Vol. 118 No. 1 July 2014

Evaluation of different treatment methods against denturestomatitis: a randomized clinical studyLidia Yileng Tay, DDS, MSc, PhD,a Janaina Habib Jorge, DDS, MSc, PhD,b

Daniel Rodrigo Herrera, DDS, MSc, PhD,c Nara Hellen Campanha, DDS, MSc, PhD,a

Brenda Paula Gomes, DDS, MSc, PhD,c and Fabio Andre dos Santos, DDS, MSc, PhDa

Ponta Grossa State University (UEPG), Ponta Grossa, Paraná, Brazil; UNESP e Univ. Estadual Paulista, Araraquara, São Paulo, Brazil; Universityof Campinas (UNICAMP), Piracicaba, São Paulo, Brazil

Objective. The aim of this clinical study was to determine the efficacy of Uncaria tomentosa (cat’s claw) against denture

stomatitis (DS).

Study Design. Fifty patients with DS were randomly assigned into 3 groups to receive 2% miconazole, placebo, or 2%

U tomentosa gel. DS level was recorded immediately, after 1 week of treatment, and 1 week after treatment. The clinical

effectiveness of each treatment was measured using Newton’s criteria. Mycologic samples from palatal mucosa and prosthesis

were obtained to determinate colony forming units per milliliter (CFU/mL) and fungal identification at each evaluation period.

Results. Candida species were identified with HiCrome Candida and API 20C AUX biochemical test. DS severity decreased in

all groups (P < .05). A significant reduction in number of CFU/mL after 1 week (P < .05) was observed for all groups and

remained after 14 days (P> .05). C albicanswas the most prevalent microorganism before treatment, followed by C tropicalis, C

glabrata, and C krusei, regardless of the group and time evaluated. U tomentosa gel had the same effect as 2% miconazole gel.

Conclusions. U tomentosa gel is an effective topical adjuvant treatment for denture stomatitis. (Oral Surg Oral Med Oral

Pathol Oral Radiol 2014;118:72-77)

Although they are often present as benign commensalorganisms in healthy individuals, Candida spp producea broad range of serious illnesses in compromisedhosts.1 Treatment has become increasingly difficult andexpensive. Oral candidiasis is the most common fungalinfection in humans, primarily among the elderly anddenture users. It can affect up to 65% of elderly peoplewho use total dentures.2,3 Denture stomatitis (DS) is aterm describing this infection when related to the use ofa dental prosthesis.

Species of Candida are the principal causative agentof DS, an inflammatory reaction of the supporting tis-sues of total and partial removable prostheses. DS ischaracterized by hyperemia and edema, sometimesaccompanied by hemorrhagic petechiae. Clinicalsymptoms of the disease may include pain, irritation,and disturbance of salivation; however, many patientswith this disease have no symptoms.4

Various treatments have been proposed against DS,and topical or systemic antifungal medications are themost common. Nystatin, amphotericin B, clotrima-zole, and miconazole are antifungal agents often used

aDepartment of Dentistry, Ponta Grossa State University (UEPG).bDepartment of Dental Materials and Prosthodontics, AraraquaraDental School, UNESP e Univ. Estadual Paulista.cDepartment of Restorative Dentistry, Endodontics Division, Piraci-caba Dental School, University of Campinas (UNICAMP).Received for publication May 16, 2013; returned for revision Mar 13,2014; accepted for publication Mar 22, 2014.� 2014 Elsevier Inc. All rights reserved.2212-4403/$ - see front matterhttp://dx.doi.org/10.1016/j.oooo.2014.03.017

72

against oral candidiasis.5,6 Treatment also includescleaning the prosthesis and verifying the need forchange in the denture.7 In addition, suspension of theuse of the prosthesis can reduce the inflammatorycomponent. However, factors such as fungal resistanceto the medicine and drug toxicity have led to thesearch for alternative treatments.8

