Effectiveness of Antimicrobial Photodynamic Therapy in the Treatment of Periodontitis: A Systematic Review and Meta-Analysis of In Vivo Human Randomized Controlled Clinical Trials

This systematic review and meta-analysis evaluated antimicrobial photodynamic therapy (aPDT) efficacy in periodontitis. The review protocol was conducted in accordance with PRISMA statements, Cochrane Collaboration recommendations and is registered in PROSPERO (CRD 42020161516). Electronic and hand search strategies were undertaken to gather data on in vivo human RCTs followed by qualitative analysis. Differences in probing pocket depth (PPD) and clinical attachment level (CAL) were calculated with 95% confidence intervals and pooled in random effects model at three and six months. Heterogeneity was analyzed, using Q and I2 tests. Publication bias was assessed by visual examination of the funnel plot symmetry. Sixty percent of 31 eligible studies showed a high risk of bias. Meta-analysis on 18 studies showed no additional benefit in split mouth studies in terms of PPD reduction (SMD 0.166; 95% CI −0.278 to 0.611; P = 0.463) and CAL gain (SMD 0.092; 95% CI −0.013 to 0.198; P = 0.088). Similar findings noted for parallel group studies; PPD reduction (SMD 0.076; 95% CI −0.420 to 0.573; P = 0.763) and CAL gain (SMD 0.056; 95% CI −0.408 to 0.552; P = 0.745). Sensitivity analysis minimized heterogeneity for both outcome variables; however, intergroup differences were not statistically significant. Future research should aim for well-designed RCTs in order to determine the effectiveness of aPDT.


1.
Limitations of scaling and root planing (SRP) have directed the research to assess alternative comprehensive treatment strategies.

2.
Antimicrobial Photodynamic therapy (aPDT) involves photo-excitation of photosensitizer dye upon illumination by a light of a matched wavelength.

3.
This systematic review and meta-analysis evaluated the effectiveness of aPDT in the treatment of periodontitis. 4.
In spite of the inconsistencies in their findings and methodological bias, the majority of the studies have demonstrated aPDT effectiveness.
• Population: Patients diagnosed with Periodontitis (CP or AgP) [26] • Intervention: Utilisation of aPDT as a monotherapy or as an adjunct to SRP • Comparison: Utilisation of SRP alone or SRP with adjunctive AB therapy • Outcome: Evaluation of clinical and/or microbiological and/or immunological profiles

Focused Research Question
Is aPDT effective as a primary mode of treatment or as an adjunct to SRP compared to SRP alone or in combination with local or systemic antibiotics (AB), in terms of clinical or microbiological or immunological profiles, in patients with Periodontitis?

Search Strategy
The search strategy only included terms relating to or describing the study's domain and intervention. The use of relevant free text keywords and medical subject heading (Mesh) terms, which were logically connected with the help of Cochrane  Related review articles and reference lists of all identified articles were searched through for further studies. Abstracts of the American Academy of Periodontology (AAP) and the European Federation of Periodontology (EFP) as well as sources for grey literature were screened to detect unpublished studies. In some instances, an attempt was made to establish a communication with the corresponding author in an attempt to obtain additional information related to the study; however, the attempts were unsuccessful. Search strategy was performed by two blinded, independent reviewers (S.D. and R.H.). In order to assess inter-reviewer reliability analysis, Kappa (κ) statistics were performed and a minimum value of 0.8 was considered acceptable [27]. In case of any disagreements, reviewers would discuss the discrepancies with a third author (S.B.), if necessary.
Subjects diagnosed with CP or AgP according to 1999 AAP Classification of Periodontal diseases and conditions [26].

2.
Studies included: In vivo human RCT's comparing the efficacy of aPDT in CP or AgP as monotherapy or adjunctive to SRP compared to SRP alone or in combination with AB. 3.

5.
No language restrictions for search strategy. 6.
Studies that have utilized any PS dye (regardless dose and incubation period) and laser wavelength combination.  7.
Studies reporting at least one of the following parameters as an outcome variable: probing pocket depth (PPD), loss of clinical attachment level (CAL), bleeding on probing (BOP), plaque index (PI), gingival index (GI), microbiological profile, or immunological profile. 8.
Studies with a minimum follow-up period of at least one month after treatment.

