Maratha Mandal’s Nathajirao G Halgekar, Institute of Dental Sciences and Research Centre, Belgaum-590010.
COURSEOF STUDY AND SUBJECT
Master of Dental Surgery
(Prosthodontics and Crown and Bridge)
DATE OF ADMISSION TO COURSE
TITLE OF THE TOPIC
Effect of melaleuca alternifolia mixed with tissue conditioners in varying doses on colonization and inhibition ofCandida albicans – an invitro study.
Brief Resume of intended work:
6.1 Need for study :
Denture stomatitis, more commonly known as ‘Denture sore mouth’, is a term used to describe certain pathologic changes such as recurrent inflammation or erythema and burning sensation of denture bearing tissues under complete or partial dentures in both the jaws. Candida albicans is the predominant oral yeast associated with denture stomatitis.1,2
Resilient denture lining materials are used to limit such injury by providing cushioning effect. But one of the most serious problems has been colonization of candida albicans and related Candida specieson soft liners.2, 3
Candida infections receive increasing attention due to the increasing prevalence worldwide. The combination of factors such as entrapment of Candida species in irregularities in the denture base and denture lining materials, poor oral hygiene and several other systemic factors are more probable causes for the onset of this infectious disease.
Many different methods have been suggested, for e.g., using antifungal agents mixed with tissue conditioners. Natural topical antiseptic with antifungal activity of Tea tree (melaleuca alternifolia) has also been suggested.7 Hence a need was felt to carry out this study as there are a very few studies in literature related to the use of natural alternatives mixed with tissue conditioners to inhibit the growth of Candida albicans.
6.2 Review of literature :
Catalan A, Pacheco JG, Martinez A, Mondaca MA. (2008)6conducted a study to investigate the effect of melaleuca alternifolia mixed with tissue conditioners (Fitt, lynal, Coe-comfort) by candida albicans. A total growth inhibition of C.albicans was revealed when Fitt and coe-comfort tissue conditioners were used with 1ml of M.alternifolia per portion of tissue conditioner or with Nystatin. But with Lynal showed inhibition only with Nystatin.
Daniluk T, Tokajuk G, Stokowska W, Fiedoruk K, Sciepuk M, Zaremba ML, et al.(2006)7 in a clinical study demonstrated that the occurrence rate of oral candida albicans in patients with dentures is higher(p=0.0003) than in patients without dentures. They also showed that continuous denture wearing facilitate denture stomatitis by increasing the local injury and the time of mucosal exposure to dental plaque.
Brosky ME, Pensun IJ, Morrison B, Hodges JS, Lai JH, Liljemark W (2003)9Conducted a study to count and speciate candida isolated from two resilient denture liners (Molloplast –B and MPDSL-SL)Molloplast-B had, on average, 5 times as many CFU/sample as MPDSL-SL, but thisdifference was not significant (p _ 0.26). A sign test gave a similar nonsignificant trend (p _ 0.057). Chromagar identified several Candida species, and confirmation was made using API 20C AUX.Candida growth on Molloplast-B was not significantly different from growth on MPDS-SL. Several yeast species were cultured from each material. The rates of culture-positive testing did not differ between the 2 resilient denture liners.
Ten cate JM, Klis FM, Pereira-Cenci T, Crielaard W, de Groot PWJ(2009)8stated that candida species adhere not only to denture surfaces, but also to other medical devices such as voice prosthesis, blood and urinary catheters, and heart valves. Therefore, the study of candida adherence to surfaces has much broader scope and relevance than for oral-dental issues. Candida present in the oral cavity serves as a reservoir for inoculation and infections elsewhere in the body.
Pavan S, Santos PHD, Filho JNA, Spolidorio DMP(2010)10evaluated the invitro adherence of pathogenic microorganisms, candida albicans, staphylococcus aureus, and pseudomonas aeruginosa, to soft lining materials and their inhibitory effect on these microorganisms. To measure adherence, specimens of Molloplast B, Ufi Gel P were inoculated (107cfu/ml) with TSB media containing microorganisms and CFU of each specimen were quantified. The surface roughness was measured with a profilometer to assess the relationship between the adherence of each microorganism and surface roughness of each material. For the inhibition test, specimens of each material were placed in the agar plates inoculated individually with the microorganisms. After 48 hours, the inhibition zones around the specimens were measured. None of the materials exhibited inhibition zones.
The number of cfu/ml of S.aureus and P.aeruginosa were significantly greater than candida albicans for both materials. The Ufi gel P exhibited greater adherence of candida albicans than Molloplast B. No correlation was observed between the adherence of microorganisms and surface roughness.
