Journal Pre-proof

A comparative clinical, microbiological and glycemic analysis of

photodynamic therapy and Lactobacillus reuteri in the treatment of
chronic periodontitis in type-2 diabetes mellitus patients

Mohamed Farouk Elsadek, Badreldin Mohamed Ahmed, Daniyah

Mohammed Alkhawtani, Adel Zia Siddiqui

PII: S1572-1000(19)30574-5
DOI: https://doi.org/10.1016/j.pdpdt.2019.101629
Reference: PDPDT 101629

To appear in: Photodiagnosis and Photodynamic Therapy

Received Date: 18 September 2019

Revised Date: 3 December 2019
Accepted Date: 18 December 2019

Please cite this article as: Elsadek MF, Ahmed BM, Alkhawtani DM, Zia Siddiqui A, A
comparative clinical, microbiological and glycemic analysis of photodynamic therapy and
Lactobacillus reuteri in the treatment of chronic periodontitis in type-2 diabetes mellitus
patients, Photodiagnosis and Photodynamic Therapy (2019),
doi: https://doi.org/10.1016/j.pdpdt.2019.101629

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Title page:

A comparative clinical, microbiological and glycemic analysis of photodynamic therapy

and Lactobacillus reuteri in the treatment of chronic periodontitis in type-2 diabetes

mellitus patients

Mohamed Farouk Elsadek1,2, Badreldin Mohamed Ahmed1, Daniyah Mohammed

Alkhawtani1, Adel Zia Siddiqui3

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Authors Details:

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1
Department of Community Health Sciences, College of Applied Medical Sciences, King Saud

University, Saudi Arabia.

2
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Nutrition and Food Science Department, Helwan University, Egypt.
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3
Dental Materials Department, Baqai Dental College, Baqai Medical University, Karachi,

Pakistan.
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Short title: PDT and probiotics in periodontitis and DM

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Corresponding Author:
Dr Mohamed Farouk Elsadek
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Department of Community Health Sciences, College of Applied Medical Sciences, King Saud

University, Saudi Arabia; Nutrition and Food Science Department, Helwan University, Egypt.

E-mail: mofaelsadek2@gmail.com

HIGHLIGHTS
  PDT shows additional benefit in deep periodontal pockets.
 PDT shows significant reduction in HbA1c levels in DM.
 PDT and PT showed equal efficacy in terms of clinical periodontal and bacterial
levels.

ABSTRACT

Background: Evidence indicates that patients with type 2 diabetes mellitus (DM) exhibit

impaired wound healing and are at higher risk for periodontal disease. In DM patients,

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adjunctive periodontal treatment provides small but statistically significant benefits in terms of

reductions in clinical periodontal parameters. This study incorporates antimicrobial and

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immune modulatory treatment approach specifically targeted at these patients.
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Purpose: To evaluate and compare the clinical periodontal, microbiological and HbA1c levels

with the use of photodynamic therapy (PDT) and probiotic therapy (PT) as adjunct to root
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surface debridement (RSD) in the treatment of periodontitis in DM.
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Materials and Methods: Demographic data was collected using a questionnaire. Treatment-

wise, chronic periodontitis with 2DM patients were subdivided into: (i) Group-A: Patients that
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underwent RSD with adjunct PDT; (ii) Group-B: Patients that underwent RSD with adjunct PT

and; (iii) Group-C: RSD alone. In all groups, probing depth (PD), plaque scores (PS), bleeding
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on probing (BOP) and clinical attachment level (CAL) gain were measured at baseline and 3

months. Microbiological data consisted the assessment of detection percentage of

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Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola.

Results: A total of 58 patients completed the trial. All clinical and microbiological parameters

showed statistically significant reduction from baseline to 3 months in all groups (p<0.05). On

inter-group comparison, the proportion of PD with ≥4 mm and ≥5 mm showed statistically

significant reduction for Group-A compared to Group-B and Group-C (p<0.01). Only Group-

A showed statistically significant reduction in percentage of HbA1c levels from baseline to 3

months (p<0.05). Groups A and B showed significantly higher reductions for all the three

bacteria compared to Group-C (p<0.05). However, this reduction was comparable between

Groups-A and B, respectively (p>0.05).

Conclusion: PDT showed additional benefit in deep periodontal pockets and slightly modest

reduction in HbA1c levels in DM patients. Further clinical trials are required with large

sample size and longer follow up duration to ascertain the findings of the present clinical

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study.

