Therapeutic Role of Nystatin Added to Tissue Conditioners for Treating Denture-Induced Stomatitis: A Systematic Review

: Denture-induced stomatitis (DIS) represents a pathological condition caused by ill-ﬁtting dentures trauma, manifested as inﬂammation of the palatal tissue beneath the denture. The fungal infections are the principal contributory factors to DIS. Although the aetiology of DIS is multifactorial, the Candida albicans fungal infection remains the main cause. The objective of the present systematic review was to evaluate the latest literature on the addition of nystatin into the tissue conditioners (TCs) in the management of DIS. To search the published literature about “the addition of nystatin in TCs for treating DIS”, electronic databases (PubMed (National library of medicine) and Cochrane Library (Wiley)) were searched (from 1975 until December 2020) using different keywords. Various studies described the effectiveness and efﬁcacy of incorporating nystatin into several TCs. The present systematic review reported that the addition of nystatin is beneﬁcial, with slight or no consequences on both the mechanical and physical features of TCs. Adding nystatin to various TCs for treating DIS can be suggested.


Introduction
Denture-induced stomatitis (DIS) is a pathological condition that affects the denture bearing mucosa as a result of trauma from ill-fitting dentures [1,2]. DIS affects a considerable proportion of denture wearers [3]. The primary causal factor of DIS include fungal infections, caused by the Candida species, particularly Candida albicans [4][5][6]. Nevertheless, the aetiology of DIS can be multifactorial, as other important risk factors, including poor oral hygiene and consistent night-time denture wearing [7], dry mouth [8,9], denture trauma [10], and variation in salivary pH [11], have been considered to be linked with DIS. Almost two-thirds (65%) of upper complete denture wearers are affected by this lesion. In the majority of the cases, it is asymptomatic but may present with symptoms of pain, halitosis, pruritus, the presence of erythema/oedema, and burning of the palatine mucosa and gingiva under the denture base [12]. The main diagnosis of the DIS is based on these clinical findings. However, according to some clinicians/researchers, the association of DIS with candidiasis is also considered essential during the diagnosis [13]. For patients with symptoms like angular cheilitis/stomatitis, and any other local/systemic lesion, additional diagnostic tests like blood pictures, smears/culture, and, rarely, biopsy of the site may be advised [14,15]. Common signs of DIS are generalised inflammation or a reddish appearance of the hard palate beneath the denture and is much more usual in complete denture wearers [2]. Among the normal oral flora, Candida albicans (C. albicans) is seen in 40% of the individuals, being part of the dental plaque formation [16]. In certain situations, C. albicans adhering to the constantly worn base of denture results in DIS [17]. In terms of oral fungal infections, C. albicans is the most abundant species and leading pathogen that contributes to the development of DIS [18,19]. Besides, Candida tropicalis and Candida glabrata [20] are usually related to the hard palate and the denture surface of healthy denture wearers [21,22].
There is an antifungal protection present in human saliva attributed to the oral antimicrobial peptides [23], but in some conditions-for example, poor oral hygiene-this antifungal defence may not be sufficient. In those cases, denture wearers may require an appropriate treatment. As a result of multifactorial aetiology, the treatment of C. albicans-related DIS is complicated [24,25]. Several therapeutic modalities have been proposed [18,24]. The conventional therapies for the treatment of DIS include a local/topical application or oral intake of various antifungal drugs, such as Fluconazole, itraconazole, nystatin, amphotericin B, ketoconazole, and clotrimazole, in addition to the use of mouth washes such as chlorhexidine digluconate (0.12%) [26]. The efficacy of these medications ranges from 77% to 100% for a clinical and microbiological cure of DIS [27]. Fluconazole and nystatin are the relatively more commonly used drugs for a DIS cure. Fluconazole showed a positive response of 89%, but many relapses were seen in the cases treated with it [28]. Nystatin, in comparison, was shown to be more potent in DIS, with a higher clinical and mycological cure rate and is now considered the standard topical treatment for oral candidiasis [27]. Fungi resistant to nystatin are rare, and also, its cost/availability makes it the drug of choice [29]. However, these treatment options are more or less supportive and may not be beneficial for every individual [30][31][32]. Photodynamic therapy and the use of nanoparticles are some of the latest/recent treatment modalities used for the cure of DIS. Methylene-blue, toluidine-blue, and porphyrin have been used as photosensitisers in these therapies, for which the results are promising [33]. Some researchers have incorporated nanomaterials such as silver-nanoparticle discs in denture bases for the prevention/treatment of DIS. The results indicated a significant reduction in the adherence of C. albicans [34].
For the conditioning purpose of the denture bearing mucosa, resilient tissue conditioners (TCs) are commonly used. TCs reduce the load endured by denture bearing mucosa, and they also work as a cushion beneath the dentures [35]. Besides, TCs are used as drug delivery carriers [35,36]-for instance, the delivery of antifungal agents for the inhibition of C. albicans [37,38]. Due to cognitive impairment, memory loss, and decreased motor activity in geriatric patients, the application of topical antifungal medicaments in DIS patients is challenging [24]. Moreover, maintaining an effective and sustained release of topical antifungal agent is also difficult. Due to regular ingestion and persistent salivary washout, the antifungal drugs are less likely to adhere with the oral mucosa [39]. To overcome these issues, a lot of of research has investigated the efficacy of TCs modified by the addition of various antifungal medicaments, such as nystatin [29,38,[40][41][42][43][44][45][46][47], derivatives of the azole group [38,[43][44][45][46][47], and chlorhexidine [45][46][47][48].
Polyenes, including nystatin, are the main choice for the management of primary oral candidal infections [49]. Nonetheless, there are associated unwanted effects, such as a bitter taste, nausea, mucosal irritation, and poor acceptance by patients [50]. Different studies have investigated the modified TCs with nystatin associated with efficacy [40,41,46,51], drug delivery [38,42], dimensional alterations and stability [52][53][54][55][56][57][58], and the stability and time period of the antifungal actions [44]. While there are plenty of studies published, the impact of nystatin in addition to TCs to treat DIS is still not conclusive. Therefore, the aim of the present review was to determine the antifungal potency of nystatin when used with different types of TCs for managing patients with DIS. Additionally, the different properties of TCs influencing its effectiveness were reviewed.

