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β-1,3 Glucan as a Test for Central Venous Catheter Biofilm InfectionAuthor(s): Jeniel Nett, Leslie Lincoln, Karen Marchillo and David AndesSource: The Journal of Infectious Diseases, Vol. 195, No. 11 (Jun. 1, 2007), pp. 1705-1712Published by: Oxford University PressStable URL: http://www.jstor.org/stable/30086160 .

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MAJOR ARTICLE

0-1,3 Glucan as a Test for Central Venous Catheter

Biofilm Infection

Jeniel Nett, Leslie Lincoln, Karen Marchillo, and David Andes

Departments of Medicine and of Medical Microbiology and Immunology, University of Wisconsin, Madison

Biofilms are microbial communities that are associated with solid surfaces such as intravascular catheters. Candida species are a major cause of medical device-associated infections. Twenty percent to 70% of all candidemias are associated with this biofilm process. Diagnosis and effective treatment of Candida device- associated infections requires removal of the involved device. The ability to identify a biofilm device infection before catheter removal may obviate removal of a substantial number of devices. Prior studies in our laboratory identified cell wall changes (specifically, increased fl-1,3 glucan) associated with biofilm, compared with planktonic C. albicans. Both in vitro and in vivo (catheter) biofilm models were used to determine whether biofilm cells secreted more f-1,3 glucan and whether these differences could be used to discern the presence of a Candida biofilm infection with 3 species (C. albicans, C. glabrata, and C. parapsilosis). A limulus lysate assay was used to quantify (-1,3 glucan in supernatants from planktonic or biofilm cultures and in the serum of rats with an intravascular catheter biofilm infection or disseminated candidiasis. fl-1,3 glucan was detected from both in vitro and in vivo models from each condition. However, the concentrations of #-1,3 glucan from the biofilm conditions were 4-10-fold greater in vitro (P<.001) and were 10-fold greater in vivo (P<.001), despite equal or fewer numbers of cells in the biofilm conditions. These results suggest the secreted polysac- charide fl-1,3 glucan may serve as a useful tool for the diagnosis of Candida biofilm and device-associated infections.

Vascular catheter infections are common and are as-

sociated with high mortality and morbidity [1-3].

Nearly 5 million vascular catheters are used annually in the United States, and it is estimated that 2%-12%

of these result in sepsis [4-7]. Among central venous

catheter (CVC)-related infections, those due to Can-

dida species are associated with an attributable mor-

tality approaching 40% [8-10]. C. albicans device in-

fections are difficult to treat and often require removal

of the device for cure. Current treatment guidelines recommend device removal for treatment of candide-

mia, although removal may be technically difficult and associated with significant risk [7].

The majority of hospital-acquired infections, includ-

ing C. albicans infections, are associated with biofilms

[11]. A biofilm is a sessile microbial community embedded in polymeric matrix, with organisms irrevers-

ibly attached to a substratum or to each other [ 11, 12]. The cells in this environment exhibit an altered phenotype with respect to antimicrobial resistance [12-16]. Candida biofilms can be up to 1000-fold more resistant to conventional antifungal agents, making the infections very difficult to control in the medical setting.

No laboratory methods exist to rapidly detect biofilm infections in vivo. Blood cultures require several days for incubation, are only positive in 50%-70% of patients with disseminated candidiasis, and do not differentiate between biofilm and nonbiofilm infection. Both semiquantitative and quantitative catheter cul-

tures have been used for diagnosis of bloodstream in-

fections, with greater specificity for catheter infection

Received 30 October 2006; accepted 21 December 2006; electronically published 17 April 2007.

Potential conflicts of interest: none reported. Reprints or correspondence: Dr. David Andes, 600 Highland Ave., Rm. H4.572,

Madison, WI 53792 ([email protected]).

The Journal of Infectious Diseases 2007;195:1705-12 a 2007 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2007/19511-0020$15.00 DO: 10.1086/517522

3-1,3 Glucan Test for Biofilm Infection * JID 2007:195 (1 June) * 1705



than blood culture alone [17-19]. Both methods involve await-

ing culture results. However, quantitative cultures do not re-

quire removal of the catheter. To avoid having to remove a

catheter for diagnosis of infection, the differential time to pos-

itivity of catheter and peripheral blood cultures has also been

used [19]. This method utilizes the higher burden of organisms in a blood culture obtained from an infected catheter and mea-

sures the earlier time to positivity, compared with that of a

peripheral culture. Although this method could circumvent the

need for catheter removal to diagnose infection, 24-72 h may be spent awaiting culture results. The only Food and Drug

Administration-approved, non-culture-based test for diagnosis of invasive candidiasis is an assay for 3-1,3 glucan [20, 21].

