Miconazole

The MIC of each antifungal drug against the various A. fumigatus strains was investigated as described previously35 (link). Tests were performed in triplicate using micafungin, amphotericin B, 5-FC, fluconazole, itraconazole, voriconazole, miconazole and posaconazole in RPMI 1640 medium (pH 7.0) at 35 °C. Assays were performed according to the Clinical and Laboratory Standards Institute broth microdilution method, document M38-A2, with some modifications (dried plates for antifungal susceptibility testing; Eiken Chemicals, Tokyo, Japan).

Biofilms were collected from the maxillary dentures of each participant at five time points, i.e., before using the solutions (baseline) and after using them. Before using each solution, the internal surfaces were stained by a disclosing solution (1% neutral red), cleaned by researchers with a specific brush (Bitufo^®, Itupeva, SP, Brazil) and neutral liquid soap (Pleasant, Perol Commercial and Industrial Ltda., Ribeirão Preto, SP, Brazil), and then returned to the patients in the same clinical initial condition. Biofilms were collected in an aseptic zone by placing each complete maxillary denture in a sterile Petri dish. Dentures were rinsed with 10 mL saline solution, and their internal surfaces were brushed (Tek, Johnson & Johnson Brazil’s Industry and Commerce Healthcare Products Ltda., São José dos Campos, SP, Brazil) for 2 min. The biofilm suspension obtained was vortexed for 2 min and diluted in decimal series (10⁰ - 10^-3). Then, 50 µL aliquots from the decimal dilutions were cultured in Petri dishes containing Mitis salivarius agar base (more bacitracin solution and 20% sucrose), MacConkey agar, and CHROMagar^® Candida for detecting S. mutans, gram-negative microorganisms, and Candida spp., respectively. Culture media were incubated at 37°C according to each respective condition: candle jar for 48 - 72 h (mutans group), or aerobiosis for 48 h (gram-negative and Candida spp).
After incubation, the number of colonies for each dilution was counted. Based on the dilution that provided 1 - 300 colonies, colony forming units (CFUs) were determined using the formula CFU/mL=number of colonies x 10ⁿ/q, where “n” is the absolute value of the dilution (0, 1, 2, or 3) and “q” is the quantity of plated suspension (0.05 mL).
For Candida spp., the number of colonies was counted and the species were identified based on the chromogenic properties of the medium. Each identified species was confirmed using yeasts kit (Candifast^®, ELITech Microbio, Signes, France); in addition, the resistance to antifungal agents, such as amphotericin B, nystatin, flucytosine, econazole, ketoconazole, miconazole, and fluconazole, was evaluated.
In order to conceal the ones involved, the solutions were dispensed in identical dark flasks and delivered without specific identification, and in the quantity to be used for a period of seven days. Each cleanser solution was used by the participants in a random sequence. Researcher P1, who was not involved in the other operational phases of the study, used a computer program to obtain a list of random numbers corresponding to the possible sequences of the treatment. Researcher P2 received these numbers and distributed the solutions to the participants. Researcher P3 implemented hygiene instructions and collected the prostheses. Researcher P4 collected the biofilms while researcher P5 washed the prostheses to ensure the complete elimination of the biofilms. Researcher P2 obtained variable information and provided it to researcher P1, who performed statistical analyses. Thus, during the study, all researchers, as well as the participants, were blinded to the applied solutions.

