 527 A a/�25 AM2001/0022 2001 UK Sternal abscess 527 A a/�26 WC01-202761 2001 Australia Blood 390 B a/�26 WC01-202762 2001 Australia Wound 391 B a/�27 WC02-202964 2002 Australia Blood 420 A a/�27 WC02-202965 2002 Australia Urine 421 A a/�28 WC02-202291 2002 Australia Blood 232 A a/�28 WC02-202292 2002 Australia Blood 232 A a/�28 WC02-202293 2002 Australia Blood 430 A a/�29 WC02-202294 2002 Australia Blood 416 B a/�29 WC02-202295 2002 Australia Blood 417 B a/�29 WC02-202296 2002 Australia Blood 418 B a/�30 WC02-201987 2002 Australia Urine 414 A a/�30 WC02-201988 2002 Australia Catheter tip 415 A a/�30 WC02-202225 2002 Australia Blood 66 A a/�

Continued on following page

3650 ODDS ET AL. J. CLIN. MICROBIOL.

the set were rescrutinized and/or the isolates were resequenced to ensure that thedifferences were reproducible.

Animal experiments. Single colonies of isolates S09 and WC02-202294 (Table1) were grown for 20 h at 30°C in NGY medium (0.1% neopeptone [Difco,Sparks, MD], 0.4% glucose, and 0.1% yeast extract [Difco]) (40). These isolateswere chosen to represent low (S09) and high (WC02-202294) levels of heterozy-gosity in the MLST data. The yeast cells were washed twice in sterile saline and

resuspended in saline, and concentrations were adjusted by hemocytometercount results. For each isolate, a single female BALB/c mouse weighing 20 � 2 gwas injected intravenously at a dose of 2 � 105 yeast cells/g body weight,confirmed by subsequent viable counts. Three days after challenge, the mice werehumanely terminated, the kidneys and brains were removed and homogenized insaline, and 10-fold dilutions were plated onto Sabouraud agar (Oxoid, Basing-stoke, United Kingdom) for incubation at 35°C. From each organ culture, six

TABLE 1—Continued

Isolateset Isolate Yr

isolatedCountry

of origina Isolated fromb: Patient detail(s)c Storage historyd DST ABCtype

MTLtype Reference

31 WC02-202861 2002 Australia Ileal conduit 419 A a/�31 WC02-202862 2002 Australia Blood 419 A a/�32 WC03-200970b 2003 Australia Catheter tip 422 B a/�32 WC03-200970d 2003 Australia Blood 423 B a/�32 WC03-200970e 2003 Australia Blood 424 B a/�32 WC03-200970h 2003 Australia Thigh 425 B a/�33 WC03-202178 2003 Australia Urine 427 A a/�33 WC03-202179 2003 Australia Blood 427 A a/�33 WC03-202180 2003 Australia Blood 427 A a/�34 WC03-200975 2003 Australia Blood 426 B a/�34 WC03-201397 2003 Australia Urine 426 B a/�35 WC03-203422 2003 Australia Blood 37 A a/�35 WC03-203424 2003 Australia Urine 428 A a/�36 AM2003/0044 2003 UK Vagina 172 C a/�36 AM2003/0045 2003 UK Vagina 172 C a/�37 AM2003/0108 2003 UK Blood 212 A a/�37 AM2003/0109 2003 UK Blood 212 A a/�38 AM2005/0131 2005 UK Blood 522 A a/�38 AM2005/0134 2005 UK Blood 522 A a/�38 AM2005/0135 2005 UK Blood 522 A a/�39 AM2005/0163 2005 UK Sputum 537 A a/�39 AM2005/0165 2005 UK Blood 537 A a/�40 AM2005/0150 2005 UK Blood 536 A a/�40 AM2005/0151 2005 UK Catheter tip 536 A a/�41 AM2005/0211 2005 UK Blood 564 A a/�41 AM2005/0212 2005 UK Blood 564 A a/�41 AM2005/0213 2005 UK Blood 565 A a/�41 AM2005/0218 2005 UK Blood 564 A a/�42 AM2005/0192 2005 UK Blood 66 A a/�42 AM2005/0195 2005 UK Blood 66 A a/�43 IHEM17156 Belgium Guinea pig penis Be1, Be2 307 B a/�43 IHEM17157 Belgium Guinea pig nose Be1, Be2 305 B a/�44 3153Abstl Recently received as