Both in vitro and CVC in vivo C. albicans models have been

used to examine drug susceptibility and pathogenesis [12-16,

22]. Using these models, the current studies identified differ-

ences in carbohydrates surrounding biofilm cells. Findings sug-

gest a dramatic increase in secreted carbohydrates, including

3-1,3 glucan. We have demonstrated the ability to differentiate between biofilm and planktonic cells in vitro and in vivo using an assay for this material.

MATERIALS AND METHODS

Organisms and culture conditions. Four clinical Candida strains obtained from patients with bloodstream infection were

chosen for these investigations. C. albicans strains K1 and 98-

17, C. glabrata 5376, and C. parapsilosis 5986 were stored in

15% (vol/vol) glycerol stock at -800C. Before experiments, strains were maintained on yeast extract-peptone-dextrose me-

dium (YPD;1% yeast extract, 2% peptone, and 2% dextrose). Fresh colonies were used for inoculation of YPD or RPMI 1640 with morpholine propanesulfonic acid (MOPS), and cultures

were allowed to incubate for 16 h at 370C with orbital shaking at 250 rpm before inoculation.

In vitro biofilm formation. Flat, sterile medical-grade silicone disks were utilized as the biofilm substrate. Before in-

fection, the disks were coated with mouse serum and incubated at 370C for 30 min. C. albicans K1 was grown in YPD at 370C on an orbital shaker to late logarithmic phase. After the con-

ditioning period, 150 ,TL of culture (106 cells/mL) was inoculated on the disk and spread evenly. The disks were then incubated with 1 mL of RPMI/MOPS for 45 min at 370C. After

the adherence period, disks were rinsed in sterile water and

then placed in 10 mL of RPMI/MOPS and incubated at 370C

with gentle shaking for varying periods of time (detailed below). Planktonic culture conditions. C. albicans K1 was grown

overnight in YPD at 370C on an orbital shaker at 250 rpm and

used for inoculation of planktonic culture. Planktonic cultures were grown in 10 mL RPMI/MOPS in a glass flask at 370C on

an orbital shaker at 250 rpm until late logarithmic phase (106

cfu/mL).

In vitro glucan determination and microbiologic enumeration.

Supernatants from C. albicans in vitro biofilm cultures and

planktonic cultures were collected for determination of 0-1,3 glucan concentration. Viable cell number was used as a nor- malization factor for both the in vitro planktonic and biofilm

systems. Viable cell burdens were measured using plate counts to ensure the cultures contained a similar number of cells and were in a similar phase of growth. Both adherent and non- adherent cells were included in microbiologic enumeration of biofilm culture. Culture supernatants were collected from both the planktonic and biofilm systems at 4, 8, and 12 h. The cultures were centrifuged at 3000 g for 20 min, and the cell- free supernatant (confirmed by light microscopy) was removed and stored at -200C until glucan analysis. Biofilm formation was confirmed by visual inspection and by confocal microscopy. Glucan concentration was determined using a limulus lysate assay, the Glucatell (1,3)-3-D-Glucan Detection Reagent Kit

(Associates of Cape Cod), in accordance with the manufac-

turer's directions. The culture experiments were repeated on 2 occasions. Three glucan assay replicates were performed for each experiment per time point.