Phytotherapeutic agents are prepared exclusivelywith plants or parts of medicinal plants. A great deal ofrecent research has focused on their effectiveness. Theyare less costly, are less toxic, and have fewer side ef-fects than many synthetics.9

Uncaria tomentosa (of the family Rubiaceae), knownas cat’s claw, is usually used in Peruvian medicine.10 Invitro and in vivo studies in animals and humans haveconfirmed its effectiveness. It is a good anti-inflam-matory arthritis treatment.11-15 Its properties are due tothe combined activity of its different components ratherthan any one isolated compound.16 In addition, it is animportant source of bioactive substances, such asmonoterpenoid oxindole alkaloids, which have immu-nomodulatory and antitumoral properties.17

Statement of Clinical Relevance

Uncaria tomentosa gel was clinically evaluated forthe treatment of denture stomatitis. It showed similarantimicrobial activity as 2% miconazole gel. How-ever, its use must be associated with hygienemethods for a successful treatment.

OOOO ORIGINAL ARTICLE

Volume 118, Number 1 Tay et al. 73

Few studies, with contradictory results, were found inthe literature regarding the effect of U tomentosa onopportunistic fungi. Ccahuana-Vasquez et al.18 observedin vitro that tested concentrations of U tomentosa did notinhibit Candida albicans. On the other hand, Herreraet al.15 indicated that 2% cat’s claw gel is effective invitro against microorganisms frequently found in the oralcavity. However, clinical studies have not tested its effecton DS. Thus, the purpose of this clinical study was todetermine the efficacy of U tomentosa gel on DS. Thehypothesis was that U tomentosa gel could reduce theinfection of dentures’ supporting tissues.

MATERIALS AND METHODSParticipantsFor determining sample size, a power analysis (signif-icance level, 5%; power, 80%) was used that was inagreement with previous studies.4,19 Calculation resul-ted in a minimal sample size of 15 patients in eachgroup.

Fifty individuals who ranged between 45 and 85years of age, had good general health, were denturewearers for at least 1 year, and had denture stomatitistype I, II, or III according to Newton’s criteria20 wereselected from the Department of Dentistry of the StateUniversity of Ponta Grossa. The selection of these pa-tients included a general health questionnaire and initialclinical examination. The procedures carried out in thestudy complied with Resolution 196/96 of the BrazilianHealth Ministry, and the protocol was approved by theEthics Committee of the Ponta Grossa State University(Protocol 17572/10). Each participant voluntarilysigned an informed consent form before enrolling.

Exclusion criteria were based on medical histories.Excluded were women of childbearing age; patientswith impaired hepatic or renal function, diabetes,xerostomia, hypoparathyroidism, immune alterations,chemotherapy, or radiation therapy; patients who hadreceived any recent treatment with antibiotics, antifun-gals, or steroidal agents within 4 weeks before thestudy; and those with poorly fitting dentures.

The selected participants were examined for oral le-sions, oral hygiene, and denture conditions. Newton’sclassifications of type I (bleeding spots), type II (diffuseerythematous areas), and type III (granular inflammation)were used. Candidiasis was identified based on the resultsof microbiologic cultures collected from the palatal mu-cosa and from the denture surface. One colony or morefrom the palatal mucosa and more than 100 colonies fromthe denture were considered as positive results.5

Study designThe study was double-blind. According to the stratifiedrandomization list, 50 patients were randomly assigned

to 1 of the 3 experimental groups as follows: group M(positive control), 2% miconazole gel (20 mg/g, Dak-tarin gel; Janssen-Cilag, São Paulo, São Paulo, Brazil);group P (placebo group), hydroxyethyl cellulose (1.5%Natrosol; Fleming Manipulações, Ponta Grossa, Paraná,Brazil); and group UT (experimental group), 2% Utomentosa gel (Fleming Manipulações). The patientsdid not know which treatment they would receive.

