Reservoir of Candida albicans infection in a vascular bypass

graft demonstrates a stable karyotype over six months

PAUL LEPHART, PATRICIA FERRIERI & JO-ANNE VAN BURIK

Departments of Medicine, Laboratory Medicine and Pathology, and Genetics, Cell Biology, and Development, University ofMinnesota, Minneapolis, Minnesota, USA

We retrospectively analyzed five Candida albicans isolates from two infection

episodes in a single patient 6 months apart. Using contour-clamped homogeneous

field electrophoresis (CHEF), random amplified polymorphic DNA (RAPD)

fingerprinting, and restriction fragment length polymorphism (RFLP) complex

probe 27A as means of molecular typing, we demonstrate an unvarying genotype

amongst the infection-causing C. albicans strains. Several months later, the patient

yielded C. glabrata in a yeast survey of oral and rectal sites. The preponderance of

C. glabrata and lack of C. albicans isolated from normal flora sites suggests that

this patient harbored the prior C. albicans bloodstream isolate on a Gore-Tex graft

for 6 months prior to the second episode of fungemia.

Keywords Candida albicans, karyotype, pulsed field gel electrophoresis

Introduction

Candida albicans is versatile as both a human com-

mensal and a human pathogen. Very little is known,

however, about the specific traits that lead to this

versatility. One of the most distinctive properties of C.

albicans is its highly variable karyotype: many clinical

isolates have a karyotype that differs from the standard

C. albicans karyotype [1]. In the clinical setting, C.

albicans fungemia usually arises from an endogenous

source after an isolate from the commensal yeast

crosses into the bloodstream from its non-sterile niche.

Sterile site bloodstream isolates that are recovered from

the same patient 6 months apart are expected to be the

result of separate events and thus to be potentially

different strains. In our clinic, a patient with blood-

stream infection caused by C. albicans developed

another fungemia 6 months later, concurrent with C.

albicans infection of an inguinal pseudoaneurysm at

the site of an endovascular graft. We hypothesized that

if the graft had been seeded during the first fungemia,

examining all the isolates with molecular typing

methods would demonstrate a single unique strain.

This paper presents evidence for the occurrence of two

widely separated incidents of systemic candidiasis in

the same patient, both caused by the same strain.

Case history

A 60-year-old man developed high fever and shaking

chills after outpatient hemodialysis. His medical history

included diabetes mellitus, peripheral vascular disease,left common femoral to popliteal artery bypass using

polytetraflouroethylene (PTFE) Gore-Tex graft, bilat-

eral above-the-knee amputations, heart transplant

secondary to ischemic cardiomyopathy, end-stage renal

disease from chronic cyclosporine use, left arm hemo-

dialysis shunt, and sacral decubitus ulcer. His medica-

tions included insulin, glipizide, enalapril, nifedipine,

atorvastatin, aspirin, cyclosporine, azathioprine, pre-dnisone, trimethoprim-sulfamethoxazole, iron, folate,

calcium acetate, vitamin formulation for renal patients,

and total parenteral nutrition (TPN).

Examination revealed no thrush in the oropharynx.

The incisions from bilateral above-the-knee amputa-

tions 2 months prior were clean without discharge.

There was mild tenderness to palpation at a central

catheter insertion site from 3 weeks prior. The catheterwas removed and venous access was gained using a

temporary catheter via the left femoral vein. His white

blood cell count was 3.4�/109 cells/mm3.

Correspondence: Jo-Anne H. van Burik, MD, MMC 250, 420

Delaware Street SE, Minneapolis, MN 55455, USA. Tel.: �/612 625

8462; Fax: �/612 625 4410; E-mail: vanbu004@umn.edu

Received 2 November 2002; Accepted 15 April 2003

– 2004 ISHAM DOI: 10.1080/13693780310001644662

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Temperatures increased as high as 103.58F. Multiple

blood cultures were negative and various studiesshowed no source of infection, including bone scan,

computed tomography (CT) of the head, chest, abdo-

men and pelvis, magnetic resonance imaging (MRI) of

the back, and ultrasound of the left forearm dialysis

fistula.

