J Clitj Periodimtiit IWS; 25: 5f>-66 Copyright © Munksgaard 1998

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JBIHHUff
perjodontolooy
LSSN 0303-6979

The effect of a one-stage CurdM. ,

Claudio Mongardini\
William Papaioannou^'^,

full-mouth disinfection on Daniei Van Steenberghe'-^ and

Marc Quirynen^-^
'Department of Periodontology, ^Research
Group tor Mjcrobial Adhesion, Catholic University

different intra-oral niches of Leuven, Belgium

Clinical and microbiological observations

Bolten CML. Motigardini C, Papaioannou IV, Van Stcenherghe D. Quirvneit M:
The effect of a one-stage full-mouth disinfectioti on different ititfa-oral niehes.
Clinical and microhiohgieal observations. J CHn Periodontol 1998: 25: 56-66.
© Munksgaard. 1998.

Abstract. A treatment for periodontnl infections often consists of consecutive

rootplanings (per quadrani, at a I- to 2-week interval), without a proper disinfec-
tion of the remaining intra-oral niches (untreated pockets, tongue, saliva, mucosa
and tonsils). Such an approach, could theoretically lead to a reinfection of pre-
viously-lreated pockets. The present ,s!udy aitns lo examine the etTect of a full-
mouth disinfection on the microhiola in the above-mentioned niches. Moreover.
the clinical benefit of such an approach Vv-as investigated. 16 patients with severe
periodoniitis were randomly allocated to a test and a control group. The palients
from the control group were scaled and rootplaned. per quadrant, at 2-week
intervals and obtained oral hygiene instructions. The patients from the tesi group
received a full-mouth disinfection consisting oi': scaling and rootplaning of all
pockets in 2 visits wiihiti 24 h, in combination with tongue brushing with \"/,>
chlorhexidine gel for I min. mouth rinsing with a 0.2'^ chlorhexidine solution
for 2 min and subgingival irrigation of all pockets (?X in 10 min) with i ' ^ chlor-
hexidine gel. Besides oral hygiene, the test group rinsed 2x daily with Q.2% chlor-
hexidine and sprayed the tonsils with a 0.2'l'u chlorhexidine for 2 months. Plaque
samples (pockets, tongue, mucosa and saliva) were taken at baseiine and after 2
and 4 months, and changes in probing depth, attachment level and bleeding on
probing were reported. The full-mouth disinfection resulted in a statistically sig-
niiicant additional reduction/elimination of periodonlopathogens, especially in the
subgingival pockets, but also in the other niches. These microhiological improve-
ments were reilected in a statistically-significani higher probing depth reduction
and attachment gain in the test patients. These findings suggest that a disinfection Key words: chlorhexidine; microbiology;
periodontitis; periodontal therapy; root planing;
of all intra-oral niches within a short time span leads to significant clinical and attachment level
microbiological improvements for up to 4 months.
Accepted for publication 5 March 1997

Periodontat infections have a multi-fac- Socransky & HalTajee 1992. Wolff el al, were found to colonise nearly all the
torial aetiology involving a susceptible 1994). An overgrowth or re-infeetion by above-mentioned niches (Van der Vel-
host, the presence of periodontopatho- pathogenic species will of course jeo- den et al. 1986, Van Winkelhoff et al.
gens. the absence of beneficial bacteria pardise the success of such a treatment. 198Sa. Asikainen et al. 1991. Mulier et
and a suitable environment. Since it Is Several studies indicated that tnost a!. 1993. Danser el al, 1994), Recent
impossible to alter the genetic suscepti- periodontopathogens colonise more microbiological studies on implants
bility of the host, a periodontal iherapy than 1 niche in the oral cavity (such as from partially edentulous patients sug-
is restricted to the reduction/elimin- the tongue, the mucosa. the saliva or gested the existence of an intra-oral
ation of the periodontopaihogens in the tonsils). In periodontitis patients, translocation (from one niche lo an-
combination with the re-establishment key-pathogens such as Aetinobaciltus other) of periodontopathogens (Apse et
of an environment suitable for a bene- aetin(n)iveeteniei>niitan.s. Porphvromo-al. 1989, Quirynen & List'garien 1990,
ficial inicrobiota (Slols & Rams 1991. nits gingivalis and Prevolelta internwdia Quiryncn et al. 1996). If such a translo-
 FulJ-numtli disinfccritin and oral niches 57

