Adherence ofPhenotypically Switched

Candida albicans toDenture Base Materials

David R. Radford, BDS, PhD, FDSRCS, MRD'Stephen f. Challacombe, BDS, PhD,

FDSRCS (Fd) FRCPatb''lohn D. Walter, BDS, DDS, FDSRCS (Fd)'

Purpose: The purpose of this study was to investigate in vitro the levels of adhesion ofphenotypically switched and nonswitched Candida albicans to the smooth and rough surfaces ofheat-cured acrylic resin, MolJoplast B, and Novus. Materials and Methods: Nonswitched andswitched cells of C a/b/cans 3153A were prepared and adhesion assays were performed.Results: There was no difference in the level of adhesion of biastospores in their nonswitched orswitched state. The adherence of hyphae to all three denture base materials was statisticallysignificantly increased when in a switched form. There was greater adhesion to the two soft liningmaterials than to the acrylic resin. Conclusions: Rough surfaces promote adhesion. There was nosignificant difference in the adhesion of switched and nonswitched biastospores, but there wasIncreased adhesion of hyphal ceils. Inl I Prosthodont 1998;! 1:75-81.

Candida albicans is classified as an asexualdiploid fungus that has a predominantly unicel-

lular method of growth and development. The maincommon feature of the genus Candida is the lack ofany sexual form. It can develop true and pseudo-hyphae, and normal cultures produce smooth,rounded, creamy white colonies ¡termed "o"smooth colony morphology). Candida albicans hasbeen associated with the etiology of denture-relatedstomatitis,^'^ a condition that has a prevalence ofbetween 11% and 67% in complete denture wear-ers.^ Phenotypic switching of C. albicans was

"Senior Lecturer, Department of Prosthetic Dentistry. UnitedMedical and Dental Schools, Cuys Dental Hospital, Universityof London, United Kingdom.

^Professor and Head, Department of Oral Pathology andMedicme, United Med'cal and Dental Schools, Guys DentalHospital. University of London, United Kingdom.

'Reader and Head. Department of Prosthetic Dentistry, UnitedMedical and Dental Schools, Guys Dental Hospital, Universityof London, United Kingdom.

Reprint requests: Dr David R Radford, Department of ProstheticDentistry. Floor 21 Guys Tower, Guys Dental Hospital, LondonSE! 9RT, United Kingdom.

rediscovered by Slutsky et al in 1985,^ and subse-quently much research has been undertaken intothis phenomenon. High-frequency phenotypicswitching is the phenomenon of cells emanatingfrom a single progenitor that express different phe-notypes resulting in variant colonial morphology,and indicates that cells can switch reversibly from"o" smooth to variant morphologies and viceversa.^ The biologic advantage of phenotypicswitching is that it may allow the organism to adaptto a changed environment. Phenotypic switchingmay also account for organisms altering from acommensal to a noncommensal state. This is im-portant for patients who wear dentures, as imprintsampling techniques have shown that 55% of com-plete denture wearers carried Candida as a com-mensal.^ It is not known why, in other subjects withdenture-related stomatitis, this common commensalorganism acts as a pathogen, but it has been shownthat the recurrence of the condition is a result of theoutgrowth of commensal strains,'

As adhesion is the first step for organisms to colo-nize a surface, one possible significance of pheno-typical switching is that the cell morphology Is as-sociated with altered adhesion properties of the

i11,Number1,I9'Sfl 75 The international Journai al Prosthodontii

ot" Candiih albkans to Oentiire Base Mate

Bridge--—-joining twin

replicates

Surfaces as processed against glass (Smooth/control)

Fig 1 Diagram ot the twin repiicates prepared to ensure iden-ticai was in Ing procedures.

organism. Investigation into the adhesion of organ-isms derived from the oral cavity has shovv'n hyphaeto be more adhesive to buccal cells than blas-tospures.^ Additionally, phenotypically switchedcells of the "white-opaque transition" were shownto adhere more readily to buccal cells.'' Despite theoriginal work on variant colony morphology in or-ganisms isolated from the oral cavity,'^ the patho-genicity of switched C. albicans in the oral cavityhas not been demonstrated conclusively as it hasbeen in other sites in the body such as the vagina.' '