1.
Subjects with systemic diseases or on medications that can influence the outcome variables.

2.
Subjects who have undergone any periodontal therapy and/or antibiotic therapy in the last six months prior to RCT enrolment. 3.
Studies utilizing low level laser therapy or laser therapy alone, as one of the intervention groups as compared to aPDT. 4.
Studies involving utilization of aPDT for residual pockets or in supportive periodontal therapy (SPT). 5.
Studies that have utilized light emitting diodes (LEDs) as a light source. 6.
No outcome variable of interest. 7.
Narrative and systematic reviews, in vitro studies, in vivo animal studies, commentaries, interviews, updates, case series and case reports.

Systematic Review Outcomes 2.7.1. Primary Outcome Measures
Changes in PPD and CAL from baseline up to the end of follow-up.

Secondary Outcome Measures
Changes in GR, BOP, PI, GI, microbiological and immunological profile from baseline up to the end of follow-up.

Data Extraction
Two reviewers independently (S.D. and R.H.) selected eligible studies from the search. They performed the review, assessment and data extraction for each eligible study. Each study received an identification with the name of the first author, year of publication and origin. A tabular representation of additional relevant information such as impact factor of journal, study design, sample size, demographics of the participants, baseline characteristics, intervention and comparator groups, type of photosensitizer used and dosage, laser parameters utilized, number of aPDT sessions performed, follow-up duration, statistical tests performed and results and conclusions, were gathered from each eligible study.

Qualitative Analysis
A qualitative assessment for each study was performed using the Revised Cochrane Risk-of-Bias (RoB) tool for Randomized trials, Version 2.0 (RoB 2) by two independent reviewers (S.D. and R.H.) [28][29][30]. Detailed assessment under the following headings was performed: 1. Bias arising from the randomization process; 2. Bias due to deviations from intended interventions; 3. Bias due to missing outcome data; 4. Bias in measurement of the outcome; 5. Bias in selection of the reported result. Depending upon fulfilment of above-mentioned criteria, the studies were determined as low, moderate or high RoB. Disagreements between the reviewers were resolved by discussion with a third author (S.B.) as well as use of 'discrepancy check' feature in RoB 2, in order to obtain consensus.

Statistical Analysis of Data
When appropriate and quantifiable data of interest were extracted from the eligible studies and combined for meta-analyses, using Stata version 15.1 software (StataCorp, Pyrmont, Australia), random effects meta-analyses were conducted to reflect the expected Pharmaceutics 2021, 13, 836 5 of 42 heterogeneity. As continuous outcomes were expected, overall treatment effects were calculated through pooled standardized mean differences (SMDs) with associated 95% confidence intervals (95% CIs) for PPD and CAL. When information was presented in median and inter-quartile ranges, means and SDs were estimated [31]. Results from SM and PG studies were pooled separately at 3 and 6 months, respectively. A pooled overall effect was considered statistically significant when p < 0.05. Consequently, statistical heterogeneity to identify outlier studies was performed by visual inspection of forest plots. Additionally, the Cochran Q test was conducted to assess statistical heterogeneity (p < 0.10) [32]. I 2 statistics for homogeneity was expressed in a range of 0-100%, with the following interpretation; 0% = no evidence of heterogeneity; 30-60% = moderate heterogeneity; 75-100% = high heterogeneity [33]. Sensitivity analysis was conducted to negate the effect of heterogeneity in between included studies by identifying the outlier studies by visual inspection of forest plots [34]. Publication bias was evaluated by visual assessment of funnel plot symmetry.