3153, Bristol, UK369 A a/�

44 3153Alds Recently received as3153, Leeds, UK

369 A a/�

44 3153ALndn2 Recently received as3153, London, UK

286 A a/�

44 3153ALndn1 1980s culture of 3153from Londonlaboratory

UK2, Be1 286 A a/�

44 90/189 Authentic 3153 UK2, Be1 54 A a/� 4144 90/195 3153, CA05 in previous

studyUK2, Be1 261 A a/� 41

44 90/198 3153, CA08 in previousstudy

UK2, Be1 262 A a/� 41

44 90/202 3153, CA12 in previousstudy

UK2, Be1 260 B a/� 41

44 90/206 3153, CA16 in previousstudy

UK2, Be1 263 B a/� 41

44 IHEM20523 3153, CA01 in previousstudy

UK2, Be1, Be2 370 A a/� 41

44 IHEM20535 3153, CA14 in previousstudy

UK2, Be1, Be2 369 A a/� 41

a UK, United Kingdom; USA, United States.b CSF, cerebral spinal fluid.c STD, sexually transmitted disease.d UK1, Department of Microbiology, University of Leeds, United Kingdom; UK2, Department of Microbiology, University of Leicester, United Kingdom; Be1,

Janssen Research Foundation, Belgium; BE2, Scientific Institute of Public Health, Brussels, Belgium.

VOL. 44, 2006 C. ALBICANS MICROVARIATION 3651

well-separated colonies were randomly selected, subcultured, and subjected toMLST.

RESULTS

Reproducibility of MLST data. Data were obtained from 17isolates fully sequenced in duplicate. Sequence reproducibilitywas perfect for 10 duplicates. For the other seven isolates,differences were at the level of a homozygous versus a het-erozygous result for a single nucleotide polymorphism (SNP)in five instances, two homozygous/heterozygous discrepanciesat a single locus in one instance, and a base-for-base (homozy-gous) SNP difference in one instance. All but one of the errorstherefore resulted from a misinterpretation of double peaks,an occasional difficulty to be expected with sequencing chro-matograms from a diploid organism.

The eight replicate differences occurred in a total of 2,329bases sequenced—an accuracy of 99.66%. Since a heterozy-gous/homozygous difference would be scored at half the valueof a homozygous difference in the calculation of distances forUPGMA analyses, the sequencing accuracy could be expressedas nine error scores among 4,658 polymorphic sites sequenced(99.8% accuracy). In the context of this study, the worst inac-curacy seen, one homozygous change or two homozygous/het-erozygous changes, would erroneously suggest nonidentity be-tween strains with a score difference of 1 across all SNPs, whichcorresponds with a p-distance difference of 0.005 by UPGMAnucleotide distance analysis. We have used as a worst-casevalue that is four times this magnitude, i.e., a p-distance dif-ference of 0.02, as a basis for describing isolates as “indistin-guishable,” although the sequence chromatograms for all iso-lates included in this study have been rescrutinized or thesequencing has been repeated to ensure that differences be-tween isolates are genuine.

Similarities and differences between multiple isolates fromsingle sources. The DSTs, ABC types, and MTL types of all theisolates studied are shown in Table 1. Figure 1 shows therelationships between the isolates in the form of a UPGMAdendrogram based on MLST sequence data together with theirisolate set numbers, ABC types, and eBURST clonal clusterassignments. In the majority of instances, isolates from thesame source coclustered, often with the same DST or with veryhigh levels of similarity by MLST (Fig. 1). Isolates in 36 of the44 sets (121 of 165 isolates) were considered indistinguishablewithin each set because they clustered in the MLST dendro-gram within our chosen limiting p distance of 0.02, they had thesame ABC type, and they fell within the same eBURST clonalcluster (when a set of isolates showed sufficient sequence vari-ation to generate a clonal cluster). This list includes 11 isolatesets (sets 12, 14, 18, 27, 28, 31, 32, 34, 35, 36, and 40) in whichsamples came from the bloodstream and a nonsterile site in thesame patient. It also includes sets 7 and 8, which came from apatient sampled on two separate admissions to the same hos-pital, 2 months apart. The set from each admission comprisedisolates clustering at the level of indistinguishability, but thetwo sets were widely separated in the UPGMA dendrogram(Fig. 1), unequivocally indicating strain replacement at all sitessampled for this patient.