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Keyword: Photodynamic therapy; probiotic, chronic periodontitis, probing depth
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1. Introduction
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There is a rapid surge in the prevalence of type-2 diabetes mellitus (DM) in the world and it is

indicated that DM is the ninth major cause of mortality [1]. The disease is manifested by a lack
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of production and/or function of insulin from the pancreatic cells or the inability of the body to

respond to the insulin (insulin resistance). Being a global epidemic, DM is associated with
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certain systemic complications including cardiovascular diseases, bone diseases, kidney

disorders and cerebral problems [2, 3].

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Ample data suggests that DM is one of the classical risk factors for chronic periodontitis

[4, 5]. Most cross-sectional studies evaluating the association between DM and periodontal

diseases have unequivocally demonstrated that clinical periodontal parameters or periodontal

disease risk disproportionally increases with rising serum levels of glycated hemoglobin
(HbA1c) [6, 7]. The underlying pathogenic mechanisms explaining the diabetes-periodontitis

interaction is that both chronic diseases are driven by enhanced inflammation and altered

immune responses [8, 9]. Raised serum levels of proinflammatory cytokines are unfailingly

observed in both the diseases [10]. The formation of advanced glycation end products that are

synthesized by oxidation of tissue proteins and lipids and nonenzymatic glycation is

particularly increased in chronic hyperglycemia which may provide additional connections of

DM and periodontitis [11-13].

The common bidirectional relationship between DM and periodontal diseases spurred

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health care professionals to seek for treating periodontal diseases in DM. There is low to

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moderate evidence on the effect of non-surgical periodontal therapy on periodontal measures

and HbA1c in DM-periodontitis patients [14-16]. These studies considered non-surgical

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periodontal therapy in the form of root surface debridement (RSD) as their gold standard
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treatment. However, RSD does not show its high efficacy in sites related to molar teeth or sites

with deep probing depth (PD) [17]. For increased efficacy of RSD, various adjunctive therapies
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have been tested including local/systemic antimicrobials, photodynamic therapy (PDT) and

probiotic therapy (PT) [18-20]. Photodynamic therapy involves a light of specific wavelength
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and power density to eradicate the microbial count in patients with deep PD [21]. On the other

hand, probiotics are live microorganisms that modulate the composition of newly formed oral
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microbes and conferring other health benefits to the host [22]. It is important to determine what

effects periodontitis and its treatment may have on diabetes. This assumption is based on
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studies that observed an improvement in diabetes glycemic control following PDT, but the

evidence is still debatable [23]. Similarly, probiotic therapy may be a targeted therapy in DM

that can modify the immune host response by competitive exclusion of harmful bacteria [24].

This needs further clinical trials as studies in this area are scarce.
 Evidence indicates that patients with DM exhibit impaired wound healing and are at

higher risk for periodontal disease. In DM patients, adjunctive periodontal treatment provides

small but statistically significant benefits in terms of reductions in clinical periodontal

parameters. This study incorporates antimicrobial and immune-modulatory treatment

approaches specifically targeted at these patients. Various prospective studies based on these

adjuncts have suggested that PDT and PT as an adjunct to RSD showed higher efficacy as

compared to RSD alone [25, 26]. However, the comparison of adjunctive therapies including

the use of PDT and PT in reducing HbA1c and periodontal parameters together have not been

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tested before. Thus the aim of this present 3 months prospective clinical trial was to evaluate

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and compare the clinical, microbiological and HbA1c levels with the use of PDT and

Lactobacillus reuteri (L. reuteri) as PT as an adjunct to RSD in the treatment of periodontitis

in DM.
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2. Materials and Methods
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2.1. Patient selection

A total of 60 patients (mean age: 48.6 ± 6.5) with known DM [27] were included in the study.
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All consecutive patients recruited were requested to sign an informed consent. The recruited

participants met the following inclusion criteria: Patients with age bracket 35–75 years; with
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diagnosis of stage III and grade C generalized periodontitis (interdental clinical attachment loss
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[CAL] ≥5 mm and radiographic bone loss extending to middle or apical third of root) [28];

presence of at least 18 teeth in the mouth except third molars. The exclusion criteria for

recruitment entailed: pregnant or lactating mothers; patients with previous periodontal therapy

of any form; systemic disease(s) (other than DM) that influence periodontal disease course;
patients on anti-inflammatory/antimicrobials/statin therapy; patients with history of

smoking/current smokers; and refusal to give signed consent.