cation occurs rapidly, a recently root- with pockets of >7 mm (/;^0.3) nor in used. The treatment always started at
planed deep pocket might be re-colon- the % sites with radiological angular de- the right upper quadrant so that there
ised by pathogenic bacteria from re- fects (p=0.5). All subjects were medi- were no differences in the healing
maining untreated pockets, or from cally healthy and none had undergone period between all experimental sites.
other intra-oral niches, bet^ore a new periodontal treatment before. More- Just prior to the 1st session of scaling
and less pathogenic ecosystem can be over, none of the participants had used and rootplaning, and 2 and 4 months
established. any antimicrobial within the 4 months later, subgingival plaque samples and
This hypothesis was suggested by the before, or during the study. Each volun- ciinica! parameters (with the exception
significant clinical and microbiological teer had at least 20 teeth with at least 2 of baseline probing depth which was
benefits of a full-moiuh disinfection, as multi-rooted and 3 single-rooted teeth scored after rootplaning. to avoid inter-
depicted in a recent pilot study (Quiry- in the first quadrant (excluding the 3rd ference with the calculus deposits) were
nenetal. 1995. Bollenetal. 1996. Vande- molar). In this quadrant, at least 2 ap- taken from the right upper quadrant,
kerckhove et al. 1996), In that study. lU proximal regions around both the single- chosen for its accessibility. At the same
patients with severe periodontitis were and multi-rooted teeth had a pocket ^ 7 moments, plaque samples from the dor-
treated either with the standard treat- mm. which bled on probing and showed sum of the tongue, the buccal mucosa
ment strategy (oral hygiene instruction radiographic evidence of severe bone and Ihe saliva, were taken.
and consecutive rootplanings per qua- loss(^ 1/2 the root length). After obtain-
drant al a two-week interval) or with a so ing informed consent, the patients were
called "full-mouth approach" consisting randomly assigned to 1 of the 2 treat- Mechanical plaque and calculus removal
of a scaling and rootplaning of all ment groups. Permission for this study
was obtained from the Ethical Commit- After local infiltration with an anaes-
pockets in 2 visits within 24 h in combi-
tee of the University Hospital, thetic, scalings and rootplanings were
nation with tongue brushing (\"/[, chlor-
performed by one investigator in both
hexidine gel for I min). mouth rinsing
the test and control group, with an as-
{0.2"A< chlorhexidine solution tor 2 min).
sortment of standard periodontal cu-
subgingiva! irrigation of all pockets f3x Experimental design
rettes (Hu-Friedy®. Chicago. Illinois).
in to min with \"A> chlorhexidine gel) at
The 8 patients of the test group received The time spent per quadrant was ap-
these 2 visits, and mouth rinsing 2x
a full-mouth scaling and rootplaning of proximately I h. All patients received
daily (O.T'Ai chlorhexidine) for 2 weeks.
all pockets, in combination with the ap- standard oral hygiene instructions im-
The present study aims to examine plication of antiseptics, within 24 h. In mediately after the 1st session of scaling
the clinical and microbiological benefits the 8 control patients in contrast, the and rooiplaning. These instructions in-
of an extended full-mouth disinfection, scaling and rootplaning was as usual cluded interdental plaque control
in a larger group of patients {n—lb). performed quadrant by quadrant, re- (toothpicks and/or interdental brushes).
Moreover, besides the microbiological sulting in a total of 4 sessions at 2-week tooth and tongue (dorsum) brushing
changes in the pockets, other intra-oral intervals. Here, no antiseptics were 2x a day. All patients were provided
niches were also investigated. In con-
trast to the previous pilot-study, the
present investigation also reports on
Ttible I. Descriptive statistics of patient population
changes in clinical attachment levels.
Age Smoking Proportion of pockets Proportion of
The full-mouth disinfection was ex- Subject Gender
(years) habit >7 mm angular defects''
tended by elongating the period of
chlorhexidine mouth rinsing (2 months Control
instead of 2 weeks) and by including an 1 45 F no 15,7 25,0
1 46
additional disinfection of the tonsils M no 17,3 14,2
3 69 M no 36,1 27.2
(2x daily using an 0,2"/i chlorhexidine
4 }5 M no 17,3 28,5
spray for 2 months). 5 42 M no 15,0 21.4
6 4S M 20 36.9 .10,7
Material and Methods 7 41 M 25 .19.1 35,7
8 32 M 2t) 27,5 16.6
Patient selection
mean and standard deviation: 25.6 (±10.5) 24,9 (±7.2)
16 volunteers. 6 women and 10 men.
Test
with ages ranging from 32 to 69 years
] 43 M 5" 20.5 4(1,0
(mean 46 years) participated in this ran- T 41 M 2t) 15.0 28.5
domised, double-blind, parallel study. 3 49 F no 25,0 41.6
All patients were referred to the depart- 4 34 F 20 19,5 14.2
ment of Periodontology of the Catholic 5 57 F no 40.6 22.2
University of Leuven for treatment of 6 68 F no 42.1 41,6
severe periodontitis. The descriptive 7 54 M no 31.2 lO.U
statistics of these participants together X 38 F no 43.4 28,5
with their disease status are summa- mean and standard deviation: 29.6 (±11.2) 28,3 (±12,3)
rised in Table I, These data showed that
Smoking habits: number of cigarettes/day (^ cigars): ^' observations for the first quadrant only;
both groups of patients are compar-
" estimaled on intra-onil long-cone radiographs. No statistical difTerence in age (/J = (),5). pro-
able: there was no statistical difference portion of siies >7 mm (y> = 0,3) and proportion of sites with radiological angular defects {p=
in age (/i-0.5), nor in the "Ai of sites 0,51 between test- and control subjects.
58 Bollen ct at.