Adhesion assays of C. aibicans to acrylic resinwere first conducted by Samaranayake andMacFarlane.'^ They undertook the assays on self-cured acrylic resin, and determined how adhesionwas affected by altering the concentration of differ-ent nutrient carbon sources and assessing the con-ditions for optimal adhesion.'^-'^ Despite subse-quent work by other authors'**"'^ many aspects ofthe adhesion of C. aibicans to denture base materi-als warrant further clarification. To ensure clinicalrelevance, the adhesion assays should be under-taken on materials routinely used in clinical den-tistry,'^ even if the complications of processingheat-curing materials adds a difficult and morecomplex initial stage compared to the use of self-cured materials. Previous studies fulfilling these cri-teria undertaken on a single clinically isolated mor-phologic variant of C. aibicans showed the reducedadherence of switched cells compared to the non-switched cells to both saliva-coated and noncoatedheat-cured acrylic resin.'^

The aim of the current study was to investigatewhether organisms in high-frequency phenotypicswitching exhibited more adhesion to smooth andrough surfaces of acrylic resin and two soft liningmaterials than similarly cultured and genetically

identical cells not undergoing high-frequency

switching.

Materials and Methods

Preparation of the Denture Base Materials

Three materials were chosen: ¡1) Trevalon, a com-monly used heat-cured poly{methyl methacrylatel(Dentsply Ltd, De Trey Division]; and two soft lin-ing materials; (2) Molloplast B (Karl Huberl, a heat-cured dimethylpolysiloxane that has been widelyresearched; and (3¡ Novus, a polyphosphazine elas-tomer (Hygenic). The two rotary instruments chosenfor the study have been widely used and producesurfaces typical of those found in prosthodonticpractice. The first instrument was a crosscut steelbur, Meisinger vulcanite cutter ISO 310 1041551 72...060 {Hager and Meisinger). The secondwas a 13-mm-diameter medium Arbor band with80-size silicon carbide grit (Bracon).

Preparation of Samples and RepresentativeSurfaces

A total of 30 twin replicates was made to allow theassay to be performed on two separate occasionsusing 15 samples on each occasion, five for eachmaterial. All samples were processed according tothe manufacturers' instructions. The samples (twinreplicates, Fig 1), were constructed so that eachtwin replicate could be placed in two adjacentwells containing different solutions, but as a twincould be washed simultaneously. During deflask-ing, nonsterile washed latex gloves were worn toavoid contact with the processed material, andsamples were handled with sterile stainless steeltweezers. The experimental surfaces used on thereplicates were:

1. Trevalon (a) processed against glass and ib) ma-chined with a steei bur

2. Molloplast B (a) processed against glass and (b)machined with an Arbor band

3. Novus (a) processed against glass and (h) ma-chined with an Arbor band

The surfaces processed against glass acted as thecontrol surfaces. The speed of the handpiece wascontrolled at 15,000 rpm using a Schick C2 hand-piece with micromotor (Schick Dental]. The proce-dure for machining the surfaces was standardized byensuring that the rotary instrument was cutting onlyalong the surface of the sample in one direction.Minimal pressure was used as in clinical practice

The International lournal of Proitiicdontii 76 Volumen,Number 1,1996

f of Candide ,-¡ibic,-¡ns to Denture iî.is

when making fine adjustments to the surface of adenture base. No attempt was made to standardizemechanically or measure the pressure applied bythe cutting instrument. Tbe time of machining couldnot be standardized because of the differing periodsrequired for the instruments to cut the various mate-rials.

Preparation of Candida albicans for AdherenceAssays

Candida albicans NCPF 3153A was taken andidentification was confirmed by germ tube for-mation in bovine serum and wilh tbe API 20 CAux system (bioMerieux). A loopful of a singlecolony from Sabouraud agar was added to 10 mLof sterile normal saline and dispersed with a me-chanical vibrator. Cells were counted in a modi-fied Euchs-Rosenthal hemocytometer, and thesuspension was adjusted to give a final concen-tration of 1 X 10^ cells/mL. A nutrient poor agarwithout zinc supplement'^ was inoculated withTOO (JL of suspension to give a density of approx-imately 100 cells per plate. The plates were incu-bated for 14 days at 25''C. Colonies with normalmorphology and five variants were selected andwere subcultured sequentially from six to eighttimes under tbe same conditions. An irregularwrinkled colony that had shown revertantsmooth colonies but wbose morphology re-mained stable over this period (Eig 2) was thenselected to provide switched cells, and the bio-chemical characteristics were reconfirmed usingthe API 20 C Aux system.