For the sets of isolates that could not be designated indis-tinguishable by the above-described criteria, the interisolate

differences were of various types. Isolates from set 32 clusteredclosely, but one isolate differed from the other two in morethan one genotype, so it did not conform to a clonal cluster(Fig. 1). The two isolates in sets 13 and 19 differed, with a pdistance of �0.02, but were matched in ABC type (Fig. 1). Inboth cases, we interpret these data to be consistent with mi-crovariation occurring in the isolate sets, which raises the num-ber of sets of isolates that were indistinguishable or very closelyrelated to 40/44 (125/165 isolates).

IHEM16346 did not cocluster with other isolates in set 1(Fig. 1), but, like the rest of the set, it belonged to the leastcommon ABC type, type C. The data therefore neither confirmnor rule out the possibility that IHEM16346 was the progeni-tor of the other isolates in set 1. Similarly, L527 did not co-cluster with the other 11 isolates in set 6 (Fig. 1), but its ABCtype, type B, matched that of the other isolates. Once again,these data do not unequivocally rule in or out an associationbetween L527 and the other isolates in set 6; our records showthat, chronologically, L527 was the last isolate received fromsurveillance cultures of the patient concerned, and the samplewas taken 5 days after the previous isolate, so strain alterationor strain replacement is a possible interpretation.

Set 18 presented the most unusual set of findings betweenisolates. The seven isolates in this set originated from samplestaken at different times from the same HIV-infected patientbut had histories of storage in different culture collections(Table 1); no detailed information on the isolates is available.Six of the isolates clustered very tightly on the basis of MLSTdata and formed a single eBURST clonal cluster (Fig. 1), butJ932575 was substantially different from these six isolates. Sixof the isolates, including J932575, were ABC type B, whileJ941383 was type A. ABC typing was repeated with theseisolates to confirm the different assignments. A similar findingapplied to the three isolates T63, T65, and T68, all of whichcame from the same patient (set 22). T63 and T65, with iden-tical DSTs (Table 1), were type B, but T68, which did notcocluster with the two other isolates, was type C.

Set 44 consisted of 11 isolates that had been supplied as“strain 3153” from various laboratories. Three isolates wererecently submitted, and the remaining isolates were part of astudy published in 1991 (41). Eight of the isolates (includingthe three recently obtained isolates) coclustered in the den-drogram (Fig. 1). Five of these isolates belonged to eBURSTclonal cluster 1. Isolate 90/206, the most distant in the dendro-gram cluster, was type B, while the other isolates were type A.Of the remaining three isolates in set 44, each of which ap-peared in isolation in the dendrogram (Fig. 1), two were typeA and one was type B.

Twelve of the 165 isolates were homozygous at the MTL:nine �/� and three a/a isolates (Table 1). The random occur-rence of MTL homozygosity among heterozygous isolates insets 9, 10, 12, 18, 21, and 24 indicates that this property is nota character of use in strain typing but is a possible indicator ofmicroadaptive change. Both oral isolates in set 3, from ahealthy female volunteer in the 1970s, and in set 19, from anAIDS patient in the 1990s, were MTL homozygous; therefore,the possibility that further isolates from these individuals mayalso have been homozygous at the MTL cannot be excluded.

