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The clinical, microbiological and systemic characteristics of periodontitis and their changesafter periodontal therapy

Bizzarro, S.

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Citation for published version (APA):Bizzarro, S. (2015). The clinical, microbiological and systemic characteristics of periodontitis and their changesafter periodontal therapy.

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risk for coronary heart disease. Arteriosclerosis Thrombosis and Vascular Biology 25,

833-838.

Pussinen, P. J., Vilkuna-Rautiainen, T., Alfthan, G., Mattila, K. & Asikainen, S. (2002)

Multiserotype enzyme-linked immunosorbent assay as a diagnostic aid for

periodontitis in large-scale studies. Journal of Clinical Microbiology 40, 512-518.

Roth, G. A., Moser, B., Huang, S. J., Brandt, J. S., Huang, Y., Papapanou, P. N., Schmidt, A.

M. & Lalla, E. (2006) Infection with a periodontal pathogen induces procoagulant

effects in human aortic endothelial cells. Journal of Thrombosis and Haemostasis 4,

2256-2261.

Saarela, M., Asikainen, S., Alaluusua, S., Pyhala, L., Lai, C. H. & Jousimies-Somer, H.

(1992) Frequency and stability of mono- or poly-infection by Actinobacillus

actinomycetemcomitans serotypes a, b, c, d or e. Oral Microbiology and Immunology

7, 277-279.

Schenkein, H. A., Barbour, S. E., Berry, C. R., Kipps, B. & Tew, J. G. (2000) Invasion of

human vascular endothelial cells by Actinobacillus actinomycetemcomitans via the

receptor for platelet-activating factor. Infection and Immunity 68, 5416-5419.

Taylor, B. A., Tofler, G. H., Carey, H. M., Morel-Kopp, M. C., Philcox, S., Carter, T. R.,

Elliott, M. J., Kull, A. D., Ward, C. & Schenck, K. (2006) Full-mouth tooth extraction

lowers systemic inflammatory and thrombotic markers of cardiovascular risk. Journal

of Dental Research 85, 74-78.

Tonetti, M. S. & Claffey, N. (2005) Advances in the progression of periodontitis and proposal

of definitions of a periodontitis case and disease progression for use in risk factor

research. Group C consensus report of the 5th European Workshop in Periodontology.

Journal of Clinical Periodontology 32 Suppl 6, 210-213.

69

CHAPTER 4

Local disinfection with sodium hypochlorite as adjunct to basic

periodontal therapy.

A randomized controlled trial

Sergio Bizzarro, Ubele Van der Velden and Bruno G. Loos

Department of Periodontology, Academic Centre for Dentistry of Amsterdam (ACTA), University of

Amsterdam and VU University of Amsterdam, The Netherlands

Submitted

Chapter 4

70

70

Abstract

Background: The aim of this study was to investigate the adjunctive clinical and

microbiological effects of local disinfection with 0.5% sodium hypochlorite (NaOCl) with or

without systemic antibiotics (amoxicillin and metronidazole) (AB) in basic periodontal

therapy (scaling and root planing and oral hygiene measures [SRP]).

Material and methods: Patients with chronic periodontitis were randomly allocated to 4

groups: SRP plus saline (SRP+S), SRP plus local disinfection with NaOCl (SRP+DIS),

SRP+DIS+AB and SRP+S+AB. Clinical measurements and subgingival microbiological

samples were analyzed at baseline, 3-month, 6-month and 12-month follow-up.

Results: 110 patients were included. All groups showed significant improvements for all

clinical and microbiological parameters throughout the total follow-up period. SRP+DIS

showed no additional improvement in comparison to SRP+S, while SRP+DIS+AB showed

fewer pockets ≥7 mm vs SRP+S only up to 6 months. In factorial analyses, additional clinical

improvements compared to SRP+S were found for AB, but not for DIS throughout the total

follow-up. In all groups, a comparable decrease of targeted bacteria was found with no

additional effects of DIS or AB. AB caused adverse events in 22% of the patients.

Conclusion: Local disinfection with NaOCl, with or without antibiotics, showed no sustained

additional effect in basic periodontal therapy for clinical and microbiological parameters after

1 year follow-up.

71

Introduction

Periodontitis is a destructive inflammatory disease of the supporting tissues of the teeth

(Pihlstrom et al. 2005). Periodontal lesions harbor complex subgingival microbiological

biofilms including the traditional periodontal pathogens Aggregatibacter

actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythia (Tf)

(Griffen et al. 2012, van Winkelhoff et al. 2002). Periodontal pockets are lined by inflamed

epithelium; it has been calculated that the periodontal inflamed surface area (PISA) of a

patient with generalized severe periodontitis can amount to 39 cm2 (Nesse et al. 2008).

Treatment of periodontitis is difficult, time consuming, costly and results are not always

predictable. Basic periodontal therapy (BPT) aims at removing supra- and sub-gingival

biofilms by means of scaling and root planing (SRP) in conjunction with meticulous

supragingival plaque control by the patient. A further reduction of microbial biofilm and

elimination of putative periodontal pathogens have been attempted by the administration of

systemic or local antimicrobials.

According to recent meta-analyses, supporting therapy with systemic antibiotics, as

metronidazole alone or in combination with amoxicillin, may improve the results of the BPT

(Keestra et al. 2015, Sgolastra et al. 2012, Sgolastra et al. 2014). However, most studies have

results only up to 6-month follow-up and the adjunct of systemic antibiotics may not be

sufficient to achieve complete resolution of periodontal inflammation and deep residual

pockets (Silva-Senem et al. 2013). Moreover, the use of systemic antibiotics can lead to

increased morbidity due to side effects (Feres et al. 2012, Machtei & Younis 2008) and there

is an increasing global attention to the problematic increment of antibiotic resistance (Hansen

et al. 2015). Thus there is the need for alternative modalities of adjunctive therapies in

patients with chronic periodontitis to obtain maximal clinical results.

Local antiseptic agents are administered professionally and therefore have the advantage to

be independent of patient’s compliance and they have little or no side effects (Quirynen et al.

2002). Subgingival antimicrobial solutions used as adjunct to BPT include chlorhexidine,

povidone-iodine, peroxides and sodium hypochlorite (NaOCl) (Slots 2002). Among these,

NaOCl has many of the ideal characteristics of an antimicrobial. It is broad spectrum and

rapidly bactericidal, it is not toxic at the concentrations that are normally used in dentistry,

and is easy and at low costs available (Slots 2002). In periodontology, NaOCl has been

investigated as a local disinfectant in different ways. It has been used as a 5.3% solution for

Chapter 4

71

70

Abstract

Background: The aim of this study was to investigate the adjunctive clinical and

microbiological effects of local disinfection with 0.5% sodium hypochlorite (NaOCl) with or

without systemic antibiotics (amoxicillin and metronidazole) (AB) in basic periodontal

therapy (scaling and root planing and oral hygiene measures [SRP]).

Material and methods: Patients with chronic periodontitis were randomly allocated to 4

groups: SRP plus saline (SRP+S), SRP plus local disinfection with NaOCl (SRP+DIS),

SRP+DIS+AB and SRP+S+AB. Clinical measurements and subgingival microbiological

samples were analyzed at baseline, 3-month, 6-month and 12-month follow-up.

Results: 110 patients were included. All groups showed significant improvements for all

clinical and microbiological parameters throughout the total follow-up period. SRP+DIS

showed no additional improvement in comparison to SRP+S, while SRP+DIS+AB showed

fewer pockets ≥7 mm vs SRP+S only up to 6 months. In factorial analyses, additional clinical

improvements compared to SRP+S were found for AB, but not for DIS throughout the total

follow-up. In all groups, a comparable decrease of targeted bacteria was found with no

additional effects of DIS or AB. AB caused adverse events in 22% of the patients.

Conclusion: Local disinfection with NaOCl, with or without antibiotics, showed no sustained

additional effect in basic periodontal therapy for clinical and microbiological parameters after

1 year follow-up.

71

Introduction

Periodontitis is a destructive inflammatory disease of the supporting tissues of the teeth

(Pihlstrom et al. 2005). Periodontal lesions harbor complex subgingival microbiological

biofilms including the traditional periodontal pathogens Aggregatibacter

actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythia (Tf)

(Griffen et al. 2012, van Winkelhoff et al. 2002). Periodontal pockets are lined by inflamed

epithelium; it has been calculated that the periodontal inflamed surface area (PISA) of a

patient with generalized severe periodontitis can amount to 39 cm2 (Nesse et al. 2008).

Treatment of periodontitis is difficult, time consuming, costly and results are not always

predictable. Basic periodontal therapy (BPT) aims at removing supra- and sub-gingival

biofilms by means of scaling and root planing (SRP) in conjunction with meticulous

supragingival plaque control by the patient. A further reduction of microbial biofilm and

elimination of putative periodontal pathogens have been attempted by the administration of

systemic or local antimicrobials.

According to recent meta-analyses, supporting therapy with systemic antibiotics, as

metronidazole alone or in combination with amoxicillin, may improve the results of the BPT

(Keestra et al. 2015, Sgolastra et al. 2012, Sgolastra et al. 2014). However, most studies have

results only up to 6-month follow-up and the adjunct of systemic antibiotics may not be

sufficient to achieve complete resolution of periodontal inflammation and deep residual

pockets (Silva-Senem et al. 2013). Moreover, the use of systemic antibiotics can lead to

increased morbidity due to side effects (Feres et al. 2012, Machtei & Younis 2008) and there

is an increasing global attention to the problematic increment of antibiotic resistance (Hansen

et al. 2015). Thus there is the need for alternative modalities of adjunctive therapies in

patients with chronic periodontitis to obtain maximal clinical results.

Local antiseptic agents are administered professionally and therefore have the advantage to

be independent of patient’s compliance and they have little or no side effects (Quirynen et al.

2002). Subgingival antimicrobial solutions used as adjunct to BPT include chlorhexidine,

povidone-iodine, peroxides and sodium hypochlorite (NaOCl) (Slots 2002). Among these,

NaOCl has many of the ideal characteristics of an antimicrobial. It is broad spectrum and

rapidly bactericidal, it is not toxic at the concentrations that are normally used in dentistry,

and is easy and at low costs available (Slots 2002). In periodontology, NaOCl has been

investigated as a local disinfectant in different ways. It has been used as a 5.3% solution for

Chapter 4

72

72

subgingival irrigation and appeared to be neutralized after 2 minutes with no histological

adverse effect on the tissues (Kalkwarf et al. 1982). 0.1% NaOCl was also used during

periodontal surgery to disinfect the wound area with exposed alveolar bone (Perova et al.

1990); again, no adverse effects at histological level could be observed. It has been shown in

vitro that 0.5% NaOCl was the lowest concentration able to eradicate bacteria within 15s

(Vianna et al. 2004). This latter concentration for subgingival disinfection has been

demonstrated to be effective in reducing gingivitis (Lobene et al. 1972). Recently, daily oral

rinse with NaOCl 0.25% showed the capacity to reduce gingival bleeding tendency in

periodontitis patients up to three months (Galvan et al. 2014, Gonzalez et al. 2015).

Although the use of NaOCl seems to be advantageous as a subgingival disinfection of

pockets in the treatment of periodontitis, there is lack of evidence about its effectiveness as

adjunct to BPT. The question is whether NaOCl may enhance the clinical and microbiological

results on itself or is specifically highly effective when it is used in combination with

systemic antibiotics during active BPT to obtain long term beneficial improvements (at least

up to one year).

Therefore, the aim of this study was to investigate the clinical and microbiological effects

of professionally administered 0.5% NaOCl to disinfect the periodontal pockets with or

without systemic antibiotics as adjunct to basic treatment of chronic periodontitis, over a

follow-up period of one year.

73

Material and methods

Consecutive chronic periodontitis patients, who were referred to the Department of

Periodontology of the Academic Centre for Dentistry of Amsterdam (ACTA) for treatment of

periodontitis, were screened for this partial double blind (patients, examiner and therapists),

parallel designed, randomized controlled clinical trial. A periodontal case was defined if

he/she had at >2 teeth interproximal attachment loss of >3 mm (Tonetti & Claffey 2005). For

this study a patient was included if he/she presented >30% alveolar bone loss at >2 teeth per

quadrant and presence of >2 teeth per quadrant with periodontal pockets >5 mm with clinical

evidence of attachment loss and at least 50% of all sites in the mouth with bleeding on

probing (BOP). Inclusion/exclusion criteria are reported in Table S1.

A patient was defined as a smoker if he/she was currently smoking, or quitted less than 6

months before baseline, and as a non-smoker if he/she had never smoked or quitted smoking

longer than 6 months before intake. Patients, who agreed to participate in the study, signed a

written informed consent. The Medical Ethical Committee of the Academic Medical Centre of

Amsterdam, The Netherlands, approved the protocol (MEC 07/264). The study was registered

at Current Controlled Trials with the number ISRCTN36043780 and the manuscript followed

CONSORT guidelines.

Subgingival treatment modalities, randomization, allocation and blinding

Treatment included SRP plus saline (S) or subgingival disinfection (DIS) and both modalities

with or without systemic antibiotics (AB). DIS consisted of subgingival irrigation with 0.5%

NaOCl and for AB the combined use of systemic amoxicillin and metronidazole (375 mg +

250 mg respectively, 3 times a day for 7 days) was prescribed (Winkel et al. 2001). Thus, at

baseline examination, every patient was allocated to one of four treatment groups:

1) SRP+S,

2) SRP+DIS,

3) SRP+DIS+AB,

4) SRP+S+AB.

Randomization was performed by a computer based generated sequence with stratification

for smoking habit. Two sets of 100 numbers were obtained for smokers and non-smokers. A

range from 1 to 4 was used, which corresponded to the 4 treatment modalities. Two different

sets of sealed encoded envelopes were subsequently prepared and put in two separated boxes,

Chapter 4

73

72

subgingival irrigation and appeared to be neutralized after 2 minutes with no histological

adverse effect on the tissues (Kalkwarf et al. 1982). 0.1% NaOCl was also used during

periodontal surgery to disinfect the wound area with exposed alveolar bone (Perova et al.