All patients (from all groups) received treatment onadmission (day 0) and were told to apply the gel on thebase of the denture after meals, 3 times a day, for 1week. Each application had 2.5 mL (1 teaspoonful) ofgel on the denture, with surplus gel kept in the mouthuntil the next application. The patients cleaned theirdentures before each application with a toothbrush andwater; they suspended the use of the denture at night,leaving it in a glass of regular water. At each visit, thepalatal mucosa was observed for local adverse re-actions, and the patients were asked whether theyexperienced any irritation, burning, nausea, vomiting,or diarrhea.

The effectiveness of treatment was clinicallyassessed using Newton’s criteria20 (1962) for scoringthe severity of DS, with 0 for a healthy palate. It wastested on day 0, day 7 (end of treatment), and day 14(7 days after treatment). At follow-up visits, visualobservations were made, and color photographs of themucosa were taken using a digital camera (EOS RebelT2i; Canon, Tokyo, Japan). The operator was alwaysthe same (L.Y.T.), as were the conditions, such asplace, light, angle, and patient position. Two indepen-dent calibrated dentists blindly analyzed the photo-graphs taken of each patient.21

Mycologic examinationFor each patient, Candida was recovered every week byrubbing oral swabs along the palatal mucosa and thetissue surface of the upper denture.22 Each swab wasplaced into a test tube containing 5 mL of brain-heartinfusion medium (Himedia Laboratories, Mumbai, In-dia). It was then vortexed for 1 minute to suspend theorganisms from the swab. An aliquot of 50 mL from thissuspension was plated on Sabouraud dextrose agar(SDA) plates (Himedia Laboratories) with 5 mg/mLchloramphenicol, another 50 mL was plated onHiCrome Candida Agar (Himedia Laboratories), andboth plates were incubated at 37�C for 48 hours. Adigital colony counter (CP 600 Plus; Phoenix Ind ComEquipamentos Científicos, Araraquara, São Paulo,Brazil) quantified the colonies on SDA and determinedthe number of colony-forming units per milliliter (CFU/mL). Values of CFU/mL from samples of the palatalmucosa were correlated with those of the tissue surfaceof the upper denture.

Fig. 1. Mean and SD of severity degree of denture stomatitis(scores) for each group (M, miconazole; P, placebo; UT,Uncaria tomentosa) in different time points. Consideringgroups in different time points: (**) P < .05 with 7 and 14days. Kruskal-Wallis and Dunn post hoc tests.

ORAL MEDICINE OOOO

74 Tay et al. July 2014

The colonies on HiCrome Candida were presump-tively identified by color and confirmed by biochemicaltests. First, 1 colony of each color type grown onHiCrome Candida was inoculated onto a fresh SDAplate. After 48 hours at 37�C, API 20C AUX (Bio-Mérieux SA, Marcy-l’Etoile, France) was used toidentify the isolates, using Apiweb software (Bio-Mérieux SA).

A 1-way analysis of variance and the Tukey multiplecomparison test compared age, gender, and time ofprosthesis use for each participant. The Friedman testand the Dunn multiple comparison test (a ¼ .05)analyzed the effectiveness of each treatment. TheKruskal-Wallis test and the Dunn multiple comparisontest (a ¼ .05) also compared clinical and microbiologicresults of the 3 groups. The Spearman r test (a ¼ .05)analyzed correlation between CFU/mL of the palatalmucosa and CFU/mL of the prosthesis. Data wereanalyzed using GraphPad Prism 6 (GraphPad SoftwareInc).

RESULTSOf the 50 patients, 2 were withdrawn from the study fornot returning to the controls. The remaining 48, whohad a mean age of 63.83 � 8.9 years, entered the trial;this group was 89.58% women and 10.41% men. Themiconazole group (group M) consisted of 13 womenand 2 men with a mean age of 65.8 years; the mean timeof use of the prosthesis was 11.4 years. The placebogroup (group P) consisted of 16 women with a meanage of 63.18 years; the mean time of use of the pros-thesis was 19.5 years. The U tomentosa group (groupUT) consisted of 14 women and 3 men with a mean ageof 62.7 years; the mean time of use of the prosthesiswas 14.76 years. There was not a significant differencein gender between the groups (P ¼ .59) or in the time ofuse of the prosthesis (P ¼ .21).