with Sensodyne" toothbrushes (no, 4 from: single-rooted teeth, multi-rooted 100 to 200 bacteria from 4 randomly se-
medium, Stafford-Miller Continental. teeth, tongue, mucosa and saliva. Per lected separate fields was classified into
Oevel. Belgium) and Sensodyne® F-gel tooth type (single- or multi-rooted). 4 morphological types (Mousques et al.
toothpaste (Stafford-Miller Continen- samples from the 3 deepest approximal 1980): coccoid cells, motile rods (other
tal. Oevcl. Belgium), Plaque control sites in the upper right quadrant were than spirochetes). spirochetes and
and oral hygiene instruction were re- pooled to get an overall idea of the others (e,g.. fusiforms. rods and fila-
peated on several occasions (month 1 microbiological situation (Christersson ments). Only the micro-organisrns that
and month 2). Moreover, at the 2- et al. 1992). Prior to sampling, the se- were part of the fluid flow were counted,
nionth recall visit, all teeth were polish- lected approximal sites were cleaned in order to avoid confusion with
ed with 3 pastes, with a decreasing or- supragingivaily (sterile curettes), iso- artefacts or dead micro-organisms.
der of abrasiveness: Zircatc Prophy lated frotn saliva (cotton rolls) and
Paste^ (Dentsply International Inc, gently dried in order to prevent con-
Milford. DE). Clean Polish"^ and Super tamination (Van der Velden et al, 1986). Culture technique and identification
Polish"* (both Hawe-Neos Dental. Biog- Per site. 4 sterile medium paperpoints All samples were transferred to the la-
gio. Switzerland). (Maillefer^. Ballaigucs. Switzerland). 2
boratory and processed in less than 6 h.
buccally and 2 liiigually were inserted.
Serial lO-lbld dilutions were prepared in
The paperpoints were kept in place for
RTF.
Adjunctive disinfection (test group only) 10 s and were then transferred into a
screw-capped vial, containing 3 till of
In the test group, immediately after in- pre-reduced transport medium (RTF, samples
strumentation of the lower jaw and Syed & Loesche 1972). The samples Aliquots of 0,1 ml of the first 3 di-
after the instrutiientation of the upper from the dorsum of the tongue were ob- lutions were plated manually on TSBV
jaw. an additional disinfection was tained by wiping a sterile cotton swab plates (Trypticasc-soy-agar (BBL*
sought, by (in chronological order): {Biomerieux S,A,. Montalieu-Vercieu. Microbiology Systems. Cockeysville.
(1) brushing the dorsum of the tongue, France) from Ihc papillae vallatae to the MD. USA) supplemented with 5 /ig/ml
for I min with chlorhexidine l"/iigel tip of the tongue along the linca me- vancomycine. 75 //g/ml bacitracin and
(Corsodyl" gel. SmithKline diana for 10 s (Danser et al. 1994). \0% sterile horse serum), for ihe selec-
Beecham Consurtier Brands SA. Samples from the mucosa were ob- tion o\' A. actinonivcetetmomilans (Slots
Genval, Belgium); tained with sterile cotton swabs from 1982). These plates were incubated for
(2) rinsing 2 x with chlorhexidine 0.2"/ii the left cheek (area of about I cm-) by .3 days in air + 5'!^i CO^^. Suspected iso-
(Corsodyl* mouthrinse. Stnith- vigorously sweeping the mucosa 5-6X lates, identified on the basis of colony
Klinc Beecham Consumer Brands in a standardised manner (Muller et a!. morphology (small colony. 1 mtn in
SA. Genvai. Belgiutn) tor 1 min; 1995). The tips of the cotton swabs were diameter, with a dark border and a
(3) applying 2x a 0.2"/ip chlorhexidine also collected in screw-capped vials, "star-" or "crossed cigars"-shaped in-
spray (nearly 0,30 mi) (Corsodyl® containing 3 ml of RTF The microbial ner structure) and positive catalase re-
spray. SmithKline Beecham Con- load in the saliva was examined by col- action, were subjected to lactose and su-
SLttncr Brands SA. Genval. Bel- lecting 10/(1 of unsiimulated saliva with crose fermentation tests for final identi-
gium) to the tonsil area; a sterile plastic loop (Biomerieux S.A., fication (Slots 1981), Moreover, the
Montalieu-Vercieu. France) atid dis- number of colony forming units per ml
(4) repeated subgingiva! irrigation (3x
persing it in 3 ml of RTF, (CFU/tnl) was calculated.
within 10 min) with chlorhexidine
1% gel (Corsodyl* gel) of all The dilutions 10"' to 10"^ were
pockets by means of a syringe with All satnples were hotnogenised by plated in duplicate by means of a spiral
a blunt needle, which was inserted vortexing for .10 s (Dahlen et al, 1992), plater (Spiral Systems'"". Inc.. Cincinna-
in the pocket until some resistance The samples were coded in such a way ti, Ohio, USA) onto a non-selective
was felt; 2 different syringes and that the treatment modality could not blood agar plate (Blood Agar Base il®.
needles were used to rinse the be detected and that the tnicrobiologi- Oxoid. Basingstoke. Englatid), supple-
pockets of the lower jaw and the cal analysis was blind. mented with hetnine (5 mg/ml). mena-
upper jaw. to reduce the possibility dione (I mg/'ml) and ^'/^ sterile horse
of recontamination of the treated blood. After 7 days of anaerobic in-
sites; this subgingival application Differential phase contrast microscopy cubation (85% N., 10% CO. and 5%
was repeated a 3rd time at day 8; (DPCM) H2) at 37°C. the total number of CFU/
(5) mouth rinsing at home 2x daily for The differential phase-contrast micro- ml was counted. For each pigmented
1 min with a 0,2".i. solution of chlor- scopy of the subgingival samples was (black, green, brown) colony type on
hexidine (Corsodyl* mouthrinse). performed less than 15 min after sam- the representative plate (containing ap-
for the following 2 months: pling, in order to preserve the motility proximately 100 colonies), every 3rd
(6) applying 2x daily a 0.2"A. chlorhe- of the micro-organisms (Petit et al. colony was subcultured on a blood agar
xidine spray (Corsodyl" spray) to 1991), The analysis was based on the plate. After 48 h of anaerobic incuba-
(he tonsil area tor the following 2 guidelines described by Listgarten & tion, the pure cultures were identihed by
months. Hellden (1978). One drop of the suspen- means of DPCM, gratn-staining. an-
sion was examined in a differential aerobiosis. and a series of biochemical
phase contrast microscope at a magni- tests (including N-acetyl-//-D-glucosa-
Sampling fication of 480X (instead of the 1200X minidase. a-glucosidase. a-galactosida-
magnification as originally suggested by se. a-fucosidase. esculine. indole and
At baseline, and at the 2- and 4-month trypsine activity) in order to identify P.
follow-ups, plaque samples were taken Listgarten & Hellden (1978)). A total of
 Fiill-nunith disinfection and oral niches 59

P. intermedia and other pig- a paircd-r-test, DilTerences between test

Results
mented Purphyromonas and Prevotella and control group however, were exam-
Differential phase contrast microscopy
species. From the original blood agar ined by means of unpaired ?-tests. For
plates, the number of CFLf/ml of Pepto- all parameters only, mean scores per pa- Botii treatment modalities resulted, up
stirpuhoccit.s micros was also estimated tient were considered so that the patient to 4 tnonths after treatment, in a signifi-
based on its typical colony morphology was the statistical unit. The threshold cant (/;<0-05) reduction in the total
(smooth or rough, white, small colo- level for significance was 0.U5, All stat- atnount of motile organisms and spiro-
nies), DPCM and gram-staining. The istical calculations were made with the chetes for both single- and multi-rooted
dilutions 10 ' to 10 ^ were also plated Fxcel" progratn of the Microsoft*' Of- teeth (Fig. I). The reduction in the test
with the spiral plater on 2 additional fice package. group, in cotnparison to the control
selective media: the Hammond medium
for the detection of Campylohucler rec-
tiis (Hamtnond & Mallonee. 1988) and
the CVE medium for the detection of
Fusnhacteriuni niiclciiium (Walker et al. ruportion.^ of Spirochtte."! and Motilcs in Single- and Multi-rootod Teeth
1979), Besides by gram-staining and of (Control (C) and Test (Tl patioiiU examined by DPC.M
DPCM analysis, C. rectus was iden- Single-Rooted Multi-lii>r
tified on the basis of its typical colony
morphology on the Hatnmond-plates
(black to dark-grey colony. Ham-
• Monies
mond & Mallonee. 1988) and F. miclea- • Spirochcles
lum was identified on its purple fluo-
rescent colony on the CVE-medium
(Walker ct al, 1979).