In vivo venous catheter biofilm model. A C. albicans CVC biofilm model was used for in vivo experiments as described elsewhere [22]. Briefly, animals were anesthetized, placed in

supine position, and prepped in a sterile fashion. A vertical incision was made in skin of the anterior neck just right of midline. The external jugular vein was identified and exposed with blunt surgical dissection, and a longitudinal incision was made in the vein wall. A sterile, heparinized (100 U/mL) poly- ethylene catheter (PE 100 [inner diameter, 0.76 mm; outer diameter, 1.52 mm]; Intramedic catalog no. 427430; Becton

Dickinson) was placed in the opening and advanced to a site above the right atrium (-2 cm). The catheter was secured with 3-0 silk ties. The proximal end of the catheter was then tunneled

subcutaneously and secured on the subscapular skin via a button. The anterior neck site was closed with surgical staples (Ethicon Endo-Surgery), and the midscapular button was secured with a 2-0 Ti-Cron suture. Recovery of the animal after the catheter surgery was assessed according to a standard protocol approved by the Veterans Administration Animal Com- mittee. After 24 h, 106 cells/mL of C. albicans strains K1 or 98-

17, C. glabrata 5376, or C. parapsilosis 5986, in late logarithmic phase and suspended in normal saline, were instilled in the catheter lumen for 4 h and then flushed with heparin. After a 12-, 24-, or 48-h incubation, blood samples were obtained from tail vein and catheter.

Tail-vein candidasis model. A tail-vein candidiasis model

was used to represent disseminated candidiasis in the absence of biofilm growth on an infected medical device [23, 24]. An- imals without catheters in place were infected by tail vein with 106 cells/mL of C. albicans strains K1 or 98-17, C. glabrata

1706 * JID 2007:195 (1 June) * Nett et al.



Figure 1. Confocal micrographs of an in vitro Candida albicans biofilm. C. albicans biofilms were grown on silicon disks and stained with FUN-1 and concanavalin A (ConA) fluorescent dyes. Metabolically active yeast cells are stained red by FUN-1, a fluorescent viability stain. ConA binds carbohydrates and results in green fluorescence of the cell walls of both yeast and hyphae. Extracellular green fluorescence is due to ConA binding to carbohydrate components of the matrix.

5376, or C. parapsilosis 5986, in late logarithmic phase and

suspended in normal saline. After 12, 24, or 48 h, serum sam-

ples were obtained for determination of 3-1,3 glucan content. To exclude the possibility of the catheter material alone causing an elevation of serum 0-1,3 glucan, several animals with catheters in place were infected by the tail-vein route. These animals had a nonbiofilm infection and a catheter in place.

In vivo glucan assay and microbiologic enumeration. Serum samples (1 mL) were frozen at -200C until analyzed for 0-1,3 glucan content using a limulus lysate assay, according

to the manufacturer's instructions (Fungitell (1,3)-f-D-Glucan Detection Reagent Kit; Associates of Cape Cod). Assays were

performed in triplicate. Kidneys were harvested and collected

at various time points (detailed above) from animals with CVC

infections and tail-vein infections for microbiologic viable bur-

den counts as an estimate of total-body organ burden [24]. Statistical analysis. Two biological replicates and 3 assay

replicates were performed at each time point for in vitro glucan

assay experiments. Three assay replicates were performed for

in vivo glucan determination. Analysis of variance and Stu-

/3-1,3 Glucan Test for Biofilm Infection - JID 2007:195 (1 June) - 1707



dent's t test were used to statistically compare glucan concentrations. Viable burden counts from both in vitro and in vivo studies were performed in duplicate. Viable counts from planktonic and biofilm conditions were compared using a Student's t test to determine significance.

Biofilm scanning electron microscopy (SEM). SEM was used to image catheters of animals with CVC infections to confirm biofilm formation and assess the extent of biofilm on the catheter luminal surface [22]. Animals with CVC infections were killed at 12, 24, or 48 h, and catheters were collected for SEM. Catheters were harvested and transected perpendicular to catheter length. Segments were then placed in fixative (4% formaldehyde and 1% glutaraldehyde in PBS) for 20 h, washed for 5 min in PBS, and placed in 1% osmium tetroxide for 30 min. After a series alcohol washes, final drying was performed by critical point drying. Catheter segments were mounted on stubs and gold coated. Samples were imaged in a scanning electron microscope (JEOL JSM-6100) in the high-vacuum mode at 10 kV. The images were assembled using Adobe Pho-

toshop (version 7.0). Confocal microscopy. Confocal microscopy was used to

confirm in vitro Candida biofilm formation. Biofilms were

grown on silicone disks as described above. Disks were cut into small sections and placed in PBS with fluorescent dyes (the FUN-1 [50 /imol/L] component of the LIVE/DEAD yeast vi-