Figure 1 shows the severity of DS. Before treatment(day 0), type II was observed in all groups, and theseverity diminished over the evaluation periods (7 and14 days) with no significant differences between thetreatments (P > .05) (Figure 2).

Candida spp were detected from each patient rubbingoral swabs along the palatal mucosa and the surface ofthe upper denture. The CFU/mL values were logarithm-transformed to achieve a normal distribution for sta-tistical analyses.

The CFU/mL values of samples taken from thepalatal mucosa were significantly lower than thosetaken from the prosthesis, but a direct correlation be-tween them was observed (r ¼ 0.452; P < .0001).Figure 3 shows the mean of CFU/mL in log based onthe total number of Candida cells from the palatalmucosa and from the surface of the upper denture.Similarly to the clinical results, the concentration of

colonies was highest on day 0 and diminished in sub-sequent evaluation periods (7 and 14 days), with nosignificant differences between the groups (P > .05).On day 7, there was a lower number of CFU in group Mthan in groups P and UT.

Table I shows the identification of Candida spp inthe patients evaluated. It can be seen that C albicanswas the most prevalent species, followed by C tropi-calis, C glabrata, and C krusei, regardless of the groupevaluated. All microorganisms reduced in numbersthroughout the trial, except C tropicalis in group UTand C glabrata in group M, whose numbers increasedfrom day 7 to day 14.

DISCUSSIONThe purpose of this clinical study was to determine theefficacy of U tomentosa gel against DS. The hypothesisof this study was that U tomentosa gel could eliminatethe infection of the supporting tissues of the dentures.We found a reduction of DS severity after 7 days oftreatment, from grade 2 to grade 1, that remained after14 days (see Figure 1). However, there were no dif-ferences among the 3 groups. Thus, it is not possible toaffirm that the U tomentosa gel was solely responsiblefor this reduction. The reduction of fungal load fromdentures and palates after treatments, associated withpatients’ compliance with the oral and denture hygiene(including denture removal during sleep), might beresponsible for the observed effect. In this study,treatment started with meticulous denture hygiene andafter removal of other local factors.23 Although notsufficient to treat DS,22,24 optimal denture and oralhygiene are essential to maintain low levels of micro-organisms on dentures and within the mouth.25-27

Consistent with this, we observed a statistically sig-nificant relationship between DS, yeast presence, anddenture cleanliness.

Fig. 3. Mean and SD of colony-forming units for each group(M, miconazole; P, placebo; UT, Uncaria tomentosa) indifferent time points. Considering time points: (*) P < .05with P and UT groups at day 7. Considering groups indifferent time points: (**) P < .05 with 7 and 14 days.Kruskal-Wallis and Dunn post hoc test.

Fig. 2. Palate of patients from 3 experimental groups. A, Patient 1 at baseline. B, Patient 1 after treatment with miconazole. C, Patient2 at baseline. D, Patient 2 after treatment with placebo. E, Patient 3 at baseline. F, Patient 3 after treatment with Uncaria tomentosa.

OOOO ORIGINAL ARTICLE

Volume 118, Number 1 Tay et al. 75

We found that U tomentosa could be an alternativetreatment for denture stomatitis, agreeing with thefindings of Paiva et al.28 They evaluated the clinicaleffectiveness of U tomentosa on oral candidiasis andfound that it was as effective as miconazole. This maybe explained by the 2 chemotypes, with different pat-terns of tetracyclic or pentacyclic oxindole alkaloids,present in U tomentosa. The pentacyclic oxindole al-kaloids have immune modulatory effects antagonisti-cally inhibited by tetracyclic oxindole alkaloids.11,29,30

Additional research is required to identify the plant’stherapeutic agents.