Samples from the mucosa. the tongue

and ihe saliva
The samples from these niches were
only plated on TSBV and non-selective
blood agar plates using the same
D.M 2M -IM OM
procedures as tnentioncd above. How-
Tirai; in months
ever, frotn the blood plates, only the
total number of CFU/ml and the num- Fig. I. Changes in the relative proportion ol' hacteriiil morpliotypcs c.vamlncd by (Jiffcrential
phase-contrast microscopy over time, by toolh type (SR: single-rooted. MR: multi-rooted)
ber CFU/ml of pigmented Porphyro-
and per treatment modality (eontrol group: C. test groupi T). The proportion of spirochetes
monas and PrcYolella species were re- and motile organisms are shown as %. (Significant differences between the control and lest
corded. groups are indicated as: *p^0.05: significant differences between difTerent time intervals in
test and control gronp are indicated :is C# or T#: /

Ciinicai parameters
At baseline, and at the 2 and 4 months
recall, the following parameters were ul';\iiacroijic Cului'y Formiin; LIiLila iirumid Single- and Mulu- Rooli'il Tttth
recorded from the teeth in the upper
right quadrant (6 sites per tooth: me- Siiiglu-Rooted I Mulli-Rixited
sial, central and distal; buccally as well
as orally) by one examiner who was
blind for the used treatment modality:
(1) the sulcus bleeding index of Mtlhie-
mann & Son (1971); (2) the Quigley &
Hein (1962) piaque index using a
plaque disclosing solution (4"/. erythro-
sine); (3) the probing depth measured
to the nearest mm using a Merrit B*'
probe (Hu-Friedy, Chicago, Ilhnois);
(4) the bleeding after probing; and (5)
the attachment level measured from a
soft thermoplastic splint using the
same Merrit B^ probe. l,00F.*Oi: -
•iM OM
Timv in miiiilhs

Statisticai anaiysis Fii-. 2. The effect over time of partial (Control) versus full-mouth disinfection (Test) on the
number of colony-forming unils per ml (CFLJ/ml in log seores) after anaerobie incubation of
Changes in time within subjects were "pooled" s;imples from single-(SR) und mLilti-(MR) rooted teeth, (Signitiuant dilTerences be-
compared with each other by means of tween control and lest group are indicitted as: *p^Q.O5. **/>«0,005).
60 Bollen e! al.

group, was however always significantly Table 2. Number of CFU/ml for specific species in subgingival samples from ,single- (SR) and
higher (p<0.05). with the smallest differ- mulli-rootcd (MR) teeth from patients treated in the standard way (CONTROL) or by means
ence for multi-rooted teeth at the 4- of i\ full-moulh iipproacli (TEST) <ts observed at baseline (0 M) or at the 2- (2 M) and 4-
month recall. If the results in one group month (4 M) follow-up
were compared over time, a statistical CONTROL A. acfirromycetemcomirans TEST
significant reduction was maintained SH
A UM
over the 4-month time period (p always 2M
; 2
} J 2
=sO,O5), Indeed,, in contrast to Ehe stan- ,1 4^^ 1
dard therapy, which was not ahle to re- MR
\ A OM 2
duec the proportion of spiroehetes and A ZM 1

motile organisms around multi-rooted } 4\I 3

teeth below the 20% threshold level for •ingiv Jlis

clinical health (Listgarten & Hellden M(
1978). Ihe full-mouth disinfection always IIM 6 6 5 5
1 " <i

JM
resulted in a reduction even below lO'Mi, 1 ' S S

1 ^5 3 JM
MX
4

! 5 = 6 nM fi 5 4
4 5 5 S :M 6 S 4 3
Culture data 5 JM fi S -1
1 .' 5

Colony forming units in siihgingival F. in term 'III II

1 SR
plaque 5 5 i 5 re. !• ft 'IM
t 6 5 5 S
The total amount of facultative anaer- <; 5 s c,
obic and strict anaerobic CFU/ml
5 5
4 5 •; / '
:M
4'l
^
15 4 4

sampled from single- and multi-rooted •'"••• 5 1 *i > fi f- (, 7 t> tp () 6 6 1 5-

teeth are shown in Fig. 2, Whereas the i 5 3 4

number of CFL'/mi remained relatively 4 4 s

3
3
5
5
!•
!-
1 -' '> 4
1
4

constant with time in the control group F. nuctea um

(a reduction of-; log), a more significant
5 5 •s I-, j •'•'! 7 • : 6 •; 3 i 5 5 5
reduction (;><0,05) was observed in the 5 i S 3 5 -I 4 4 4 4 J
test group (> I log for single- and multi- 4 s •% ^ (••
•IM j 5 i ^ 1 2
Ml!
rooted teeth), especially at month 2. At 5 5 5 ^ ;i c, u 6 6 6 5
iiM U
a3 >.i -.

month 4. the difference between both 3 5 3 5 S ,5 3 (, ;M 3 5 4 4 3 3

4 5 :> 3 S ; fi fi JM 6 • 3 3 4 4 4 3
groups further increased for the single-
rooted teeth (with a reduction of 1.5 log micr •)X

in the test group). Around the multi-

1 5 5 5 r> •iM 7 <•,
S • 5 4
rooted teeth, this difference became less 4 5 5 5 5 G :' 1

significant due to some relapse in the test 4 4 4 JM 5 4 4 4

' 1
Mli
group, though the differences over time i 3 3 5 6 'IM t' , 6 6 fi
in comparison to the baseline remained i 5 S fi ;M 5 3 3 5 p-^
5 IM 5 3 4
statistically significant (/)=0,04 after 2 4 -•>

months and/.'=0.03 after 4 months). C. reclus

SR
1 4 4 4 4 4 4 UM <•- ID 5 5 4 4
2 4 1\\ 4 4 :? i
Pathogenic bacteria 2 ; 4 4 i 4 1
2 1 4 4 A i 5 4 4 J 3 i -!
The number of CFU/ml for several Mil
pathogenic micro-organisms {A. aetiiw- 4 4 4 1 A S 5 (i\l •ft- .; 5 5 i 5 4 4
1 1 4 4 4 4 4 ;M j < 5 4 4
myci'temcomitans. P. gingivalis. P. inter- 4 4 4 4 4 4 'i JM (-• 5 4 4 4
media, F. nueleatuni. P. micro.s and C.
rectus) from both single- and multi-
The numbers indicate the log number of every micro-organism as found in ilie positive sites.
rooted teeth in test and control group The data are not linked to the specific patients. (Dala presentation adapted from Mombelli
are depicted in Table 2. The changes for el al, 1995), The darker the shadowing of the boxes, the higher the amount of bacteria.
patients in the control group were negli-
gible, whereas patients from the test
group showed (with the exception for
A. aetinomyeefemconiitans) significant CFU/ml), Afler therapy, there was the test and the control group (Fig, 3).
reductions (>2 log) in the CFU/ml up nearly no change in the conirol group, Only in the test group, could a small
to 4 months, for both single and multi- but in the test group, only 4 samples re- but not significant, reduction (/J=0, 1), be
rooted teeth. For some patients from mained positive (± 10-^ CFU/ml). detected for samples from the saliva at
the test group, several bacteria were the 2-month follow-up visit. However,
even no longer detectable by means of Colony forming imit.s in other niches when the amount of pigmented Porphy-
culturing. especially at the 2-months re- The total amount of CFU/ml in romonas and Prevotclla species in these
call, P. gingivalis. for example, was ini- samples from the tongue, the buccal niches were considered (Fig, 4). the con-
tially detected in 10 samples from the mucosa or the saliva remained nearly trol group showed only small improve-
test group (±10*' CFU;ml) and 6 constant (changes within 0,5 log) dur- ments, whereas significant reductions in
samples from the control group (ilO** ing the experimental period, both for the test group could be reported
 Full-mouth disinfection and oral niches 61