ability kit [Molecular Probes] and concanavalin A [ConA] con-

jugate [200 mmol/L; Alexa Fluor 488 conjugate]) and processed according to the manufacturer's instructions, as described elsewhere [22]. Imaging of FUN- 1-stained cells was accomplished by using a protocol with an excitation wavelength of 488 nm and an emission band at 617 nm for red. The ConA protocol included excitation and emission wavelengths of 490 and 528 nm, respectively. Biofilms were observed with a Nipkow disk- based confocal microscope (Axiovert 200; Zeiss) equipped with a mercury arc lamp. The objectives used included X20 and X40 differential interference contrast oil immersion objectives. Confocal images of green (ConA) and red (FUN-1) fluorescence were conceived simultaneously using the Z-stack mode.

Images were processed for display by using Axiovision software (version 3.x; Zeiss).

RESULTS

In vitro biofilm confocal microscopy. We used confocal microscopy to confirm and characterize the presence of biofilm on the silicone disks in our in vitro biofilm model (figure 1). We selected FUN-1 and ConA fluorescent dyes to stain yeast cells, hyphae, and matrix. At 24 h, yeast cells, hyphae, and matrix components were present on the silicone disk. Live, metabolically active yeast cells processed the FUN-1 viability stain, resulting in red fluorescence within intravacuolar struc- tures. Glucose and mannose residues of cell wall polysaccha-

rides were visualized as a green fluorescence due to ConA bind-

ing. The cell walls of both yeast and hyphae were visualized by this dye. The carbohydrate component of the matrix was also visualized by ConA dye and was seen as an extracellular green fluorescence. Because of colocalization of both fluorescent dyes, the metabolically active cells appeared yellow to orange during multichannel image capture.

In vitro biofilm glucan assay. We collected supernatants from the planktonic culture and the in vitro biofilm model described above to compare concentrations of /3-1,3 glucan. Glucan was detected in supernatants from both cultures and

temporally increased with rising cell numbers (figure 2). Using

microbiologic viable counts, we confirmed that cultures con-

tained the same number of viable cells. Although microbiologic

A

-J E

2 o

-,- O

7

6

5

4

3

2

Biofilm * Planktonic

2 4 6 8 10 12 14 16 18 20

Time (h)

B

E

r-

C!

250

200

150

100 -

50 i

0-

Biofilm Planktonic

4 8 12

Time (h)

Figure 2. In vitro biofilm and planktonic supernatant 0-1,3 glucan. Candida albicans biofilms were grown on medical-grade silicone disks. Supernatants were collected from planktonic and biofilm culture conditions, and p-1,3 glucan content (picograms per milliliter) was determined using limulus lysate assay (B). The black bars represent 0-1,3 glucan concentration from biofilm culture supernatant in picograms per milliliter, and the gray bars represent 0-1,3 glucan concentration from planktonic conditions. The viable burden of cells (colony-forming units per milliliter) from both conditions was determined by microbiologic enumeration (A). Error bars represent SDs.




A. C. albicans K1 B. C. albicans 98-17 E. C. glabrata 5376

C. C. glabrata 5376 D. C. parapsilosis 5986

........ ........ ......

Figure 3. Scanning electron micrographs (SEMs) of in vivo Candida species biofilms. Catheter segments from the rat venous catheter model were prepared for SEM. Panels A-D show biofilm on the luminal surface of the catheter segments from C. albicans K1, C. albicans 98-17, C. glabrata 5376, and C. parapsilosis 5686 at x50 magnification. Panel E shows the C. glabrata biofilm at x1000 magnification.

viable counts demonstrated similar numbers of cells in each

culture, biofilm culture supernatants were found to contain

significantly higher concentrations of 0-1,3 glucan (P<.001). At each of the time points, the supernatant from the biofilm

system contained 2-10-fold more glucan. The difference was

most pronounced at the 8-h time point, when biofilm supernatant contained >150 pg/mL 0-1,3 glucan and the planktonic

supernatant content was <25 pg/mL (P<.001).

In vivo biofilm SEM. Our next experiments were intended

to determine whether the enhanced release of /3-1,3 glucan from

biofilm cells observed in vitro occurs in vivo as well. We used

our in vivo CVC model to mimic device-associated infection

involving formation of a biofilm. We utilized SEM to verify biofilm formation and extent on the infected CVCs. We cap- tured images of the luminal surface of multiple catheter segments 24 h after inoculation (figure 3). C. albicans strains KI

01

A

6

5.

