Study design
A single-arm non-randomized clinical trial was conducted among twenty study participants who met the inclusion and exclusion criteria at the Eastman Institute for Oral Health, University of Rochester. The study protocol was approved by the University of Rochester Research Subject Review Board (#STUDY00004638). This study is registered at the Trials.gov (#NCT04550546). This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.
Participants
Study participants were recruited from the existing pool of patients at the University of Rochester Medical Center (URMC) Eastman Institute for Oral Health (EIOH) clinics and from the larger community at Rochester NY.
The inclusion criteria include: a) 18 years and older; b) positive oral Candida detection with sufficient oral Candida burden to meet the laboratory criteria for a diagnosis of oral candidiasis, ≥ 400 Colony Forming Unit (CFU) /mL of salivary Candida [20]; c) ≥ 10,000 CFU/mL of S. mutans in the saliva.
The exclusion criteria include: a) Visible signs of candidiasis on the mucosa or tongue at screening; b) With systemic diseases, such as HIV, cancer or diabetes; c) History of using local (oral) or systemic antibiotics or antifungal medication within the last 3 months; d) Women who are currently pregnant or reported that she is currently breast feeding. A pregnancy test (urine test) was conducted to exclude participants who are pregnant; e) With more than 8 missing teeth (third molars and orthodontically extracted teeth are not included); f) With more than 4 decayed teeth; g) With removable dental prosthesis that are used to restore missing teeth; h) Allergy to Nystatin.
Study procedures
Study participants who entered the trial were instructed to rinse the mouth with (6 ml of 100,000 IU/mL) nystatin suspension, without swallowing the suspension, at the frequency of four times per day (every six hours) for one week. The Investigational Drug Services dispensed the Nystatin oral suspension at the University of Rochester Medical Center. The nystatin suspension bottle was returned to quantify the unused quantity of nystatin and assess the adherence to measure. In addition, a treatment adherence log was filled out by the study participants.
Study flow
The study flow in demonstrated in Fig 1. The study participants were assessed at 3 time points: 1) Baseline visit (V1); 2) 1 week after the completion of nystatin oral rinse (V2); 3) 3 months after the completion of nystatin oral rinse (V3).
All eligible study participants were enrolled and received a 1-week supply of nystatin oral suspension at baseline visit. Study participants were followed at two additional visits post antifungal treatment.
Data collection, examination and sample collection
At the baseline visit, data on demographic and socioeconomic background were collected using a questionnaire. Data on the medical background and medications were self-reported and confirmed by electronic medical records. The medical background included: 1) physician-diagnosed systemic diseases (Y/N), such as hypertension, diabetes, asthma, anxiety, depression, kidney disease, etc.; 2) medications that subjects were taking at the baseline study visit; 3) smoking status (Y/N). The oral hygiene practice was collected using a questionnaire.
The comprehensive examination was performed at the baseline and each study visit by a dentist in a dedicated examination room at the URMC, using standard dental examination equipment, materials, and supplies. Caries was scored using DMFT (decayed, missing, and filled teeth). Bleeding on probing (BOP) was evaluated to assess the gingival inflammation. The periodontal tissue was assessed to the bottom of the clinical pocket or sulcus with a periodontal probe. Interproximal sites for every existing tooth, except the third molars, were scored from the buccal and the lingual sides [21]. Dental plaque accumulation was assessed using the Plaque Index (PI) described by Löe [22]. Each of the four gingival areas of the tooth was given a score of 0-3. A score of 0 indicates no plaque in the gingival area, a score of 1 indicates the presence of a film of plaque adhering to the free gingival margin, a score of 2 indicates moderate accumulation of soft deposits within the gingival pocket, on the gingival margin and or adjacent tooth, a score of 3 indicates an abundance of soft matter within the gingival pocket and or on the gingival margin and adjacent tooth structure.
Methods used for saliva and plaque sample collection were detailed previously [9, 10]. Approximately 2 ml of whole non-stimulated saliva samples were collected by spitting into a sterile 50 ml centrifuge tube. Study subjects were instructed not to eat, drink or brush their teeth two h before the oral sample collection prior to their study visit. Supragingival plaques from the whole dentition were collected using a sterilized periodontal scaler. The plaque samples were suspended in 1ml of a 0.9% sodium chloride solution in a sterilized Eppendorf tube.
Identification and quantification of Candida spp. and S. mutans
The clinical samples (saliva and plaque) were stored on ice and transferred to the lab located at the Center for Oral Biology, UR within 2 h for laboratory testing. The saliva and plaque sample were gently vortexed and sonicated to break down the aggregated plaque before plating. The sonication cycle was repeated three times, with 10 s sonication and 30 s rest on ice. BBL™ CHROMagar™ Candida (BD, Sparks, MD, USA) was used to isolate and identify C. albicans. S. mutans was isolated using Mitis Salivarius with Bacitracin selective medium and identified by colony morphology [23, 24]. Both Candida spp. and S. mutans were incubated at 37 °C, 5% CO2, for 48 h before identification. Colony PCR was used for a further identification of those Candida spp. and S. mutans that were unable to be identified by colony morphology [25]. The CFU values of Candida spp. and S. mutans on each plate were recorded.