Resistance to drug combinations was assayed in 96-well microtiter plates, as previously described [16] (link), [21] (link). Minimum inhibitory concentration (MIC) assays were set up to a final volume of 0.2 ml/well. MICs were performed in the absence of fluconazole (Sequoia Research Products) or with a constant concentration of fluconazole or miconazole (Sigma–Aldrich Co.), as indicated in the figures. All gradients were two-fold dilutions per step, with the final well containing no drug. The starting concentration of geldanamycin (Invivogen) gradients was 50 µM for S. cerevisiae strains and 5 µM for C. albicans strains. The starting concentration of FK506 (A.G. Scientific) gradients was 6 µM for S. cerevisiae strains and 100 µM for C. albicans strains. The starting concentration of radicicol (A.G. Scientific) gradients was 25 µM for both S. cerevisiae and C. albicans strains. The starting concentration of cyclosporin A (Calbiochem) gradients was 50 µM for both S. cerevisiae and C. albicans strains. The cell densities of overnight cultures were determined and diluted to an inoculation concentration of ∼10³ cells/well. Plates were incubated at 30°C in the dark for the period of time specified in the figure legend. Cultures were resuspended and absorbance at 600 nm was determined using a spectrophotometer (Molecular Devices) and corrected for background of the corresponding medium. OD measurements were standardized to either drug-free or azole-only control wells, as indicated. Data was plotted quantitatively with colour using Java Treeview 1.1.3 (http://jtreeview.sourceforge.net/). Resistance phenotypes were assessed on multiple occasions and in duplicate on each occasion with concordant results, validating that the phenotypes are reproducible and stable.
Dose response matrices, or checkerboard assays, were performed to a final volume 0.2 ml/well in 96-well microtiter plates, as previously described [74] (link). Two-fold dilutions of fluconazole were titrated along the X-axis from a starting concentration of 256 µg/ml, with the final row containing no fluconazole. Along the Y-axis, either geldanamycin or FK506 was titrated in two-fold dilutions with the final column containing no geldanamycin or FK506. The starting concentration of geldanamycin was 5 µM for checkerboards with either S. cerevisiae or C. albicans strains. The starting concentration of FK506 was 4 µM for checkerboards with S. cerevisiae and 40 µM for checkerboards with C. albicans strains. Concentrations were selected to cover a range that spanned from no effect on growth to near complete inhibition of growth. Plates were inoculated and growth assessed as was performed for MIC assays.
Fluconazole was dissolved in sterile ddH₂O. The Hsp90 inhibitors geldanamycin and radicicol and the calcineurin inhibitors FK506 and cyclosporin A were dissolved in DMSO. Myriocin (Sigma) was dissolved in methanol.

From the 24 h incubated slant nutrient agar of each bacterial species, a loop full of microorganisms was injected into a tube containing 4 to 5 mL of tryptic soy broth (TSB). The broth culture was kept at 35 °C for 2–6 h to obtain the required turbidity of 0.5 McFarland BaSO₄. A 625 nm spectrophotometer was used to measure the standard turbidity density. With different bacterial cultures, the sensitivity test of the Moringa extracts was determined [61 (link)]. Cotton swabs were utilized to create the bacterial cultures from TSB, and Petri dishes were produced with 20 mL of nutrient agar. The discs were positioned on the seeded plates using sterile forceps. Tetracycline (500 g/mL) was utilized as a positive control, and DMSO was used as a negative control. The following day, inoculated plates were incubated at 37 °C for 24 h. At the conclusion of the incubation period, inhibition zones were measured and represented as the diameter of the clear zone including the diameter of the disc.
The fungi strains were plated onto potato dextrose agar (PDA) and cultured at 25 °C for 5 days. “YES” medium Petri dishes were evenly spread with a sterile L-glass rod after being inoculated with 0.05 mL of each fungus culture. The extract-loaded discs were positioned on the seeded plates using sterile forceps. DMSO was utilized as a negative control, while the commercial fungicide Miconazole (1000 unit/mL) was administered as a positive control. The inoculation plates were incubated at 25 °C for 24–48 h. By measuring the inhibition zone (mm) against the tested fungus at the conclusion of the time, the antifungal activity was determined [62 (link)]. Three replicas of each treatment were used to calculate the averages of the results of the experiments.

In each group, chemotherapy and the induction of oral mucositis were carried out as previously described by Katagiri et al. [1 (link)], and inoculation with Candida albicans was performed as described by Takakura et al. [9 (link)]. Briefly, for chemotherapy, 7 mg/kg cisplatin on day 1 and 10 mg/kg 5-fluorouracil on days 1, 2, 3, and 4 were intraperitoneally injected into the mice. For C. albicans inoculation, 5.0 × 10⁶ cells/25 µL of the ATCC 48130 strain were orally administered at day 2, 3, and 5 of chemotherapy. Prophylactic treatment in Groups A, B, and C was performed on days 3, 4, and 5 (Figure 1). In Group A, the mouth was carefully cleaned with sterile cotton and saline until no removable stains remained in the entire mouth. In Group B, 2% miconazole gel was administrated orally at 60 mg/kg with sterile cotton (Mochida Pharmaceutical, Tokyo, Japan). In Group C, fluconazole 1 mg/mL (Pfizer, Tokyo, Japan) was administrated intravenously at 0.52 mg/kg. All mice were sacrificed on day 6 with a lethal dose of general anesthesia. At the same time, a blood sample for culture and polymerase chain reaction (PCR) was taken from the cardiac apex without any contamination.