The positive results in the placebo group could beexplained by the fact that simply letting patients know

about their disease can raise their concern andcompliance to improve oral hygiene.31 In addition, theapplication of placebo gel could have preventedretention of yeasts on the denture surface.23 Becausethe gel formulation keeps the active substance in con-tact with the lesion for a longer time, it has shown moresuccess on its application than other oral antifungalssuch as nystatin. The product is applied directly to thepreviously cleaned prosthesis, which acts as a tray,allowing longer contact time with the lesion, potentiallycausing a better response and faster regression ofsymptoms.

On day 7, there were fewer CFU/mL in group M thanin groups P and UT. Thus, miconazole seems to bemore efficacious in reducing the number of coloniesthan U tomentosa (experimental group) and Natrosol(placebo group). Miconazole is a scientifically provenantifungal.5 Azoles have a cytostatic or cytotoxic effect,inhibiting the metabolism of fungal cell membranecomponents.32,33 In addition, the azole stimulatesintracellular reactive oxygen species that are inducers ofapoptosis in yeasts.33,34 Despite miconazole’s provenantifungal action, some clinical isolates have shownresistance,34 probably owing to the protective effect ofantioxidants during miconazole treatment.33 Thus, theuse of alternative treatments such as phytotherapeuticagents is promising.

There are a variety of methods for identifying yeastsfrom clinical samples. These include traditionalmethods, such as the germ-tube test, morphologystudies, and carbohydrate utilization; rapid methods,such as enzymatic and fluorogenic tests; commerciallyavailable methods; automated systems; and molecular

Table I. Number and percentage of patients in whom Candida species were found

C albicans C tropicalis C glabrata C krusei

Day 0 7 14 0 7 14 0 7 14 0 7 14Group M (n ¼ 15) 14 (93.3) 7 (46.6) 3 (26.6) 3 (20) 2 (13.3) 1 (6.6) 2 (13.3) 0 2 (13.3) 1 (6.6) 0 0Group P (n ¼ 16) 15 (93.7) 7 (43.7) 6 (37.5) 5 (31.2) 4 (25) 1 (6.2) 3 (18.7) 1 (6.2) 2 (12.5) 1 (6.2) 0 0Group UT (n ¼ 17) 15 (88.2) 8 (47.0) 3 (17.6) 5 (29.4) 3 (17.6) 5 (29.4) 1 (5.8) 1 (5.8) 1 (5.8) 3 (17.6) 0 1 (5.8)

ORAL MEDICINE OOOO

76 Tay et al. July 2014

typing techniques.35 This study used HiCrome Candidaagar and biochemical test API 20C AUX. C albicanswas the most prevalent species, followed by C tropi-calis, C krusei, and C glabrata, regardless of the groupevaluated. These results are in agreement with severalstudies.35-39 A large variation in species frequency hasbeen reported in different regions of the world.Although several investigators found C tropicalis oneof the most common non-albicans species isolated inBrazil and South America, C glabrata is found morefrequently in North America.40,41

The week-to-week reduction of microorganisms had2 exceptions, C tropicalis in group C and C glabrata ingroup A, which grew back by day 14 after decreasingby day 7, suggesting that treatments were not effectivefor this species. C glabrata exhibits quite clinicallysignificant cross-resistance to older azole drugs (flu-conazole and itraconazole) and to voriconazole.42 Onthe other hand, C tropicalis cells obtained from the oralcavity of denture wearers with denture stomatitis aremore adherent to buccal epithelial cells than those ob-tained from patients without signs of disease.43 Theability of C tropicalis strains to form biofilm ondifferent surfaces is another potential virulence trait,which may increase their resistance to antifungaltreatment.44 Although these factors may explain theresistance by C glabrata and C tropicalis in the presentstudy, further investigations are required to examineand better understand the mechanism of pathogenicityof these species of Candida.