Culture of drainage at the skin site where the

temporary femoral central venous catheter was placed

grew a C. albicans strain that was numbered T308.Blood cultures 11 and 12 days later grew C. albicans

strains numbered T330 and T332, respectively. The

patient was started on fluconazole treatment on day 13,

and then switched to an investigational intravenous

echinocandin FK463 (Micafungin; Fujisawa, Chicago

IL, USA) from days 15 to 21, after which oral

fluconazole 200 mg/day was resumed from days 22 to

36. A transthoracic echocardiogram revealed no vege-tations. Blood cultures were negative on days 40, 45, 58,

60, and 89.

Six months later, the patient noted rapid swelling in

the left thigh and pain in his left leg. Ultrasound

showed a 9�/5.5�/5.5 cm left groin pseudoaneurysm.

Pus and blood were drained operatively, and infected

Gore-Tex was removed. Sample of the pus grew out C.

albicans (Strain T838). One day after surgery, C.

albicans was cultured from blood (Strain T840).

Six months later, during a yeast survey of oral and

rectal sites, Candida glabrata was recovered from the

carriage site samples.

Materials and methods

Organisms

Control strains included American Type Culture Col-

lection (ATCC) (Manassas, VA, USA) strains ATCC

10231, ATCC 10261, ATCC 32354 (also called B311)

and ATCC 62376 (FC18) and laboratory strains CBS

5736 (Centraalbureau voor Schimmelcultures, Utrecht,

The Netherlands) and SC5314. ATCC 62376 is a clear

example of the standard C. albicans karyotype.

Pulsed field gel electrophoresis

Yeast chromosomal DNA for pulsed field gel electro-

phoresis (PFGE) was prepared as described previously

[2]. PFGE was carried out by the contour clamped

homogeneous electric field (CHEF) method using a

CHEF DRII or DRIII system (Bio-Rad, Hercules, CA,USA) as was essentially described by Chibana et al . [3].

The conditions for separation were as follows: 0.7%

agarose gel, 60�/120 s, 6 V/cm, 1208 included angle for

24 h; then 120�/300 s, 4.5 V/cm, 1208 included angle for

12 h; then 800�/1200 s, 2 V/cm, 1068 for 12 h.

PCR fingerprinting

DNA polymorphisms generated by the polymerase

chain reaction were used to genotype the strainscollected for this study. The core sequence of phage

M13 (5?-3?: GAGGGTGGCGGTTCT), the 10-mer

oligonucleotide AP3 (5?-3?: TCACGATGCA), the

tRNA intergenic spacer derived T3B (5?-3?:AGGTCGCGGGTTCGAATCC) and the simple re-

peat (GACA)4 were used as single primers in the PCR

experiments [4,5]. PCR reactions used primers M13

core and AP3 at a final concentration of 0.5 mmol/l,T3B at 0.2 mmol/l and (GACA)4 at 0.1 mmol/l; dNTPs

(Fisher) at 200 mmol/l; 2.5 units Taq (Sigma, St Louis,

MO, USA); 1�/PCR buffer; and 1.5 mmol/l MgCl2.

Genomic DNA from the extraction was adjusted to

10 ng/ml and 2.5 ml were used for the AP3 and M13 core

PCR while only 1.0 ml was used for the T3B and

(GACA)4 PCR. PCR reactions were conducted in a

total volume of 50 ml. Thermocycler (MJ ResearchPTC-200, Waltham, MA, USA) conditions for AP3

were as follows, with superscripted notations indicating

numbers of cycles: 958C for 5 min, (958C for 15 s, 368Cfor 30 s, 728C for 1 min 20 s)45, 728C for 6 min and hold

at 48C. T3B cycling conditions were: (958C for 20 s,

528C for 30 s, 728C for 1 min 20 s)32. M13 cycling

conditions were: (958 for 20 s, 508C for 1 min, 728C for

20 s)27. (GACA)4 cycling conditions were: (958 for 20 s,508C for 1 min, 728C for 20 s)35.

