F1000ResearchF1000Research2046-1402F1000 Research LimitedLondon, UK10.12688/f1000research.125097.1Research ArticleArticlesAntifungal activity of propolis extract against
Candida albicans in patients with vulvovaginal candidiasis
[version 1; peer review: 1 not approved]
Esteves PairazamanAmbrocio TeodoroConceptualizationData CurationFormal AnalysisMethodologySupervisionhttps://orcid.org/0000-0003-4168-0850a1Collanque PintoJesus DanielMethodologySupervisionWriting – Review & Editing1Alvarado ChávezBrittWriting – Review & Editing1Leon ApacGabrielWriting – Review & Editing1Horna SandovalConsuelo BertaWriting – Review & Editing1Malpartida QuispeFederico MartinWriting – Review & Editing1Collanque MezaVadir Ali JesúsWriting – Review & Editing2Ibarra FretellWalter GregorioWriting – Review & Editinghttps://orcid.org/0000-0003-4276-53893Rodríguez LoyolaGina AndreaInvestigationMethodologyValidationVisualizationWriting – Review & Editing1
Universidad Privada Norbert Wiener, Lima, 15072, Peru
Instituto SISE, Lima, 15046, Peru
Universidad Cesar Vallejo, Lima, Peruambrocio.esteves@uwiener.edu.pe
Background: The aim was to evaluate the antifungal activity of propolis extract against
Candida albicans (or
C. albicans) in patients with vulvovaginal candidiasis.
Methods: The research presents a quantitative approach with an experimental design, with a population of
C. albicans strains isolated from patients diagnosed with vulvovaginal candidiasis who were admitted to the Gynecology Service of the Maria del Socorro Clinic, district of Ate, Lima; a sample of 34 strains was chosen in total, considering three repetitions per strain, obtaining 102 experimental units.
The antifungal susceptibility test was carried out by the disc diffusion plate method using a single concentration of propolis extract, with fluconazole as a positive control.
Results: Propolis extract was shown to inhibit all strains of
C. albicans, as was fluconazole, with the effect of the latter being significantly greater than that of the product under investigation.
Conclusions: The conclusion is that propolis extract does show antifungal activity against
C. albicans in patients with vulvovaginal candidiasis.
Propolis extractCandida AlbicansAntifungal activityVulvovaginal candidiasisThe author(s) declared that no grants were involved in supporting this work.Introduction
Candidiasis affects approximately 138 million women worldwide each year, and is the second most common type of vaginal infection.
1,2 Studies show that approximately 6000 women from different countries have a 9% prevalence of recurrent vulvovaginal candidiasis (VVC) and the likelihood of progression is high
3; about 75% of women experience an episode of VVC during their childbearing years; about 40-50% experience a second VVC infection, and 5-8% of adult women have four or more episodes per year.
4,5
The World Health Organization (WHO) considers
C. albicans as one of the most recurrent causes of vaginal infections, because it is responsible for 80 to 92% of the cases of VVC.
6 It is known that in the regions of Europe and Latin America, infection rates are 20 to 25%, of which 75% of adult women have contracted
C. albicans at least once
7; a study carried out in a Tertiary Care Hospital in India showed that out of 180 patients who underwent microbiological tests, 76-89% of the population studied had
C. albicans as the most frequent pathology-causing species.
8 In regions of the Americas, such as Mexico, a study of CVV was conducted on 412 people and found that 25% of them were positive. In the United States, 65% of hospitalised patients had fungal infections, and 78% of them are known to reach mortality depending on the risk factors presented by the patient.
9 It is worth noting that
C. albicans is considered to be the second most common cause of vaginal infections.
10
In Peru, the most frequent species causing VVC is
C. albicans, with a prevalence of 40-60% in adult patients.
11 An obstetrician-gynecologist from the Instituto Especializado Materno Perinatal in Peru, after conducting a study, mentioned that, of all the cases found, 50% had vaginal discharge due to VVC and the vast majority were due to self-medication and overdose.