Nanibpr ofAoaiTobic Culoiiy Conning I'oils on Tongup, Mucosa and Saliva iiver TIIIIP nificantly higher reductions {p<0.05) of
I,OQE+O8 Mucosa Saliva all parameters at month 2. and of
bleeding on probing at month 4.

Probing depth and attachment level

l,00E+07
The reduction in probing depth for
deep (^7 mm) and medium (5 to 6 mm)
pockets in the upper right quadrant,
"S 1.00E*06 over time, is depicted per treatment mo-
dality and per tooth type in Fig, 5A
(single-rooted teeth) and 5B (multi-
rooted teeth). Both treatment modali-
ties showed significant reductions in
probing depth after 2 and 4 months.
When deep (^7 mm) pockets were con-
sidered, the full-tiiouth disinfection
group revealed,, compared to the stan-
dard therapy, an additional probing
Fig. 3. The effect over time of partial (CONTROL) versus full-mouth disinfection (TEST) on depth reduction of 1,4 mm for mulli-
the number of colony forming units per ml (CFU/ml in log scores) after anaerobic incubation rooted teeth (/J<0,()05) and 2,3 mm for
of samples from the tongue, the buccul mucosa und the saliva. the single-rooted teeth (/)<0,005) after
4 months. When medium pockets (5 to
6 mm) were examined, an additional re-
N u m b e r of Colony Forming I,'nits ti( BLick-[jigmt'ntyd I'orphyriimvnaa anri frevotdla duction of 0.7 mm for multi-rooted
on Tongue, Mucosa and in Saliva teeth (y><0,05) and 0,9 mm for single-
1,OOE+08 Saliva rooted teeth (/j<0.05) in iest group
could be noticed.
1.00E+07
The changes in attachment level are
depicted in Fig. 6A for the single-root-
ed and Fig. 6B for the multi-rooted
teeth with a distinction between deep
and medium pockets. Again, the test
group showed significantly better re-
sults than the control group over 2 as
well as over 4 months, especially if deep
pockets were considered (2 months.
/j<0.005 for single-rooted, /7<0.O05 for
multi-rooted: 4 months, /3<O.O5 for
l.OOE+02 single-rooted,/'<0,01 for multi-rooted).
OM 2M 4M •IM 4M
Deep pockets around single-rooted
Time in m o n t h s
leeth showed a gain in attachment of
Fig. 4. The effect over time of partial (CONTROLl- versus full-mouth disinfection (TEST) 1.3 and 1.7 mm at months 2 and 4 in
on the CFU/ml of black/brown/green-pigmented Porphyronuinas and Prevotella species (log the test group, and only 0.3 to 0.4 mm
scores) after iinaerobic incubation of samples from the tongue, ihe buccal mucosa and the
in the control group. When multi-root-
saliva, (Significant differences between control Lind lest group are indicated as: V
ed teeth were considered, a gain of 1.6
to 1.3 mm over 2 and 4 months was
found in the test group, whereas the
(;j<0.05) lor all niches at month 2. atid ferences could be detected between the control group even showed an initial
for the mucosa up to month 4. When patient groups (gingivitis index, /)^0.8; loss of attachment of 0.3 mm at month
the treatments were compared with plaque index, p-0.9: bleeding on prob- 2 and a negligible gain at rnonth 4.
each other, the full-mouth disinfection ing, /'=U.l), Both therapies resulted in Comparable, but smaller changes were
resulted in slatistically significant a significant decrease in all parameters recorded around initially medium
(/j<0,05) higher reduction in pigmented at the 2-month follow-up visit. Al- pockets. Also here, the test group re-
anaerobic Porphyromonas and Prevotel- though the plaque index slightly in- acted more favourably (2 months.
la species on the tongue at month 2 and creased between month 2 and month 4, /xO.005 for single-rooled, /'<0,005 for
in the saliva at months 2 and 4. the gingivitis index and the bleeding multi-rooted; 4 months, /)<0,05 for
frequency remained nearly constant single-rooted,/»<0.U5 for multi-rooted).
with time for both groups. All these
Clinical data changes were statistically significant
Periodontal indices over time, except for the bleeding on
probing on month 4 in the control Discussion
The changes in plaque and gingivitis in-
dices and in the bleeding on probing are group (/)-().07), When the therapies Several papers clearly indicated that
were compared with each other, the most periodontopathogens (with the ex-
depicted per treatment modality in
full-mouth disinfection resulted in sig- ception of spirochetes) can colonise, be-
Table 3. At baseline, no significant dif-
62 Bollen cl al.