Loss of heterozygosity associated with strain microvaria-tion. Among the sets of multiple single-source isolates where

3652 ODDS ET AL. J. CLIN. MICROBIOL.

MLST sequence diversity was most evident, differences wereseen mainly as heterozygous/homozygous changes between iso-lates rather than as homozygous base exchanges. In set 32,isolate WC03-200970b, which differed most markedly from itsthree coisolates, had 15 heterozygous SNPs, compared with 4,9, and 10 for isolates WC03-200970d, WC03-200970e, and

WC03-200970h, respectively. In the pair of isolates in set 13,L516 had 20 heterozygous SNPs, compared with 25 in L513.Similarly, in set 19 J942148 had 4 heterozygous SNPs andJ942149 had 10 heterozygous SNPs. For isolate set 18,IHEM16346, which may or may not have been the progenitorisolate, had 18 heterozygous SNPs, which was more than those

FIG. 1. UPGMA dendrogram showing clustering of 165 C. albicans isolates from 44 sources together with information on ABC type and clonalcluster. The dendrogram has been split to facilitate text legibility, with the top half at the left and the continuation at the right.

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for IHEM3742 (11 SNPs) or RV4688 (nine SNPs). Similarly, inset 22, isolate T68 had 33 heterozygous SNPs, which was manymore than the 16 heterozygous SNPs in its two sister isolates,T63 and T65.

eBURST analysis of MLST data generates “clonal clusters”of isolates in which each differs from another by a single ge-notype among the seven sequenced. For each clonal cluster,the eBURST software determines the founder DST as the onewith the greatest number of other DSTs that can be linked insteps by single locus changes (single locus variants). In thepresent study, most eBURST clonal clusters comprised onlytwo isolates, but clusters 1 and 2 (Fig. 2) contained severalisolates. The isolates in clonal cluster 2 (Fig. 2c) all came froma single patient. The isolates in cluster 1 (Fig. 2a) representedeight different isolate sets. The implication of clonal clusterdata is of evolutionary patterns that normally show paths ofisolate derivation from putative parental isolates. However, thepathways inferred from clonal cluster 1, if correct, indicateboth gain and loss of heterozygosity as evolutionary mecha-

nisms for C. albicans. For example, DST 254 in Fig. 2a differsfrom the putative founder DST 278 by the loss of all heterozy-gous SNPs at the MPIb locus. The step to DST 79 from DST254, however, involves a gain of two heterozygous SNPs fromthe ACC1 locus that was fully homozygous in DST 254. Simi-larly, the path from DST 278 to DST 415 via DST 66 involvesthe gain of heterozygosity in MPIb in the final step, and thepath from DST 278 to DST 37 via DST 151 requires a gain ofheterozygosity in DST 37. An alternative explanation for thechanges is shown in Fig. 2b, in which connecting lines betweenisolates from different sets have been removed so that isolatescoming from a single patient or other source remain con-nected. The links within each set now show how the isolatescould all have evolved within each patient by LOH from adifferent putative parent, without the clonal relationship im-plied by the eBURST analysis. The validity of disconnectingclonal cluster 1 in this way is emphasized by the sometimesconsiderable geographical distance between the isolate sources:sets 20 and 23 came from North America; sets 30, 31, and 35came from came Australia; and sets 15 and 42 came from theUnited Kingdom. (Isolates in set 44 were laboratory strains.)

Table 2 summarizes the nature of the sequence differencesbetween all the sets of isolates that gave nonidentical sequenc-ing results for one or two of the test loci. The table shows thathomozygous SNP changes (indicated with the letter C in thetable) accounted for only four of the 38 differences found; theremaining changes were all changes from heterozygosity tohomozygosity at a polymorphic base position. The loss of asingle heterozygous base position, indicated with the letter Ain Table 2, included those instances with only a single heterozy-gous base position in the entire genotype. When two or moreheterozygosities were found in the sequence for one isolate

TABLE 2. Details of differences in sequences between isolatesfrom the same source

Set Clonalcluster

Nature of differencea between isolates in gene fragment

AAT1a ACC1 ADP1 MPIb SYA1 VPS13 ZWF1b Note

1 4 A B2 15 C3 6 B5 10 A6 9 A7 A B8 7 C A9 13 B11 14 B12 B A18 2 A A A A See Fig. 220 1 B See Fig. 221 5 C C23 1 A A See Fig. 224 A A26 3 B27 A A28 8 B29 3 B A30 1 B A See Fig. 232 16 B35 1 A See Fig. 241 A B43 12 A44 See Fig. 2

a A, heterozygous/homozygous difference at one SNP; B, difference at allheterozygous SNPs; C, single homozygous SNP change.