Methodology
This systematic review followed the updated Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) reporting guidelines [59].

Focus Question
The focus question designed for this study was "What is the antifungal potential of "Nystatin" added to different TCs for the management of DIS?" The PICOS format was used to develop the research question.

Literature Search
A systematic search was conducted using electronic databases, PubMed (National Library of Medicine), and the Cochrane Library (Wiley) from January 1975 up to and including December 2020. The following free text keywords were used to carry out the search: "denture induced stomatitis" OR "sore mouth" AND "prosthesis" OR "denture" AND "tissue conditioner" AND "antifungal" OR "nystatin". The bibliographies of the most relevant papers were also searched manually for the identification of eligible studies. Additionally, grey literature was searched using Open Grey.

Eligibility Criteria
The systematic review included original research and clinical studies investigating nystatin added to TCs for antifungal activity. Any research performed using permanent denture base materials, resilient soft liners, and chemically cured acrylics were excluded. Any systematic reviews and narrative reviews, letter to the editor, unpublished data, and opinions were also excluded.
Two independent reviewers (MSS and MSZ) screened the titles, abstracts, and full texts of the eligible papers against the inclusion and exclusion criteria. Any disagreement was discussed and solved by consensus. The kappa coefficient (κ) was used to evaluate the inter-rater agreement between the study authors [60].

Data Extraction
The following data was extracted from the included studies: • Publication year and authors details. • Experimental group(s) reported by the study. • TC used with nystatin.

•
Main results of the study.

Quality Assessment
The quality assessment of the included studies was analysed by the Risk Of Bias In Non-randomized Studies-of Interventions (ROBINS-I), a novel tool for appraising the risk of bias from studies not using randomisation to assign units to comparison groups [61]. For the animal interventional study, Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) (a risk of bias tool) was used [62]. Two authors (MSS and MSZ) independently performed the quality assessment of the included papers.

Data Collection and Analysis
Questionnaire surveys are a popular approach to obtain data for semi-quantitative analysis, while interviewing is often chosen for qualitative studies. In most studies, either

Reason for Exclusion References
Nystatin was not incorporated into tissue conditioner [30,64,68,69,73] Nystatin was used as a mouth rinse [67][68][69][70][71] Full text not available [65] Nystatin not used in the study [48] Nystatin-alginate composite was used [77] Cohen's Kappa score is always less than or equal to one. A value of one indicates a perfect agreement, and values less than one imply less than perfect agreement. It is possible that Cohen's Kappa score is in the negatives, suggesting that the two reviewers agreed less than would be expected just by chance. The extent of inter-rater reliability between the two reviewers was tested using Cohen's Kappa coefficient. The calculated scores of Cohen's Kappa statistic κ were 0.74, 0.80, and 0.85 (Figure 1), indicating substantial to almost perfect agreement [60]. The characteristics of the included papers are demonstrated in Table 2. Table 2. Characteristics of the studies included in the present review.