1990); again, no adverse effects at histological level could be observed. It has been shown in

vitro that 0.5% NaOCl was the lowest concentration able to eradicate bacteria within 15s

(Vianna et al. 2004). This latter concentration for subgingival disinfection has been

demonstrated to be effective in reducing gingivitis (Lobene et al. 1972). Recently, daily oral

rinse with NaOCl 0.25% showed the capacity to reduce gingival bleeding tendency in

periodontitis patients up to three months (Galvan et al. 2014, Gonzalez et al. 2015).

Although the use of NaOCl seems to be advantageous as a subgingival disinfection of

pockets in the treatment of periodontitis, there is lack of evidence about its effectiveness as

adjunct to BPT. The question is whether NaOCl may enhance the clinical and microbiological

results on itself or is specifically highly effective when it is used in combination with

systemic antibiotics during active BPT to obtain long term beneficial improvements (at least

up to one year).

Therefore, the aim of this study was to investigate the clinical and microbiological effects

of professionally administered 0.5% NaOCl to disinfect the periodontal pockets with or

without systemic antibiotics as adjunct to basic treatment of chronic periodontitis, over a

follow-up period of one year.

73

Material and methods

Consecutive chronic periodontitis patients, who were referred to the Department of

Periodontology of the Academic Centre for Dentistry of Amsterdam (ACTA) for treatment of

periodontitis, were screened for this partial double blind (patients, examiner and therapists),

parallel designed, randomized controlled clinical trial. A periodontal case was defined if

he/she had at >2 teeth interproximal attachment loss of >3 mm (Tonetti & Claffey 2005). For

this study a patient was included if he/she presented >30% alveolar bone loss at >2 teeth per

quadrant and presence of >2 teeth per quadrant with periodontal pockets >5 mm with clinical

evidence of attachment loss and at least 50% of all sites in the mouth with bleeding on

probing (BOP). Inclusion/exclusion criteria are reported in Table S1.

A patient was defined as a smoker if he/she was currently smoking, or quitted less than 6

months before baseline, and as a non-smoker if he/she had never smoked or quitted smoking

longer than 6 months before intake. Patients, who agreed to participate in the study, signed a

written informed consent. The Medical Ethical Committee of the Academic Medical Centre of

Amsterdam, The Netherlands, approved the protocol (MEC 07/264). The study was registered

at Current Controlled Trials with the number ISRCTN36043780 and the manuscript followed

CONSORT guidelines.

Subgingival treatment modalities, randomization, allocation and blinding

Treatment included SRP plus saline (S) or subgingival disinfection (DIS) and both modalities

with or without systemic antibiotics (AB). DIS consisted of subgingival irrigation with 0.5%

NaOCl and for AB the combined use of systemic amoxicillin and metronidazole (375 mg +

250 mg respectively, 3 times a day for 7 days) was prescribed (Winkel et al. 2001). Thus, at

baseline examination, every patient was allocated to one of four treatment groups:

1) SRP+S,

2) SRP+DIS,

3) SRP+DIS+AB,

4) SRP+S+AB.

Randomization was performed by a computer based generated sequence with stratification

for smoking habit. Two sets of 100 numbers were obtained for smokers and non-smokers. A

range from 1 to 4 was used, which corresponded to the 4 treatment modalities. Two different

sets of sealed encoded envelopes were subsequently prepared and put in two separated boxes,

Chapter 4

74

74

labeled for smokers and non-smokers, and ordered according to the randomization sequence.

The allocation procedure was performed and recorded by a researcher who was neither

involved in the clinical recordings nor in the treatment. At the baseline visit, before the

clinical and microbiological examinations, the independent researcher picked the first

envelope from one of the boxes, according to the smoking habit of the patient and he then

unsealed the envelope. All envelopes contained a prescription for the use of a 0.12%

chlorhexidine rinse (2 x day x 28 days). Envelopes encoded for the groups SRP+DIS+AB and

SRP+S+AB contained also a prescription for the use of the antibiotics. During this session,

the independent researcher gave oral and written instructions about the use of the medications

and rinse. Patients in the SRP+S and SRP+DIS received no placebo tablets for the antibiotics.

Patients were blinded for the subgingival disinfection with NaOCl but not for the use of

antibiotics. Patients were carefully instructed by no means to inform the examiner and the

therapists about the content of the envelops. The examiner and the therapists involved in the

periodontal therapy were blinded for treatment allocation during the whole study period.

Clinical examination and microbiological sampling

After patient allocation, the following clinical measurements at 6 sites per tooth were

recorded for all teeth: plaque (presence/absence), bleeding on probing (BOP)

(presence/absence), PPD (mm) and CAL (mm). From these parameters the PISA (cm2) was

calculated (Nesse et al. 2008).

The deepest pocket in each quadrant at non-furcated sites was selected for microbiological

sampling (Mombelli et al. 1991). After isolating the site with cotton rolls, supragingival

plaque was removed with a Gracey curette and the site was dried with gentle airflow.

Subsequently 2 medium-sized paper points (Henry Schein, Almere, The Netherlands) were

inserted in the pocket for 10 s. All paper points were pooled, transferred to a reduced transport

medium (Syed & Loesche 1972) and processed within 3 hours for culturing.

All clinical measurements and microbiological sampling procedures were repeated at 3, 6

and 12 months after completion of periodontal treatment. All side effects and compliance with

the prescribed medications were recorded, by means of a face-to-face interview, at the 3-

month follow-up examination by the same independent researcher responsible for the

randomization.

75

Power calculation and intra-examiner reproducibility.

A sample of 24 patients per treatment group was calculated based on a difference of clinical

attachment level (CAL) of 1.0 mm between the groups with a standard deviation (SD) of 1.0

mm (α = 5%, β = 20%) (Del Peloso Ribeiro et al. 2008). In order to compensate for patient

dropout, the study sample was enlarged up to 110. All clinical measurements were carried out

by the same experienced periodontist (SB). Examiner reproducibility took place before the

start of the study. In 4 patients, duplicated measurements were recorded and an intraclass

correlation was obtained for probing pocket depth (PPD) of 0.94 and for CAL of 0.95.

Microbiological laboratory assessments

Culturing of subgingival bacterial samples was performed according to a protocol described

previously (Boutaga et al. 2005). The following bacteria were identified: Aa, Pg, Tf,

Prevotella intermedia (Pi), Parvimonas micra (Pm), Fusobacterium nucleatum (Fn) and

Campylobacter rectus (Cr) (van Winkelhoff et al. 2002).

Periodontal therapy

Patients were assigned to one of 3 experienced dental hygienists of the Department of

Periodontology at ACTA, before treatment allocation took place, according to scheduling

availability. The 3 therapists performed the treatment in two consecutive days, with a standard

time of 2.5 hours on each of these days. All dental hygienists followed a standardized

treatment protocol that was set up prior the start of the study. Both an ultrasonic device (Hu-

Friedy EMS piezon®, Hu-Friedy, Chicago, IL, USA) and hand instruments were used. BPT

consisted of full-mouth supra- and subgingival debridement, oral hygiene instructions with a

powered toothbrush (Sonicare®, Philips Oral Healthcare, Bothell, WA, USA) and interdental

aids on a personal need. Patients were asked to bring the bottle of chlorhexidine rinse and the

packages of the antibiotics to the first appointment of the treatment. Patients allocated in the

SRP+S+AB and SRP+DIS+AB group took the first dose of systemic antibiotics just before

the start of the treatment under supervision of the independent researcher. All patients were

instructed to start using chlorhexidine rinse at the evening of the first treatment day. Three

days after the last session of BPT, disinfection with NaOCl or saline solution was performed

by another experienced and specifically trained dental hygienist in a 1.5-hour session. Four

weeks after disinfection, patients were recalled for an extra session of supra- and subgingival

scaling, polishing and oral hygiene reinforcement. Patients were subsequently enrolled in a 3-

Chapter 4

75

74

labeled for smokers and non-smokers, and ordered according to the randomization sequence.

The allocation procedure was performed and recorded by a researcher who was neither

involved in the clinical recordings nor in the treatment. At the baseline visit, before the

clinical and microbiological examinations, the independent researcher picked the first

envelope from one of the boxes, according to the smoking habit of the patient and he then

unsealed the envelope. All envelopes contained a prescription for the use of a 0.12%

chlorhexidine rinse (2 x day x 28 days). Envelopes encoded for the groups SRP+DIS+AB and

SRP+S+AB contained also a prescription for the use of the antibiotics. During this session,

the independent researcher gave oral and written instructions about the use of the medications

and rinse. Patients in the SRP+S and SRP+DIS received no placebo tablets for the antibiotics.

Patients were blinded for the subgingival disinfection with NaOCl but not for the use of

antibiotics. Patients were carefully instructed by no means to inform the examiner and the

therapists about the content of the envelops. The examiner and the therapists involved in the

periodontal therapy were blinded for treatment allocation during the whole study period.

Clinical examination and microbiological sampling

After patient allocation, the following clinical measurements at 6 sites per tooth were

recorded for all teeth: plaque (presence/absence), bleeding on probing (BOP)

(presence/absence), PPD (mm) and CAL (mm). From these parameters the PISA (cm2) was

calculated (Nesse et al. 2008).

The deepest pocket in each quadrant at non-furcated sites was selected for microbiological

sampling (Mombelli et al. 1991). After isolating the site with cotton rolls, supragingival

plaque was removed with a Gracey curette and the site was dried with gentle airflow.

Subsequently 2 medium-sized paper points (Henry Schein, Almere, The Netherlands) were

inserted in the pocket for 10 s. All paper points were pooled, transferred to a reduced transport

medium (Syed & Loesche 1972) and processed within 3 hours for culturing.

All clinical measurements and microbiological sampling procedures were repeated at 3, 6

and 12 months after completion of periodontal treatment. All side effects and compliance with

the prescribed medications were recorded, by means of a face-to-face interview, at the 3-

month follow-up examination by the same independent researcher responsible for the

randomization.

75

Power calculation and intra-examiner reproducibility.

A sample of 24 patients per treatment group was calculated based on a difference of clinical

attachment level (CAL) of 1.0 mm between the groups with a standard deviation (SD) of 1.0

mm (α = 5%, β = 20%) (Del Peloso Ribeiro et al. 2008). In order to compensate for patient

dropout, the study sample was enlarged up to 110. All clinical measurements were carried out

by the same experienced periodontist (SB). Examiner reproducibility took place before the

start of the study. In 4 patients, duplicated measurements were recorded and an intraclass

correlation was obtained for probing pocket depth (PPD) of 0.94 and for CAL of 0.95.

Microbiological laboratory assessments

Culturing of subgingival bacterial samples was performed according to a protocol described

previously (Boutaga et al. 2005). The following bacteria were identified: Aa, Pg, Tf,

Prevotella intermedia (Pi), Parvimonas micra (Pm), Fusobacterium nucleatum (Fn) and

Campylobacter rectus (Cr) (van Winkelhoff et al. 2002).

Periodontal therapy

Patients were assigned to one of 3 experienced dental hygienists of the Department of

Periodontology at ACTA, before treatment allocation took place, according to scheduling

availability. The 3 therapists performed the treatment in two consecutive days, with a standard

time of 2.5 hours on each of these days. All dental hygienists followed a standardized

treatment protocol that was set up prior the start of the study. Both an ultrasonic device (Hu-

Friedy EMS piezon®, Hu-Friedy, Chicago, IL, USA) and hand instruments were used. BPT

consisted of full-mouth supra- and subgingival debridement, oral hygiene instructions with a

powered toothbrush (Sonicare®, Philips Oral Healthcare, Bothell, WA, USA) and interdental

aids on a personal need. Patients were asked to bring the bottle of chlorhexidine rinse and the

packages of the antibiotics to the first appointment of the treatment. Patients allocated in the

SRP+S+AB and SRP+DIS+AB group took the first dose of systemic antibiotics just before

the start of the treatment under supervision of the independent researcher. All patients were

instructed to start using chlorhexidine rinse at the evening of the first treatment day. Three

days after the last session of BPT, disinfection with NaOCl or saline solution was performed

by another experienced and specifically trained dental hygienist in a 1.5-hour session. Four

weeks after disinfection, patients were recalled for an extra session of supra- and subgingival

scaling, polishing and oral hygiene reinforcement. Patients were subsequently enrolled in a 3-

Chapter 4

76

76

monthly maintenance program for SRP at sites >3 mm, polishing and oral hygiene

reinforcement until the end of the study.

Subgingival disinfection procedure

In order to achieve a maximal killing effect on microorganisms in the periodontal pockets,

subgingival biofilm was disrupted before the subgingival disinfection with an ultrasonic

device. Subgingival disinfection was carried out with 100 ml of 0.5% NaOCl in the SRP+DIS

and SRP+DIS+AB groups or with 100 ml sterile saline solution (0.9% NaCl) in the SRP+S

and SRP+S+AB groups. The solutions were applied by using a sterile syringe (Terumo®,

Terumo Europe, Leuven, Belgium) with a plastic needle (capillary tip, Ultradent® product,

South Jordan, UT, USA). The capillary tip was gently placed into the pocket until resistance

at the bottom was felt. Then the tip was slightly withdrawn and moved along the surface of

the tooth while ejecting the solution. The subgingival irrigation was performed per quadrant

starting at the disto-buccal aspect of the most distal tooth, going to the midline, followed by

the lingual side. In this manner all pockets in all quadrants were irrigated three times, in order

to ensure sufficient bactericidal effect of NaOCl (Oosterwaal et al. 1990).

Statistical analysis

Statistical analyses were performed by the first author on the basis of consultations with a

statistician, who was blinded for the treatment allocations. Statistical analyses were carried

out with SPSS version 20.0 (SPSS, IBM, New York, NY, USA). The primary outcome was

CAL at 12 month follow-up; secondary outcomes were PPD, BOP, PISA and mean numbers

and mean frequencies of pockets with a given residual PPD at 12-month time point. Both

primary and secondary outcomes were tested also at the 3- and 6-month time visits. All

variables were checked for normality with Kolmogorov-Smirnoff test. Baseline differences in

background and clinical characteristics between the treatment groups were tested with

Analysis of Variance (One-way ANOVA) or Chi-square test where appropriate. Data were

explored with Little’s Missing Completely At Random test (Groenwold et al. 2012) and

missing data were imputed using the Expectation-Maximization method (Elashoff et al.