Susceptibility of C. albicans isolated to Nystatin in vitro
Antifungal susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 broth microdilution method using (RPMI 1640; Gibco) growth medium (with glutamine and without bicarbonate). The medium was buffered with 3-(N-morpholino) propanesulfonic acid (MOPS: Fisher Scientific) at a pH of 7.0 and to a final concentration of 0.165 mol/l. Clinical isolated of C. albicans were recovered from frozen stock using Yeast Peptones Dextrose Agar (YPD), and incubated overnight at 37° C with 5% CO2. The antifungal agent tested was nystatin (VWR International, Sanborn, NY, USA). A stock drug was prepared (100 times the higher concentration) and aliquots in small volumes for future use. Two-fold serial dilutions were prepared, yielding final concentrations ranging from 0.06 to 32 ug/ml. The yeast inoculums were prepared by picking up young colonies of appropriate size from the 24-hour old fresh cultures and suspending them in 5 mL of RPMI 1640 medium. The resulting suspensions cell density was adjusted to 0.5 McFarland standard, corresponding to a stock inoculum of 1-5x106 CFU/ml. Alternatively, the cells were counted using a hemocytometer. The suspension was diluted twice: first 1:20 and then 1:50 with RPMI medium to obtain a final working density of 1-5x103 CFU/ml. Subsequently, 100 ul of this working inoculum solution and 100 ul of antifungal solution (at a working concentration twice higher than the desired final concentration) were seeded into a sterile, disposable, flat-bottomed flat 96-well plate (Greiner Bio-One). In each well, the final inoculum concentration was 0.5-2.5x103 CFU/ml. The 96-well plates were incubated in ambient air without agitation at 35° C and read spectrophotometrically at 600 nm after 24 h and 48 h using a 96-well microtiter plate reader (infinite M200 PRO, Tecan). In the case of the spectrophotometer readings, the MIC50 and MIC90 were determined. C. albicans SC 5314 was used as a reference for quality control in all the experiments.
Susceptibility to Nystatin was assessed using the MIC of each isolate. The isolates were classified as susceptible (S), susceptible dose-dependent (SDD), and resistant (R) and expressed visually as heatmaps. The interpretive criteria used was according to CLSI guidelines: nystatin: resistant ≥ 16 ug/ml.
Salivary cytokine level
Cytokine/chemokine assessment for 24 analytes was performed in the University of Rochester Human Immunology Center Core Lab facility on saliva samples collected at 4 time points (Baseline, 1-week, and 3-month after Nystatin rinse). Samples were centrifuged for 5 minutes at 125000 rcf at 4oC prior to incubation of the sample at neat concentration overnight at 4oC in two multiplexed magnetic bead array assays, an 17-Plex Milliplex MAP high sensitivity human cytokine panel (Cat#HSTCMAG-28SK) for GM-CSF, IFNg, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-17A, IL-21, IL-23, ITAC, Fractalkine, and TNFa and a 7-Pex Milliplex MAP human cytokine/chemokine panel (Cat#HCYTOMAG-60K) for Eotaxin, IL-1a, IL-1RA, IL-15, IP-10, MDC, and MCP-1. Both assays were performed following kit instructions and read on a Luminex 200 instrument. Results were reported in pg/mL based on standard curve values.
Statistical analysis
We grouped the study participant into two groups (responded and did not respond to nystatin oral rinse) based on their salivary C. albicans status at the 1-week follow up visit. The characteristics of the study participants from these two groups were compared using t-test for normal distributed continuous data (age, DFMT, and DMFS), Mann-Whitney U test for non-normal distributed data (weighted and non-weighted sweet/non-sweet indices), and Chi-square or Fisher’s exact tests for categorical data including demographic characteristics (race, ethnicity, education level, marital status), medical background (hypertension, allergy to penicillin, long-term use of antibiotics, and smoking), oral health condition (tooth brushing frequency and use of nightguard), and detection of non-albicans Candida species. The Wilcoxon matched-pair signed-rank test was used to compare the changes of oral health conditions (BOP and PI) between the follow-up visits and baseline visit. Pair-wise t-test was used to compare the carriage of S. mutans (converted to natural log value), C. albicans (converted to natural log value), and salivary cytokines (converted to natural log value) between the follow-up visits and baseline visit. A multivariate logistic regression was used to analyze the factors (race, gender, ethnicity, age, educational level, hypertension, smoking, tooth brushing frequency, night guard usage, adherence to Nystatin rinse, weighted sweet index, and non-sweet index) associated with the response to nystatin oral rinse. Responded to oral rinse is defined as no detection of C. albicans in saliva at the 1-week follow-up visit. All statistical tests were two-sided with a significant level of 5%. SPSS IBM was used for statistical analyses.