4.

3.

2

1-

0-

Biofilm Disseminated

12 h 24 h 48 h

-J E c3, CL

cm

co 03

")" e3.

300

250

200

150

100

50

0

B Biofilm catheter Biofilm peripheral Disseminated

12 h 48 h

Figure 4. Serum 0-1,3 glucan concentrations (picograms per milliliter) in Candida albicans biofilm and disseminated (nonbiofilm) infections. Central venous catheters were surgically implanted in rats and infected with C. albicans. The viable burden of cells (log,,0 colony-forming units/organ) from the kidneys of animals with disseminated and biofilm infections were determined at various time points to estimate systemic burden of infection (A). Serum samples were obtained at 12 and 48 h, and 0-1,3 glucan content was determined using a limulus lysate assay (B). Black vertical bars represent 0-1,3 glucan content in picograms per milliliter in serum samples obtained from the catheter. Light gray bars represent 0-1,3 glucan content in the peripheral serum sample (tail vein) obtained from an animal with a biofilm catheter infection. Dark gray bars represent /3-1,3 glucan content of a serum sample from an animal with a disseminated infection. Error bars represent 1 SD.

03-1,3 Glucan Test for Biofilm Infection e JID 2007:195 (1 June) * 1709



and 98-17 formed extensive biofilms consisting of a network

of hyphae and yeast cells embedded in a heterogeneous matrix.

C. glabrata and C. parapsilosis, however, formed biofilms con-

sisting primarily of yeast cells. Like C. albicans, these biofilms also were heterogeneous and contained abundant matrix

material.

In vivo biofilm glucan assay. To determine whether biofilm formation is associated with elevated concentrations of 0-1,3

glucan in vivo, we compared the CVC (biofilm) rat model to

a traditional disseminated candidiasis (nonbiofilm) model. An-

imal catheters were inoculated with C. albicans K1, and biofilm

formation was confirmed using SEM at 24 h (figure 3A). After

catheter inoculation, serum samples were collected both directly from the catheter and the tail vein at 12 and 48 h. In a second

group of rats, infection was produced by injection of identical

inoculum via the tail vein. Blood samples were collected at the

various time points. We measured serum glucan content as well

as the viable cell burden in kidneys. As anticipated, the viable

burden in the kidneys, as an estimate of the systemic burden

of cells, was greater in the disseminated model (figure 4). De-

spite the in vivo presence of more cells, the serum from the

biofilm catheter infection contained nearly 10-fold more 0-1,3

glucan (P< .01) (figure 4). Also, tail-vein serum samples taken from animals with a biofilm infection contained high concen-

trations of 0-1,3 glucan, indicating a systemic release of glucan

during biofilm infection. Animals with catheters were also in-

fected by tail-vein route. Serum samples from these animals did not have elevation of 03-1,3 glucan, indicating that the catheter material does not result in the elevated serum glucan levels

without the presence of a biofilm (data not shown). Next, we extended our investigated of 3-1,3 glucan secretion

to other Candida species. We used clinical isolates of C. glabrata, C. parapsilosis, and an additional C. albicans isolate for infection of the biofilm CVC model. SEM confirmed the presence of biofilm on the luminal surface at 24 h (figure 3B-3E). Similar to our previous results, animals infected by biofilm route had 2-10-fold higher serum 0-1,3 glucan concentrations (figure 5). However, the magnitude of the difference varied by species. Biofilm infection with C. glabrata was associated with the high- est serum 0-1,3 glucan content. The disseminated model produced >10-fold less glucan using this same strain. Interestingly, a serum sample taken from a site distant from the biofilm

infection contained only low levels of glucan. The C. parapsilosis and C. albicans biofilm infections produced 2-8-fold higher serum 1-1,3 glucan concentrations, compared with the re-

spective disseminated models.