2008). Data were analyzed with the intention-to-treat (ITT) approach.

Differences within groups during the follow-up period were analyzed with ANOVA for

repeated measures. Differences between groups after treatment at every follow-up were tested

with Univariate analysis of Co-variance (ANCOVA) where the parameters were used as

77

dependent variables, the treatment modalities as fixed factor and the corresponding parameter

at baseline as co-variate; post-hoc testing was performed by means of the Bonferroni

correction method for multiple comparisons. In order to test the interaction between AB and

DIS, an additional analysis was used, employing a two-way ANOVA (factorial design). The

same dependent variables and co-variate of the previously described ANCOVA model were

imputed and antibiotic usage and disinfection were used as fixed factors. Finally, differences

between groups for frequency of patients with residual pockets of >6 mm were tested with

Fisher’s Exact Test.

A therapist effect was also investigated. Therapist allocation was added as fixed factor to

the previously described ANCOVA model. The primary and secondary periodontal variables

were explored across the three therapists at baseline and at 12 months with one-way ANOVA.

Microbiological species were analyzed as % of total Colony Forming Units (CFU). Due to

the non-normal distribution, changes within groups in % of CFU for the specific bacteria were

tested with Friedman’s Two-Way ANOVA and Wilcoxon Signed Rank test for multiple

comparisons at the different time points. Changes between groups were tested with Kruskal-

Wallis tests. Changes in prevalence of bacteria per patient within groups were analyzed with

the Cochrane Q test; the McNemar test was used for multiple comparisons. Changes in

prevalence of bacteria between groups at the various time points were tested with Fisher’s

Exact Test.

The level of significance were set at p<0.05. For the pairwise comparisons of

microbiological data, the level of significance was set at p<0.0167.

Chapter 4

77

76

monthly maintenance program for SRP at sites >3 mm, polishing and oral hygiene

reinforcement until the end of the study.

Subgingival disinfection procedure

In order to achieve a maximal killing effect on microorganisms in the periodontal pockets,

subgingival biofilm was disrupted before the subgingival disinfection with an ultrasonic

device. Subgingival disinfection was carried out with 100 ml of 0.5% NaOCl in the SRP+DIS

and SRP+DIS+AB groups or with 100 ml sterile saline solution (0.9% NaCl) in the SRP+S

and SRP+S+AB groups. The solutions were applied by using a sterile syringe (Terumo®,

Terumo Europe, Leuven, Belgium) with a plastic needle (capillary tip, Ultradent® product,

South Jordan, UT, USA). The capillary tip was gently placed into the pocket until resistance

at the bottom was felt. Then the tip was slightly withdrawn and moved along the surface of

the tooth while ejecting the solution. The subgingival irrigation was performed per quadrant

starting at the disto-buccal aspect of the most distal tooth, going to the midline, followed by

the lingual side. In this manner all pockets in all quadrants were irrigated three times, in order

to ensure sufficient bactericidal effect of NaOCl (Oosterwaal et al. 1990).

Statistical analysis

Statistical analyses were performed by the first author on the basis of consultations with a

statistician, who was blinded for the treatment allocations. Statistical analyses were carried

out with SPSS version 20.0 (SPSS, IBM, New York, NY, USA). The primary outcome was

CAL at 12 month follow-up; secondary outcomes were PPD, BOP, PISA and mean numbers

and mean frequencies of pockets with a given residual PPD at 12-month time point. Both

primary and secondary outcomes were tested also at the 3- and 6-month time visits. All

variables were checked for normality with Kolmogorov-Smirnoff test. Baseline differences in

background and clinical characteristics between the treatment groups were tested with

Analysis of Variance (One-way ANOVA) or Chi-square test where appropriate. Data were

explored with Little’s Missing Completely At Random test (Groenwold et al. 2012) and

missing data were imputed using the Expectation-Maximization method (Elashoff et al.

2008). Data were analyzed with the intention-to-treat (ITT) approach.

Differences within groups during the follow-up period were analyzed with ANOVA for

repeated measures. Differences between groups after treatment at every follow-up were tested

with Univariate analysis of Co-variance (ANCOVA) where the parameters were used as

77

dependent variables, the treatment modalities as fixed factor and the corresponding parameter

at baseline as co-variate; post-hoc testing was performed by means of the Bonferroni

correction method for multiple comparisons. In order to test the interaction between AB and

DIS, an additional analysis was used, employing a two-way ANOVA (factorial design). The

same dependent variables and co-variate of the previously described ANCOVA model were

imputed and antibiotic usage and disinfection were used as fixed factors. Finally, differences

between groups for frequency of patients with residual pockets of >6 mm were tested with

Fisher’s Exact Test.

A therapist effect was also investigated. Therapist allocation was added as fixed factor to

the previously described ANCOVA model. The primary and secondary periodontal variables

were explored across the three therapists at baseline and at 12 months with one-way ANOVA.

Microbiological species were analyzed as % of total Colony Forming Units (CFU). Due to

the non-normal distribution, changes within groups in % of CFU for the specific bacteria were

tested with Friedman’s Two-Way ANOVA and Wilcoxon Signed Rank test for multiple

comparisons at the different time points. Changes between groups were tested with Kruskal-

Wallis tests. Changes in prevalence of bacteria per patient within groups were analyzed with

the Cochrane Q test; the McNemar test was used for multiple comparisons. Changes in

prevalence of bacteria between groups at the various time points were tested with Fisher’s

Exact Test.

The level of significance were set at p<0.05. For the pairwise comparisons of

microbiological data, the level of significance was set at p<0.0167.

Chapter 4

78

78

Results

From the screening of 1409 eligible patients, 134 patients met the selection criteria and 110

volunteered to participate in this study and signed the informed consent. Patients were

recruited between May 2008 and April 2013. Recruitment was stopped when the 110th patient

was included. Based on the randomization, 29 patients were allocated in the SRP+S group, 27

in the SRP+DIS group, 25 in the SRP+DIS+AB group and 29 in SRP+S+AB group. At 12-

month follow-up a total of 99 patients completed the study, with a dropout rate of 10%

(Figure 1). At baseline, the four groups showed comparable background (Table 1) and

periodontal characteristics (Table 2), with no significant difference for CAL or any of the

secondary variables analyzed.

Clinical results

Each treatment modality showed a significant improvement for CAL and all other clinical

parameters at all follow-up visits, in comparison with baseline (for all parameters p<0.001 at

every follow-up moment in comparison to baseline) (Table 3).

Overall differences between groups were found for CAL as well as PPD and PISA at 3 and

6 months after therapy. For CAL, after pairwise comparisons between groups at 3 and 6

months after therapy, more gain was found in the group SRP+S+AB compared to SPR+DIS.

This significant difference was not anymore present at 12 months (Table 3). For number of

residual PPD >6 and >7 mm, overall differences between groups were found at 3, 6 and 12

months (Table 3). Post-hoc testing revealed that SRP+DIS+AB showed smaller number of

PPD >7 mm in comparison to SRP+S at 3 months (mean difference 2.72; 95% CI 0.14,5.30;

padj=0.033) and 6-months (mean difference 3.04; 95% CI 0.11,5.97; padj=0.037), but not for

the 12-month visit (mean difference 3.06; 95% CI -0.85,6.97; padj=0.227) (Table S1). No

statistical difference was found at all follow-up visits for SRP+DIS in comparison with

SRP+S for CAL and all other clinical parameters tested. The SRP+S+AB group showed, in

comparison to SRP+DIS, more CAL gain after treatment at 3 and 6 months and smaller

number of PPD >6 mm and PPD >7 mm at 3 months (Table 3). Furthermore, in comparison

to SRP+S, the SRP+S+AB group showed less number of PPD >5 mm at the 3-month follow-

up, less number of PPD >6 mm and PPD >7 mm at 3 and 6-month visits. None of these

differences remained significant at the 12-month follow-up (Table 3). There was no difference

for any of the other secondary variables between the four groups (Table 3, Table S2).

79

The two-way ANOVA (factorial analysis) showed that there was no significant interaction

between AB and DIS, no significant main effect of DIS and a significant main effect of AB at

12 months for PPD, PISA and number of PPD >5, 6 and 7 mm (Table 4).

Non-parametric analysis, for numbers of patients having residual pockets ≥6 mm per

treatment modality, showed that SRP+DIS+AB group had less patients having >3 residual

pockets of >6 mm at 12 months, but this difference was not significant (p=0.162) (Table 5).

When we explored for a therapist effect, we found that the treatments of therapist #1

resulted in less gain in CAL, and less reduction in PD, PISA and number of PD >6 mm.

However at baseline the one-way ANOVA showed a difference in the periodontal parameters

across the 3 therapists. Therapist #1 treated patients with lower mean CAL, PPD, PISA and

number of pockets >5, 6 and 7 mm, and higher percentage of sites with visible plaque. There

was no significant difference between the therapists for the periodontal variables at 12 months

(Table S3).

Microbiological results

Seven microbial species, being the traditional putative periodontal pathogens, were analyzed

by anaerobic culture (Table S4). There was no statistical significant difference at baseline

between the four treatment groups. At 12 months, all treatment modalities showed a

significant reduction of patients being positive for Tf. All treatment modalities, except

SRP+S+AB, showed a significant decrease of patients positive for Pg and all treatment

modalities, except SRP+S, showed a significant decrease of patients positive for Pm; for

SPR+DIS+AB this decrease was significant only up to 6 months. The prevalence of Pi was

significantly decreased in the SRP+DIS+AB group only at 3 months. SRP+DIS+AB showed a

higher reduction of patients being positive for Tf at 3 months (p<0.0167). There was no

significant decrease of patients positive for Aa, Fn, and Cr for any of the 4 treatment

modalities. SRP+S+AB showed a significant higher reduction of patients being positive for

Pm in comparison with SRP+S at 3 months (p<0.0167) and at 12 months after therapy

(p<0.0167). No other significant difference was found between the groups (Table 6).

Compliance and adverse effects

None of the patients reported adverse effects for the professionally applied NaOCl irrigation.

All patients in the groups SRP+S+AB and SRP+DIS+AB completed the antibiotic therapy.

Eight patients in SRP+S+AB (27.5%) and four in SRP+DIS+AB (16.0%) experienced

Chapter 4

79

78

Results

From the screening of 1409 eligible patients, 134 patients met the selection criteria and 110

volunteered to participate in this study and signed the informed consent. Patients were

recruited between May 2008 and April 2013. Recruitment was stopped when the 110th patient

was included. Based on the randomization, 29 patients were allocated in the SRP+S group, 27

in the SRP+DIS group, 25 in the SRP+DIS+AB group and 29 in SRP+S+AB group. At 12-

month follow-up a total of 99 patients completed the study, with a dropout rate of 10%

(Figure 1). At baseline, the four groups showed comparable background (Table 1) and

periodontal characteristics (Table 2), with no significant difference for CAL or any of the

secondary variables analyzed.

Clinical results

Each treatment modality showed a significant improvement for CAL and all other clinical

parameters at all follow-up visits, in comparison with baseline (for all parameters p<0.001 at

every follow-up moment in comparison to baseline) (Table 3).

Overall differences between groups were found for CAL as well as PPD and PISA at 3 and

6 months after therapy. For CAL, after pairwise comparisons between groups at 3 and 6

months after therapy, more gain was found in the group SRP+S+AB compared to SPR+DIS.

This significant difference was not anymore present at 12 months (Table 3). For number of

residual PPD >6 and >7 mm, overall differences between groups were found at 3, 6 and 12

months (Table 3). Post-hoc testing revealed that SRP+DIS+AB showed smaller number of

PPD >7 mm in comparison to SRP+S at 3 months (mean difference 2.72; 95% CI 0.14,5.30;

padj=0.033) and 6-months (mean difference 3.04; 95% CI 0.11,5.97; padj=0.037), but not for

the 12-month visit (mean difference 3.06; 95% CI -0.85,6.97; padj=0.227) (Table S1). No

statistical difference was found at all follow-up visits for SRP+DIS in comparison with

SRP+S for CAL and all other clinical parameters tested. The SRP+S+AB group showed, in

comparison to SRP+DIS, more CAL gain after treatment at 3 and 6 months and smaller

number of PPD >6 mm and PPD >7 mm at 3 months (Table 3). Furthermore, in comparison

to SRP+S, the SRP+S+AB group showed less number of PPD >5 mm at the 3-month follow-

up, less number of PPD >6 mm and PPD >7 mm at 3 and 6-month visits. None of these

differences remained significant at the 12-month follow-up (Table 3). There was no difference

for any of the other secondary variables between the four groups (Table 3, Table S2).

79

The two-way ANOVA (factorial analysis) showed that there was no significant interaction

between AB and DIS, no significant main effect of DIS and a significant main effect of AB at

12 months for PPD, PISA and number of PPD >5, 6 and 7 mm (Table 4).

Non-parametric analysis, for numbers of patients having residual pockets ≥6 mm per

treatment modality, showed that SRP+DIS+AB group had less patients having >3 residual

pockets of >6 mm at 12 months, but this difference was not significant (p=0.162) (Table 5).

When we explored for a therapist effect, we found that the treatments of therapist #1

resulted in less gain in CAL, and less reduction in PD, PISA and number of PD >6 mm.

However at baseline the one-way ANOVA showed a difference in the periodontal parameters

across the 3 therapists. Therapist #1 treated patients with lower mean CAL, PPD, PISA and

number of pockets >5, 6 and 7 mm, and higher percentage of sites with visible plaque. There

was no significant difference between the therapists for the periodontal variables at 12 months

(Table S3).