Selection Criteria
Several inconsistencies were observed amongst the included studies [2,3,5,17,, which have been outlined in Table 1, in which 21 out of 31 studies included patients with CP [17,70,[75][76][77][78][79][80][81][82]84,86,[88][89][90][91][92][93][94][95][96], whereas the remaining 10 studies included patients with AgP [2,3,5,[71][72][73][74]83,85,87]. Significant improvement in BOP was noted with aPDT group (p = 0.03). PDT increased the expression of RANK and OPG, which could indicate a reduction in osteoclastogenesis. Furthermore, the use of PDT in conjunction with conventional treatment significantly increased the expression of FGF2, which has an important role in the periodontal repair process. In the AB group, patients showed a statistically significant (p = 0.01) decrease of MMP-8 GCF level at both 3-and 6-months post treatment. In the PDT group, the change of MMP-8 GCF level was not statistically significant. Both groups showed at 3 and 6 months a decrease in MMP-9 levels. However, this change did not reach statistical significance. SRP+AB is more effective in reducing GCF MMP-8 levels compared to SRP+aPDT.   PD values decreased significantly in both groups after 1 month (p = 0.001) and 3 months (p = 0.001) in the SRP and (p = 0.001) in the PDT groups the inter-group differences were not significant after 1 (p = 0.25) and 3 months (p = 0.51). Clinical measurements showed significant decreases after 1 and 3 months at both sites, without inter-group differences, except for BOP after 1 (p = 0.004) and 3 months (p = 0.0001).  PD and CAL showed statistically significant reduction in the test group on evaluation at 3 months and 6 months as compared to the control group (p < 0.05). A statistically significant improvement in GI and GBI was seen for the test group after 2 weeks and 1 month of aPDT (p < 0.01), whereas the improvement in GI and GBI at 3 months and in plaque index at 2 weeks after aPDT was less (p < 0.05). In addition, a significant difference was detected for the test group at 1 month in terms of halitosis, which did not persist for long (p < 0.05). There was a significant reduction in PI, GI, PD, CAL and microbiologic parameters in test group, following SRP and PDT, when compared with SRP alone in control group (p < 0.001). SRP+aPDT has shown additional improvement in periodontal parameters when compared to SRP alone and has a beneficial effect in chronic periodontitis patients. PI, reddening, PD, BOP, CAL, GR Quantitative analysis for F.n.

Qualitative Assessment
Qualitative assessment was performed using the RoB 2 tool, designed for in vivo human RCTs, as depicted in Figures 4 and 5. The most recent version of this tool was utilized to perform a qualitative assessment for both randomized PG and SM human RCTs [29,30]. Figure 4 represents a risk of bias assessment summary of all eligible studies. Figure 5 is a  (Table 1).

Qualitative Assessment
Qualitative assessment was performed using the RoB 2 tool, designed for in vivo human RCTs, as depicted in Figures 4 and 5. The most recent version of this tool was utilized to perform a qualitative assessment for both randomized PG and SM human RCTs [29,30]. Figure 4 represents a risk of bias assessment summary of all eligible studies. Figure 5 is a graphical representation of percentage RoB score for each risk domain, which has been evaluated, using the abovementioned tool. Furthermore, 53.1% of included trials were at a high risk of inadequate randomization, whereas 40.6% and 6.3% of included trials were at a low risk or had some concerns, respectively. In addition, 50% of included studies were at a high risk of deviations from intended interventions, whereas 43.7% and 6.3% of them were at a low risk or had some concerns, respectively. All included papers reported substantial evidence (100%) for reporting missing outcome data and, hence, were at a low risk. Although a majority of studies were free of bias arising from reporting outcome measurement (71.9%), 28.1% were at a high risk. In terms of selective reporting of the results, inferences are as follows: 59.4% studies were at a high risk, 37.5% studies were at a low risk, and 3.1% studies had some concerns. Overall, 60% studies reported a high risk of bias, while 35% studies had a low risk of bias, and the final 5% studies had some concerns. It should be noted that information provided in these figures represents the consensual answers verified using the 'Discrepancy check' feature of RoB 2 tool, across two independent reviewers (S.D. and R.H.) (inter-reviewer agreement, κ = 0.94), and, in case of any disagreements, a third author (S.B.) was consulted. graphical representation of percentage RoB score for each risk domain, which has been evaluated, using the abovementioned tool. Furthermore, 53.1% of included trials were at a high risk of inadequate randomization, whereas 40.6% and 6.3% of included trials were at a low risk or had some concerns, respectively. In addition, 50% of included studies were at a high risk of deviations from intended interventions, whereas 43.7% and 6.3% of them were at a low risk or had some concerns, respectively. All included papers reported substantial evidence (100%) for reporting missing outcome data and, hence, were at a low risk. Although a majority of studies were free of bias arising from reporting outcome measurement (71.9%), 28.1% were at a high risk. In terms of selective reporting of the results, inferences are as follows: 59.4% studies were at a high risk, 37.5% studies were at a low risk, and 3.1% studies had some concerns. Overall, 60% studies reported a high risk of bias, while 35% studies had a low risk of bias, and the final 5% studies had some concerns. It should be noted that information provided in these figures represents the consensual answers verified using the 'Discrepancy check' feature of RoB 2 tool, across two independent reviewers (S.D. and R.H.) (inter-reviewer agreement, κ = 0.94), and, in case of any disagreements, a third author (S.B.) was consulted.