2.2. Study groups

Sixty patients were divided into three groups based on the treatment provided: Group-A;

Patients were rendered methylene blue mediated PDT with RSD, Group-B; Patients

administered with probiotic and RSD, and Group-C; Patients rendered with RSD alone.

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2.3. Study design

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Demographic data were collected using a questionnaire. All recruited patients underwent

history taking, blood and plaque sample analysis, full-mouth periapical radiographs,
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motivational interviewing on oral hygiene measures with toothbrush, interproximal brushes,

dental floss and a toothpaste as gift. All patients were given supragingival prophylaxis. In
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addition, all patients were instructed to continue with oral hypoglycemic drugs and seeing their

physician with no modifications in the diet and lifestyle.

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All patients underwent four 1-hour sessions of RSD using Gracey curettes (Hu-Friedy®

Manufacturing Inc., Chicago, IL, USA) and ultrasonic scaler machine (Acteon Suprasson
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Xinetic Scaler, Saudi Arabia). Each patient was treated over a maximum of 4 weeks period.
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Patients were followed up twice at 3 months. All metabolic, periodontal, microbiological

assessments and supragingival prophylaxis techniques were repeated, oral hygiene care was

reinforced and new oral hygiene samples distributed.

2.4. Periodontal measurements

All patients underwent full-mouth clinical periodontal assessment around all teeth but

excluding third molars by a single calibrated examiner. The measurements were taken at six

sites per tooth (distofacial, mesiofacial, facial, distolingual, mesiolingual and lingual) using a

standard North Carolina periodontal probe (Hu-Friedy® Manufacturing Inc.). Clinical variables

included the measurements of plaque scores (PS) [29], bleeding on probing (BOP), PD,

gingival recession and clinical attachment level (CAL). To avoid any bias, the periodontal

examiner was not involved in periodontal treatment.

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2.5. Treatments

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Phenazathionium chloride trihydrate (methylene blue) with a concentration of 0.005% (w/v)

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was used as photosensitizer (Periowave™ Treatment Kit, Ondine Biopharma Corporation,

Vancouver, BC, Canada). The photosensitizer was suspended in phosphate-buffered saline

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along with mucoadhesive viscosity agent (hydroxymethylcellulose). The photosensitizer was

applied directly into the worst pocket using a blunt-ended 23 gauge side-port irrigating needle
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(Dentsply, York, PA, USA) that allowed complete irrigation until the base of the pocket. A 670

nm wavelength and 150 mW maximum power diode laser (Periowave™) was used. An
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autoclavable handpiece was used through which a flexible optic fiber cable was attached and

accommodated a light-diffusing tip (configured similar to a periodontal probe) for access to

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periodontal PD. A uniform light was emitted from the most apical part of the tip that allowed
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complete light distribution throughout the periodontal pocket for 60 seconds per site and power

density of 20 J/cm2 per site. The tip was gently moved from apical to the coronal direction in

the pocket.

The patients in Group-B received probiotic tablets in the form of two strains L. reuteri

at a standard dose of 2 x 108 CFU/tablet (DSM-17938 and ATCC PTA5289). Subsequently,

the patients were instructed to use two lozenges for over 24 hours for 3 weeks. Patients were

instructed to use the lozenges after every tooth brushing [30].

2.6. Metabolic recordings

For the confirmatory purpose, all metabolic parameters were assessed to confirm the diabetic

status. For this study, only the serum levels of HbA1c were considered and analyzed for data.

The normal levels ranging from 4.8 to 6% were considered normal. Serum levels of HbA1c

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were quantified using high-resolution liquid chromatography with L-9100 hemoglobin

analyzer (Hitachi, Merck, Japan).