12 In addition, the prevalence of VVC was found in 24% of pregnant women in the areas of Monsefu (Chiclayo) and 29.8% in Inkawasi (Ferreñafe),
C. albicans always stood out as the aetiological agent for all these cases.
11 In the Arzobispo Loayza National Hospital in Lima, of the total population investigated, 42.2% had a prevalence of vaginal infections.
13
Current VVC treatment regimens have lost relevance in the face of antifungal resistance challenges,
14 because the treatment of fungal infections such as VVC lacks specific drugs, unlike bacterial infections. These shortcomings require the production of a vaginal drug delivery system (VDDS) capable of attenuating, treating, and eliminating CVV clinically and effectively, thus avoiding the risk of disease recurrence and prolongation.
15
Currently, amphotericin B and fluconazole are readily available azoles frequently used to treat candidiasis; amphotericin B is considered to be an excellent antifungal, but its level of toxicity is very high. However, their efficacy is poor due to the development of natural resistance by strains of
C. albicans and non-
albicans.16
Antifungal resistance is considered a consequence of inappropriate drug use, inadequate dosage, and incomplete treatment, as resistance to antifungal drugs has been increasing by up to 40% in recent years.
16,17 These data are alarming in addition to the symptoms, recurrent misuse of medications, self-medication,
18 short medication cycles or incomplete treatment of antifungal drugs
19; these affect women who suffer from VVC as they have a poor quality of life that is unfavorable to their health.
18 Therefore, the aim is to provide a solution to this common problem in women by considering the use of alternatives from natural products.
The aim of this study was to evaluate the antifungal activity of propolis extract against strains of
Candida albicans isolated from patients diagnosed with acute and recurrent VVC, and to compare the antifungal activity of propolis extract and fluconazole.
Methods
The research was hypothetico-deductive, with a quantitative approach. The type of research was basic, and the design was experimental.
20–22
The population consisted of
Candida albicans strains isolated from patients diagnosed with VVC who were admitted to the gynaecology department of the María del Socorro Clinic in the district of Ate in Lima.
The sample size was 34 strains, a figure derived from the interaction between 17 strains of
C. albicans (nine from acute VVC and eight from recurrent VVC) and two treatments (propolis extract and fluconazole), considering three replicates per strain, a total of 102 experimental units were obtained.
23
It was suggested that there may be a significant difference between the antifungal activity of propolis extract and fluconazole against
C. albicans strain types, this being the study hypothesis which will be tested by analysis of variance (ANOVA).
Reliability
In the present investigation, to measure antifungal activity, the manual for susceptibility testing indicated by the Clinical and Laboratory Standards Institute (CLSI) in document M44 - A was used. Inocula of clinical strains of
C. albicans were prepared and adjusted to the turbidity of the scale at 0.5 McFarland. Following the standardised susceptibility protocol established by Bauer
et al.,24 the technique used was direct observation of the susceptibility of
C. albicans strains.
The technique used was direct observation of inhibition halos and the instrument used was a data collection sheet to record the information obtained.
Data processing and analysis
Prior to data processing and analysis, the following was applied:
Propolis extract
The propolis extract was collected with the support of beekeepers in the Oxapampa district, Cerro de Pasco region, Peru. Once the sample was obtained, we began with the separation of some components that were not part of the analysis, such as dust, wood chips, wood remains, among others that may alter the composition of the extract.
25
Preparation of the extract
The characterization and preparation of the propolis at 50% concentration were done under aseptic conditions in a laminar flow cabinet at room temperature where impurities were discarded; then the sample was chopped and pulverized with the aid of a pestle and mortar.
The extract was prepared using 50 g of propolis in 100 mL of 70% ethanol, then the mixture was subjected to maceration for eight days at 37°C without contact with light. Subsequently, the sample was filtered with the help of Whatman No. 60 filter paper and then taken to a rotary evaporator at 60°C for the ethanol to be evaporated. The total extracts were transferred to the rotary evaporator until the solvent was eliminated and the solid obtained was placed in an oven for two hours at 70°C.