Microbiological results

Seven microbial species, being the traditional putative periodontal pathogens, were analyzed

by anaerobic culture (Table S4). There was no statistical significant difference at baseline

between the four treatment groups. At 12 months, all treatment modalities showed a

significant reduction of patients being positive for Tf. All treatment modalities, except

SRP+S+AB, showed a significant decrease of patients positive for Pg and all treatment

modalities, except SRP+S, showed a significant decrease of patients positive for Pm; for

SPR+DIS+AB this decrease was significant only up to 6 months. The prevalence of Pi was

significantly decreased in the SRP+DIS+AB group only at 3 months. SRP+DIS+AB showed a

higher reduction of patients being positive for Tf at 3 months (p<0.0167). There was no

significant decrease of patients positive for Aa, Fn, and Cr for any of the 4 treatment

modalities. SRP+S+AB showed a significant higher reduction of patients being positive for

Pm in comparison with SRP+S at 3 months (p<0.0167) and at 12 months after therapy

(p<0.0167). No other significant difference was found between the groups (Table 6).

Compliance and adverse effects

None of the patients reported adverse effects for the professionally applied NaOCl irrigation.

All patients in the groups SRP+S+AB and SRP+DIS+AB completed the antibiotic therapy.

Eight patients in SRP+S+AB (27.5%) and four in SRP+DIS+AB (16.0%) experienced

Chapter 4

80

80

adverse effects (Table S4). These adverse effects were fungal infection (1), flatulence (1),

rash and itching (1), dark urine (1), diarrhea (4), nausea (3), acidic reflux (1). All patients in

groups SRP and SRP+DIS used the chlorhexidine rinse as prescribed, while 26 (89.7%) in

SRP+S+AB and 22 (88%) in SRP+DIS+AB complied with the mouthwash prescription. The

percentage of subjects in the four groups reporting adverse effect of chlorhexidine varied

between 27.5% and 44% (Table S4). These adverse effects were taste change (31), black

tongue (10), tooth staining (12) and dry mouth (3). There was no statistical difference in the

distribution of the compliant patients between the 4 groups and in the distribution of patients

presenting adverse effects for either the chlorhexidine rinse or for systemic antibiotics

(SRP+S+AB and SRP+ DIS+AB only) (Table S4).

81

Discussion

In the current study we tested the hypotheses whether the adjunctive use of subgingival

disinfection with 0.5% NaOCl could enhance the long term (>6 months) clinical and

microbiological effect of BPT, and if the local antiseptic effect of NaOCl could work

synergistically with systemic antibiotics. Using a 4-arm parallel design, this RCT showed

that, in comparison to SRP alone, SRP in combination with disinfection with 0.5% NaOCl did

not show any significant adjunctive effect for CAL (primary outcome) and any other

parameter tested. The two treatment groups who used additionally systemic antibiotics

showed more improvement for CAL and PPD at 3 and 6 months, but not statistically

significant at 12 months. Up to 6 months, SRP+DIS+AB and SRP+S+AB showed a lower

number of residual pockets >6 and >7 mm but not at 12 months. In contrast, when we tested

for main effect and interaction of the use of antibiotics and the disinfection, we found that the

antibiotics held a small significant better improvement for PPD, PISA and PPD >6 and >7

mm up to 12 months. However, antibiotics still did not account for a better significant

reduction for CAL at 12 months. We conclude that the systemic antibiotics accounted for the

main additional effect of these two therapies, especially for PISA and PPD, while the

disinfection with NaOCl played no role. We suggest that the antimicrobial effect of NaOCl is

only short lived and at long term disappeared by the possible bacterial recolonization of the

pockets (Magnusson et al. 1984, Rhemrev et al. 2006).

When we consider the clinical results (CAL, PPD and BOP) of the two groups of chronic

periodontitis patients treated with systemic antibiotics, we see that the current clinical results

are in line with previous similar investigations with a follow-up up to 6 months (Cionca et al.

2009, Keestra et al. 2015, Sgolastra et al. 2012, Winkel et al. 2001) and with two other studies

with 12-month follow-up or longer (Feres et al. 2012, Goodson et al. 2012). Our dosage of

antibiotics was originally published and tested by van Winkelhoff and co-workers (van

Winkelhoff et al. 1989, Winkel et al. 2001) and lower compared to the one used in other

investigations (Cionca et al. 2009, Feres et al. 2012, Goodson et al. 2012). To date there are

no RCT studies available that prove that higher and/or prolonged dosages of these antibiotics

than those in the original publication tested, lead to a better long term and sustainable clinical

results in the treatment of periodontitis. Nevertheless, in a review paper, Feres et al. (2014)

suggested that a 14 days prescription of amoxicillin and metronidazole does result in better

pocket depth reduction at 3 months in comparison to a 7-day prescription. In our study

Chapter 4

81

80

adverse effects (Table S4). These adverse effects were fungal infection (1), flatulence (1),

rash and itching (1), dark urine (1), diarrhea (4), nausea (3), acidic reflux (1). All patients in

groups SRP and SRP+DIS used the chlorhexidine rinse as prescribed, while 26 (89.7%) in

SRP+S+AB and 22 (88%) in SRP+DIS+AB complied with the mouthwash prescription. The

percentage of subjects in the four groups reporting adverse effect of chlorhexidine varied

between 27.5% and 44% (Table S4). These adverse effects were taste change (31), black

tongue (10), tooth staining (12) and dry mouth (3). There was no statistical difference in the

distribution of the compliant patients between the 4 groups and in the distribution of patients

presenting adverse effects for either the chlorhexidine rinse or for systemic antibiotics

(SRP+S+AB and SRP+ DIS+AB only) (Table S4).

81

Discussion

In the current study we tested the hypotheses whether the adjunctive use of subgingival

disinfection with 0.5% NaOCl could enhance the long term (>6 months) clinical and

microbiological effect of BPT, and if the local antiseptic effect of NaOCl could work

synergistically with systemic antibiotics. Using a 4-arm parallel design, this RCT showed

that, in comparison to SRP alone, SRP in combination with disinfection with 0.5% NaOCl did

not show any significant adjunctive effect for CAL (primary outcome) and any other

parameter tested. The two treatment groups who used additionally systemic antibiotics

showed more improvement for CAL and PPD at 3 and 6 months, but not statistically

significant at 12 months. Up to 6 months, SRP+DIS+AB and SRP+S+AB showed a lower

number of residual pockets >6 and >7 mm but not at 12 months. In contrast, when we tested

for main effect and interaction of the use of antibiotics and the disinfection, we found that the

antibiotics held a small significant better improvement for PPD, PISA and PPD >6 and >7

mm up to 12 months. However, antibiotics still did not account for a better significant

reduction for CAL at 12 months. We conclude that the systemic antibiotics accounted for the

main additional effect of these two therapies, especially for PISA and PPD, while the

disinfection with NaOCl played no role. We suggest that the antimicrobial effect of NaOCl is

only short lived and at long term disappeared by the possible bacterial recolonization of the

pockets (Magnusson et al. 1984, Rhemrev et al. 2006).

When we consider the clinical results (CAL, PPD and BOP) of the two groups of chronic

periodontitis patients treated with systemic antibiotics, we see that the current clinical results

are in line with previous similar investigations with a follow-up up to 6 months (Cionca et al.

2009, Keestra et al. 2015, Sgolastra et al. 2012, Winkel et al. 2001) and with two other studies

with 12-month follow-up or longer (Feres et al. 2012, Goodson et al. 2012). Our dosage of

antibiotics was originally published and tested by van Winkelhoff and co-workers (van

Winkelhoff et al. 1989, Winkel et al. 2001) and lower compared to the one used in other

investigations (Cionca et al. 2009, Feres et al. 2012, Goodson et al. 2012). To date there are

no RCT studies available that prove that higher and/or prolonged dosages of these antibiotics

than those in the original publication tested, lead to a better long term and sustainable clinical

results in the treatment of periodontitis. Nevertheless, in a review paper, Feres et al. (2014)

suggested that a 14 days prescription of amoxicillin and metronidazole does result in better

pocket depth reduction at 3 months in comparison to a 7-day prescription. In our study

Chapter 4

82

82

population, only 2 of a total of 8 patients in SRP+DIS+AB and SRP+S+AB became negative

for Aa at 12 months after treatment. This is in contrast with previous studies, where it was

shown that the combination therapy currently used, was highly effective in suppressing Aa

(Ehmke et al. 2005, Flemmig et al. 1998, Pavicic et al. 1994, Winkel et al. 2001). This finding

may also suggest that, currently, this dosage of antimicrobials may be less effective than one

or two decades ago, due to a possible increasing bacterial antibiotic resistance (Rams et al.

2014). Nevertheless, our used dosage is sufficient to obtain better clinical effects for several

secondary parameters.

Considering that the average difference in full mouth CAL gain after therapy between the

groups is limited to <1 mm, and that 22.2% of the patients allocated in the antibiotic groups

(SRP+DIS+AB and SRP+S+AB) reported adverse effects, we still do not support the

routinely use of antibiotics in the treatment of chronic periodontitis. We agree with the

conclusions of the most recent meta-analysis, which recommends the prescription of systemic

antibiotics in combination with BPT only in specific clinical situations (Keestra et al. 2015).

Similarly it was also stated by the 6th European Workshop on Periodontology, that the adjunct

of antibiotics should be considered only on case specific situations (Sanz et al. 2008). Also

the professional irrigation with NaOCl as local disinfectant was not effective in the long term

to enhance reduction of residual inflammation or improving any of the other clinical and

microbiological parameters.

In conclusion, the local disinfection with 0.5% NaOCl in addition to SRP, failed to show

adjunctive effects for CAL and the secondary periodontal and microbiological parameters

both with or without the use of systemic antibiotics.

Source of funding

This study was funded by several sources: by the authors’ institution, by an unconditional

grant from Philips Oral Healthcare and by a grant from the University of Amsterdam for the

focal point “Oral infection and inflammation”.

Acknowledgments

We thank Efthimis Arvanitidis, Arti Abhilakh Missier, Sandra Douwes, Juliette Groot,

Hendrik Jan Hansma, Wiebke Houcken, Tiong Oei, Martijn Rosema, Arne Scholten, Wijnand

Teeuw, Wouter van Wesemael, for their extremely valuable help during this research project.

83

We thank dr. Irene Aartman and prof. Geert van der Heijden for their valuable statistical

advices.

Chapter 4

83

82

population, only 2 of a total of 8 patients in SRP+DIS+AB and SRP+S+AB became negative

for Aa at 12 months after treatment. This is in contrast with previous studies, where it was

shown that the combination therapy currently used, was highly effective in suppressing Aa

(Ehmke et al. 2005, Flemmig et al. 1998, Pavicic et al. 1994, Winkel et al. 2001). This finding

may also suggest that, currently, this dosage of antimicrobials may be less effective than one

or two decades ago, due to a possible increasing bacterial antibiotic resistance (Rams et al.

2014). Nevertheless, our used dosage is sufficient to obtain better clinical effects for several

secondary parameters.

Considering that the average difference in full mouth CAL gain after therapy between the

groups is limited to <1 mm, and that 22.2% of the patients allocated in the antibiotic groups

(SRP+DIS+AB and SRP+S+AB) reported adverse effects, we still do not support the

routinely use of antibiotics in the treatment of chronic periodontitis. We agree with the

conclusions of the most recent meta-analysis, which recommends the prescription of systemic

antibiotics in combination with BPT only in specific clinical situations (Keestra et al. 2015).

Similarly it was also stated by the 6th European Workshop on Periodontology, that the adjunct

of antibiotics should be considered only on case specific situations (Sanz et al. 2008). Also

the professional irrigation with NaOCl as local disinfectant was not effective in the long term

to enhance reduction of residual inflammation or improving any of the other clinical and

microbiological parameters.

In conclusion, the local disinfection with 0.5% NaOCl in addition to SRP, failed to show

adjunctive effects for CAL and the secondary periodontal and microbiological parameters

both with or without the use of systemic antibiotics.

Source of funding

This study was funded by several sources: by the authors’ institution, by an unconditional

grant from Philips Oral Healthcare and by a grant from the University of Amsterdam for the

focal point “Oral infection and inflammation”.

Acknowledgments

We thank Efthimis Arvanitidis, Arti Abhilakh Missier, Sandra Douwes, Juliette Groot,

Hendrik Jan Hansma, Wiebke Houcken, Tiong Oei, Martijn Rosema, Arne Scholten, Wijnand

Teeuw, Wouter van Wesemael, for their extremely valuable help during this research project.

83

We thank dr. Irene Aartman and prof. Geert van der Heijden for their valuable statistical

advices.

Chapter 4

84

84

Figure 1: Flow chart presenting the study design and the rate of dropouts. 1 patient in the

SRP+AB group did not receive intervention because he did not attend the appointments and 1

patient in the SRP+DIS+AB group withdrew before treatment. All other patients (n = 9)

stopped because they did not wish to attend the follow-up visits.

85

Chapter 4

85

84

Figure 1: Flow chart presenting the study design and the rate of dropouts. 1 patient in the

SRP+AB group did not receive intervention because he did not attend the appointments and 1

patient in the SRP+DIS+AB group withdrew before treatment. All other patients (n = 9)

stopped because they did not wish to attend the follow-up visits.

85

Chapter 4

86

Tabl

e 1.

Gen

eral

cha

ract

eris

tics o

f the

four

gro

ups a

t bas

elin

e. V

alue

s are

mea

ns ±

stan

dard

dev

iatio

ns o

r num

bers

(%).

SRP+

S SR

P+D

IS

SRP+

DIS

+AB

SR

P+S+

AB

p-

valu

e*

N=2

9 N

=27

N=2

5 N

=29

Age

(yea

rs)

48

.2 ±

7.7

47

.7 ±

11.

2 46

.9 ±

8.5

48

.2 ±

9.6

0.

951

Gen

der

0.

207

mal

e

21 (7

2)

15 (5

5)

11 (4

4)

16 (5

5)

fe

mal

e

8 (2

8)

12 (4

4)

14 (5

6)

13 (4

4)

Et

hnic

ity

0.74

0 D

utch

-Cau

casi

an

23

(79)

18

(67)

18

(72)

22

(76)

Non

-Dut

ch-C

auca

sian

6 (2

1)

9 (3

3)

7 (2

8)

7 (2

4)

Ed

ucat

ion

0.

631

< hi

gh sc

hool

7 (2

4)

7 (2

6)

8 (3

2)

5 (1

7)

>

high

scho

ol

22

(76)

20

(74)

17

(68)

24

(83)

Smok

ing

0.