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2.7. Microbiological procedures – PCR assays
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For plaque sampling, teeth were isolated with cotton rolls and air-dried. Plaque samples were

taken from the deepest pocket using sterile Gracey curettes. Samples were treated in the
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laboratory for DNA extraction protocol as described by Doyle and Doyle [31] and estimated

in a spectrophotometer at wavelength set at 260 nm and stored at -20 ºC until PCR analysis.
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The technique involves the samples to be mixed in lysing solution and later purified with

chloroform:isoamil-alcohol. Later, DNA precipitation was carried out using isopropanol and
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75% ethanol. The precipitated DNA was resuspended in TE buffer solution. PCR was

performed to identify microbial molecule using specific primers for Porphyromonas gingivalis
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(P. gingivalis), Tannerella forsythia (T. forsythia) and Treponema denticola (T. denticola)

(Table 1). Following conditions were set for PCR amplification: P. gingivalis = an initial

denaturation step at 94ºC for 5 minutes, followed by 30 cycles of denaturation at 94ºC for 60

s, annealing at 70ºC for 60 s, an extension at 72ºC for 60 s, and a final elongation step at 72ºC

for 2 minutes; T. forsythia = an initial denaturation step at 95ºC for 2 minutes, followed by 36
cycles of denaturation and extension at 95ºC for 30 seconds, annealing at 60ºC for 60 s, an

extension at 72ºC for 60 s, and a final elongation step at 72ºC for 2 minutes; T. denticola = an

initial denaturation step at 95ºC for 2 minutes, followed by 32 cycles of denaturation at 95ºC

for 30 seconds, annealing at 60ºC for 60 s, an extension at 72ºC for 1 minute, and a final

elongation step at 72ºC for 2 minutes. The PCR products were analyzed using the

electrophoresis technique in 1.75% agarose gel using Tris-borate-EDTA buffer. A total of 1-

kilobase DNA ladder was included in each gel. Lastly, each DNA was stained using ethidium

bromide and visualized under ultraviolet illumination.

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2.8. Statistics

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Statistical analysis was performed using statistical software (SPSS v20, Chicago, IL. USA).

All tests were computed on patient-level data and not site-level. Computing of normality test
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using Shapiro-Wilk indicated that microbiological (detection frequency of P. gingivalis, T.

forsythia, and T. denticola) and metabolic (HbA1c levels) variables were normally distributed.
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The normality testing for clinical periodontal data indicated non-normal distribution. Means

and standard deviations were reported for clinical and HbA1c data for each time point. Chi-
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square test and one-way analysis of variance (ANOVA) (group factor: PDT/PT/RSD; time

factor: baseline and 3 months) with repeated measures at periods was computed for
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microbiological variables. Two-way repeated measure ANOVA with a post hoc test was used
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for comparing the change of clinical parameters (PD, CAL, the proportion of PD >4 mm, or

proportion of PD >5 mm) after the treatments (PDT/PT/RSD). Intragroup differences were

compared performing a paired t-test. P-value <0.05 were reported as significant.

3. Results
Demographic characteristics are described in Table 2. One patient each (both refused to return

for follow-up assessments) lost to follow up from both Group-B and Group-C, respectively.

None of the patient's complaint of side effects from either PDT, PT or RSD. The mean age of

patients in Groups A, B and C were 52.16 years, 51.87 years and 52.88 years, respectively.

Generally, males were higher in number among groups. The mean duration of diabetes ranged

from 15.7 year to 18.2 years among groups. The diabetic therapy consisted of diet and exercise,

oral hypoglycemic drugs, insulin therapy and a combination of oral hypoglycemics and insulin.

The majority of patients took oral hypoglycemic drugs for antidiabetic therapy.

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Mean clinical periodontal and HbA1c levels of the study groups are shown in Table 3.

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It is noted that all the variables were statistically comparable at baseline. Plaque scores and

BOP showed a statistically significant reduction from baseline to 3 months post-treatment

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(p<0.05). Mean PD, proportions of PD with ≥4 mm and ≥5 mm and CAL also showed
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statistically significant improvement in all groups at 3 months post-treatment. On inter-group

comparison, the proportion of PD with ≥4 mm and ≥5 mm showed a statistically significant

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reduction for Group-A (PDT + RSD) compared to Group-B and Group-C (p=0.006). The

percentage of HbA1c showed decreased from baseline to 3 months post-treatment. This

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reduction was statistically significant in Group-A only (p=0.039).

Microbiological data is plotted in Figure 1. All groups showed a significant reduction

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in the detection frequency of P. gingivalis, T. forsythia, and T. denticola. Group A showed

significantly higher reductions for P. gingivalis (p=0.016), T. forsythia (p=0.0021) and T.