Publication Bias
Visual inspection of funnel plots revealed noticeable asymmetry in SM-study analysis for PPD reduction indicating a probable risk of publication bias in SM studies included in this meta-analysis. However, slight asymmetries for PG studies were noted in corresponding funnel plots suggestive of a low risk of publication bias in the same ( Figure 6).
Heterogeneity: Q = 9.7; DF = 3; P = 0.22; I 2 = 0.00% Figure 6. (a,b) Funnel plots illustrating the publication bias in overall PPD reduction in SM and PG studies, respectively; (c,d) funnel plots illustrating the publication bias in overall CAL gain in SM and PG studies, respectively. Each circle represents a single included study, the y-axis and x-axis represent the standard error of the effect estimate and the results of the study respectively and the graphical plot resembles an inverted funnel with scatter due to sampling variations.

Discussion
Based on the hypothesis that aPDT monotherapy or as an adjunct to NSPT can enhance the clinical or microbiological or immunological profile in comparison to conventional SRP, or SRP+AB, a critical appraisal of the available scientific evidence was conducted. After meticulous scrutiny, 31 studies were included in the present systematic review [2,3,5,17,. Owing to the methodological discrepancies, only 18 out 31 studies were eligible for a meta-analysis [17,71,[73][74][75][76]80,81,[84][85][86][88][89][90][91][92][93]96]. This report is the first to evaluate the role of SRP+aPDT compared to SRP alone or SRP+AB in SM and PG studies in AgP as well as in CP patients. The results of this meta-analysis indicated that, in comparison to SRP alone or SRP+AB, SRP+aPDT failed to show any additional benefit in the management of periodontitis up to six months. A significant heterogeneity was reported, arising from confounders in aPDT protocols. Subsequently, after omitting outlier studies [74,86,90,92,93], sensitivity analysis was able to eliminate heterogeneity completely but Figure 6. (a,b) Funnel plots illustrating the publication bias in overall PPD reduction in SM and PG studies, respectively; (c,d) funnel plots illustrating the publication bias in overall CAL gain in SM and PG studies, respectively. Each circle represents a single included study, the y-axis and x-axis represent the standard error of the effect estimate and the results of the study respectively and the graphical plot resembles an inverted funnel with scatter due to sampling variations.

Discussion
Based on the hypothesis that aPDT monotherapy or as an adjunct to NSPT can enhance the clinical or microbiological or immunological profile in comparison to conventional SRP, or SRP+AB, a critical appraisal of the available scientific evidence was conducted. After meticulous scrutiny, 31 studies were included in the present systematic review [2,3,5,17,. Owing to the methodological discrepancies, only 18 out 31 studies were eligible for a meta-analysis [17,71,[73][74][75][76]80,81,[84][85][86][88][89][90][91][92][93]96]. This report is the first to evaluate the role of SRP+aPDT compared to SRP alone or SRP+AB in SM and PG studies in AgP as well as in CP patients. The results of this meta-analysis indicated that, in comparison to SRP alone or SRP+AB, SRP+aPDT failed to show any additional benefit in the management of periodontitis up to six months. A significant heterogeneity was reported, arising from confounders in aPDT protocols. Subsequently, after omitting outlier studies [74,86,90,92,93], sensitivity analysis was able to eliminate heterogeneity completely but failed to report statistically significant improvements in primary outcome variables. Furthermore, risk of publication bias was reported indicating a possible selective outcome reporting in eligible published studies. In some instances, missing outcomes could not be detected by comparing the published report with the respective study protocol due to unavailability of the latter. Until now, seven meta-analyses have been reported to assess the role of aPDT in periodontitis [6][7][8][9][10][11][12]. The present review protocol is in accordance with the existing reviews [8,[10][11][12]. Azaripour et al. is the only other systematic review and meta-analysis that has assessed the efficacy of SRP+aPDT as compared to SRP alone for SM and PG studies [8]. While three reviews report short-term benefits of aPDT up to 6 months [7][8][9], four have reported otherwise [6,[10][11][12]. Our findings are in accordance with findings of the latter scientific reports. In order to gain an insight on merits and inadequacies of each included study, a comprehensive and systematic investigation was performed as follows:

Role of Baseline Characteristics
A key feature of RCTs is the application of balanced baseline characteristics in treatment arms of the trial in order to achieve unbiased treatment outcomes [97]. Most often, researchers provide a tabular representation of relevant variables to confirm an impartial baseline evaluation. In case of missing information on baseline characteristics, a 'selection bias' can be suspected [98]. All eligible studies have provided this vital information and were free from any kind of 'selection bias'. Additionally, evidence-based studies have suggested the potential harmful effects of smoking on the onset and progression of periodontitis, for which smokers were excluded [99,100]. Likewise, the inter-relationship of periodontitis and its systemic manifestations are well-established, resulting in the exclusion of patients with systemic diseases [101,102]. Utilization of 'placebo/sham' procedures to enhance clinical outcomes of the trial is an evidence-based verified concept [103]. In the present systematic review, only six out of 31 studies [71,78,[92][93][94]96] have utilized a 'placebo/sham' procedure as an adjunct to conventional SRP. Furthermore, the role of SRP+aPDT+AB, compared to SRP+AB and SRP+aPDT as well the efficacy of SRP+PS compared to the conventional SRP and SRP+ aPDT have been assessed in this review [83,95]. Differences in study designs were apparent and the majority of clinicians have utilized the SM study design in oral health research [104]. Hence, this meta-analysis included both SM and PG studies in order to assess whether the estimated intervention effect has differed between them.

Assessment Methods for Various Parameters and Their Inferences to Determine aPDT Efficacy
A decrease in periodontal inflammation is directly proportional to a decrease in the incidence of BOP and detectable plaque levels [105]. Furthermore, the endpoints of a comprehensive periodontal therapy include PPD reduction and CAL gain, both of which are crucial for determining treatment success [106]. Clinical evidence has proven that there is a direct correlation between initial severity of PPD and CAL values and the amount of post-operative differences [107][108][109]. In terms of disease severity at baseline evaluation, a lack of homogeneity in pre-treatment values of PPD and CAL in the data extracted from various studies was observed (Table 1). Therefore, variations in level of significance across clinical parameters were noted, thus making it difficult to provide a cumulative result ( Table 5). Utilization of a narrow laser optic fibre tip in deep periodontal pockets facilitates easy and atraumatic periodontal pocket probing ultimately resulting in the PPD and GR reduction post-operatively [3,88]. Furthermore, evidence-based research has proven that clinical outcome remains unaffected by the type of instrumentation utilized in SRP [76,110], which was observed to vary across all eligible studies (Table 1).
An imbalance amongst the local etiological factors such as dental plaque and calculus, inflammation, and a host defense system can have a great impact on the disease severity and progression [17]. Hence, it is essential to monitor the microbiological and the molecular changes of various growth factors and proinflammatory cytokines. De Oliveira and coworkers have demonstrated, through their studies, the importance of SRP in reducing bacterial load from tooth surface and the failure of aPDT monotherapy in reducing the bacterial counts of A.a. periodontal disease activity as well as bone resorption [2,5].