To ensure the cells of the adhesion assays werecultured identically, the cells were derived fromboth switched and nonswitched colonies after 3weeks growth at 25°C on agar. The final prepara-tion of the yeast cells for the adherence assaysbroadly fol lowed the technique described bySamaranayake and MacFarlane.'-^ A loopful of cul-ture botb of switcbed cells and nonswitched cellswas streaked onto Sabouraud agar and incubated at37°C for 18 to 20 hours. Four loopfuls of this freshyeast growth was then transferred into a filter-sterilized glucose supplemented (50 mM) yeast ni-trogen base broth (0.2 pm pore size filter, Minisarl,Sartorius). Ibe culture was incubated at 37°C on anorbital shaker at 150 rpm for 18 to 20 hours to sta-tionary phase. The culture was centrifuged at 2,500g for 10 minutes, and the resultant cell pellet waswashed twice with phospbate-buffered saline (PBS).Cells were counted optically, and the suspensionwas adjusted to give a finai suspension of 1.0 X 10'yeasts/mL.

Fig 2 irregular wrinkle colony type of switched C aiblcans(31S3A) The irreguiar wrinkied colonies were used in the ad-hesion assays as ceils in "high frequency switching." (Originalmagnification x 10.)

Method for the Adhesion Assays

Twin replicafes were placed in 24-weN sérologieplates (Corning), one replicate of eacb twin in a sus-pension of nonswitched cells and the other in a sus-pension of switched cells. For each material, a con-trol replicate was placed in a solution of PBS. Thesuspension was incubated for 1 hour at 37°C on anorbital shaker at 80 rpm and then carefully pipettedfrom each well in rotation. Each twin replicate washeld with tweezers by the bridge and removed fromthe sérologie plate. With a single sweep, twin repli-cates were slowly passed for 3 seconds eachthrough three containers of 100 mL PBS. The solu-tion was changed after each group of replicates of asingle material had been washed. The replicateswere air dried for 2 hours at 37°C and left for 16hours at room temperature before mounting onslides and staining. Replicates were mounted onglass slides using double-sided tape and stainedwith 0.5% crystal violet solution and 1.0% iodinesolution for 5 minutes each.

Adherent yeast cells (blastospores) werecounted using a stratified sample technique at 400X magnification with a graticule covering an areameasuring 0.025 X 0.025 mm. Five fields for each

77 Tlie Irrer nations I Journal of frostliodomics

Adhe ä ol C.mdidù ûlbkans to Demure Base MaleriaU

Table 1 Adhesion Assays of Blastospores of C. atbicans in Switched andNonswitched Forms on Control and Rough Surfaces

Materiai

Acrylic resin

Moilopiast B

Novus

Surface

Control

Controi

Rough

Rough

Controi

Confroi

Rough

Rough

Controi

Control

Rough

Rough

Candida

Nonswitched

Switched

Nonswitched

Switched

Nonswitched

Switched

Nonswitched

Switched

Nonswitched

Switched

Nonswitched

S switched

Assay 1ceil counts

2.60.505.00.68

16.41 17

11.61.08

10.90.88

13.81.12

29.51.41

23.11.336.30.84

11.01.04

35.71 50

26.91.43

(1.7)(0.23](4.1)(0.32)(9.3)(0.26)(4.7)(0.161(9.3)(0.471(8.1)(0-21)

(17.5)(0.26)

(12.4)(0.20)(2.6)(0.16)(1.9)(0.21)

(18.3)(0.25)

(14.2)(0.20)

Assay Iicell <

6.40.826.30.82

24.61.37

28.01.417.10.848.30.92

26.71.41

29.31.457.70.887.90.90

32.41.50

29.81.47

Munts

(3.3)(0.20)(3.3)(0.20)

(11.2)(0.35)

(14.1)(0.21)(3.9)(0.281(3.4)(0.22)

(10.9)(0.18)

(10.8)(0.16)(3.5)(0.26)(3.4)(0.26)

(10.2)(0.15)

(10.1)(0.15)

This tabie sliows the results o( the two assays (Assays i S ii) undertaken on separate occasicns using tliesame protocol. The figures in boid are the mean yeast cell counts per tield over 20 fields. Standard devia-tions are given in parentheses The iog,^ transformation is siiown in roman type beicw tiie mean yeast cell

surface of each material were counted. Because ofthe reduced number of adherent hyphal ceils, thehyphai count was undertaken at a reduced magni-fication of X 100 with the graticule covering anarea measuring 0.1 x 0.1 mm. At this low magni-fication il was difficult lo distinguish the filamentsfrom machining strialions in ihe material surface,so the assay was evaiuated oniy on the smoothsurfaces.