Tubk- 5. Plaque (Quigley & Hcin. 1%2) and gingivits indices (Mahlemann & Son 1971). and sides the periodontal pockets, difierent
bleeding on probing in ihe upper right qii;idrani per trealmcnt modality ;it baseline and after
other tiiches in the oral cavily: the dor-
2 and 4 months (means iind SD) sum of the tongue, the buccal mucosa.
PI GI Bleedmg probing the tonsils and/or the saliva (Van der
rime mean SD mean SD me;i n SD
Veldeti et al. I9S6. Van Winkelhoff et al,
1986. Asikainen et al, 1991. Danser et
OM 2.20 (J,48 0.89 0.48 0,5(1 0,49 al. 1994. Petit et al, 1994), Moreover, re-
control 2M 1,05 0,60 0,14 0,08 0,4(1 0,49 search on partially edentulous patients,
4M 1,24 0,55 0.14 0.15> 0,47 0.50
rehabilititted with inipkml-supportcd
OM 2,20 0,?5 0.83 \).51 0,80 0.39 bridges, suggested the existence oi an
test 2M 0,19 0,13 0.04 0,02 0,19 0,39 intra-oral transmission (translocation
4M 0,87 0,32 0.05 0,04 0.20 0,40 from one niehe to another) of these
PI: in tesl and control group/» always <0.05 between 0 and 1 months and between 0 and 4 periodontopathogens (Apse et al. 1989.
months. When test group is compared to conirol group, only at month 2: /?<0,05, GI: in test Quirynen & Listgarten 1990. Quirynen
and control group p always <0.05 between 0 and 2 months and belwccn 0 and 4 months. et al, 1996. Papaioannou et al, 1996a),
When lest group is compared to control group, only at month 2: /'<0,05, Bleeding on probing: The following vehicles are considered
in test group ;> always <(l,05 between 0 iind 2 months and between 0 and 4 months. In conirol [OT that transmission: the salivary flow
group, p only between 0 and 2 months <0,05, When test group is compared to control group, (Van Winkelhoff et al. i988b).' peri-
at month 2 and month 4:/?<0,05, odontal probes (Barnett et al, 1982.
Christersson et al, 1985. Papaioannou
et al, 1996b). caries explorers (Cau-
Evolution of the Pruhing Depth for Single-Ibioted Teeth over Time field & Gibbons 1979. Loesche et al.
1979) or even oral hygiene aids (Miillcr
et al. 19X9. Preus et al, 1993). The rela-
tive importance of the above-mentioned
intra-oral niehes in the development
and maintenance of periodontal infec-
tions is still very questionable. However,
a study by Danser et al, (1994) showed
that an extraction of all the retiiaining
leeth in an oral cavity resulted in a .sig-
nificant reduction in the proportion of
pathogenic bacteria in all the remaining
niehes. This suggests a predominant
role for periodontal pockets in the con-
tamination of the oral cavity.

2M
Because the results of a periodontal
Time in month,';
therapy depend on Ihe degree of elimi-
nation of the so-called exogetious
Evolution of the Probing Depth for Multi-Root eil Ttnth nver Time
pathogens and/or the reduction in the
proportion of endogenous species
(Slots & Rams 1990. Pavicie et al.
1994), a more full-mouth oriented ap-
proach in the treatment of periodontal
infections seems to be reasonable. Such
a more global approach was already in-
troduced by Axelsson et al. (1987).
They reported significant improvements
(less recurrence of caries, elimination of
Siveplococcus mulans) in their treat-
ment of children with poly-caries after
a full-mouth disinfection using ehlor-
hexidine gels and mouthrinses.
The full-mouth disinfection for pa-
tients with periodontal infections was
2M first suggested in a pilot study by Qui-
Time in months rynen et al. {1995), An optimal disinfec-
tion of the oral cavity was attempted in
Fig. 5. (A) The elTcct over lime of partial (CONTROL) versus full-mouth disinfection (TEST)
the iirst place by scaling and rootpla-
on the mean probing depth (including standard deviation) for deep (>7 mm) and medium (5
to 6 mm) initial pockets around single-rooted teeth, (B) Mean probing depth (including stan- ning all teeth in less than 24 h, in order
dard deviation) for deep (>7 mm) and medium (5 to 6 mm) around multi-rooted teeth at to reduce the number of subgingival
baseline, and monihs 2 and 4 (significant differences between control and tesl group are indi- pathogenic bacteria (Walsh el al. 1986.
cated as: '/i^O.Og; ** Loos et a!. 1988). thereby reducing the
 Fiill-tnotith disinfection and oral niches 63

Change in Clinical Attachment Level around Single-Rooted Teeth over Time ieally. the probing depth for tnedium and
deep pockets even decreased more than
in the pilot study. Also mierobiologi-
2,5 - cally. the results of this recent study ex-
ceed those of the former one. A possible
explanation ean be found in the ex-
tended one-stage full-mouth approach.
All test patients in this study maintained
a good oral hygiene and contintied their
rinsing and spraying with chlorhexidine
0.2% for up to 2 months post therapy.
This might have coincided with the time
0,5 - needed to establish a neu' and less
favourable environment for pathogenic
bacteria. In the pilol study, in contrast, a
0- 2M 0-4M decrease in the oral hygiene level could
-0.5
be detected al the 4-month examination,
Time in munths
which led to a deterioration of the micro-
biological situation around the single- as
Change in Clinical Attachment li'vel around Multi-Rooted Teeth over Time well as around the multi-rooted teeth.
2,5 -,
The present paper also shows data at
the clinical attachment level. The test
Cnntml'tlepp group showed significantly more gain in
attachtnent Ihan the eontrol group (a
mean gain of 1.5 mm versus U.3 mm for
1,5
deep pockets, and 0.9 mm versus 0,0 mm
for medium pockets).
Since 4 out oi the 8 control and test
patients had an aggressive form of perio-
dontitis and 2 out of the 8 test and eon-
0,5 -
trol patients were diagnosed as having
"generalised early onset periodontitis"
and sinee 3 test and 3 eontroi patients
0-4M were heavy smokers, a less favourable
outcome of the treatment would have
-0,5 been not surprising in comparison with
B Time in months
"classical studies". Furthermore, no
Fig. 6. (A)Theefiect over time of partial (CONTROL)- versus ruU-moulh disinfection (TEST) subgingival instrumentation was per-
on the glinigal altachtnent level (including slandard deviaiion) for deep (>7 mm) and mcdiutn formed after the initial treatment,
(5 to 6 mm) initial pockets around single rooted teeth, (B) Mean clinical attachment level whereas most other studies reporting on
(including standard deviation) for deep (>7 mm) and medium (5 to 6 tntiil around mulii- the same topic, used repeated subgingi-
rooted leeth at baseline, and months 2 and 4. (Significant differences between control and test
group are indicated as: */j^0,()5; **/)^'J,005),
val instrumentation. Recently Soskolne
et al. (1997) reported almost sitnilar re-
sults in probing depth and clinical
attachment level after 6 months.
bacterial load in the other niches. In or- infected by means of a topically applied When the changes for single-rooted
der to reduce the number of remaining 0.2'/^, spray of chlorhexidine. a mode of teeth (1,7 mm at month 4) are eom-
bacleria in the pockets, they were irri- application with nearly the same anti- pared to other studies, the additional
gated 3X within 10 min with a 1% mierobial effect as a 0,2'/- ehiorhexidiiie beneficial effect of this new concept ts
chlorhexidine gel. a protocol with rinse (Kalaga et al, 1989), In order to again illustrated, Badersten et al. (1982)
which Oosterwaal et al. (1991) were delay the subgingival re-eolonisation, indeed reported a mean gain in attach-
able to reduce the stibgingival flora up the patients were asked to rinse 2X ment of 1.2 mm after 24 months, while
to 99%. The tongue of the patients (an daily with chlorhexidine for 2 months Lindhe et al. (1982) showed a mean
important reservoir of black-pigmented (Magnusson et al, 1984. Bollen & Qui- gain in attachment of 0.25 mm after 4
bacteria; Van Winkelhoff ct al. 1988a) rynen 1996), monlhs. For multi-rooted teeth, the lat-
was brushed with chlorhexidine 1% gel The 8-months data of the previous pi- ter study reported an attachment gain
to reduce the number of bacteria on this lot studies showed that the full-mouth of 0,1 mm after 4 months, a change
surface (Gilmore & Bhaskar 1972. disinfection resulted in an additional which is inferior to our observations.
Wolffe & Van der Velden 1987). The beneficial clinical and microbiologieal The microbiological effect of this
number of bacteria in the saliva was re- effeet over a elassieal treatment even over treatment on the other inlra-oral niches
duced by rinsing (2X 1 min) with a 0,2'Mi a longer period (Bollen et al. 1996. Van- (tongue, buccal surface and saliva) is am-
ehlorhexidine solution (Rindom-Sehiott dekerckhove et al. 1996). These findings biguous. If only the total amount of an-
et al. 1976). Finally, the tonsils were dis- are confirmed by the present study. Clin- aerobie eultured baeteria is eonsidered.
64 Bollen e! al.