Representation of the Treatment Outcomes
Positive outcomes of any treatment strategy are governed by several factors such as evaluation of disease status at carefully planned follow-up visits, signs of unevent-ful healing, role of supportive periodontal therapy (SPT) and patient compliance with treatment [111][112][113]. The majority of included studies performed follow-up assessment for up to 3 or 6 months, whereas results of a longer follow-up ranging up to 1-2 years was lacking. Collective data obtained from longitudinal studies have confirmed the role of long-term follow up visits in greater reduction of clinical parameters. These findings have been confirmed by three studies included in the present review [73,77,95]. With regard to healing outcomes, 24 out of 31 studies reported no uneventful healing associated with the absence of any postoperative complications, after application of aPDT [2,3,5,[70][71][72][73][74][75][77][78][79][80][81][83][84][85][86][87][88][89][90]92,95]. While six studies have failed to provide any information on healing outcomes [17,76,82,91,93,94], one study [96] has reported a gastrointestinal complication in one patient of the aPDT group. This happens to be unique evidence that has not been registered elsewhere. The presence of residual plaque and calculus resulting in a relapse of periodontal disease severity being inevitable with aPDT monotherapy. Hence, the role of SPT is quintessential. In the present systematic review, apart from the three sequential studies conducted by De Oliveira and co-workers [2,3,5] which utilized aPDT monotherapy, all of the other studies have utilized SRP+aPDT. In the former group of studies, a supragingival professional tooth cleaning was performed 14 days prior to the application of aPDT monotherapy in the treatment sites, which have extensively improved the post-operative clinical findings. Likewise, amongst the studies that have assessed the efficacy of SRP+aPDT, only seven studies [71,75,82,84,90,92,95] have mentioned a planned SPT protocol, which was implemented throughout their study period. Furthermore, information on oral hygiene instructions tailored according to respective study criteria has been specified in all included studies. Co-relation of patient's compliance in adhering to hygiene instructions cannot be overlooked, since it is vital for treatment success and prevention of disease recurrence [114]. Quantitative measurement of plaque by means various indices can aid in monitoring compliance towards therapy. In connection to this, the assessment of plaque levels was performed in 21 studies included in this review [3,17,71,[73][74][75][77][78][79]81,82,84,85,[88][89][90][91][92][93][94]. Therefore, an inconsistency in the representation of treatment outcomes in this review is evident.

Role of Laser Parameters
Apart from study methodology, laser parameters are crucial in determining treatment outcome. Calibration of therapeutic power output with a power-meter can aid in regulating low output power for achieving the desired aPDT effect at treatment sites [115]. This technique can also prevent any inadvertent damage caused by utilization of high output power, resulting in a photothermal effect. However, none of the included studies have utilized a power-meter to calibrate the power output. Furthermore, some other parameters that have been overlooked were: emission mode, contact/non-contact mode, energy/treated site, power output, spot size/fibre diameter, fluence and irradiance. Diode lasers have a high affinity to pigment, which, in the case of aPDT, is the PS. However, inflamed periodontal tissues are rich in blood and high levels of proteins, which are also rich in pigments. Traumatic instrumentation can lacerate the sulcus lining and elicit bleeding [23]. Consequently, the overall aPDT effect, which could be achieved by an effective PS dye-laser wavelength combination, will be compromised [23,116]. Additionally, placement of the fibre tip inside the gingival sulcus needs to be performed judiciously, in order to avoid further trauma to inflamed gingival sulcus, serving as a niche for plaque accumulation and favoring disease relapse [3,88].
Bacterial re-colonization after SRP has been proven to occur after three weeks [117] and, hence, multiple aPDT sessions can help to delay this pathological process. Annaji et al., 2016 [46] have compared the efficacy of three sessions (0, 7th and 21st day) versus a single session (0 day) of aPDT in the management of CP, and concluded that the group receiving multiple sessions demonstrated superior treatment outcomes. Nine studies included in this review conducted multiple aPDT sessions [17,[71][72][73][74][75]79,90,96], and have unanimously concluded that the utilization of multiple aPDT sessions has positive healing effects. However, the frequency of aPDT application varies in all these studies ( Table 2). As a result, a conclusion on the ideal number of sessions and frequency of application of aPDT cannot be drawn.
Additionally, PS concentration and pre-irradiation time (wash-out or PS incubation time) are governed by the binding capacity of PS to target cells [118]. It was observed in 13 out of 31 studies, whereby the PS concentration was not reported [5,70,[72][73][74]76,81,[84][85][86]91,93,94], whereas a range of PS concentrations have been utilized in the remaining eligible studies. Furthermore, four studies have failed to mention PS pre-irradiation time [5,75,77,90], whereas the remaining 27 studies have reported this time, which ranged from 1-5 min for different PS. An in vitro study by Fumes et al. [119] evaluated the effect of different PS pre-irradiation time periods (1, 2 and 5 min) in aPDT on the biofilms formed by Streptococcus mutans and Candida albicans, by monitoring the microbial load and have successfully demonstrated that the efficacy of all pre-irradiation times was equal. They emphasized patient discomfort associated with longer pre-irradiation times and thus have advised the use of shorter pre-irradiation times (1 min) in future studies. Up until now, there have been no studies that have determined the minimal duration of PS incubation as well as its role against periopathogens. Hence, further studies on this subject should be sought after. Moreover, discrepancies in the reported data, in terms of the number of sites receiving aPDT application per tooth as well the irradiation time, were noticed amongst the eligible studies (Table 2). These voids have raised concerns regarding the reliability of existing literature, which lacks a reproducible methodology and ultimately hampers the rational use of aPDT in non-surgical management of periodontitis.