546

non

smok

er

13

(45)

14

(52)

8

(32)

13

(45)

curr

ent

16

(55)

13

(48)

17

(68)

16

(55)

Alc

ohol

0.83

5 >

2 un

its/d

ay

9

(31)

9

(33)

8

(32)

7

(24)

< 2

units

/day

20 (6

9)

18 (6

7)

17 (6

8)

22 (7

6)

Num

ber o

f tee

th

26.5

± 2

.6

26.6

± 3

.3

27.1

± 2

.4

27.0

± 2

.6

0.80

0

*One

-way

AN

OV

A a

nd C

hi-s

quar

ed te

st w

here

app

ropr

iate

.

Abb

revi

atio

ns: S

RP+

S: S

calin

g an

d ro

ot p

lann

ing

+ sa

line;

SR

P+D

IS: S

calin

g an

d ro

ot p

lann

ing

+ di

sinf

ectio

n w

ith 0

.5%

sodi

um h

ypoc

hlor

ite

(NaO

Cl);

SR

P+D

IS+A

B: S

calin

g an

d ro

ot p

lann

ing

+ di

sinf

ectio

n w

ith 0

.5%

NaO

Cl +

ant

ibio

tics (

amox

icill

in a

nd m

etro

nida

zole

); SR

P+S+

AB

: sc

alin

g an

d ro

ot p

lann

ing

+ sa

line

+ an

tibio

tics B

MI =

Bod

y M

ass I

ndex

.

Tabl

e 2.

Per

iodo

ntal

cha

ract

eris

tics o

f the

four

gro

ups a

t bas

elin

e. V

alue

s are

mea

ns ±

stan

dard

dev

iatio

ns.

SRP+

S SR

P+D

IS

SRP+

DIS

+AB

SR

P+S+

AB

p-va

lue*

N

=29

N=2

7 N

=25

N=2

9

CA

L (m

m)

4.

4 ±

0.8

4.1

± 0.

8 4.

2 ±

1.1

4.2

± 1.

2

0.75

8

PPD

(mm

)

3.9

± 0.

6 3.

7 ±

0.6

3.9

± 0.

7 4.

0 ±

0.7

0.

669

PISA

(cm

2 )

17.9

± 4

.1

16.6

± 5

.7

17.9

± 6

.4

18.6

± 6

.4

0.

645

BO

P (%

)

66.3

± 1

4.9

65.7

± 1

4.8

67.3

± 1

4.4

69.9

± 1

7.2

0.

749

Plaq

ue (%

)

60.6

± 2

4.6

67.6

± 2

0.5

61.9

± 2

4.8

68.6

± 2

6.3

0.

511

# PP

D >

5 m

m

% si

tes P

PD >

5 m

m

51

.3 ±

21.

4

32.3

± 1

3.2

45.3

± 2

2.6

28.3

± 1

2.5

51.2

± 2

5.6

21.9

± 1

3.3

53.3

± 2

4.7

33.1

± 1

5.2

0.

618

0.60

5

# PP

D >

6 m

m

% si

tes P

PD >

6 m

m

34

.0 ±

17.

7

21.3

± 1

1.0

30.3

± 1

8.3

18.7

± 1

0.2

35.8

± 2

2.1

21.9

± 1

3.3

34.6

± 2

0.9

21.3

± 1

2.6

0.

764

0.77

1

# PP

D >

7 m

m

% si

tes P

PD >

7 m

m

19

.7 ±

13.

4

12.4

± 8

.3

16.5

± 1

3.9

10.2

± 8

.0

20.2

± 1

6.5

12.3

± 9

.8

18.9

± 1

4.5

11.5

± 8

.5

0.

795

0.77

1

*One

-way

AN

OV

A.

Abb

revi

atio

ns: s

ee T

able

1 a

nd: C

AL

= C

linic

al A

ttach

men

t Los

s; P

PD =

Pro

bing

Poc

ket D

epth

; PIS

A =

Per

iodo

ntal

Infla

med

Sur

face

Are

a

BO

P =

Ble

edin

g on

Pro

bing

; # =

num

ber o

f poc

kets

with

a g

iven

dep

th.

Chapter 4

87

Tabl

e 2.

Per

iodo

ntal

cha

ract

eris

tics o

f the

four

gro

ups a

t bas

elin

e. V

alue

s are

mea

ns ±

stan

dard

dev

iatio

ns.

SRP+

S SR

P+D

IS

SRP+

DIS

+AB

SR

P+S+

AB

p-va

lue*

N

=29

N=2

7 N

=25

N=2

9

CA

L (m

m)

4.

4 ±

0.8

4.1

± 0.

8 4.

2 ±

1.1

4.2

± 1.

2

0.75

8

PPD

(mm

)

3.9

± 0.

6 3.

7 ±

0.6

3.9

± 0.

7 4.

0 ±

0.7

0.

669

PISA

(cm

2 )

17.9

± 4

.1

16.6

± 5

.7

17.9

± 6

.4

18.6

± 6

.4

0.

645

BO

P (%

)

66.3

± 1

4.9

65.7

± 1

4.8

67.3

± 1

4.4

69.9

± 1

7.2

0.

749

Plaq

ue (%

)

60.6

± 2

4.6

67.6

± 2

0.5

61.9

± 2

4.8

68.6

± 2

6.3

0.

511

# PP

D >

5 m

m

% si

tes P

PD >

5 m

m

51

.3 ±

21.

4

32.3

± 1

3.2

45.3

± 2

2.6

28.3

± 1

2.5

51.2

± 2

5.6

21.9

± 1

3.3

53.3

± 2

4.7

33.1

± 1

5.2

0.

618

0.60

5

# PP

D >

6 m

m

% si

tes P

PD >

6 m

m

34

.0 ±

17.

7

21.3

± 1

1.0

30.3

± 1

8.3

18.7

± 1

0.2

35.8

± 2

2.1

21.9

± 1

3.3

34.6

± 2

0.9

21.3

± 1

2.6

0.

764

0.77

1

# PP

D >

7 m

m

% si

tes P

PD >

7 m

m

19

.7 ±

13.

4

12.4

± 8

.3

16.5

± 1

3.9

10.2

± 8

.0

20.2

± 1

6.5

12.3

± 9

.8

18.9

± 1

4.5

11.5

± 8

.5

0.

795

0.77

1

*One

-way

AN

OV

A.

Abb

revi

atio

ns: s

ee T

able

1 a

nd: C

AL

= C

linic

al A

ttach

men

t Los

s; P

PD =

Pro

bing

Poc

ket D

epth

; PIS

A =

Per

iodo

ntal

Infla

med

Sur

face

Are

a

BO

P =

Ble

edin

g on

Pro

bing

; # =

num

ber o

f poc

kets

with

a g

iven

dep

th.

Chapter 4

88

Tabl

e 3.

Res

ults

of t

he tr

eatm

ent a

t 3-,

6- a

nd 1

2-m

onth

s fol

low

-up.

Val

ues a

re m

eans

± st

anda

rd d

evia

tions

.

SR

P+S

SR

P+D

IS

SR

P+D

IS+A

B

SR

P+S+

AB

p adj

val

ue†

N

=29

N

=27

N

=25

N

=29

Prim

ary

outc

omes

CA

L (m

m)

ba

selin

e

4.

4 ±

0.8*

4.1

± 0.

8*

4.

2 ±

1.1*

4.2

± 1.

2*

3 m

onth

s

3.

9 ±

0.9

3.

7 ±

1.0

3.

6 ±

0.9

3.

4 ±

1.1a

0.

015

6 m

onth

s

3.

8 ±

1.0

3.

7 ±

0.9

3.

6 ±

0.9

3.

4 ±

1.1b

0.

041

12 m

onth

s

3.

8 ±

0.9

3.

7 ±

1.1

3.

6 +

0.9

3.

5 ±

1.2

0.

243

Seco

ndar

y ou

tcom

es

P

PD (m

m)

ba

selin

e

3.

9 ±

0.6*

3.7

± 0.

6*

3.

9 ±

0.7*

4.0

± 0.

7*

3 m

onth

s

2.

9 ±

0.4

2.

9 ±

0.6

2.

7 ±

0.4

2.

7 ±

0.4

0.

024

6 m

onth

s

3.

0 ±

0.4

2.

9 ±

0.5

2.

7 ±

0.3

2.

7 ±

0.4

0.

027

12 m

onth

s

2.

9 ±

0.5

2.

8 ±

0.7

2.

7 ±

0.4

2.

8 ±

0.4

0.

143

PISA

(cm

2 )

base

line

17.9

± 4

.1*

16

.6 ±

5.7

*

17.9

± 6

.4*

18

.6 ±

6.4

*

3

mon

ths

4.9

± 3.

5

5.2

± 3.

3

3.8

± 2.

7

3.4

± 2.

1

0.03

3

6

mon

ths

5.4

± 3.

9

4.6

± 2.

8

4.1

± 3.

3

3.4

± 3.

1

0.10

9

12

mon

ths

5.4

± 3.

9

5.2

± 4.

1

4.1

± 2.

9

4.0

± 3.

1

0.20

6

B

OP

(%)

ba

selin

e

66

.3 ±

14.

9*

65

.7 ±

14.

8*

67

.3 ±

14.

4*

69

.9 ±

17.

2*

3 m

onth

s

21

.5 ±

12.

9

26.0

± 1

3.3

21

.2 ±

16.

7

19.6

± 1

1.0

0.

142

6 m

onth

s

24

.3 ±

14.

4

23.1

± 1

0.9

21

.4 ±

17.

0

18.8

± 1

3.0

0.

308

12 m

onth

s

24

.1 ±

14.

9

24.7

± 1

4.7

21

.3 ±

14.

7

22.7

± 1

5.5

0.

635

Plaq

ue (%

)

base

line

60.6

± 2

4.6*

67.6

± 2

0.5*

61.9

± 2

4.8*

68.6

± 2

6.3*

3

mon

ths

21.5

± 1

9.6

20

.1 ±

18.

3

19.4

± 1

8.9

19

.7 ±

16.

9

0.63

4

6

mon

ths

27.7

± 2

2.0

21

.5 ±

18.

6

24.8

± 2

3.0

21

.0 ±

20.

6

0.16

6

12

mon

ths

19.7

± 1

4.1

20

.8 ±

17.

3

24.1

± 2

1.3

24

.6 ±

19.

4

0.63

8

#

PPD

>5

mm

base

line

51.3

± 2

1.4*

45.3

± 2

5.6*

51.2

± 2

5.6*

53.3

± 2

4.7*

3

mon

ths

19.8

± 1

4.0

18

.6 ±

17.

7

14.9

± 1

1.5

13

.2 ±

10.

4c

0.01

8

6

mon

ths

19.5

± 1

4.8

16

.5 ±

16.

0

12.6

± 1

0.4

12

.2 ±

8.8

d

0.01

5

12

mon

ths

20.1

± 1

7.1

17

.5 ±

20.

8

13.3

± 1

1.6

12

.7 ±

10.

5

0.05

5

#

PPD

>6

mm

base

line

34.0

± 1

7.7*

30.3

± 1

8.3*

35.8

± 2

2.1*

34.6

± 2

0.9*

3

mon

ths

9.4

± 9.

9

8.7

± 11

.2

5.

6 ±

5.5

3.

8 ±

4.1e,

f

0.00

5

6

mon

ths

9.5

± 10

.9

7.

6 ±

11.4

5.0

± 6.

0

3.4

± 3.

8g

0.00

8

12

mon

ths

10.1

± 1

1.5

9.

1 ±

16.7

4.7

± 6.

5

4.2

± 5.

0

0.02

3

# PP

D >

7 m

m

ba

selin

e

19

.7 ±

13.

4*

16

.5 ±

13.

9*

20

.2 ±

16.

5*

18

.9 ±

14.

5*

3 m

onth

s

4.

1 ±

5.4

3.

2 ±

4.3

1.

4 ±

2.5h

0.

9 ±

1.5i,

l

0.00

1

6

mon

ths

4.3

± 5.

7

3.0

± 6.

0

1.3

± 2.

6m

1.

1 ±

2.2n

0.

007

12 m

onth

s

4.

4 ±

6.5

4.

0 ±

9.4

1.

5 ±

2.5

1.

0 ±

2.9

0.

021

*Sig

nific

ant d

iffer

ence

bet

wee

n ba

selin

e an

d th

e ot

her f

ollo

w-u

p tim

e po

ints

(p <

0.0

01) (

AN

OV

A fo

r rep

eate

d m

easu

res)

. † A

NC

OV

A fo

r diff

eren

ces b

etw

een

grou

ps, a

djus

ted

for t

he c

orre

spon

ding

val

ue a

t bas

elin

e.

Lette

rs: P

airw

ise

com

paris

on b

etw

een

grou

ps (p

-val

ues a

djus

ted

for t

he c

orre

spon

ding

val

ue a

t bas

elin

e).

a, S

RP+

S+A

B v

s SR

P+D

IS, p

adj=

0.02

0

b, S

RP+

S+A

B v

s SR

P+D

IS, p

adj =

0.04

6

c, S

RP+

S+A

B v

s SR

P+D

IS, p

adj =

0.04

2 d,

SR

P+S+

AB

vs S

RP+

S, p

adj =

0.0

42

e,

SR

P+S+

AB

vs S

RP+

DIS

, pad

j =0.

023

f,

SRP+

S+A

B v

s SR

P+S,

pad

j =0.

021

g, S

RP+

S+A

B v

s SR

P+S,

pad

j =0.

016

h,

SR

P+D

IS+A

B v

s SR

P+S,

pad

j =0.

033

i,

SRP+

S+A

B v

s SR

P+D

IS, p

adj =

0.04

1 l,

SRP+

S+A

B v

s SR

P+S,

pad

j =0.

005

m, S

RP+

DIS

+AB

vs S

RP+

S, p

adj =

0.03

7

n, S

RP+

S +A

B v

s SR

P+S,

pad

j =0.