FIG. 2. Clonal clusters 1 (a) and 2 (c) generated by eBURST anal-ysis of the MLST data. For each cluster, the putative founding isolateis shown as a hollow circle. Diameters of the circles are proportionateto the numbers of isolates with the DST indicated. With each DST, thegene locus and SNP difference from the putative predecessor isolatealong the path from the founder isolate are specified. The lengths ofthe joining lines are arbitrary. b shows clonal cluster 1 with the joininglines removed to leave different isolate sets separate; set numbers areshown next to DSTs. Only sets 30 and 42 share a common DST.

3654 ODDS ET AL. J. CLIN. MICROBIOL.

and all were homozygous in the other, this was regarded ascomplete LOH for the locus concerned and is indicated withthe letter B in Table 2. Thus, the 34 instances of putative LOHinvolved 21 instances in which single heterozygosities changedand 13 instances of complete LOH for a sequenced locus.

MLST differences among isolates recovered from experi-mentally infected animals. Because the MLST result for iso-late RV2LK from set 01, recovered from the left kidney of amouse infected with RV4688 (set 01), showed a minor differ-ence (two more heterozygous SNPs in the recovered isolatethan in the originally typed isolate), we conducted a prelimi-nary investigation into possible MLST variations in isolatesrecovered from infected tissues. Isolates S09 and WC02-202294 were first spread to single colonies on Sabouraud agar,and six clones on each plate were subjected to MLST. All sixclones of S09 were indistinguishable by MLST, whereas threeDSTs were found among the six clones of WC02-202294.These resulted from a heterozygous/homozygous difference atone SNP in ACC1 in two instances and a similar single-sitedifference in ADP1 in one instance. Cloned inocula of the twostrains were each injected intravenously into two mice. After 3days, the mice were sacrificed, and colonies of C. albicans werereisolated from homogenates of kidney and brain. In repeats ofMLST for five or six colonies from each organ, no differencesin the DST of each strain were found.

DISCUSSION

Our study used MLST, the most dependable system for C.albicans strain differentiation, with a discriminatory power of0.9996 (84), to investigate strain relationships in multiple iso-lates from single sources. None of the isolates included in thisstudy happened to represent the most commonly encounteredDST, type 69 (84), or a close relative. MLST has confirmedthat sets of such isolates most often belong to a single or highlyrelated strain type; 36 (82%) of 44 strain sets were very closelyrelated by MLST data and had identical ABC types. MLSTthus agrees with many other strain typing systems that havesimilarly shown the persistence of a single strain type perpatient to be the most common situation. All 11 sets of isolatesthat included cultures from blood and nonsterile sites in thesame patient fell within the 36 most closely related sets. Theseresults clearly indicate that adult patients (none of the isolatestested came from pediatric sources) with bloodstream infec-tions are usually infected with their own commensal isolates.This observation does not, of course, show whether theportal of endogenous infection was the patient’s digestivetract or another route, such as an intravenous catheter.Results similar to ours were obtained in a recently publishedstudy from Taiwan in which both MLST and electrophoretickaryotypes of multiple isolates from nine patients in inten-sive care units showed high relatedness but were not alwaysindistinguishable (13). However, multiple oral isolates takenfrom three HIV-positive patients over several years showedevidence of strain replacement. Our own findings are sup-ported in the case of isolates from set 21, which were pre-viously found by Ca3 DNA fingerprinting to be highly sim-ilar but not indistinguishable (87).

All but 2 of the 44 sets of isolates that we tested came fromhuman sources. Among these were two sets of surveillance

culture isolates (sets 7 and 8) from a patient who was admittedtwice to the same hospital in March and May of 1985. Whileeach of these individual sets of isolates clustered within thisstudy’s bounds of indistinguishability, the two sets were suffi-ciently different, including a change of ABC type, to provideunequivocal evidence of strain replacement between the pa-tient’s hospital admissions. No other similar, unambiguous ex-amples of strain replacement were found. It is possible thatisolate L527 (set 1) might also represent strain replacement,but the level of dissimilarity of this isolate from the rest of theset may have resulted from mishandling errors in the course ofmany years of maintenance. A third possibility is that thepatient carried two strain types and that the less populous onewas the randomly picked colony from the isolation plate onlyfor the L527 sample.