This was a cross-sectional study of the prescription patterns of systemic antibiotics used for patients treated in hospitals with a confirmed diagnosis of COVID-19; the patients were identified from reports of confirmed cases by polymerase chain reaction (RT–PCR) or antigen tests performed by the Ospedale Group network in eight clinics located in the cities of Armenia, Barranquilla, Bogotá, Cali, Cartagena, Manizales, Pereira, and Popayán in Colombia. These are highly complex clinics that are reference centers and also treat patients referred from other cities.
From this population, patients of any age, gender, and city of residence with a first confirmed diagnosis of SARS-CoV-2 between March 6, 2020, and August 31, 2022, and treated in the emergency department, a general ward, or the ICU, were selected. With this selection, information on the use of medications was obtained through the dispensing company (Audifarma SA, Pereira, Colombia). A database was designed that allowed the following groups of patient variables to be collected:

Sociodemographic data: gender, age (<18 years, 18–39 years, 40–64 years, 65 years or older), and city of origin. City of origin was categorized by department based on the regions of Colombia, taking into account the classification of the National Administrative Department of Statistics (Departamento Administrativo Nacional de Estadística—DANE) of Colombia: Caribbean, Central, Bogotá-Cundinamarca, Pacific and Amazonia, and Orinoquia—Oriental;

Clinical symptoms (cough, dyspnea, fever, fatigue, odynophagia, chest pain, and asthenia/adynamia, among others), comorbidities (cardiovascular, respiratory, digestive, endocrine, neurological, psychiatric, rheumatological, and oncological), and mortality;

Treatment: The management received by the patients was established from the dispensing of the medications.

Place of care: emergency department, general ward or ICU;

Supplemental oxygen: oxygen requirement, mechanical ventilation, and need for tracheostomy;

Antibiotics: classified by therapeutic group (aminoglycosides, cephalosporins, fluoroquinolones, macrolides, penicillins, and tetracyclines, among others) and by the WHO AWaRe (Access, Watch, and Reserve) classification (Supplementary Table S1) [16 ];

Antifungals: azoles (fluconazole, miconazole, voriconazole, posaconazole, itraconazole, and ketoconazole), polyene antibiotics (amphotericin B and nystatin), echinocandins (caspofungin, micafungin, and anidulafungin), and others;

Comedications, grouped into the following categories: (a) antidiabetes drugs, (b) antihypertensive and diuretic drugs, (c) lipid-lowering drugs, (d) antiulcer drugs, (e) systemic glucocorticoids, (f) vasopressors and inotropes, (g) anticoagulants, (h) analgesics and anti-inflammatories, and (i) bronchodilators and inhaled glucocorticoids, among others.

The protocol was approved by the Bioethics Committee of the Universidad Tecnológica de Pereira (Technological University of Pereira) in the category of “research without risk” (approval code: 30-070421). The principles of confidentiality of information established by the Declaration of Helsinki were respected.
The data were analyzed using the statistical package SPSS Statistics, version 26.0 for Windows (IBM, Armonk, NY, USA). A descriptive analysis was performed; qualitative variables are presented as frequencies and proportions, and quantitative variables are presented as measures of central tendency and dispersion (medians and interquartile ranges). Quantitative data were compared using the Mann–Whitney U test and categorical data were compared using X² test or Fisher’s exact test. Multivariate binary logistic regression models were developed, which included the associated variables in the bivariate analyses as well as variables with sufficient plausibility or a reported association to identify variables that are associated with the prescription of antibiotics (yes/no) and the prescription of antibiotics from the Watch/Reserve categories (yes/no). A p < 0.05 was considered statistically significant.