025

Abb

revi

atio

ns: s

ee T

able

s 1 a

nd 2

Chapter 4

89

Plaq

ue (%

)

base

line

60.6

± 2

4.6*

67.6

± 2

0.5*

61.9

± 2

4.8*

68.6

± 2

6.3*

3

mon

ths

21.5

± 1

9.6

20

.1 ±

18.

3

19.4

± 1

8.9

19

.7 ±

16.

9

0.63

4

6

mon

ths

27.7

± 2

2.0

21

.5 ±

18.

6

24.8

± 2

3.0

21

.0 ±

20.

6

0.16

6

12

mon

ths

19.7

± 1

4.1

20

.8 ±

17.

3

24.1

± 2

1.3

24

.6 ±

19.

4

0.63

8

#

PPD

>5

mm

base

line

51.3

± 2

1.4*

45.3

± 2

5.6*

51.2

± 2

5.6*

53.3

± 2

4.7*

3

mon

ths

19.8

± 1

4.0

18

.6 ±

17.

7

14.9

± 1

1.5

13

.2 ±

10.

4c

0.01

8

6

mon

ths

19.5

± 1

4.8

16

.5 ±

16.

0

12.6

± 1

0.4

12

.2 ±

8.8

d

0.01

5

12

mon

ths

20.1

± 1

7.1

17

.5 ±

20.

8

13.3

± 1

1.6

12

.7 ±

10.

5

0.05

5

#

PPD

>6

mm

base

line

34.0

± 1

7.7*

30.3

± 1

8.3*

35.8

± 2

2.1*

34.6

± 2

0.9*

3

mon

ths

9.4

± 9.

9

8.7

± 11

.2

5.

6 ±

5.5

3.

8 ±

4.1e,

f

0.00

5

6

mon

ths

9.5

± 10

.9

7.

6 ±

11.4

5.0

± 6.

0

3.4

± 3.

8g

0.00

8

12

mon

ths

10.1

± 1

1.5

9.

1 ±

16.7

4.7

± 6.

5

4.2

± 5.

0

0.02

3

# PP

D >

7 m

m

ba

selin

e

19

.7 ±

13.

4*

16

.5 ±

13.

9*

20

.2 ±

16.

5*

18

.9 ±

14.

5*

3 m

onth

s

4.

1 ±

5.4

3.

2 ±

4.3

1.

4 ±

2.5h

0.

9 ±

1.5i,

l

0.00

1

6

mon

ths

4.3

± 5.

7

3.0

± 6.

0

1.3

± 2.

6m

1.

1 ±

2.2n

0.

007

12 m

onth

s

4.

4 ±

6.5

4.

0 ±

9.4

1.

5 ±

2.5

1.

0 ±

2.9

0.

021

*Sig

nific

ant d

iffer

ence

bet

wee

n ba

selin

e an

d th

e ot

her f

ollo

w-u

p tim

e po

ints

(p <

0.0

01) (

AN

OV

A fo

r rep

eate

d m

easu

res)

. † A

NC

OV

A fo

r diff

eren

ces b

etw

een

grou

ps, a

djus

ted

for t

he c

orre

spon

ding

val

ue a

t bas

elin

e.

Lette

rs: P

airw

ise

com

paris

on b

etw

een

grou

ps (p

-val

ues a

djus

ted

for t

he c

orre

spon

ding

val

ue a

t bas

elin

e).

a, S

RP+

S+A

B v

s SR

P+D

IS, p

adj=

0.02

0

b, S

RP+

S+A

B v

s SR

P+D

IS, p

adj =

0.04

6

c, S

RP+

S+A

B v

s SR

P+D

IS, p

adj =

0.04

2 d,

SR

P+S+

AB

vs S

RP+

S, p

adj =

0.0

42

e,

SR

P+S+

AB

vs S

RP+

DIS

, pad

j =0.

023

f,

SRP+

S+A

B v

s SR

P+S,

pad

j =0.

021

g, S

RP+

S+A

B v

s SR

P+S,

pad

j =0.

016

h,

SR

P+D

IS+A

B v

s SR

P+S,

pad

j =0.

033

i,

SRP+

S+A

B v

s SR

P+D

IS, p

adj =

0.04

1 l,

SRP+

S+A

B v

s SR

P+S,

pad

j =0.

005

m, S

RP+

DIS

+AB

vs S

RP+

S, p

adj =

0.03

7

n, S

RP+

S +A

B v

s SR

P+S,

pad

j =0.

025

Abb

revi

atio

ns: s

ee T

able

s 1 a

nd 2

Chapter 4

90

Tabl

e 4.

Res

ults

at 1

2 m

onth

s of m

ain

effe

ct a

nd in

tera

ctio

n of

the

treat

men

ts w

ith a

ntib

iotic

s and

/or d

isin

fect

ion.

Val

ues a

re m

eans

± st

anda

rd

devi

atio

ns

D

IS+

D

IS-

p adj

va

lue*

A

B+

A

B-

p adj

va

lue†

p adj

va

lue¶

N

=52

N

=58

N

=54

N

=56

Prim

ary

outc

omes

CA

L (m

m)

ba

selin

e

4.

2 ±

1.0

4.3

± 1.

0

4.2

± 1.

1 4.

3 ±

0.8

12 m

onth

s

3.

6 ±

1.0

3.7

± 1.

0 0.

354

3.5

± 1.

0 3.

7 ±

1.0

0.07

8 0.

783

Seco

ndar

y ou

tcom

es

PP

D (m

m)

ba

selin

e

3.

8 ±

0.6

3.9

± 0.

6

3.9

± 0.

7 3.

8 ±

0.6

12 m

onth

s

2.

8 ±

0.5

2.9

± 0.

6 0.

641

2.7

± 0.

5 2.

9 ±

0.6

0.02

3 0.

877

PISA

(cm

2 )

base

line

17.2

± 6

.0

18.2

± 5

.4

18

.3 ±

6.4

17

.3 ±

4.9

12

mon

ths

4.7

± 3.

6 4.

7 ±

3.6

0.90

8 4.

0 ±

3.0

5.3

± 4.

0 0.

036

0.80

1

B

OP

(%)

ba

selin

e

66

.5 ±

14.

5 68

.1 ±

16.

0

68.7

± 1

5.9

66.1

± 1

4.8

12 m

onth

s

23

.1 ±

14.

7 23

.4 ±

15.

1 0.

926

22.0

± 1

5.0

24.4

± 1

4.7

0.19

9 0.

822

Plaq

ue (%

)

base

line

64.8

± 2

2.6

64.6

± 2

5.5

65

.5 ±

25.

6 64

.0 ±

22.

8

12

mon

ths

22.4

± 1

9.2

22.1

± 1

7.0

0.92

8 24

.4 ±

20.

1 20

.2 ±

15.

6 0.

229

0.58

7

#

PPD

>5

mm

ba

selin

e

48

.1 ±

24.

0 52

.3 ±

22.

9

52.3

± 2

5.0

48.4

± 2

2.0

12 m

onth

s

15

.5 ±

17.

0 16

.4 ±

14.

5 0.

901

13.0

± 1

0.9

18.9

± 1

8.9

0.00

7 0.

724

# PP

D >

6 m

m

base

line

33.0

± 2

0.2

34.3

± 1

9.2

35

.2 ±

21.

3 32

.2 ±

18.

0

12

mon

ths

7.0

± 12

.9

7.1

± 9.

1 0.

975

4.4

± 5.

7 9.

6 ±

14.1

0.

002

0.93

6

#

PPD

>7

mm

ba

selin

e

18

.3 ±

15.

1 19

.3 ±

13.

8

19.5

± 1

5.3

18.2

± 1

3.6

12 m

onth

s

2.

4 ±

3.6

2.5

± 4.

2 0.

927

1.1

± 2.

0 3.

7 ±

4.9

<0.0

01

0.46

5

* M

ain

effe

ct o

f tre

atm

ent w

ith lo

cal d

isin

fect

ion

with

NaO

Cl (

Two-

way

AN

OV

A)

† M

ain

effe

ct o

f tre

atm

ent w

ith a

ntib

iotic

s (Tw

o-w

ay A

NO

VA

) ¶ M

ain

effe

ct w

ith in

tera

ctio

n of

trea

tmen

ts w

ith a

ntib

iotic

s + d

isin

fect

ion

(Tw

o-w

ay A

NO

VA

)

Abb

revi

atio

ns: s

ee T

able

s 1 a

nd 2

Chapter 4

91

Plaq

ue (%

)

base

line

64.8

± 2

2.6

64.6

± 2

5.5

65

.5 ±

25.

6 64

.0 ±

22.

8

12

mon

ths

22.4

± 1

9.2

22.1

± 1

7.0

0.92

8 24

.4 ±

20.

1 20

.2 ±

15.

6 0.

229

0.58

7

#

PPD

>5

mm

ba

selin

e

48

.1 ±

24.

0 52

.3 ±

22.

9

52.3

± 2

5.0

48.4

± 2

2.0

12 m

onth

s

15

.5 ±

17.

0 16

.4 ±

14.

5 0.

901

13.0

± 1

0.9

18.9

± 1

8.9

0.00

7 0.

724

# PP

D >

6 m

m

base

line

33.0

± 2

0.2

34.3

± 1

9.2

35

.2 ±

21.

3 32

.2 ±

18.

0

12

mon

ths

7.0

± 12

.9

7.1

± 9.

1 0.

975

4.4

± 5.

7 9.

6 ±

14.1

0.

002

0.93

6

#

PPD

>7

mm

ba

selin

e

18

.3 ±

15.

1 19

.3 ±

13.

8

19.5

± 1

5.3

18.2

± 1

3.6

12 m

onth

s

2.

4 ±

3.6

2.5

± 4.

2 0.

927

1.1

± 2.

0 3.

7 ±

4.9

<0.0

01

0.46

5

* M

ain

effe

ct o

f tre

atm

ent w

ith lo

cal d

isin

fect

ion

with

NaO

Cl (

Two-

way

AN

OV

A)

† M

ain

effe

ct o

f tre

atm

ent w

ith a

ntib

iotic

s (Tw

o-w

ay A

NO

VA

) ¶ M

ain

effe

ct w

ith in

tera

ctio

n of

trea

tmen

ts w

ith a

ntib

iotic

s + d

isin

fect

ion

(Tw

o-w

ay A

NO

VA

)

Abb

revi

atio

ns: s

ee T

able

s 1 a

nd 2

Chapter 4

92

92

Table 5. Distribution of patients according to the frequency of numbers of residual pockets >6

mm at 12-month follow-up. Values are numbers of patients (%).

N=110 SRP+S SRP+DIS SRP+DIS+AB SRP+S+ABN=29 N=27 N=25 N=29 p-value*

Frequency of # PPD >6 mm

0.162

0 4 (13.8) 6 (22.2) 6 (24.0) 8 (27.6)

1-2 3 (10.3) 2 (7.4) 8 (32.0) 4 (13.8)

>3 22 (74.9) 19 (70.4) 11 (44.0) 17 (58.6)

*Fisher’s Exact Test.

Abbreviations: see Table 1 and 2 and.

93

Table 6. Results from microbiological analyses. Values are numbers of subjects (%) culture

positive for the targeted bacterial species and mean ± standard deviation of % Colonies

Forming Units in culture positive subjects.

SRP+S SRP+DIS SRP+DIS+AB SRP+S+ABN=29 N=27 N=25 N=29

Aa+ baseline 3 (10.3)1.1 ± 1.5

5 (18.5) 5.7 ± 6.0

2 (8.0)0.04 ± 0.04

6 (20.7)15.6 ± 36.6

3 months 3 (10.3)0.04 ± 0.02

4 (14.8)9.3 ± 12.9

0 (0.0) 0.0

2 (6.9)0.2 ± 0.2

6 months 2 (6.9)0.5 ± 0.6

4 (14.8)1.7 ± 2.9

1 (4.0) 3.0

3 (10.3)0.1 ± 0.2

12 months 2 (6.9)10.1 ± 12.6

2 (7.4)0.4 ± 0.2

2 (8.0)16.2 ± 22.2

4 (13.8)12.6 ± 22.9

Pg+ baseline 11 (37.9)31.7 ± 27.0

16 (59.3)35.7 ± 24.4

13 (52.0)25.9 ± 19.9

8 (27.6)34.1 ± 28.2

3 months 4 (13.8)12.7 ± 9.8

5 (18.5)3.2 ± 2.6

2 (8.0)6.2 ± 6.8

3 (10.3)3.5 ± 5.0

6 months 4 (13.8)9.7 ± 6.0

4 (14.8)21.3 ± 29.4

4 (16.0)5.5 ± 5.2

4 (13.8)6.6 ± 3.5

12 months 5 (17.2)17.7 ± 27.5

3 (11.1)20.3 ± 14.7

5 (20.0)9.8 ± 8.0

2 (6.9)1.9 ± 8.0

Pi+ baseline 18 (62.1)9.7 ± 17.5

11 (40.7)6.4 ± 8.0

11 (44.0)12.5 ± 12.9

14 (48.3)5.6 ± 6.8

3 months 13 (44.8)5.4 ± 5.5

8 (29.6)4.9 ± 5.4

3 (12.0)0.6 ± 0.7

9 (31.0)2.2 ± 2.4

6 months 11 (37.9)7.9 ± 5.8

5 (18.5)2.3 ± 1.8

6 (24.0)1.4 ± 1.6

11 (37.9)8.6 ± 17.0

12 months 15 (51.7)2.5 ± 1.5

6 (22.2)2.2 ± 1.9

9 (36.0)2.5 ± 1.5

13 (44.8)7.7 ± 11.1

Tf+ baseline 26 (89.7)8.0 ± 10.1

25 (92.6)4.9 ± 6.2

24 (96.0)8.8 ± 9.1

22 (75.9)5.7 ± 4.7

3 months 13 (44.8)1.9 ± 1.8

8 (29.6)2.0 ± 3.1

3 (12.0)*2.4 ± 2.7

7 (24.1)1.9 ± 1.3

6 months 14 (48.3)2.6 ± 2.1

12 (44.4)2.4 ± 3.3

5 (20.0)1.6 ± 0.9

11 (37.9)3.9 ± 6.5

12 months 14 (48.3)2.9 ± 2.6

11 (40.7)4.7 ± 5.7

9 (36.0)4.2 ±5.5

13 (44.8)4.8 ± 5.4

Pm+ baseline 25 (86.2)12.6 ± 12.4

26 (96.3)11.3 ± 13.8

24 (96.0)9.8 ± 9.5

23 (79.3)11.0 ± 9.1

3 months 25 (86.2)13.2 ± 12.9

19 (70.4)12.8 ± 16.5

14 (56.0)9.7 ± 10.9

13 (44.8)*9.7 ± 7.2

6 months 22 (75.9)12.5 ± 12.5

18 (66.7)18.0 ± 14.7

12 (48.0)10.7 ± 7.9

19 (65.5)7.1 ± 7.7

12 months 25 (86.2)13.6 ± 15.3

17 (63.0)16.5 ±17.5

20 (80.0)8.2 ± 8.1

14 (48.3)*10.3 ± 9.4

Chapter 4

93

92

Table 5. Distribution of patients according to the frequency of numbers of residual pockets >6

mm at 12-month follow-up. Values are numbers of patients (%).