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denticola (p=0.037) compared with Group C. Similarly, Group B showed significantly higher

reductions for P. gingivalis (p=0.042), T. forsythia (p=0.0013) and T. denticola (p=0.027)

compared with Group C. However, this reduction was comparable between Groups-A and B,

respectively (p>0.05).
4. Discussion

To the authors' information from the online database, this is the first study that evaluates and

compares the clinical efficacy of PDT and probiotic therapy as an adjunct to non-surgical

periodontal debridement in the treatment of chronic periodontitis among type 2 DM patients.

The outcomes of the present 3 months prospective clinical trial showed that PDT and probiotic

showed a significant reduction in the clinical periodontal parameters and detection frequency

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of bacteria. However, PDT showed additional benefit in deep periodontal pockets and slightly

modest reduction in HbA1c levels.

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The results of the present study corroborate with a recent study that evaluated the

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clinical periodontal measurements and serum HbA1c levels among chronic periodontitis

patients with type 2 DM [19, 25]. A study by Al-Zahrani et al. evaluated and compared three
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treatment modalities including PDT, antimicrobial and scaling and root planing alone [23].

They reported that the use of systemic doxycycline significantly improved the levels of HbA1c
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in DM patients. This finding could be considered an important finding that shows antibiotic

therapy may be used to reduce HbA1c levels in diabetic patients. However, in our study, PDT
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showed a significant reduction in serum HbA1c compared to probiotic therapy and RSD alone.

There could be two possible explanations for this. Firstly, the level of baseline serum HbA1c
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levels in Group-A was not as much as compared to the patients in Group-B and C. Secondly,
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the duration of diabetes was comparatively lesser among the same group of patients. In addition

to this, the levels of serum HbA1c may have reduced further through any non-modification of

the dietary habits and regular exercise with the intake of anti-hypoglycemic drugs in Group-A.

Some important findings need to be highlighted. It is observed that the proportions of

PD with ≥5 mm statistically significantly reduced in the PDT group compared to probiotic

therapy and mechanical debridement alone. This finding is mainly translated by the significant

reduction of the pathogenic bacteria (including P. gingivalis, T. forsythia, and T. denticola) in

this study which is involved in the pathogenesis of the chronic periodontitis. In classical

periodontitis infection, the range of microbiota increases as the disease advances and

progresses and it is these numbers that increase if left inside the deep pockets for prolonged

periods [32, 33]. However, important issues including how much the level of the disease (in

terms of deep pockets) is retained for longer duration is still uninvestigated. Future studies are

needed to investigate this hypothesis.

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Research indicates that the serum and local levels of AGEs may be an important

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contributing factor for periodontal destruction in DM patients [34, 35]. These end-products are

responsible for soft tissue and bone destruction. In addition, the increased levels of AGEs may
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cause the production of increasing levels of pro-inflammatory biomarkers that further add to
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the periodontal insult [36]. Although this study did not evaluate the levels of AGEs or

proinflammatory cytokines, however, the true impact of therapeutic modality may have been
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proven by the evolution of the levels of these biomarkers with time. Moreover, there was no

repeated applications of PDT sessions in this study. We hypothesize that several sessions of
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PDT may have led to higher improvements in periodontal and metabolic outcomes compared

to an only single application of PDT as described by a recent animal study by Longo et al [37].
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Further studies are needed to test this.

Some important limitations are described in the present study. The present study was
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based on a short follow-up period. Long follow-up durations may have given different

outcomes in both periodontal, metabolic and microbiological parameters. Also, the study

included patients with HbA1c levels 8.81% to 9.38%. The inclusion of various glycemic levels

according to mild, moderate and severe DM may also help to indicate the true efficacy of the

treatment modalities in DM patients. Furthermore, the present study did not analyzed advanced
molecular investigations such as serum levels of AGEs and relied on HbA1c levels. The local

reduction of AGEs with PDT or probiotics may further help to understand the molecular basis

of the association and its impact on treatment.

5. Conclusions

Within the limitations of the study, PDT showed additional benefit in deep periodontal pockets

and a slightly modest reduction in HbA1c levels. PDT showed additional benefit in deep

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periodontal pockets and a slightly modest reduction in HbA1c levels in DM patients. Further

clinical trials are required with large sample size and longer follow up period to ascertain the

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findings of the present clinical study.