One finding from this study concerns the storage and trans-mission of isolates between collections. The earliest isolates ofcertain date in our 44 sets were a pair of successive vaginalisolates first cultured in Leeds, United Kingdom, in 1973. Over30 years of storage, the with removal and occasional reculturefrom the first author’s collection in Leicester, United King-dom, Beerse, Belgium, and now Aberdeen, United Kingdom,these isolates differed by only a single polymorphism: a changefrom A to G in the ADP1 sequence. Twelve other isolate setsdating from the late 1970s to the late 1980s had also beentransferred between several locations and in some instanceshad been returned after storage in other collections. All ofthese isolate sets coclustered to high levels of similarity byMLST and ABC type, suggesting that their storage and main-tenance had been adequate for long periods of time. By con-trast, several of the isolates that had originally been receivedinto the Leicester, United Kingdom, collection from otherlaboratories under the label 3153 (set 44 in the present study)were obviously subjected to handling errors at some stage intheir history. In the original interlaboratory studies of theseisolates (32, 41), restriction fragment length polymorphism andmultilocus enzyme electrophoresis data already suggested thatseveral of them differed from the pattern of the majority of theisolates. The MLST results now show that at least three of theisolates cannot be regarded as authentic examples of strainNCPF3153. The data for the most recently received examplesof NCPF3153 define the strain as DST 369 and ABC type A.Isolate 90/189, which we received as the “authentic” strain3153 in the 1980s, differed from this DST in two genotypes butwas still type A and remained closely clustered with six otherexamples of “strain 3153” by MLST (Fig. 1).

The differences between the coclustered NCPF3153 isolatesin Fig. 1 are of the same order of magnitude as those weinterpret as microvariation in other isolate sets. The nature ofthese changes most often involves the loss of one or all het-erozygous SNPs from one or two of the sequenced genes (Ta-ble 2). LOH as a general mechanism for microvariationchanges has been previously described (16, 21, 70, 85, 88).However, the precise mechanism underlying LOH events isunknown. The list of possible mechanisms includes recombi-nation and gene conversion. The possibility of chromosomeploidy changes must now be added to this list, since aneu-ploidy, including segmental aneuploidy of subchromosomal re-gions, has now been elegantly demonstrated in C. albicans (77).The loss of all or part of a chromosome followed by redupli-

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cation has been invoked as a mechanism underlying a range ofphenotypic properties in the fungus, including sorbose utiliza-tion and fluoroorotic acid resistance (26, 94) and the genera-tion of MTL-homozygous variants (98). If spontaneous partialploidy changes occur as frequently in clinical isolates as wasshown for the laboratory strain CAI-4 (77), the high frequencyof heterozygosity changes seen among the isolates that wesequenced may not be surprising. This comment is furthersupported by the occasional appearance of isolates homozy-gous at the MTL in a temporal series of isolates that wereotherwise MTL heterozygous. Wu et al. previously studieddistantly located heterozygous markers on chromosome 5 onboth sides of the MTL and found that whole-chromosome lossand replacement accounted for spontaneous conversion toMTL homozygosity in 15/16 isolates studied, with a recombi-national event in only one isolate (98). We speculate that acomplete or partial chromosome loss and reduplication, com-bined with occasional genetic exchange through recombinationand inefficient DNA repair, account for the apparent geneticplasticity of C. albicans evidenced in our data, including ap-parent diploid homozygous base switches, which would be in-explicable by chance mutation. Future investigations of karyo-types and of sequences neighboring and distant from theMLST genes on the same chromosome will provide evidencefor recombination, gene conversion, or aneuploidy in strainsets showing LOH and single-base homozygous changes.