The literature reports several alternative treatmentsand different times of treatment against DS withoptimal results ranging between 1 and 4 weeks, with theeffectiveness depending on medical therapy or pros-thesis treatment.21,22 In the present study, 1 treatmentweek was sufficient to be effective. However, furtherstudies increasing the time of treatment with Utomentosa gel are needed.

CONCLUSIONSU tomentosa gel was an effective topical adjuvanttreatment (i.e., it had the same effect as 2% micona-zole gel); however, the influence of the prescribedhygiene methods should be evaluated in future studies.In addition, U tomentosa, as an herbal treatment,might be more economical than other treatments forpatients.

REFERENCES1. Rex JH, Rinaldi MG, Pfaller MA. Resistance of Candida species

to fluconazole. Antimicrob Agents Chemother. 1995;39:1-8.2. Rabiei M, Kasemnezhad E, Masoudi rad H, Shakiba M,

Pourkay H. Prevalence of oral and dental disorders in institu-tionalized elderly people in Rasht, Iran. Gerodontology. 2010;27:174-177.

3. Jorge J Jr, Almeida OP, Bozzo L, Scully C, Graner E. Oralmucosal health and disease in institutionalized elderly in Brazil.Community Dent Oral Epidemiol. 1991;19:173-175.

4. Altarawneh S, Bencharit S, Mendoza L, et al. Clinical and his-tological findings of denture stomatitis as related to intraoralcolonization patterns of Candida albicans, salivary flow, and drymouth. J Prosthodont. 2013;22:13-22.

5. Amanlou M, Beitollahi JM, Abdollahzadeh S, Tohidast-Ekrad Z.Miconazole gel compared with Zataria multiflora Boiss gel in thetreatment of denture stomatitis. Phytother Res. 2006;20:966-969.

6. Redding S, Bhatt B, Rawls R, Siegel G, Scott K, Lopez-Ribot J.Inhibition of Candida albicans biofilm formation on denturematerial. Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2009;107:669-672.

7. Aldana L, Marker VA, Kolstad R, Iacopino AM. Effects ofCandida treatment regimens on the physical properties of dentureresins. Int J Prosthodont. 1994;7:473-478.

8. Casaroto AR, Lara VS. Phytomedicines for Candida-associateddenture stomatitis. Fitoterapia. 2010;81:323-328.

9. Valerio LG Jr, Gonzales GF. Toxicological aspects of the SouthAmerican herbs cat’s claw (Uncaria tomentosa) and maca(Lepidium meyenii): a critical synopsis. Toxicol Rev. 2005;24:11-35.

10. Mammone T, Akesson C, Gan D, Giampapa V, Pero RW.A water soluble extract from Uncaria tomentosa (cat’s claw) is apotent enhancer of DNA repair in primary organ cultures of hu-man skin. Phytother Res. 2006;20:178-183.

11. Keplinger K, Laus G, Wurm M, Dierich MP, Teppner H. Uncariatomentosa (Willd.) DC.dethnomedicinal use and new pharma-cological, toxicological and botanical results. J Ethnopharmacol.1999;64:23-34.

12. Lemaire I, Assinewe V, Cano P, Awang DV, Arnason JT.Stimulation of interleukin-1 and -6 production in alveolar mac-rophages by the neotropical liana, Uncaria tomentosa (uña degato). J Ethnopharmacol. 1999;64:109-115.

13. Sandoval M, Okuhama NN, Zhang XJ, et al. Anti-inflammatoryand antioxidant activities of cat’s claw (Uncaria tomentosa andUncaria guianensis) are independent of their alkaloid content.Phytomedicine. 2002;9:325-337.

14. Santos Araújo Mdo C, Farias IL, Gutierres J, et al. Uncariatomentosadadjuvant treatment for breast cancer: clinical trial[published online June 28, 2012]. Evid Based ComplementAlternat Med. 2012;2012:676984, http://dx.doi.org/10.1155/2012/676984.