Study Experimental Group(s) TC and Nystatin Key Outcomes
In-vitro studies Douglas and Clarke (1975) [63] Nystatin + Coe comfort Control Coe comfort Improved physical and mechanical properties of the denture liners by the addition of nystatin. Thomas

Antifungal Potential of Nystatin When Added to TCs
The use of nystatin added to the TCs was investigated against C. albicans for their effectiveness and efficacy in decreasing the DIS. This combination has been reported for its effectiveness and stability [40,41,45,46,74]; however, the duration of the efficiency remains debatable. One study reported a maximum effectiveness for 48 h [75], two studies for 72 h [40,44], one study for 1 week [41], and six studies reported an effectiveness for up to 2 weeks [45,[56][57][58]72,74].
For the treatment of DIS, various combinations of nystatin and TCs were investigated for their efficacy in reducing the growth of fungus and colony numbers. Most of the studies contemplated it as considerably successful if there was candidal growth inhibition after the topical nystatin application added with the TCs [40,46,52,66]. Otherwise, the antifungal action of the TCs added with nystatin was evaluated. A reduction or absence in candidal colonies after applying nystatin TC was considered beneficial [38,41,43,51,58,76]. A few studies evaluated the fungal adherence to the denture base or soft liners. A decrease in the adhesion or formation of a microbial biofilm was considered successful [29,45]. Recently, one study investigated the growth kinetics of C. albicans and reported that nystatin was the most effective antifungal in reducing the fungal metabolic activity [75]. While the criteria for the meaningful outcomes were not homogenous in the papers included, a majority of the studies established that a C. albicans reduction may considerably influence the management of DIS.

Effect of Nystatin on the Properties of TCs
Several studies reported the impact of nystatin on the properties of TCs, such as tensile strength [53,58,74], particle size and distribution [54], shore A hardness and roughness [55], water sorption [56], porosity [57], peel bond strength [70], and surface properties [72]. Most of the studies demonstrated no significant changes in the mechanical, as well as physical, properties of the TCs added with nystatin, showing no harmful effects of the antifungal drug [53,[56][57][58]70,72,74]. In one study, minor changes with the passage of time in the hardness and roughness of the TC was reported [55]. More recently, an in vivo study demonstrated that an application of nystatin added to a TC to rat palatal mucosa did not significantly induce histopathological changes [47], suggesting its in vivo biocompatibility.

Quality Assessment
The quality assessment of the clinical studies is shown in Figure 2 using the ROBINS-I tool [61]. All the three clinical studies [38,41,51] in this analysis were classified to have a moderate risk of bias. The SYRCLE tool [62] used for the quality assessment of one animal study [47] is given in Figure 3, and it was categorised as an unclear risk of bias.