Of particular note in our study are the few examples amongour isolates of strains that were very closely related by MLSTbut that differed in ABC type (notably in sets 18 and 44). ABCtype, based on the presence of one or both ITS1 sequences inrRNA genes, is usually a very stable epidemiological marker inC. albicans isolates and is sufficient information to demonstrategeographical and temporal differences among C. albicans iso-lates (46). It has been suggested that type C, the least commonABC type, may represent an intermediate form that is gainingor losing the type A or B ITS1 sequence (25). On this basis, andin view of our finding of two examples where types A and Bwere represented among types similar by MLST, we regardABC typing as a very helpful confirmatory test in instanceswhere isolates differ by more than one or two SNPs in MLST,but it should not be regarded as a definitive test for isolaterelatedness in all instances. The rRNA genes in C. albicans arelocated on chromosome R; among the MLST genes, onlyACC1 is located on this chromosome.

eBURST analysis of MLST data provides a useful basis fortracing putative evolutionary patterns among microbial iso-lates. However, in the present study, this analytical approachmay have suggested invalid relationships between strains. Themajor clonal cluster found among our isolates (Fig. 2) suggeststhat there is a set of evolutionary paths between isolates thatrequires successive gains and losses of heterozygosity at one ofthe sequenced loci to be sustainable. Clonal cluster 1 is there-fore unlikely to represent a true clonal relationship betweenthese isolates of widely disparate geographical origins. Oneobvious mechanism for the gain of heterozygosity would bemating, but it remains uncertain to what extent mating be-tween C. albicans isolates occurs naturally (5). If mating is thebasis for the regaining of allelic heterozygosity, then the matingprocess would need to involve strains with dissimilar alleles; inother words, the C. albicans cells colonizing or infecting a site

within a patient would need to include homozygous strains ofthe opposite MTL type and with different MLST DSTs. Thissituation appears to be quite different from the high tendencytoward clonal reproduction evidenced by most isolate sets inthis study as well as those described previously in other publi-cations (66, 85). The disadvantage of the eBURST approachand other statistical methods that examine only genotype andDST assignations is that they do not distinguish whether iso-lates differ in sequence at a locus by a single SNP or by multipleSNPs. Hence, its value becomes limited with an organism suchas C. albicans, which regularly undergoes “mutations” at mul-tiple nucleotides within a single locus.

One hypothesis that would accommodate the otherwise con-flicting evidence suggesting both clonal strain maintenance andtemporal gain and loss of heterozygosities and even ABC typeswould be that C. albicans populations commonly exist in a stateof high genetic plasticity. The phenomenon of hypermutable(“mutator”) strains is well recognized among some species ofbacteria, particularly gram-negative bacilli (18, 59, 82). Hyper-mutability is associated with the appearance of multiple colonyforms and other phenotypic variations, and it is possible thatsimilarly variable strains exist among pathogenic Candida spe-cies. Almost all the isolates that we have studied began assingle clones picked from primary isolation plates in clinicallaboratories. When successive isolates from the same site inthe same patient are found to be sometimes MTL homozygousand sometimes MTL heterozygous (sets 18 and 21), the mostreasonable explanation is not that the clones picked randomlyfrom a given isolation plate represent all colonies from thatsample but rather that the sample contained a mixture of types.The possibility that C. albicans colonies on isolation platesoften differ has been suggested by the minority of colonies thatare able to undergo a white-opaque transition (36), by minorintercolony differences in DNA fingerprint patterns (the orig-inal finding characterized as “microevolution”) (35), and bycolony-to-colony differences in azole antifungal susceptibility(75). In the present study, three DSTs were found among thesix clones of WC02-202294 (from set 29) randomly sampledfrom a spread plate. If either some strains at all times or allstrains in certain environments undergo recombination, genereplacement, or partial ploidy changes at elevated frequencies,the resulting mixture of strain types in the population isolatedwould explain the occasional superficially aberrant result in thepresent study. Our pilot experiment to demonstrate this phe-nomenon by resampling infected tissues infected with clonedC. albicans isolates failed to confirm the hypothesis; however,we plan to investigate a broader range of isolates in vivo toassess the reality, or otherwise, of strain diversity as a naturalprocess for C. albicans. We are also investigating natural ge-netic diversity between colonies on primary isolation plates.

ACKNOWLEDGMENTS

This study was supported by grants from the Wellcome Trust (69615and 74898).

We thank individuals who have provided us with isolates, particu-larly James Anderson of the University of Toronto and Nicole Nolardand Francoise Symoens of the Scientific Institute of Public Health inBrussels.

3656 ODDS ET AL. J. CLIN. MICROBIOL.

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