15. Herrera DR, Tay LY, Rezende EC, Kozlowski VA Jr, Santos EB.In vitro antimicrobial activity of phytotherapic Uncaria tomen-tosa against endodontic pathogens. J Oral Sci. 2010;52:473-476.

16. Ribeiro AF, de Oliveira Rezende RL, Cabral LM, de Sousa VP.Poly e-caprolactone nanoparticles loaded with Uncaria tomentosa

OOOO ORIGINAL ARTICLE

Volume 118, Number 1 Tay et al. 77

extract: preparation, characterization, and optimization using theBox-Behnken design. Int J Nanomedicine. 2013;8:431-442.

17. Heitzman ME, Neto CC, Winiarz E, Vaisberg AJ, Hammond GB.Ethnobotany, phytochemistry and pharmacology of Uncaria(Rubiaceae). Phytochemistry. 2005;66:5-29.

18. Ccahuana-Vasquez RA, Santos SS, Koga-Ito CY, Jorge AO.Antimicrobial activity of Uncaria tomentosa against oral humanpathogens. Braz Oral Res. 2007;21:46-50.

19. Uludamar A, Ozkan YK, Kadir T, Ceyhan I. In vivo efficacy ofalkaline peroxide tablets and mouthwashes on Candida albicansin patients with denture stomatitis. J Appl Oral Sci. 2010;18:291-296.

20. Newton AV. Denture sore mouth: a possible aetiology. Br Dent J.1962;112:357-360.

21. Sanitá PV, Machado AL, Pavarina AC, Massucato EMS,Colombo AL, Vergani CE. Denture microwave disinfectionversus nystatin in treating well-controlled type 2 diabetics withdenture stomatitis: a randomized clinical trial. Int J Prosthod.2012;25:232-244.

22. Neppelenbroek KH, Pavarina AC, Palomari Spolidorio DM,Sgavioli Massucato EM, Spolidorio LC, Vergani CE. Effective-ness of microwave disinfection of complete dentures on thetreatment of Candida-related denture stomatitis. J Oral Rehabil.2008;35:836-846.

23. Könsberg R, Axéll T. Treatment of Candida-infected denturestomatitis with a miconazole lacquer. Oral Surg Oral Med OralPathol. 1994;78:306-311.

24. Barnabé W, de Mendonça Neto T, Pimenta FC, Pegoraro LF,Scolaro JM. Efficacy of sodium hypochlorite and coconut soapused as disinfecting agents in the reduction of denture stomatitis,Streptococcus mutans, and Candida albicans. J Oral Rehabil.2004;31:453-459.

25. Ramage G, Zalewska A, Cameron DA, et al. A comparative invitro study of two denture cleaning techniques as an effectivestrategy for inhibiting Candida albicans biofilms on denturesurfaces and reducing inflammation. J Prosthodont. 2012;21:516-522.

26. Kanli A, Demirel F, Sezgin Y. Oral candidosis, denture cleanli-ness and hygiene habits in an elderly population. Aging Clin ExpRes. 2005;17:502-507.

27. Kulak-Ozkan Y, Kazazoglu E, Arikan A. Oral hygiene habits,denture cleanliness, presence of yeasts and stomatitis in elderlypeople. J Oral Rehabil. 2002;29:300-304.

28. Paiva LCA, Ribeiro RA, Pereira JV. Avaliação clínica e labo-ratorial do gel da Uncaria tomentosa (unha de gato) sobre can-didose oral [Clinical and laboratory evaluation of Uncariatomentosa gel (cat’s claw) gel on oral candidiasis] [in Portu-guese]. Brazilian Journal of Pharmacognosy. 2009;19:423-428.

29. Wagner H, Kreutzkamp B, Jurcic K. Die Alkaloide von Uncariatomentosa und ihre Phagozytose-steigernde Wirkung [The alka-loids of Uncaria tomentosa and their phagocytosis-enhancingeffect] [in German]. Planta Med. 1985;51:419-423.