Restriction fragment length polymorphism (RFLP)fingerprinting of C. albicans isolates with probe 27A

The complex DNA fingerprinting probe 27A was used

to assess the genetic relatedness of C. albicans isolates

[1,6]. Equivalent amounts of genomic DNA were

digested with EcoRI and electrophoresed in a 0.8%

gel for 5 h at 80 V. The gel was stained with ethidium

bromide to compare loading amongst lanes and to

evaluate the efficiency of digestion. DNA was thentransferred onto Hybond-N�/ membrane (Amersham)

and hybridized with a random-primer-labeled

([32P]dCTP) 27A probe overnight at 658C in Hybridi-

zation buffer (1 mmol/l ethylene diamine tetraacetic

acid [EDTA], 0.25 mol/l sodium phosphate pH 7.2, 7%

SDS). The membrane was washed twice at room

temperature in 2�/ SSC 0.1% sodium dodecyl sulfate

(SDS); twice at 378C in 1�/ SSC 0.1% SDS; and onceat 658C in 0.1�/ SSC 0.1% SDS. The membrane was

then exposed to X-Omat AR film (Eastman Kodak,

Rochester, NY, USA) for 2 days at �/808C.

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Fluconazole susceptibility testing

In the clinical microbiology laboratory, macrobroth

dilution methods were used for determining minimum

inhibitory concentrations of clinical fungal isolates

T330, T838 and T840 to amphotericin B and flucona-

zole within 1 week of their recovery in culture.In the research setting, fluconazole susceptibility

testing was performed at a later point in time for all

the clinical strains, the standard BG2 C. glabrata

strain, and one isolate each from the oral and rectal

C. glabrata normal flora isolates. Etest strips (Biodisk,

Solna, Sweden) were used after growth on modified

casitone phosphate agar for 24 h.

Results

Electrophoretic karyotypes for the invasive infection

and control strains were determined on a CHEF gel

with a program designed for maximum separation of

chromosomes 5�/7. Figure 1 shows the karyotypic

identity of the clinical isolates and also two distinguish-

ing characteristics from the standard karyotype. These

characteristics are the separable chromosome five

homologues and separable chromosome seven homo-

logues. The control strains show both the standard

karyotype (ATCC 62376, as noted previously) and

some of the variability found in common isolates.

In addition to demonstrating similarity amongst the

clinical strains through karyotyping, we used four

DNA fingerprint techniques to confirm strain identity.

PCR fingerprinting based on the AP3 primer (Fig. 2a)

and the M13 core primer (Fig. 2b) showed identical

PCR profiles amongst the clinical isolates. It also

showed the difference between clinical and control

strains, as well as intraspecific variation within the

control strains. The two primers generated different sets

of amplification products, and these varied in band

positions and intensities for non-identical strains. Key

differences between clinical isolates and control strains

in the AP3 fingerprinting are shown in the regions

denoted A�/D (Fig. 2a). The strong presence of bands

Fig. 1 Contour-clamped homogenous electric field electrophoresis

(CHEF) of five Candida albicans isolates (lanes 1�/5) from one

patient strain and six laboratory comparison strains (lanes 6�/11).

The five clinical isolates show identical karyotypes. Lane 1 shows

clinical isolate T308; lane 2, T330; lane 3, T332; lane 4, T838; lane 5,

T840. Note that in patient strains chromosome five and seven

homologues are separable. Laboratory strains used for comparison

are: lane 6, ATCC 10231; lane 7, ATCC 10261; lane 8, ATCC 32354;

lane 9, SC5314; lane 10, ATCC 62376 (commonly referred to as

FC18); lane 11, CBS5736. Control strains show some of the

variability common in C. albicans karyotypes.