DISCUSSION

Candida infections are common in hospitalized patients, par- ticularly those with implanted medical devices [4, 6, 7]. CVCs

become infected at an estimated rate of 1.2-4.5/1000 patient- catheter days [25, 26]. The diagnosis of a Candida CVC infection is problematic, and the decision to remove a catheter is difficult. Replacement of a CVC is costly, associated with patient discomfort, and carries a mechanical complication rate of

6.3%-19.4% [25]. The Candida cells growing as biofilms are different from their

free-floating counterparts [11]. Sessile and embedded in a ma-

a, C

0

0T -J

5 -

4-

3-

2-

1-

0-

A

Biofilm kidney Disseminated kidney

CA-98-17 CG-5376 CP-5986

B

-J,

0a

C~ U,

400

300

200

100

0

I Biofilm catheter I Biofilm peripheral IDisseminated

CA-98-17 CG-5376 CP-5986

Figure 5. Serum 0-1,3 glucan concentrations (picograms per milliliter) from Candida albicans (CA-98-17), C. glabrata (CG-5376), and C. parapsilosis (CP-5986) biofilm and disseminated infections. Central venous catheters were surgically implanted in rats and infected with various Candida species. Serum samples were obtained at 24 h, and 3-1,3 glucan content was determined using limulus lysate assay (B). Black vertical bars represent 0-1,3 glucan content in picograms per milliliter in serum samples obtained from the catheter. Light gray bars represent 0-1,3 glucan content in the peripheral serum sample obtained from an animal with a biofilm catheter infection. Dark gray bars represent 3-1,3 glucan content of a serum sample from an animal with a disseminated infection. The viable burden of cells from the kidneys of animals with disseminated and biofilm infections were determined at various time points to estimate systemic burden of infection (A). Error bars represent 1 SD.




trix, they exhibit a drug-resistant phenotype, making removal

of an infected catheter often necessary for cure [7, 10-12, 15]. The ability to detect a Candida biofilm could have a great

impact on the treatment on candidemia. Of patients diagnosed with candidemia, 30%-70% are reported to have a catheter-

related bloodstream infection [9, 10, 27]. However, studies in-

cluding high numbers of patients with neutropenia have dem-

onstrated lower incidence of CVC-associated candidemia and,

thus, biofilm infection (18%-47%) [6, 28]. Many devices in

these patients are surgically implanted, making removal and

replacement more difficult. Current guidelines recommend de-

vice removal for all patients with candidemia [7]. A test allow-

ing detection of a biofilm device-associated infection would

identify patients who would benefit most from device removal.

Those with candidemia and a negative biofilm test would be

able to avoid device removal.

In these investigations, we have proposed 0-1,3 glucan as a

biofilm marker for Candida. Previous studies have dem-

onstrated differences in secreted carbohydrate composition be-

tween C. albicans biofilm and planktonic cultures [29, 30]. Our

recent laboratory study has demonstrated a greater concentra-

tion of 0-1,3 glucan in the cell wall of Candida biofilm cells

[30]. We reasoned that the unidentified matrix carbohydrate could be a 0-glucan molecule. To test this hypothesis, we com-

pared the C. albicans biofilm conditions to planktonic condi-

tions and demonstrated increased 10-1,3 glucan concentrations

surrounding Candida biofilms both in vitro and in vivo. Using an in vivo model, which closely mimics a patient CVC infection, we identified significantly higher serum levels of 0-1,3 glucan,

compared with animals with disseminated infections. Our find-

ings suggest that serum 10-1,3 glucan may be a potential target to differentiate biofilm and nonbiofilm infections in patients.

The limulus lysate assay used in the present studies is ap-

proved for diagnosis of invasive fungal infection including Can-

dida [20, 31-34]. A cutoff of 60 pg/mL glucan has been used

for diagnosis of an invasive fungal infection [20, 34]. However, in one study, the serum 10-1,3 glucan concentration in patients with candidemia ranged from 60 to >2000 pg/mL [20]. Although a difference in systemic viable burden may account for variation

in serum glucan concentrations in these patients, it is also possible that patients with higher values had a biofilm catheter infection.

In the present experiments, 1-1,3 glucan served as a useful marker

for in vitro and in vivo biofilm formation. Using the glucan assay as an indicator of biofilm infection may markedly improve the

specificity of this test. Further investigation of the 1-1,3 glucan

assay in patients with Candida biofilm infections would be im-

portant to determine whether this assay, or a modified glucan

assay, may be able to diagnose a biofilm or device-associated

infection in a heterogeneous patient population.

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