Finally, the ethanolic dry extract of propolis was obtained using the dilution and filtration technique to obtain propolis at a concentration of 50%. The selected propolis was visually identified according to its organoleptic characteristics, being predominantly brown in colour and lacking a defined aroma, to ensure its quality and purity.
18
Method for obtaining clinical strains of Candida albicans
Permission was requested from the gynaecology service of the Clínica María del Socorro in the district of Ate in Lima to work with volunteer patients, who were previously informed of the study and agreed to sign the informed consent form.
Once authorization was obtained, samples of vaginal exudate were collected from the patients and taken by the doctor in charge of the area, who used a sterile dry vaginal swab to obtain the sample from the bottom of the vaginal sac, identifying the patients who presented acute and recurrent VVC; then, the samples were labelled with the corresponding type of infection and transported in sterile containers in a vertical position, in a cool environment and protected from light, to the laboratory of the aforementioned clinic. For isolation of the strains, the technique of depletion-streaking was used in Petri dishes with Sabouraud Dextrose Agar (SDA), and then incubated in an oven at 37°C.
11
Candida albicans identification tests
After incubation, growth was observed on the surface of SDA, then a colony was taken for seeding on Chromogenic
Candida agar to differentiate
C. albicans from other
Candida species, and microscopic observation was performed (see
Figure 1 and
Figure 2).
Identification of the strains of
<italic toggle="yes">C. Albicans.</italic>
Note. a and b) Isolation on chromogenic agar.
Process flowchart on general data processing and analysis of
<italic toggle="yes">C. albicans</italic> strains.
In addition, for confirmation purposes, the growth of
Candida sp. was validated with the germ tube test and then tested with the API 20 C AUX
Candida test.
10
Germ tube testing:
The germ tube test is highly useful in differentiating
C. albicans and non
-albicans Candida species. The procedure was performed with 0.5 mL of fresh human serum using the culture loop, inoculated with the strain under study and incubated at 37 °C. After three hours, a wet mount was prepared to observe under the light microscope, with 40× objective; the presence of the germ tube consisted of the filamentous extension of the yeast, where both
C. albicans and
C. dubliniensis formed a small extension similar to a “hand mirror” (Reynolds - Braude phenomenon). For this reason, the API 20 C AUX
Candida test was used for confirmation.
15
API 20 C AUX
Candida test
The API 20 C AUX
Candida test is an accurate yeast identification system consisting of 20 microtubes containing dehydrated substrates. These microtubes are inoculated into a minimal medium of API C medium and grow only yeasts capable of utilizing these substrates.
With 2 mL of saline and a young yeast culture, a suspension was made to a turbidity of McFarland scale 2.0. Then 100 uL of this suspension was added to the ampoule with API C Medium to proceed to fill the domes, always avoiding the formation of bubbles. The suspension was then kept in a humid chamber and incubated for 48-72 hours at 37°C.
14
Finally, once the incubation time was over, the results were read and interpreted, observing growth by turbidity and with the help of the manufacturer's own numerical profile and the Apiweb identification program, the exact identification of the yeasts was carried out.
Preparation of culture environment
To measure antifungal activity, Müller Hinton Agar medium was used, which is validated for susceptibility testing as indicated by the Clinical and Laboratory Standards Institute (CLSI) M44 - A. Inocula of
C. albicans clinical strains were prepared and adjusted to scale turbidity at 0.5 McFarland. The wavelength was 610 mm in the spectrophotometer for measuring the turbidity of the inoculum of the strains; it was then inoculated onto the agar surface with the help of a swab by streaking in three directions, allowed to dry for about 10 minutes and placed on fluconazole discs (positive control) and distilled water (negative control).
9
Preparation of control groups
Positive control: Fluconazole (25 mg/100 mL distilled water).
Negative control: Distilled water (100 mL).