N=110 SRP+S SRP+DIS SRP+DIS+AB SRP+S+ABN=29 N=27 N=25 N=29 p-value*

Frequency of # PPD >6 mm

0.162

0 4 (13.8) 6 (22.2) 6 (24.0) 8 (27.6)

1-2 3 (10.3) 2 (7.4) 8 (32.0) 4 (13.8)

>3 22 (74.9) 19 (70.4) 11 (44.0) 17 (58.6)

*Fisher’s Exact Test.

Abbreviations: see Table 1 and 2 and.

93

Table 6. Results from microbiological analyses. Values are numbers of subjects (%) culture

positive for the targeted bacterial species and mean ± standard deviation of % Colonies

Forming Units in culture positive subjects.

SRP+S SRP+DIS SRP+DIS+AB SRP+S+ABN=29 N=27 N=25 N=29

Aa+ baseline 3 (10.3)1.1 ± 1.5

5 (18.5) 5.7 ± 6.0

2 (8.0)0.04 ± 0.04

6 (20.7)15.6 ± 36.6

3 months 3 (10.3)0.04 ± 0.02

4 (14.8)9.3 ± 12.9

0 (0.0) 0.0

2 (6.9)0.2 ± 0.2

6 months 2 (6.9)0.5 ± 0.6

4 (14.8)1.7 ± 2.9

1 (4.0) 3.0

3 (10.3)0.1 ± 0.2

12 months 2 (6.9)10.1 ± 12.6

2 (7.4)0.4 ± 0.2

2 (8.0)16.2 ± 22.2

4 (13.8)12.6 ± 22.9

Pg+ baseline 11 (37.9)31.7 ± 27.0

16 (59.3)35.7 ± 24.4

13 (52.0)25.9 ± 19.9

8 (27.6)34.1 ± 28.2

3 months 4 (13.8)12.7 ± 9.8

5 (18.5)3.2 ± 2.6

2 (8.0)6.2 ± 6.8

3 (10.3)3.5 ± 5.0

6 months 4 (13.8)9.7 ± 6.0

4 (14.8)21.3 ± 29.4

4 (16.0)5.5 ± 5.2

4 (13.8)6.6 ± 3.5

12 months 5 (17.2)17.7 ± 27.5

3 (11.1)20.3 ± 14.7

5 (20.0)9.8 ± 8.0

2 (6.9)1.9 ± 8.0

Pi+ baseline 18 (62.1)9.7 ± 17.5

11 (40.7)6.4 ± 8.0

11 (44.0)12.5 ± 12.9

14 (48.3)5.6 ± 6.8

3 months 13 (44.8)5.4 ± 5.5

8 (29.6)4.9 ± 5.4

3 (12.0)0.6 ± 0.7

9 (31.0)2.2 ± 2.4

6 months 11 (37.9)7.9 ± 5.8

5 (18.5)2.3 ± 1.8

6 (24.0)1.4 ± 1.6

11 (37.9)8.6 ± 17.0

12 months 15 (51.7)2.5 ± 1.5

6 (22.2)2.2 ± 1.9

9 (36.0)2.5 ± 1.5

13 (44.8)7.7 ± 11.1

Tf+ baseline 26 (89.7)8.0 ± 10.1

25 (92.6)4.9 ± 6.2

24 (96.0)8.8 ± 9.1

22 (75.9)5.7 ± 4.7

3 months 13 (44.8)1.9 ± 1.8

8 (29.6)2.0 ± 3.1

3 (12.0)*2.4 ± 2.7

7 (24.1)1.9 ± 1.3

6 months 14 (48.3)2.6 ± 2.1

12 (44.4)2.4 ± 3.3

5 (20.0)1.6 ± 0.9

11 (37.9)3.9 ± 6.5

12 months 14 (48.3)2.9 ± 2.6

11 (40.7)4.7 ± 5.7

9 (36.0)4.2 ±5.5

13 (44.8)4.8 ± 5.4

Pm+ baseline 25 (86.2)12.6 ± 12.4

26 (96.3)11.3 ± 13.8

24 (96.0)9.8 ± 9.5

23 (79.3)11.0 ± 9.1

3 months 25 (86.2)13.2 ± 12.9

19 (70.4)12.8 ± 16.5

14 (56.0)9.7 ± 10.9

13 (44.8)*9.7 ± 7.2

6 months 22 (75.9)12.5 ± 12.5

18 (66.7)18.0 ± 14.7

12 (48.0)10.7 ± 7.9

19 (65.5)7.1 ± 7.7

12 months 25 (86.2)13.6 ± 15.3

17 (63.0)16.5 ±17.5

20 (80.0)8.2 ± 8.1

14 (48.3)*10.3 ± 9.4

Chapter 4

94

94

Fn+ baseline 22 (75.9)3.0 ± 4.8

21 (77.8)3.3 ± 3.4

23 (92.0)2.1± 2.0

23 (79.3)3.1 ± 3.3

3 months 27 (93.1)2.0 ± 2.6

24 (88.9)2.1 ± 4.4

19 (76.0)2.9 ± 3.8

21 (72.4)3.3 ± 3.4

6 months 21 (72.4)2.1 ± 3.2

21 (77.8)2.1 ± 4.4

16 (64.0)5.1 ± 4.8

19 (65.5)1.8 ± 1.6

12 months 25 (86.2)2.5 ± 2.8

19 (70.4)2.8 ± 3.3

22 (88.0)2.6 ± 3.9

25 (86.2)2.9 ± 3.3

Cr+ baseline 4 (13.8)2.7 ± 2.3

4 (14.8)3.9 ± 3.3

5 (20.0)15.1 ± 15.8

5 (17.2)5.3 ± 4.4

3 months 9 (31.0)3.0 ± 2.3

3 (11.1)0.6 ± 0.3

1 (4.0)(0.3)

3 (10.3)0.5 ± 0.3

6 months 7 (24.1)5.4 ± 4.7

6 (22.2)2.7 ± 1.6

5 (20.0)2.1 ± 2.0

4 (13.8)0.7 ± 0.2

12 months 5 (17.2)3.3 ± 3.2

5 (18.5)12.2 ± 15.8

5 (20.0)2.5 ± 2.7

6 (20.7)3.4 ± 4.2

*p < 0.0167 vs SRP (Fisher’s exact test).

Values in bold represent statistical significant differences within groups from baseline (p <

0.0167, McNemar test).

Abbreviations: see Table 1 and

Aa: Aggregatibacter actinomycetemcomitans

Pg: Porphyromonas gingivalis

Pi: Prevotella intermedia

Tf: Tannerella forsythia

Pm: Parvimonas micra

Fn: Fusobacterium nucleatum

Cr: Campylobacter rectus.

95

Table S1: Inclusion and exclusion criteria.

Inclusion 2 teeth per quadrant with pocket ≥5 mm with clinical evidence of

attachment loss

>50% bleeding on probing

≥20 teeth present

Exclusion Any systemic disease

Chronic use of medications

Use of systemic antibiotics in the last 6 months

Previous periodontal treatment in the last 2 years

Pregnancy/lactation

Presence of implants or orthodontic appliances

Chapter 4

95

94

Fn+ baseline 22 (75.9)3.0 ± 4.8

21 (77.8)3.3 ± 3.4

23 (92.0)2.1± 2.0

23 (79.3)3.1 ± 3.3

3 months 27 (93.1)2.0 ± 2.6

24 (88.9)2.1 ± 4.4

19 (76.0)2.9 ± 3.8

21 (72.4)3.3 ± 3.4

6 months 21 (72.4)2.1 ± 3.2

21 (77.8)2.1 ± 4.4

16 (64.0)5.1 ± 4.8

19 (65.5)1.8 ± 1.6

12 months 25 (86.2)2.5 ± 2.8

19 (70.4)2.8 ± 3.3

22 (88.0)2.6 ± 3.9

25 (86.2)2.9 ± 3.3

Cr+ baseline 4 (13.8)2.7 ± 2.3

4 (14.8)3.9 ± 3.3

5 (20.0)15.1 ± 15.8

5 (17.2)5.3 ± 4.4

3 months 9 (31.0)3.0 ± 2.3

3 (11.1)0.6 ± 0.3

1 (4.0)(0.3)

3 (10.3)0.5 ± 0.3

6 months 7 (24.1)5.4 ± 4.7

6 (22.2)2.7 ± 1.6

5 (20.0)2.1 ± 2.0

4 (13.8)0.7 ± 0.2

12 months 5 (17.2)3.3 ± 3.2

5 (18.5)12.2 ± 15.8

5 (20.0)2.5 ± 2.7

6 (20.7)3.4 ± 4.2

*p < 0.0167 vs SRP (Fisher’s exact test).

Values in bold represent statistical significant differences within groups from baseline (p <

0.0167, McNemar test).

Abbreviations: see Table 1 and

Aa: Aggregatibacter actinomycetemcomitans

Pg: Porphyromonas gingivalis

Pi: Prevotella intermedia

Tf: Tannerella forsythia

Pm: Parvimonas micra

Fn: Fusobacterium nucleatum

Cr: Campylobacter rectus.

95

Table S1: Inclusion and exclusion criteria.

Inclusion 2 teeth per quadrant with pocket ≥5 mm with clinical evidence of

attachment loss

>50% bleeding on probing

≥20 teeth present

Exclusion Any systemic disease

Chronic use of medications

Use of systemic antibiotics in the last 6 months

Previous periodontal treatment in the last 2 years

Pregnancy/lactation

Presence of implants or orthodontic appliances

Chapter 4

96

96

Table S2. Pairwise comparisons between treatment groups at the 12-month follow-up time

point. Parameters in the rows minus parameters in the columns. Values are patient mean

differences, based on adjusted means (95% CI) between treatment modalities from the

ANCOVA model (adjusted for the corresponding parameter at baseline).

SRP+DIS SPR+DIS+AB SRP+S+AB

CAL (mm)

SRP+S -0.069

(-0.466, 0.328)

0.089

(-0.315, 0.493)

0.216

(-0.172, 0.604)

SRP+DIS 0.158

(-0.252, 0.568)

0.285

(-0.110, 0.680)

SRP+DIS+AB 0.127

(-0.276, 0.568)

PPD (mm)

SRP+S 0.025

(-0.279, 0.329)

0.224

(-0.085, 0.533)

0.174

(-0.123, 0.471)

SRP+DIS 0.199

(-0.116, 0.514)

0.149

(-0.156, 0.453)

SRP+DIS+AB -0.050

(-0.360, 0.259)

PISA (cm2)

SRP+S 0.09

(-2.40, 2.58)

1.33

(-1.21, 3.86)

1.57

(-0.87, 4.01)

SRP+DIS 1.24

(-1.35, 3.82)

1.48

(-1.02, 3.99)

SRP+DIS+AB 0.24

(-2.29, 2.78)

BOP (%)

SRP+S 0.80

(-10.70, 9.10)

3.20

(-6.90, 13.30)

2.80

(-6.90, 12.60)

SRP+DIS 4.0

(-6.30, 14.30)

3.70

(-6.30, 13.60)

SRP+DIS+AB -0.30(-10.5, 9.80)

Plaque (%)

SRP+S 1.40 -4.0 -2.0

97

(-10.1, 12.9) (-15.70, 7.70) (-13.3, 9.30)

SRP+DIS -5.40

(-17.3, 6.60)

-3.40

(-14.90, 8.10)

SRP+DIS+AB 2.0

(-9.80, 13.70)

# PPD >5 mm

SRP+S 0.59

(-9.18, 10.37)

6.81

(-3.12, 16.74)

8.05

(-1.51, 17.62)

SRP+DIS 6.21

(-3.92, 16.36)

7.46

(- 2.35, 17.27)

SRP+DIS+AB 1.24

(-8.70, 11.18)

# PPD >6 mm

SRP+S 0.92

(-6.94, 7.13)

5.83

(-1.34, 12.99)

6.04

(-0.86, 12.93)

SRP+DIS 5.73

(-1.59, 13.05)

5.94

(- 1.10, 12.99)

SRP+DIS+AB 0.21

(-6.96, 7.38)

# PPD >7 mm

SRP+S -0.15

(-3.99, 3.70)

3.06

(-0.85, 6.97)

3.27

(-0.49, 7.04)

SRP+DIS 3.21

(-0.79, 7.20)

3.42

(- 0.42, 7.26)

SRP+DIS+AB 0.21

(-3.70, 4.13)

No statistical significant difference was found in any of the pairwise comparisons between the groups.

Abbreviations: see Table 1 and Table 2.

Chapter 4

97

96

Table S2. Pairwise comparisons between treatment groups at the 12-month follow-up time

point. Parameters in the rows minus parameters in the columns. Values are patient mean

differences, based on adjusted means (95% CI) between treatment modalities from the

ANCOVA model (adjusted for the corresponding parameter at baseline).