almost no changes could be detected. pletion of the scaling and rootplaning kosa und Tonsillen), Theorilisch konnte ein
However, if the proportion in pathogenic within 24 h, had a significant impact. solches Kon/ept 7U einer Reinfektion vorah
speeies is considered (number of pig- The fuil-mouth approach in perio- behandeher Taschen f"iihren. Die hier vorlie-
mented Porphyrottionas and Prevotella gende Studte beabsichtigt. den tiniluB einer
dontology has also been tested by Lang
vollstiindigon Desinfektion der Mundhohle
speeies) some amelioration eould be de- and co-workers (Lehmann et al. 1995. auf die Mikroflora der oben genannten Ni-
tected in the test group. It is difficult to Tonetti et al. 1995). They reported a schen zu untersuchen. AuBerdem wurde der
conclude whether this improvement is beneficial effect when tetracycline fibres klinische Nut2cn cincs solchen Konzepts un-
due to the fuil-mouth approach (with an were applied to all pockets above 3 mtn tersuchl, 16 Patienlen mit profunder Paro-
improved subgingival disinfection), or (fuil-mouth approach) instead of only dontitis wurden zufitllig in eine Versuchs- und
due to intensive mouth rinsing with the 2 deepest sites. Kontrollgriippe eingeteiU. Bei den Patienten
ehlorhexidine (Roberts & Addy 1981, der Kontrollgruppe wurde der Zahnstein in 2
However, several questions remain wochentlichen Abstiinden quadrantcnwcise
Minhas & Greentnan 1989. Southard et unanswered. First oi all. it is still de-
al, 1989. Jenkins etal. 1990), Howcvcr.it entfernt, Wurzclglaltung vorgenommen und
batable whether the supplementary ef- man crlcille Mundhygieiieatiweisungen, Bei
is clear that the changes in the other fect oi Ihe full-moulh disinfection is den Patienten der Ver such sgriippe wurde eine
niches are better than those obtained by caused by the full-mouth approach as vollsliindige Desinteklion der Mundhohle
Danser et al. (1996) with a standard peri- such, or by the extra antimicrobial ef- vorgenommen. die bei 2 Einbesiellungen in-
odontal therapy. They found that a peri- fect of the chlorhexidine (Hennessey nerhalb von 24 Stunden aus: Scaling, Wurzel-
odontal treatment, consisting of an ex- 1977), the latter being suggestive of a glattung in alien Tasclieii iind daran ansehlie-
tensive scaling and rootplaning in com- Bender Reinigung der Ziinge durch 1 Minute
more extensive reduction in mierobial
bination with an oral hygiene instruction langes Biirsten mil 1 "nigemChlorhcxidingel.2
load by the treatment so that the re- Minuien langer Mundspiilung mit einer
and periodonta] surgery, had a signifi- growth was retarded. A final answer to 0.2"'nigen ChlorhexidiuUisung und subgingl-
cant effeet on the subgingival prevalenee this question can only be obtained by valer Irrigation allcrTasehen {3x withrcnd 10
of A. actinomycetemcomitans., P. gitigi- new studies in which the same fuil- Minuien) mit ]"'iiigem Chlorhexidingel be-
vali.s and P. intermedia, but no influence mouth protocol as deseribed above is stand, Voti der Mundhygiene abgesehen. spul-
was seen on the prevalence of these tar- followed, but without any supplemen- le die Verstiehsgruppe 2 tnal liiglich mit
get speeies eolonising the oral mucous tary chlorhexidine applieation. An- 0.2'/^jigem Chlorhexidin und spruhte die Ton-
membranes. other open question is what the el'fect sillen tnii einem (),2""igen Chlorhexidinspray
2 Monate hmg, Plaqueproben (Taschen. Zun-
of this approach would be in the most gc, Mukosa und Speichel) wurden eingangs
Even though all patients started lo
difficult periodontal infection eases, und nach 2 imd 4 Monaicn cntttommen und
brush their tongue at baseline, improve-
the early onset patients. More researeh Veranderungen der sondierlen Tasehentielcn.
ments in this niche were not detected.
is also needed in this aspect. der Attaehmentchene und des Blutens nach
This observation is in agreement with a
Sondierung angegcben. Die vollstandige Des-
previous paper of Gilmore & Bhaskar In summary, a one-stage full-mouth infeklion der Mundhohle halte vor allem in
(1972) who also failed to demonstrate disinfection results in a significant clin- den subgingivalen Tascben. aber aucb in den
differences in bacterial load on the ical and mierobiologieal improvement anderen Nischen eine sUitistisch abgesicherU*
tongue afler the introduction of tongue over the classical periodontal therapy. Reduktion/Elimination der Parodontopatho-
brushing. on a medium-term basis (4 months) in gene zur Folge, Bei den Versucbspalienten
patients with chronic periodontitis. fanden dicse mikrohiologischen Verbesserun-
One ean only speeulate on the rela-
gen in einer statistisch signifikani hiiheren Re-
tive importance of each oi the above- duktion derSondierungstiefcn ttnd in Allach-
mentioned steps in the tesl protocol, mentgewinn ihren klinischen Ausdruck, Die
since all oi them are known lo reduce Acknowledgements Befunde vermittein den Eiiidruck. daB die
the pathogenicity of the subgingival This study was supported by the Ca- Desinfeklion der inlraoralen Nischen eine sig-
flora. However, some have only a short- nifikante klinische und mikrobiologische Ver-
tholic Liniversity of Leuven (grant: OT''
to tnedium-term effect. Several reports besserung erreicht. die bis 4 Monale anhall.
95/28). by the Belgium National Fund
have indicated ihat, after a single ses- for Scientific Research (grant:
sion of sealing and rootplaning. the 3,0179.96) and by the Ministry of Edu-
subgingival flora rapidly returns to cation of the Flemish Comtiiunity in
baseline values, "quantitatively as well Resume
co-operation with ihe Ministry of Fo-
as qualitatively" (Mousques et al, 1980, reign Affairs of Italy, The authors wish Effcl dc Id desinfectioii hticcale roralc sur dif-
Van Winkelhoff et al, 1988b. Sbordone to thank Dr, Martinc Pauwels for her Icrentes niches intra-huccales. Observation.s
el al, 1990), Moreover, several studies cliniqtiL's et microhiohf^itjiw.s
teehnica! assistance. Le trailement des infections parodontales
have illustrated that an irrigation of the
pockets with a chlorhexidine solution consiste souvent en des siirfavages suivis (par
quadrant a I on 2 semaines d'intervallel sans
failed to improve the effect of mecha-
une desinfection propre des niebes inlra-buc-
nical debridement at either a miero- Zusammenfassung cales restitntes (pochcs parodontales non-
scopic or a clinical level over a 6-month traitees. langue, salive. nuiqueuse et amygda-
Der Finflup einer voJIstandigen Desinfektion
observation period (Braalz et al. 1985. iler Miiiulliiihlf ciiifvi'r.'\chictlfi)i' intriioriile Ni- les), line tclle approehe pourrail theoritique-
MaeAlpine et al. 1985, Lander et al, .scheti. Klini.schf wnl niikrohiolai'ischc Beob- meni etre suivie d'utie reinfection des poches
1986, Wennstrom et al. 1987). More- Iraitces preeedemment. L'etiide presente exa-
over, the effect of oral rinsing on the Oft besteht die Bchandlung parodonialer In- mine I'clTet d'une desinfection buceale lotaie
subgingival flora, however, is negligible fektionen aus aufeinander folgenden Wurzel- sur la microflore des niches ci-dessus men-
(Flotra et al. 1972). These observations gliittungcn (tjundrantenweise. in .Ahstanden lionttees, De plus le betiefice clinique d'une
von 1 his 2 Wochen) ohne eigentliche Desin- leiie approehe a ete analyse, 16 patients avee
therefore suggest that the full-mouth parodoniite severe ont ete repartis au hasard
approach, and especially the com- fektion der resllichen intraoralcn Nischcn
dans le groupe lest on eontrole. Les patients
(unbehandelteTaschen. Zunge. Speichel, Mu-
 Full-moulh disinfection and oral niches 65