Quality control of the growth of C. albicans strains
One litre of Müller Hinton Agar was prepared according to the recommendation of the DIFCO manual
26 by adding 20 g of glucose and 100 μL of stock solution of methylene blue. The latter solution is prepared by adding 0.1 g of methylene blue to 20 mL of distilled water (obtaining 5 μ/mL concentration). It was then autoclaved for 15 minutes at a temperature of 121°C and 15 lbs of pressure; following this, we let the temperature drop to about 50°C and poured 27 mL per Petri dish to reach 4 mm thickness, in 90 mm diameter plates. Two plates were considered for sterility control at 35°C for 24 hours.
Next, the previously grown
C. albicans ATCC 90028 strain was taken to be seeded on agar. As described above, susceptibility testing was performed by adjusting the strain inoculum to 0.5 turbidity on the McFarland scale, seeding in three directions and placing a 25 μg disc of fluconazole for reading after 24 hours. To ensure quality control of strain growth, inhibition halos were observed.
27
Antifungal susceptibility testing
Once the
C. albicans strains were obtained, the disc diffusion plate method was used, for which filter paper discs containing 200 μL of propolis extract and the respective controls were used, placing each embedded disc on a Petri dish with Müller Hinton agar and incubated for 24 hours at 37°C.
10
After the incubation period, the antifungal growth inhibition halos were analyzed; the results were expressed by measuring the halos formed in the Petri dish, where the diameter of each zone was measured in millimeters, following the standardized susceptibility protocol established by Bauer,
et al.24
Once the experimental procedure was completed, the data obtained were processed and a flow chart was elaborated for general data processing and analysis of the
C. albicans strains.
Data collection
The data collection process was carried out during September, October and November 2021, using a data collection sheet, and then tabulating the information in Microsoft Office Excel v. 2019 software, in which the information was recorded in a dataset.
Authorization and prior arrangements for data collection
The project was approved by Resolution N°1561-2022 by the president of the Institutional Research Ethics Committee of the Norbert Wiener Private University.
At the same time, written authorization was obtained from the Clinic María del Socorro to obtain samples and information through official letter N°017-2021-GMO-CMS.
Application of data collection instruments
The data collection instrument was applied at the time of measuring the growth inhibition halos of
C. albicans strains by the effect of the antifungal activity of propolis extract and fluconazole, as well as recording the type of vvc and whether the causative agent was
C. Albicans.
Statistical analysis methods
For the statistical analysis of the data, SPSS version 23.0 statistical software was used, in which analysis of variance (ANOVA) was used in order to establish whether the antifungal activity of propolis extract and fluconazole was equal in all studied strains; this was complemented with the Tukey significance test (α = 0.05) to define the differences in antifungal activity according to the types of
C. albicans strains and the treatments used.
Informed consent
Participants were informed about the aim of the study, and those who agreed to participate signed an informed consent form, where they gave their approval for the use of the strains to be processed for the purpose of the study. All patients, carriers of the strain, were informed that their personal data would remain anonymous according to Law No. 29733 “Law on the protection of personal data”.
28
Results
Table 1 describes the antifungal activity of propolis extract and fluconazole against
C. albicans strains isolated from patients with vulvovaginal candidiasis (VVC), showing mean inhibition halos of 15.2 mm for propolis extract and 18.0 mm for fluconazole.
Antifungal activity of propolis extract against
<italic toggle="yes">Candida albicans</italic> strains isolated from patients diagnosed with VVC as represented by inhibition halos.
Candidiasis
Strains
Inhibition halos (mm)
Propolis extract
Fluconazole
Acute
A-1
16.7
18.2
A-2
15.3
19.3
A-3
17.2
19.2
A-4
19.2
20.5
A-5
19.2
22.6
A-6
18.7
20.9
A-7
17.9
20.8
A-8
16.9
19.8
A-9
18.2
19.2
Recurrent
R-1
11.8
13.7
R-2
11.8
12.5
R-3
12.6
16.6
R-4
11.1
14.3
R-5
14.9
18.7
R-6
13.2
17
R-7
11.5
15.3
R-8
12.8
17.4
Average
15.2
18.0
Table 2 shows the existence of a statistically significant difference between the types of
C. albicans strains that were inhibited by propolis extract and fluconazole. Thus,
C. albicans strains isolated from acute VVC showed inhibition halos of a larger size on average than those from recurrent infection.