30. Wurm M, Kacani L, Laus G, Keplinger K, Dierich MP. Penta-cyclic oxindole alkaloids from Uncaria tomentosa induce humanendothelial cells to release a lymphocyte-proliferation-regulatingfactor. Planta Med. 1998;64:701-704.

31. De Amici D, Klersy C, Ramajoli F, Brustia L, Politi P. Impact ofthe Hawthorne effect in a longitudinal clinical study: the case ofanesthesia. Control Clin Trials. 2000;21:103-114.

32. Van den Bossche H, Willemsens G, Cools W, Marichal P,Lauwers W. Hypothesis on the molecular basis of the antifungal

activity of N-substituted imidazoles and triazoles. Biochem SocTrans. 1983;11:665-667.

33. Vandenbosch D, Braeckmans K, Nelis HJ, Coenye T. Fungicidalactivity of miconazole against Candida spp. biofilms.J Antimicrob Chemother. 2010;65:694-700.

34. Kobayashi D, Kondo K, Uehara N, et al. Endogenous reactiveoxygen species is an important mediator of miconazole antifungaleffect. Antimicrob Agents Chemother. 2002;46:3113-3117.

35. Campanha NH, Neppelenbroek KH, Spolidorio DM,Spolidorio LC, Pavarina AC. Phenotypic methods and commer-cial systems for the discrimination between C. albicans and C.dubliniensis. Oral Dis. 2005;11:392-398.

36. Sanitá PV, Pavarina AC, Giampaolo ET, et al. Candida spp.prevalence in well controlled type 2 diabetic patients with denturestomatitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2011;111:726-733.

37. Da�gistan S, Aktas AE, Caglayan F, Ayyildiz A, Bilge M. Dif-ferential diagnosis of denture-induced stomatitis, Candida, andtheir variations in patients using complete denture: a clinical andmycological study. Mycoses. 2009;52:266-271.

38. Dorko E, Jenca A, Pilipcinec E, Danko J, Svický E, Tkáciková L.Candida-associated denture stomatitis. Folia Microbiol (Praha).2001;46:443-446.

39. Cross LJ, Williams DW, Sweeney CP, Jackson MS, Lewis MA,Bagg J. Evaluation of the recurrence of denture stomatitis andCandida colonization in a small group of patients who receiveditraconazole. Oral Surg Oral Med Oral Pathol Oral RadiolEndod. 2004;97:351-358.

40. Colombo AL, Perfect J, DiNubile M, et al. Global distributionand outcomes for Candida species causing invasive candidi-asis: results from an international randomized double-blindstudy of caspofungin versus amphotericin B for the treatment ofinvasive candidiasis. Eur J Clin Microbiol Infect Dis. 2003;22:470-474.

41. Pfaller MA, Moet GJ, Messer SA, Jones RN, Castanheira M.Geographic variations in species distribution and echinocandinand azole antifungal resistance rates among Candida bloodstreaminfection isolates: report from the SENTRY Antimicrobial Sur-veillance Program (2008 to 2009). J Clin Microbiol. 2011;49:396-399.

42. Panackal AA, Gribskov JL, Staab JF, Kirby KA, Rinaldi M,Marr KA. Clinical significance of azole antifungal drug cross-resistance in Candida glabrata. J Clin Microbiol. 2006;44:1740-1743.

43. Lyon JP, Resende MA. Evaluation of adhesion to buccalepithelial cells in Candida species obtained from denture wearersafter exposure to fluconazole. Mycoses. 2007;50:21-24.

44. Ramage G, Tomsett K, Wickes BL, López-Ribot JL,Redding SW. Denture stomatitis: a role for Candida biofilms.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004;98:53-59.

Reprint requests:

Janaina Habib Jorge, DDS, MSc, PhDUniversidade Estadual Paulista (UNESP)Rua Humaitá1680, AraraquaraSP 14801-903BrazilJanainahj@bol.com.br; janainahj@foar.unesp.br