Fig. 2 Genomic DNA extracts of five Candida albicans isolates

(lanes 1�/5) from one patient and six laboratory comparison strains

(lanes 6�/11) were amplified using the AP3 primer (a) and M13 core

primer (b). The five clinical isolates show identical AP3 fingerprint

patterns: lane 1 shows the pattern for T308; lane 2, T330; lane 3,

T332; lane 4, T838; lane 5, T840. Laboratory strains used for

comparison are: lane 6, ATCC 10231; lane 7, ATCC 10261; lane 8,

ATCC 32354; lane 9, SC5314; lane 10, ATCC 62376 (commonly

referred to as FC18); lane 11, CBS 5736. Lane 12, control sample

without genomic DNA. Lane MW, molecular size marker in kb. Key

differences between clinical isolates and laboratory strains are shown

in the regions denoted A�/D.

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Candida albicans karyotype strain variation 257

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A and C and lack of bands in B and D in the clinical

strains serve to differentiate these strains from controls.

Key differences between clinical and control isolates in

the M13 core fingerprinting are shown in the regions

denoted A�/D (Fig. 2b). The strong presence of bands

A and B, the decreased intensity of band D and the lack

of a band at position C serve to differentiate the clinical

strains from the control strains. PCR fingerprinting

based on the T3B and (GACA)4 primers was also able

to differentiate between clinical and control isolates

(data not shown). The pattern of relatedness shown by

karyotyping and by random amplified polymorphic

DNA (RAPD) probes was verified by RFLP analysis

utilizing the complex probe 27A (Fig. 3). Key differ-

ences between clinical and control isolates in this RFLP

analysis are shown in the regions denoted A�/C (Fig. 3).

The strong presence in the clinical isolates of the two

bands in A, the triplet of bands in B and the two bands

in C constitutes a unique pattern.

Yeast flora isolates were collected from the patient

approximately 6 months after the second treatment

course and compared with the invasive infection isolate

T308 using CHEF karyotyping. Twenty oral and 20

rectal colonies were subjected to karyotyping and

found to be similar (data not shown). The karyotypes

of these isolates strongly resembled that of a standard

C. glabrata strain and were markedly different from the

karyotype of the C. albicans strains from the same

patient (data not shown). API yeast identification stripsconfirmed that the yeast strains karyotyped were C.

glabrata.

Amphotericin B drug susceptibility did not differ

between C. albicans isolates from the two invasive

infections that occurred 6 months apart (Table 1). The

same series of isolates showed a trend toward reduced

susceptibility to fluconazole in both the macrobroth

dilution and Etest methods. The C. glabrata strainsfrom the normal flora showed an expectedly low degree

of susceptibility to fluconazole.

Discussion

The clinical strains that were collected from the patient

are probably the same strain, based on karyotyping,

RAPD fingerprints, and RFLP analysis. One possibleexplanation for these results is that the patient was

colonized by only one strain of C. albicans prior to the

first episode of candidiasis and in the time preceding

and including the second episode. Thus, this strain may

have been the only endogenous strain present to cause

disease. The presence of only a single C. albicans strain

in the normal yeast flora (monoassociation) has been

noted before in immunocompromized patients [7].A second possibility is that the strain identified as the

infectious agent in both episodes was more virulent and

more able to initiate disease than other, undetected

endogenous strains present in the patient. However,

hypervirulence is not thought to be a major contributor

to recurrent disease in C. albicans [8]. No evidence has

been put forward to implicate a specific genotype with

increased pathogenesis. Comparison of the virulence ofthe recovered C. albicans clinical strains with normal

flora C. albicans strains from the patient could indicate

whether the clinical strains were more virulent.