6.3 Objectives :
To evaluate the colonization of Candida albicans in selected commercially available denture lining materials by mixing them with varying doses of tea tree oil.
To evaluate the inhibition of Candida albicans in selected commercially available denture lining materials by mixing them with varying doses of tea tree oil.
Materials and Methods :
Following materials will be used:
Visco Gel (DENTSPLY, India) - Self cure denture lining material.
The study will be conducted at the Department of Prosthodontics and Crown and Bridge and Department of Microbiology& Molecular Biology, MarathaMandal’sNathajirao G. Halgekar Institute of Dental Science and Research Centre, Belgaum.
Four different doses of Melaleuca alternifolia will be added to the two pre-selected denture lining material liquids and then these liquids will be mixed with the powder according to the manufacturer’s instructions.
7.2 Method of collection of data :
Preparation of test discs of denture lining materials :
A total of 180 test discs of 10mm diameter and 1.5 mm thickness will be processed with the help of denture flasks (160-experimental & 20-control). For this, discs of modeling wax will be punched out from the wax sheet, Dental stone will be poured into the shallow part of the dental flask, and the punched wax disks will be placed on the surface of the stone. After setting, separating medium will be applied, upper part of the flask will be placed into the position and dental stone will be poured into it. After de-waxing, the voids formed will be filled by the denture lining materials in following manner ;
Doses of Melaleuca alternifolia (0.5, 1.0, 1.5, 2.0 ml) will be added to the conditioner liquids and homogenized in a sterile glass beaker for 30 seconds, so that the concentration of tea tree oil in four different groups, namely 10, 20, 30, 40% vol/vol.
Immediately afterwards the conditioning powder will be added and mixed and filled into the voids left by the wax discs according to the manufacturer’s instructions. .
This procedure will produce 10 discs for each concentration of tea tree oil added for each material of denture lining material which will then be polymerized, trimmed and kept in polyethylene containers in a dry environment until required.
Testing for colonization of Candida albicans on test discs of denture lining material:
Test discs of denture lining materials in sterile artificial saliva will be inoculated with Candida albicans and incubated for 6 weeks. After this the candida cells on the test discs will be fixed, dehydrated and air dried. Appropriate controls without addition of tea tree oil will be set up for each group. The test discs will be sectioned and viewed via fluorescence microscopy. The penetration into the denture lining material will be measured by the amount of hyphae present in each field.
Testing for the inhibition of Candida albicans by test discs of denture lining materials :
This will be tested using diagnostic sensitivity testing agar for each material with Nystatin test discs as control.
The data will be tabulated as under:
ANTIFUNGAL AGENTS USED
(colony forming units)
GC SOFT LINER
(Tea tree oil)
The data will be subjected to statistical analysis byTwo way ANOVA (Analysis of variance) followed byDunnet’s post hoc multiple comparison test. Differences will be considered statistically significant for p<0.05.
7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If, so please describe briefly.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Anto Jose, Brent JC, Steven M, Beth Y, David FL, Jeremy B,et al. Reducing the incidence of Denture stomatitis: Are denture cleaners sufficient? Journal of Prosthodontics 2010;19:252-57.
Bulad K, Taylor RL, Verran J, Fraser J. Colonization and penetration of denture soft lining materials by candida albicans. Dental Materials 2004;20:167-75.
Williamson RT, Clinical application of soft denture liner: a case report. Quintessence Int 1995;26:413-18.
Catalan A, Pacheco JG, Martinez A, Mondaca MA. Invitro and Invivo activity of Melaleuca alternifolia mixes with tissue conditioner on Candida albicans. Oral Surg Oral Med Oral Pathol Oral RAdiol Endod 2008;105:327-32.
Daniluk T, Tokajuk G, Stokowska W, Fiedoruk K, Sciepuk M, Zaremba ML et al. Occurance rate of oral candida albicans in denture wearer patients. Advances in Medical Sciences 2006;51:77-80.
Ten Cate JM, Klis FM, Pereira-Cenci T, Crielaard W, de GGroot PWJ. Molecular and Cellular Mechanisms that lead to candida biofilm formation. JDEnt Res 2009;88:105-15.
Brosky ME, Pensun IJ, Morrison B, Hodges JS, Lai JH, Liljemark W Clinical Evaluation of Resilient Denture Liners.Part 2: CandidaCount and Speciation.J Prosthodont 2003;12:162-67.
Pavan S, Santos PHD, Filho JNA, Spolidorio DMP. Colonisaation of soft lining materials by microorganisms. Gerodontology 2010;27:211-16.