Statistical Methods

Descriptive data were analyzed using a main frameSPSS (Statistical Package for the Social Sciences,SPSS). Logarithmic Iransformation to the base 10was undertaken for the data, and this provided nor-mai distributions. Factorial analysis of variance(ANOVAl was performed using the Censtal pack-age (Censtat 5, Numerical Algorithms Group) toevaluate the effecl of various faclors on the microbi-ologie counls. Subsequently, one-way ANOVA andthe Scheffe test, were used to explore differencesbetween the categorical levels of the individual fac-tors where these exceeded two.

Results

The results presented in Tables 1 and 2 show themean number of cells within each field for 20 indi-viduai graticule counts for each of the surfaces in-vestigated. Mean data is also represented by bargraphs for each assay (Fig 3). Factorial ANOVA ofthe data from Tabie 1 shows there was no signifi-cant difference in adhesion of switched and non-switched biastospores on all three materials.Significantly more organisms adhered to the rough(machined) surfaces than to the control surfaces (P< 0.01), which had been processed against glassand therefore were smooth. There was iess adhe-sion to acrylic resin than to Moilopiast B or Novus.

Adhesion of hyphal cells over a larger field area(0.1 X 0.1 mm) demonstrated that there was a sig-nificant increase in number of hyphae that had ad-hered (Table 2). Factorial ANOVA of the hyphalcounts showed that significantly (P < 0.001) morehyphae had adhered in the assays with switchedrather than nonswitched organisms (Table 2). Therewas no significant difference in adhesion of hypbaeto the three different denture base materials.

The International lournal of Prosthodontii 78 Voiumel1,Number1, 1998

Adiierence of Candida sibicans lo Denture Base Materiais

Table 2 Results of the Hyphal Counts for the Adhesion Assays of C aibicar)s inSwitched and Nonswitched Forms on Control Surfaces.

Material

Acrylic resin

Moliopiast B

Candida

Nons witched

Switched

Monswitobed

Switched

Nonswitciied

Switcbed

Assay 1ceil counts

Mean

1.30.266.90.831.20.27e.20.891.10.256.60.82

SD

(1.9)(0.28)(4.7)(0.24)(1.2)(0.24)(5.9)(0 26)(1.2)(0.23)(4.1)(0.24)

Assay liceil (

Mean

1.00.257.30.861.30.319.90.982,10.389,10.95

:ounts

SD

(1.0)(0.23¡(1-7)(0.24)(0.9)(0.19)(5,9)(0.25)(2,1)(0,31)(4.2)(0.23)

The tigure in bold is the mean hypiial count per field over 20 fieids, FieW size for the cell counts was in-creased to 0.1 X D.1 mm. Standard deviations are given in parentheses. The log,„ trarstcrmatlon is shownin roman type below the mean hyphal counts

Fig 3 This experiment showed some variation in ranked order between tfie two assays. Reproducibility across the two assaysshowed there was no significant variation in adhesion between tbe switctiad and nonswitched forms of yeast ceils. Tbere was a signiti-cant effect resulting trom surtace roughness.

Discussion

The aim of the current study was to investigate theeffect of high-frequency switching on the adhesionof C aibicans to the smooth and rough surfaces ofheat-cured acrylic resin and two soft lining materi-als. Having established that variant colony mor-phology is associated with increased numbers oftrue and pseudohyphal cclls,'^ it was important todetermine in vitro whether increased adhesion todenture base materials was related to switching.

Candida alhicans is of significant interest toprosthodontists because of the association betweenthe organism and denture-related stomatitis, a con-dition that is often recurrent and affects patientswho wear either partial or complete dentures.Candida aibicans has no possibility of sexual repro-duction, and bigh-frequency switching as expressed

by colony morphology is a possible mechanism ofreversible phenotypic change.^^ This phenomenonmay allow the organism to adapt quickly to a newenvironment, which would not occur in the ab-sence of meiosis.

Previous research has shown the organism'sability to form hyphae for dissemination in the tis-sues.^" Other research^' has suggested that theability to switch may facilitate multistage invasion,and it is therefore reasonable to speculate that thismultistage invasion may be one of the reasons C.aibicans in high-frequency switching mode andproducing hyphae is more pathogenic than the or-ganism in a nonswitched form. Certainly the inva-sion of vaginal epithelial cells by hyphae, one ofthe pathogenic factors attributed to C. aibicans, hasbeen shown by transmission electron mi -croscopy.^"

.,e11,Nijrrber1,1i Journal uf Prosthodontics

No difterence in adhesion was shown in assays Iand II, which compared adhesion betweenswitched and nonswitched biastospores, but greaternumbers of hyphae had attached in the assays withthe switched cells. This differed from previously re-ported findings'-'' that showed a reduction of adhe-sion with a variant clinical isolate of Candida. Theresults of the present study also differed from the re-sults of adhesion of switched cells to epithelium.'-^In vitro investigation of the 31 53A switching systemshowed that switched cells adhered in smallernumbers to epithelial cells.^'