Neither of these possibilities is consistent with the

fact that following the patient’s two episodes of

candidiasis, normal flora sampling indicated that the

patient’s normal yeast flora is dominated by C.

glabrata . C. glabrata has relatively high resistance tofluconazole, and this may have led to its establishment

as the dominant element of the normal yeast flora

subsequent to the patient’s first treatment with fluco-

nazole. Fluconazole treatment was initiated upon

isolation of C. albicans from the patient’s blood and

continued for 2 weeks. The bulk of the patient’s normal

C. albicans flora may have been overwhelmed by the

fluconazole treatment and by growth competition fromC. glabrata , resulting in the predominance of C.

glabrata in the normal flora. As this shift would have

occurred prior to the second episode of candidiasis, the

Fig. 3 Southern blot of EcoRI digests of the genomic DNA of five

Candida albicans isolates (lanes 1�/5) from one patient strain and six

laboratory comparison strains (lanes 6�/11) hybridized with the 27A

probe. The five clinical isolates show identical RFLP patterns. Lane 1

shows the pattern for isolate T308; lane 2 shows the pattern for T330;

lane 3, T332; lane 4, T838; lane 5, T840. Laboratory strains used for

comparison are: in lane 6, ATCC 10231; lane 7, ATCC 10261; lane 8,

ATCC 32354; lane 9, SC5314; lane 10, ATCC 62376; lane 11, CBS

5736. Lane MW, molecular size marker in kb. Key differences

between clinical isolates and laboratory strains are shown in the

regions denoted A�/C.

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source of the second infection would likely not be from

a normal flora site. Of note, we may have failed to

detect C. albicans from these carriage sites because we

did not plate the samples on CHROMagar to facilitate

detection of numerically rare yeast types.

A third possible interpretation is that after the initial

bloodborne infection, a strain consistent with T330 and

T332 was harbored in an immune and drug-privileged

site, from which it later emerged and caused the second

infection. The apparent identity among the clinical

strains isolated, as well as the complete resolution of

infection after the first incident caused by strains T308,

T330 and T332, makes the presence of an asympto-

matic reservoir likely. The detection of only C. glabrata

in the patient’s normal flora as well as resolution of the

symptoms of the initial infection suggests that fluco-

nazole treatment was successful in suppressing, if not

eradicating, the patient’s C. albicans flora. Yet, the

same strain of C. albicans that caused the first infection

was able to re-emerge and cause a second incident of

disease in the patient despite seemingly effectual drug

treatment. Due to limitations in the number of colonies

that were tested from the patient’s normal flora sites, it

is formally possible that the second incident of disease

was caused by a small reservoir of the original strain

harbored in a normal flora site. However, a more likely

scenario is that involving establishment of C. albicans

in a drug-privileged reservoir elsewhere in the patient.

C. albicans has been shown to form biofilms on

prosthetic devices [9,10] and biofilms are known to

protect microbes from antimicrobial agents [11�/14].

Furthermore, C. albicans is thought to be highly

adherent to Gore-Tex. Thus, the Gore-Tex bypass

could have provided the proposed drug-privileged site

in the present case. The re-emergence of C. albicans

infection at the site of the graft is further evidence for

that scenario. Interestingly, the proposed biofilm in

which C. albicans persisted seemed to restrict the

resultant infection to the graft and the infection was

only identified as systemic the day after the surgery to

remove the infected graft. It was likely this surgery,

which disrupted the C. albicans biofilm on the graft,

resulted in a release of yeast into the bloodstreamcausing the second incident of candemia.

Acknowledgements

This work was supported, in part, by Grant AI-01411

from the National Institutes of Health. We thank Paul

T. Magee for critical review of this manuscript, Anja

Forche for optimized DNA fingerprinting protocolsand Brendan Cormack for the C. glabrata BG2 strain.

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Table 1 Antifungal susceptibility testing (minimum inhibitory concentrations, mg/ml) of clinical isolates obtained in the present case of

recurrent candiasis.

Candida isolates Amphotericin B

(clinical lab)

Fluconazole

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Fluconazole

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C. albicans T308, drainage from catheter onto skin 2

C. albicans T330, bloodstream 0.25 0.25 2

C. albicans T332, bloodstream 3

C. albicans T838, groin abscess 0.25 0.5 3

C. albicans T840, bloodstream 0.25 1.0 4

C. glabrata BG2 8

Oral flora isolate 1, C. glabrata 32

Rectal flora isolate 1, C. glabrata 32

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