SRP+DIS SPR+DIS+AB SRP+S+AB

CAL (mm)

SRP+S -0.069

(-0.466, 0.328)

0.089

(-0.315, 0.493)

0.216

(-0.172, 0.604)

SRP+DIS 0.158

(-0.252, 0.568)

0.285

(-0.110, 0.680)

SRP+DIS+AB 0.127

(-0.276, 0.568)

PPD (mm)

SRP+S 0.025

(-0.279, 0.329)

0.224

(-0.085, 0.533)

0.174

(-0.123, 0.471)

SRP+DIS 0.199

(-0.116, 0.514)

0.149

(-0.156, 0.453)

SRP+DIS+AB -0.050

(-0.360, 0.259)

PISA (cm2)

SRP+S 0.09

(-2.40, 2.58)

1.33

(-1.21, 3.86)

1.57

(-0.87, 4.01)

SRP+DIS 1.24

(-1.35, 3.82)

1.48

(-1.02, 3.99)

SRP+DIS+AB 0.24

(-2.29, 2.78)

BOP (%)

SRP+S 0.80

(-10.70, 9.10)

3.20

(-6.90, 13.30)

2.80

(-6.90, 12.60)

SRP+DIS 4.0

(-6.30, 14.30)

3.70

(-6.30, 13.60)

SRP+DIS+AB -0.30(-10.5, 9.80)

Plaque (%)

SRP+S 1.40 -4.0 -2.0

97

(-10.1, 12.9) (-15.70, 7.70) (-13.3, 9.30)

SRP+DIS -5.40

(-17.3, 6.60)

-3.40

(-14.90, 8.10)

SRP+DIS+AB 2.0

(-9.80, 13.70)

# PPD >5 mm

SRP+S 0.59

(-9.18, 10.37)

6.81

(-3.12, 16.74)

8.05

(-1.51, 17.62)

SRP+DIS 6.21

(-3.92, 16.36)

7.46

(- 2.35, 17.27)

SRP+DIS+AB 1.24

(-8.70, 11.18)

# PPD >6 mm

SRP+S 0.92

(-6.94, 7.13)

5.83

(-1.34, 12.99)

6.04

(-0.86, 12.93)

SRP+DIS 5.73

(-1.59, 13.05)

5.94

(- 1.10, 12.99)

SRP+DIS+AB 0.21

(-6.96, 7.38)

# PPD >7 mm

SRP+S -0.15

(-3.99, 3.70)

3.06

(-0.85, 6.97)

3.27

(-0.49, 7.04)

SRP+DIS 3.21

(-0.79, 7.20)

3.42

(- 0.42, 7.26)

SRP+DIS+AB 0.21

(-3.70, 4.13)

No statistical significant difference was found in any of the pairwise comparisons between the groups.

Abbreviations: see Table 1 and Table 2.

Chapter 4

98

98

Table S3. Periodontal status at baseline and three months of patients assigned to

the three therapists.

Therapist 1 Therapist 2 Therapist 3 p value

N=37 N=30 N=43

Primary outcomes

CAL (mm) baseline 3.9 ± 1.1* 4.4 ± 0.9 4.4 ± 0.8 0.03212 months 3.7 ± 1.1 3.6 + 0.9 3.5 ± 1.2 0.912

Secondary outcomes

PPD (mm) baseline 3.6 ± 0.6* 4.0 ± 0.6 4.0 ± 0.5 0.00112 months 2.8 ± 0.5 2.9 ± 0.7 2.8 ± 0.5 0.490

PISA (cm2) baseline 15.5 ± 5.8* 19.0 ± 6.6 18.8 ± 4.3 0.01312 months 5.0 ± 2.9 5.5 ± 4.8 3.8 ± 2.9 0.116

BOP (%) baseline 66.4 ± 16.1 68.3 ± 14.9 67.5 ± 15.1 0.88212 months 26.6 ± 15.7 23.9 ± 16.5 19.9 ± 12.2 0.129

Plaque (%) baseline 73.8 ± 17.6† 57.1 ± 26.6 62.2 ± 25.1 0.01212 months 25.2 ± 16.4 21.7 ± 19.1 20.1 ± 18.6 0.446

# PPD >5 mm baseline 38.8 ± 23.3* 57.0 ± 24.4 55.6 ± 19.4 0.00112 months 14.7 ± 13.3 20.6 ± 22.8 13.8 ± 10.3 0.159

# PPD >6 mm baseline 22.3 ± 17.1* 40.3 ± 22.5 38.8 ± 15.1 <0.00112 months 6.2 ± 7.8 10.7 ± 17.5 5.2 ± 6.5 0.096

# PPD >7 mm baseline 12.4 ± 12.6 23.7 ± 17.3 21.0 ± 11.7 0.00212 months 2.4 ± 3.9 4.4 ± 9.9 3.6 ± 0.5 0.207

One way-ANOVA

*significant different from Therapist 2 and 3 (Bonferroni correction)

† significant different from Therapist 2 (Bonferroni correction)

99

Table S4. Distribution of patients who complied with the medications and who presented

adverse effects of adjunctive therapies. Values are numbers of patients (%).

SRP+S SRP+DIS SRP+DIS+AB SRP+S+AB

N=29 N=27 N=25 N=29

Compliance with Antibiotics N/A N/A 25 (100) 29 (100)

Compliance with Chlorhexidine 29 (100) 29 (100) 22 (88.0) 26 (89.7)

Adverse effects of NaOCl (DIS) N/A 0 0 N/A

Adverse effects antibiotics N/A N/A 4 (16.0) 8 (27.5)

Adverse effects chlorhexidine 12 (41.3) 10 (37.0) 11 (44.0) 8 (27.5)1 adverse effect 7 (24.1) 6 (22.2) 9 (36.0) 5 (17.2)

>1 adverse effects 5 (17.2) 4 (14.8) 2 (8.0) 3 (10.3)

Abbreviations: see Table 1 and

N/A = non applicable.

Chapter 4

99

98

Table S3. Periodontal status at baseline and three months of patients assigned to

the three therapists.

Therapist 1 Therapist 2 Therapist 3 p value

N=37 N=30 N=43

Primary outcomes

CAL (mm) baseline 3.9 ± 1.1* 4.4 ± 0.9 4.4 ± 0.8 0.03212 months 3.7 ± 1.1 3.6 + 0.9 3.5 ± 1.2 0.912

Secondary outcomes

PPD (mm) baseline 3.6 ± 0.6* 4.0 ± 0.6 4.0 ± 0.5 0.00112 months 2.8 ± 0.5 2.9 ± 0.7 2.8 ± 0.5 0.490

PISA (cm2) baseline 15.5 ± 5.8* 19.0 ± 6.6 18.8 ± 4.3 0.01312 months 5.0 ± 2.9 5.5 ± 4.8 3.8 ± 2.9 0.116

BOP (%) baseline 66.4 ± 16.1 68.3 ± 14.9 67.5 ± 15.1 0.88212 months 26.6 ± 15.7 23.9 ± 16.5 19.9 ± 12.2 0.129

Plaque (%) baseline 73.8 ± 17.6† 57.1 ± 26.6 62.2 ± 25.1 0.01212 months 25.2 ± 16.4 21.7 ± 19.1 20.1 ± 18.6 0.446

# PPD >5 mm baseline 38.8 ± 23.3* 57.0 ± 24.4 55.6 ± 19.4 0.00112 months 14.7 ± 13.3 20.6 ± 22.8 13.8 ± 10.3 0.159

# PPD >6 mm baseline 22.3 ± 17.1* 40.3 ± 22.5 38.8 ± 15.1 <0.00112 months 6.2 ± 7.8 10.7 ± 17.5 5.2 ± 6.5 0.096

# PPD >7 mm baseline 12.4 ± 12.6 23.7 ± 17.3 21.0 ± 11.7 0.00212 months 2.4 ± 3.9 4.4 ± 9.9 3.6 ± 0.5 0.207

One way-ANOVA

*significant different from Therapist 2 and 3 (Bonferroni correction)

† significant different from Therapist 2 (Bonferroni correction)

99

Table S4. Distribution of patients who complied with the medications and who presented

adverse effects of adjunctive therapies. Values are numbers of patients (%).

SRP+S SRP+DIS SRP+DIS+AB SRP+S+AB

N=29 N=27 N=25 N=29

Compliance with Antibiotics N/A N/A 25 (100) 29 (100)

Compliance with Chlorhexidine 29 (100) 29 (100) 22 (88.0) 26 (89.7)

Adverse effects of NaOCl (DIS) N/A 0 0 N/A

Adverse effects antibiotics N/A N/A 4 (16.0) 8 (27.5)

Adverse effects chlorhexidine 12 (41.3) 10 (37.0) 11 (44.0) 8 (27.5)1 adverse effect 7 (24.1) 6 (22.2) 9 (36.0) 5 (17.2)

>1 adverse effects 5 (17.2) 4 (14.8) 2 (8.0) 3 (10.3)

Abbreviations: see Table 1 and

N/A = non applicable.

Chapter 4

100

100

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metronidazole as an adjunct to full-mouth scaling and root planing of chronic

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chronic periodontitis: a clinical, microbiological and immunological study. Journal of

Clinical Periodontology 35, 789-798.

Ehmke, B., Moter, A., Beikler, T., Milian, E. & Flemmig, T. F. (2005) Adjunctive

antimicrobial therapy of periodontitis: long-term effects on disease progression and

oral colonization. Journal of Periodontology 76, 749-759.

Elashoff, R. M., Li, G. & Li, N. (2008) A joint model for longitudinal measurements and

survival data in the presence of multiple failure types. Biometrics 64, 762-771.

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non-surgical treatment of chronic periodontitis: a 1-year double-blinded, placebo-

controlled, randomized clinical trial. Journal of Clinical Periodontology 39, 1149-

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Feres, M., Figueiredo, L. C., Silva Soares, G. M., Faveri, M. (2015) Systemic antibiotics in

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Clinical Periodontology 25, 380-387.

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pocket depth reduction at treated sites. Journal of Clinical Periodontology 39, 526-

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Lobene, R. R., Soparkar, P. M., Hein, J. W. & Quigley, G. A. (1972) A study of the effects of

antiseptic agents and a pulsating irrigating device on plaque and gingivitis. Journal of

Periodontology 43, 564-568.

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metronidazole as an adjunct to full-mouth scaling and root planing of chronic

periodontitis. Journal of Periodontology 80, 364-371.

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Casati, M. Z., (2008) Periodontal debridment as a therapeutic approach for severe

chronic periodontitis: a clinical, microbiological and immunological study. Journal of

Clinical Periodontology 35, 789-798.

Ehmke, B., Moter, A., Beikler, T., Milian, E. & Flemmig, T. F. (2005) Adjunctive

antimicrobial therapy of periodontitis: long-term effects on disease progression and

oral colonization. Journal of Periodontology 76, 749-759.

Elashoff, R. M., Li, G. & Li, N. (2008) A joint model for longitudinal measurements and

survival data in the presence of multiple failure types. Biometrics 64, 762-771.

Feres, M., Soares, G. M., Mendes, J. A., Silva, M. P., Faveri, M., Teles, R., Socransky, S. S.

& Figueiredo, L. C. (2012) Metronidazole alone or with amoxicillin as adjuncts to

non-surgical treatment of chronic periodontitis: a 1-year double-blinded, placebo-

controlled, randomized clinical trial. Journal of Clinical Periodontology 39, 1149-

1158.

Feres, M., Figueiredo, L. C., Silva Soares, G. M., Faveri, M. (2015) Systemic antibiotics in

the treatment of periodontitis. Periodontology 2000 67, 131-186.

Flemmig, T. F., Milian, E., Karch, H. & Klaiber, B. (1998) Differential clinical treatment

outcome after systemic metronidazole and amoxicillin in patients harboring

Actinobacillus actinomycetemcomitans and/or Porphyromonas gingivalis. Journal of

Clinical Periodontology 25, 380-387.

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pilot study. Journal of Periodontal Research 49, 696-702.

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Goodson, J. M., Haffajee, A. D., Socransky, S. S., Kent, R., Teles, R., Hasturk, H., Bogren,

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pocket depth reduction at treated sites. Journal of Clinical Periodontology 39, 526-

536.

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periodontitis and health revealed by 16S pyrosequencing. International Society for

Microbial Ecology Journal 6, 1176-1185.

Groenwold, R. H., Donders, A. R., Roes, K. C., Harrell, F. E., Jr. & Moons, K. G. (2012)

Dealing with missing outcome data in randomized trials and observational studies.

American Journal of Epidemiology 175, 210-217.

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Periodontology 43, 564-568.

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subgingival microbiota following scaling in deep pockets. Journal of Clinical

Periodontology 11, 193-207.

Mombelli, A., McNabb, H. & Lang, N. P. (1991) Black-pigmenting gram-negative bacteria in

periodontal disease. II. Screening strategies for detection of P. gingivalis. Journal of

Periodontal Research 26, 308-313.

Nesse, W., Abbas, F., van der Ploeg, I., Spijkervet, F. K., Dijkstra, P. U. & Vissink, A. (2008)

Periodontal inflamed surface area: quantifying inflammatory burden. Journal of

Clinical Periodontology 35, 668-673.

Oosterwaal, P. J., Mikx, F. H. & Renggli, H. H. (1990) Clearance of a topically applied

fluorescein gel from periodontal pockets. Journal of Clinical Periodontology 17, 613-

615.

Pavicic, M. J., van Winkelhoff, A. J., Douque, N. H., Steures, R. W. & de Graaff, J. (1994)

Microbiological and clinical effects of metronidazole and amoxicillin in

Actinobacillus actinomycetemcomitans-associated periodontitis. A 2-year evaluation.

Journal of Clinical Periodontology 21, 107-112.

Perova, M. D., Lopunova Zh, K., Banchenko, G. V. & Petrosian, E. A. (1990) [A clinico-

morphological assessment of the efficacy of sodium hypochlorite in the combined

therapy of periodontitis]. Stomatologiia (Mosk) 69, 23-26.

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CHAPTER 5

Biomarkers of metabolic syndrome during periodontal therapy:

a 12-month observational study.

Sergio Bizzarro1, Ubele van der Velden1, Wijnand J. Teeuw1, Victor E. A. Gerdes2, Bruno G. Loos1

1Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of

Amsterdam and VU University Amsterdam, 1081LA, Amsterdam, the Netherlands.2Department of Internal Medicine, Slotervaart Hospital, 1066EC, Amsterdam, the Netherlands

Submitted