du groupe eontrole ont ete dctartres et ont therapy: A review of the literature. Journal (II), Salivary bacterial counts. Journal oJ
subi un surfai;iige par quadrant a 2 semaines of Feriodoiuahgy 67, I i4.''-| 158- Clinical Fvriodontology 17. 698-701,
d'intervalle. et ont rei^u des instriiciions en Boilen. C M, L,. Vandekerckhove. B. N, A,, Kalaga. A,. Addy. M, & Hunter. B, (1989)
hygiene buccalc. Par contre les patients du Papaioannou. W, Van Eldere, J, & Quiryn- Comparison of chlorhexidine delivery by
groupe test ont regu une desinfection buceale en, M. (1996), Full- versus partial-mouth mouthwash and spray on plaque accutnu-
totale eonsistant en: detartrage et surfaijagc disinfection in the treatment of periodontal lation. Journal of Feriodonlologv 60. 127-
radiculaire de loutes les poches en 2 visites infections, A pilot study. Long-term micro- 130.
sur 24 heures en association avec le brossagc biological observations. Journal of Clinical Lander. P E.. Newcomb. G, M.. Seymour, G.
de !a langue avec un gel de cblorhexidine l'/n Feriodontohgy 23, ^60-970. J, & Powell, R. N, (1986) The anti-
durant I min. un rin(;age huccal avec une so- Braatz. L,, Garrett. S,. ClalTey, N, & Egei- microbiai and clinical effects of a single
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et une irrigation sous-gingivale de toutes les deep pockets to supplement non-surgical advanced periodontal lesions. Journal of
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chlorhexidine l"/.i, A parl I'hygiene buceale le Journal of Clinttcd Feriodontologv 12, 630 Lehitiann, B,. Mombelli. A,. Tonetti, M, S. &
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chlorhexidine 0,2''ii et recevait un spray sur
Caufield, P W, & Gibbons. R, J. (1979) Sup- cal delivery of tetracycline in relation to
les amygdales avec dc ia chlorhexidine 0.2%
pression of Streptococcus mutans in tlie overall conditions of the other teeth.
pendant 2 mois, Des echantillons de plaque
mouihs of humans by a dental prophyiaxis Journal of Dental Researeh 74 (spec. Iss,),
(poches, langue, muqueuse et salive) ont ete
and topically applied iodine. Journal of abstr. 64 L
pris tors de I'examen initial et apres 2 ei 4
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couvertes suggereni que la desinfection des dontilis. Journal of Feriodontologv 63, R, (1979) Intra-oral transmission of
niches intra-buccales s'accompagne d'ame- 418 425, Streptococcus mutans by a dental ex-
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Calholie University of Leuven
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Belgium
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