The clear result of the present study of increasedadhesion to rough surfaces is of interest and hasbeen reported previously.-"* Although greater levelsof plaque formation on rough surfaces in vivo havebeen shown,-"' plaque formation is much morecomplex than in vitro adhesion assays.-^ One of thereasons for the greater accumulation of plaque onrough surfaces intraorally may be the greater adher-ence of the organism in the first instance, as hasbeen shown in this present in vitro study. This ad-hesion could be caused by the larger surface areapresented by rough surfaces, or, more importantly,because the organisms are protected from the ef-fects of washing in the in vitro adhesion assays orfrom the action of saliva in vivo.

Cleaner working procedures were required in thisinvestigation than those that are normally practicedwhen performing routine dental laboratory proce-dures. Extra precautions included the use of latexgloves free from powder during deflasking, and themachining of replicates. The control surface had tobe as smooth as possible, and this was achieved byprocessing against glass. It has been shown thatpacking denture base material against vacuum-spatulated stone did not produce an adequately flatand smooth surface.-'' Previous adhesion studies re-ported in the literature have appeared to address in-adequately the smoothness of study surfaces,'"'^'

In this investigation, pilot experimentation hadshown considerable variation of adhesion betweenreplicates as a result of variations inherent in thewashing technique. This has also been discussed byVerran and Maryan,--^ who stated that the washingprocedure should be standardized and specified. Inthe current experiment, an assay had to be devisedthat would allow similar surfaces that had been ex-posed to two different suspensions of organisms tobe washed in an identical fashion and not just in astandardized way. It was therefore necessary to de-sign a twin replicate that could be placed at thesame time into two wells of a sérologie plate andthen washed simultaneously.

Except for one study reported in 1989,^'' little at-tention has been paid to the poor adhesion ofCandida to inert surfaces and the consequences forin vitro experimentation. It was shown that afterthree sequential immersions for 5 seconds each inPBS, only 20% of the original adherent organismswere still present. After a further three identicalwashings, this was reduced to 5% of the original or-ganisms still adhering. It is therefore apparent that thewashing regime in adherence experiments with C.albicans is critical in producing a reproducible assay.Although great care was exercised in designing thewashing procedure, there was still a risk of minor dif-ferences in the assays with the "twin" replicates. Thismay have accounted for some of the variation in re-sults between the assays in this study. The produc-tion of the twin replicates as used in this study wastime consuming but permitted a satisfactory washingregime, as it was critical to design experimentationso that the test surfaces under comparison were onone replicate to be washed together.

Conclusions

The main conclusions of this study were:

1. Nonswitched biastospores were equally adher-ent to acrylic resin, Molloplast B, and Novus asbiastospores derived from switched organisms.

2. There was greater adherence of hyphae ofswitched cells than nonswitched cells.

3. Hyphal adhesion was at approximately thesame level on all three materials investigated.

4. There was greater adherence to rough soft liningmaterials than to smooth acrylic resin.

Acknowl edgments

The authois would like to acknowledge the help ot Dr S. Sweetin the niicrohiologic procedures and Dr R. F. Wi lson for helpwith fhe statistical analysis.

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Literature Abstract

Some clinical and radiographical features of submerged and non-sub-merged titanium impiants. A 5-year foilow-up study

This is the 5-year repcri of a study on 11 patients previousiy reported atter 18 months. Asplit-moutli approach was used in the mandibie: in the right side a convenlionai 2-step im-plant surgery technique was used; in the left side a 1 -step technique was used. At the 5-year ¡oliow-up examination ali 61 implants present at the examination atter 18 months werestill in service and tound clinically stabie irrespective of surgical procedure. Radio graphic ail ythere was no signiticant ditference between the treatment groups in marginai bone levelsduring the observation inten/ai. At the 5-year examination, one to four retaining gold screwshad to be retjghtened in five out of the 11 patients. This tinding is in agreement with datareported by Kalius 8 Bessing (IntJ Orai Maxiliofac Impianls t994;9:169-178).

Ericsson I. Random K, Nilner K, Peterson A. Clin Orai tmpiants Res 1997,8.422-426. Reterences: 26.Reprints: Ingvai Ericsson, Department ot Prosthetic Dentistry. Faculty of Odontciogy, Lund University,Carl Gustavs vâg 34. S-ai4 31 Malmd, Sweden—SP

,Number 1,1998 81 i of Prosthodortics