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Conflict of interest: None declared in this manuscript.
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Acknowledgment

The authors extend their appreciation to the Deanship of Scientific Research at King Saud
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University for funding this work through research group no (RG-1439-81)

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Figure Legends:

Figure 1. Detection frequency of Pg, Tf, and Td at baseline and 3 months after treatments.

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Table 1. Primers used in PCR analysis.

Bacteria Primer sequence Size of PCR Thermal conditions

Pg 5`-ATGCCAACTTGACGTTAAT-3`
5`-AATCGTAACGGGCGACACAC-3`
Tf 5`-GGGTTGCTCCTTCATCATAC-3`
5`-GCGTATGTAACCTGCCCGCA-3`
Td `-ATGCGGGACCTGAAGTGCAT-3`
5`-AATGCCCATAGGTAACACAC-3`
product
593 bp 30 cycles – 94 ºC for 1 min, 70 º
for 1 min and 72 ºC for 1 min
641 bp 36 cycles – 95 ºC for 30 s, 60 º
for 1 min and 72 ºC for 1 min
610 bp 32 cycles – 93 ºC for 1 min, 70 º
for 1 min and 72 ºC for 1 min

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Table 2. Characteristics of study groups.

Characteristics Group-A Group-B Group-C

(PDT + RSD) (PT + RSD) (RSD alone)
Number of patients (n) 20 19 19
Mean age (years) 52.16 51.87 52.88
Male/female 11/9 10/10 13/7
Duration of diabetes (years) 15.7 ± 8.6 17.2 ± 9.4 18.2 ± 10.3
Diabetic therapy
 Diet and exercise 5 2 5
 Oral hypoglycemic drugs 13 15 10
 Insulin - 1 -
 Oral hypoglycemic drugs + insulin 2 2 5

P value for differences among groups using the Chi2 test and one-way ANOVA for categoric and continuous

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variables, respectively.

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 Table 3. Mean clinical periodontal and metabolic parameters of the study groups. All data presented in mean ± SD.

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Parameters Group-A Group-B Group-C
(PDT + RSD) (PT + RSD) (RSD alone)
Baseline 3 months Difference Baseline 3 months Difference Baseline 3 months Difference
Plaque scores (0/1) 0.86 ± 0.16 0.53 ± 0.25§ –0.33 ± 0.05 0.84 ± 0.18 0.51 ± 0.21§ –0.33 ± 0.07 0.82 ± 0.15 0.55 ± 0.18§ –0.27 ± 0.04

pr
Bleeding on probing 0.74 ± 0.22 0.51 ± 0.31§ –0.23 ± 0.07 0.83 ± 0.13 0.63 ± 0.33§ –0.20 ± 0.06 0.69 ± 0.25 0.45 ± 0.27§ –0.31 ± 0.08
(0/1)
Probing depth (mm) 3.14 ± 0.42 2.65 ± 0.31* –0.49 ± 0.06 3.29 ± 0.47 2.81 ± 0.25¶ –0.48 ± 0.02 3.36 ± 0.62 2.74 ± 0.75* –0.62 ± 0.06

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Proportion of PD ≥4 0.31 ± 0.14 0.12 ± 0.13¶ –0.19 ± 0.05 0.28 ± 0.21 0.14 ± 0.06§ –0.14 ± 0.01 0.26 ± 0.20 0.15 ± 0.16§ –0.11 ± 0.03
mm
Proportion of PD ≥5 0.12 ± 0.07 0.04 ± 0.06¶ –0.08 ± 0.02 0.15 ± 0.12 0.09 ± 0.04§ –0.06 ± 0.03 0.15 ± 0.15 0.09 ± 0.12§ –0.06 ± 0.04

Pr
mm
Clinical attachment 4.35 ± 1.18 3.88 ± 1.19* 0.47 ± 0.01 4.06 ± 0.83 3.54 ± 0.48¶ 0.52 ± 0.03 4.71 ± 1.35 4.29 ± 1.44* 0.42 ± 0.09
level (mm)
HbA1c (%) 8.81 ± 1.96 8.03 ± 1.89§ –0.78 ± 0.04 9.38 ± 2.62 8.85 ± 2.77 –0.53 ± 0.06 9.16 ± 1.72 8.68 ± 0.98 –0.48 ± 0.05
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The paired t test and one-way ANOVA, respectively, were used to compare within- and between-group differences.
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* P <0.001
¶ P <0.01
§ P <0.05
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