Discussion
The results from the studies evaluated suggest that the majority of them showed favourable outcomes for adding nystatin to TCs. The treatment of DIS is both multifactorial and complicated, taking into consideration the contributing and aetiological factors [12,18,24,25,32]. The denture base materials with added nystatin proved to be a successful treatment modality for cases with DIS [29,38,40,43,45,47,58]. Douglas and Walker (1973) introduced the concept of using nystatin in denture liners around four decades ago. Following that, a significant level of research has been performed to enhance the effectiveness, as well as efficacy, of nystatin added to TCs [78]. The use of nystatin has been commonly documented for the treatment of oral candida infections such as DIS [79][80][81][82]. Likewise, nystatin has been widely used with a variety of TCs [38,[41][42][43]45,46] and exhibited very favourable results related to the reduction of fungal growth.
A study conducted four decades ago showed an effective antifungal activity of nystatin added to various TCs [78]. In another study, nystatin was steadily released from the TCs to the saliva, lessening the salivary yeasts for a limited time period [41]. Furthermore, in a recent animal study, the in-vivo biocompatibility of DIS was determined due to the fact that incorporating nystatin into the TC did not induce histopathological variations in the rat palatal mucosa [47]. More lately, the growth kinetics of C. albicans were investigated, and the results showed a maximum effectiveness for the antifungal nystatin rather than the other antifungals [75]. Concerning the effective stability and time duration of nystatin added to TCs, the studies ranged between three days to one week [40,41,44,70] or to a maximum of two weeks [45,[56][57][58]72,74].
The stability and duration were directly controlled by several factors, including the chemical nature and concentration [38,45,52,72]. Regarding the nystatin concentration, different studies used different concentrations, varying from 500,000 U to 1,000,000 U; therefore, no agreement has been given related to an effective nystatin concentration [29,41,42,55,66]. However, most of latest studies reported different units of measurement for the nystatin concentrations, i.e., 0.032 g [56,57,70,72,74]. This variability between the doses and units of measurements in nystatin made it difficult to conduct a meta-analysis.
For the treatment of mucosal lesions, nystatin is considered to be both a safe and potent antifungal for topical, as well as systemic, applications. Nystatin is one of the drugs of choice to treat oral infections of fungal origin. Though the topical application of nystatin is considered to be effective and safe, a prolonged systemic use can lead to detrimental effects on the liver and kidneys [29,38,[41][42][43]45]. The topical application of nystatin is potent against a fungal infection invading the superficial tissues, but this can be linked to a few adverse effects, such as bad taste and the requirement of repeated applications. This, in turn, may lead to inadequate patient compliance, influencing the treatment outcomes further [38,40,41,51].
The topical application of nystatin four times per day for two weeks was effective in the treatment of DIS [18,19]. Furthermore, the concurrent administration of suspension and nystatin tablets for 15 days yielded a higher rate of clinical, as well mycological, cures in comparison to a monotherapy [27]. A topical nystatin application in combination with amphotericin B also demonstrated a considerable clinical result [84]. Finally, while comparing the nystatin and fluconazole efficacy in the management of DIS, it was summarised that nystatin is the standard topical treatment for oral candidiasis, with the total inhibition of both the binding and colonisation of C. albicans [29]. The uncommon presence of nystatinresistant fungi and its affordable cost are the other advantages of this drug compared to other antifungals [41]. Hota et al. [47] advocated the biocompatibility and feasibility of the incorporation of chlorhexidine and nystatin to the TCs for the treatment of DIS, as no histopathological changes were observed in the palatal mucosa of rats.
Moreover, the studies reported different effective durations, which were based on the type of TCs used with nystatin, such as two days [75], three days [40,44], one week [41], and two weeks [45,[56][57][58]72,74]. Taking into consideration the results of the analysed papers, an efficacious time period of one to two weeks may be recommended to get adequate results of the nystatin therapy. Nevertheless, an ideal time period of nystatin added to the TCs for achieving the desired outcomes may not be determined to date and necessitates more research in this area. A study reported that the yeasts completely disappeared following 15 days of nystatin treatment [85]. Nevertheless, few studies reported a faster fungal regrowth in both the oral cavity and alimentary tract [85,86]. Following two weeks of nystatin withdrawal, the fungal colonies on the base of the dentures and denture-bearing mucosa were similar to those found initially. This fungal regrowth can be reduced if nystatin is administered over a longer period, such as four-six weeks [87]. Therefore, for the treatment of DIS, the exposure of the antifungal for a long time period is needed. Adding to that, the overuse of nystatin is not free from adverse effects and the development of resistant strains of the fungus [88][89][90]. Among the various medicaments, nystatin and chlorhexidine remain the most incorporated medicaments in the TCs for the treatment of DIS [91].
The studies included in the present review analysed altered TCs by their nystatin addition. This addition may interfere the setting reaction of TCs or alter the mechanical and physical characteristics. For achieving the ideal results, the properties of TCs should not be disrupted because of a nystatin addition. Certain studies demonstrated no alterations in the mechanical properties-for example, tensile strength [53,58,74], water sorption and solubility [56], porosity [57], peel bond strength [70], and hardness and roughness [72]. On the contrary, one study exhibited slight alterations in the hardness, as well as roughness, with the passage of time [55].
This variation in the outcomes may be attributed to the different concentrations and materials of the applied nystatin. Apart from that, the additive particle size may also impact the drug filtering from the plasticised matrix of the TCs [54]. For instance, the high surface area and reactivity of nanoparticles may lead to an acceleration of the drug release [92]. While nystatin and TCs with varied strengths were examined, the outcomes of the papers revealed no-to-slight modifications in the mechanical, as well as physical, characteristics of the TCs. Nonetheless, additives may probably affect the properties of materials if they impede the TC polymerisation or react chemically.
Few limitations pertain to this current systematic review. The studies reported a broad range of heterogeneity considering the methodology, outcomes, nystatin concentrations, and dimensional variations of the TCs. These variations did not permit a meta-analysis and, consequently, the application of the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach, which is the most commonly adopted tool for grading the quality of evidence and for making recommendations of quantitative data for each outcome. Out of the included studies, only a limited number of studies were in-vivo experiments (one animal and three clinical) [38,41,51]. Most of the studies were in-vitro laboratory-based studies with or without imitating clinical situations. Moreover, a quality assessment of the in-vitro studies could not be conducted, as there was no standard tool available for a critical appraisal of the in-vitro studies.

Conclusions
From the above-mentioned discussion, it can be summarised that the addition of nystatin into TCs can be advocated for treating patients with DIS. Nevertheless, for the future, more studies are necessary to solve the still-open questions-for instance, optimum dosage, drug release control, and TC stability. Furthermore, randomised controlled clinical trials are much needed for improving the antifungal efficacy for DIS patients.