Role of RoB Assessment
A vast majority of bias were raised from inadequate randomization, deviations from intended interventions and selective reporting of results (Figures 4 and 5). Another key finding of this systematic review was the presence of a potential conflict of interest in 10 out of the 31 studies [2,3,5,[72][73][74]76,79,80,94]. Therefore, the results of the included studies are questionable, and their biased methodology cannot be relied upon.

Limitations of the Present Systematic Review
The majority of the included studies have assessed the efficacy of SRP+aPDT in comparison to aPDT monotherapy resulting in a lack of meta-analysis on the latter. Utilization of a limited number of teeth (mostly anterior teeth) or on specific sites (interproximal sites in case of deep pockets) or on any two quadrants of the dental arch that could facilitate cross contamination from the untreated sites and overshadow the putative benefits of aPDT was noted. In this review, efficacy of aPDT was monitored in systemically healthy non-smokers only, and its benefits in their immunocompromised counterparts were not established. A lack of long-term follow-up in order to determine the stability in healing after aPDT was also observed. The number of studies included in the meta-analysis was nearly half of the studies eligible for a systematic review due to paucity and inconsistency in available literature. Owing to the aforesaid drawbacks, the objective of this review could not be accomplished.

Future Scope
Future investigations should compare aPDT monotherapy versus SRP+aPDT and provide details of all appropriate laser and PS parameters. Efficacy of aPDT in smokers with various systemic diseases in CP and AgP should be established. The role of patient related outcomes and SPT should be emphasized upon. Nevertheless, future RCTs must have a robust methodology with balanced baseline characteristics, performed by experienced, masked and calibrated clinicians, which will reduce bias. Owing to the evident benefits of a PG-study design, clinicians should prefer its utilization in order to minimize potential 'carry-over' effects. Additionally, researchers should conduct a full mouth study protocol with a long-term follow-up assessment of minimum 6 months duration, consisting of, but not limited to, a vast range of clinical, microbiological, radiographic as well as, immunehistological profiles.

Conclusions
Within the limits of the present systematic review and meta-analysis, it can be concluded that the efficacy of aPDT in improving treatment outcomes, when it is utilized in the non-surgical management of periodontitis, remains debatable. However, the results of a majority of included studies have demonstrated the effectiveness of aPDT, and this role is more pronounced for SRP+aPDT rather than aPDT monotherapy. A careful and critical appraisal was performed which helped to obtain a qualitative assessment of eligible studies, thus highlighting the substantial flaws that prevent a reproducible methodology. Data on standardized aPDT study protocol, ideal PS dye-laser combination, optimal laser and PS parameters remain inconsistent and inconclusive amongst the prevalent literature owing to a highly inferior RoB in many studies. Finally, future research should aim for well-designed, robust and preferably PG-RCTs that will overcome the abovementioned limitations and confounders, in order to achieve palpable progress in this field of research while ensuring the use of an appropriate local laser safety protocol.