Comparison of antifungal activity between
<italic toggle="yes">C. albicans</italic> strains isolated from patients diagnosed with acute and recurrent vulvovaginal candidiasis (VVC) using Tukey's significance test.
Type of
C. albicans strain
Mean
n
E.E.
Acute VVC
18.87
54
0.20
A
Recurrent VVC
14.08
48
0.22
B
Note. Means with a common letter are not significantly different (p > 0.05).
Table 3 shows the existence of a statistically significant difference between the antifungal activity of propolis and fluconazole, the latter showing the largest average size of inhibition halos on
C. albicans strains compared to propolis extract.
Comparison of antifungal activity of propolis extract and fluconazole against
<italic toggle="yes">C. albicans</italic> strains isolated from patients diagnosed with vulvovaginal candidiasis (VVC) using Tukey's significance test.
Product
Mean
n
E.E.
Fluconazole
17.86
51
0.21
A
Propolis extract
15.10
51
0.21
B
Note. Means with a common letter are not significantly different (p > 0.05).
H
0-1: There is a significant difference between the antifungal activity of propolis extract and fluconazole against
C. albicans strain types.
H
0-2: There is no significant difference between the antifungal activity of propolis extract and fluconazole against
C. albicans strain types.
In
Table 4, the observed p-value was <0.0001, which is less than 0.05; therefore, we rejected the null hypothesis and affirm that there is a significant difference between the antifungal activity of propolis extract and fluconazole against the types of
Candida albicans strains.
Analysis of variance (ANOVA) of the inhibition halos (mm) generated in C. albicans strains by the effect of propolis extract and fluconazole.
FV
SC
GL
CM
F
p-value
Decision
Model
777.42
2
388.71
174.73
<0.0001
Strain type
582.78
1
582.78
261.97
<0.0001
Reject H
0-1
Treatment
194.64
1
194.64
87.49
<0.0001
Reject H
0-2
Error
220.24
99
2.22
Total
997.65
101
Note: SC: Total squares, GL: Degrees of freedom, MC: Mean square, F: Test, P: Probability.
Discussion and conclusions
The antifungal activity of propolis extract was evaluated against
Candida albicans strains isolated from patients diagnosed with VVC; in the present investigation it was determined that propolis extract at a concentration of 50% showed antifungal activity on
C. albicans strains. Studies by De La Cruz,
13 and Joya
et al.,3 who used ethanolic extracts of propolis against strains of the fungal species in question, differed from the present study in that they did consider using higher and lower concentrations of the product. The above suggests that a key factor in the sensitivity of
C. albicans is its chemical composition, which is represented by proteins and polysaccharides. In addition, these microorganisms are known to have a cell wall, which is mainly composed of polysaccharides such as glucan and chitin. In addition, these species have a cytoplasmic membrane, where the antifungal agents carry out their function. In addition, the cytoplasmic membrane is composed of large amounts of carbohydrates and proteins in smaller proportions, which allow the entry and exit of substances such as secondary metabolites
e.g., flavonoids, tannins, terpenes, and alkaloids.
11,29
Regarding the specific objective of evaluating the antifungal activity of propolis extract against
C. albicans strains isolated from patients diagnosed with acute and recurrent VVC, it was found that propolis extract showed inhibition of
C. albicans strains isolated from acute and recurrent VVC. This finding is related to that reported by Adjapong
et al.30 who compared the susceptibility of
C. albicans strains isolated from acute and recurrent VVC, where the former showed greater sensitivity to fluconazole compared to those with recurrent infection, applying three doses of the antifungal agent.
30 In the case of propolis, precise studies comparing its effect on both types of strains could not be found; however, it can be suggested that, in recurrent candidiasis,
C. albicans expresses resistance to antifungal agents such as azoles, and therefore also to metabolites that induce their inhibition.
31
Additionally, to compare the antifungal activity of
C. albicans strains isolated from patients diagnosed with acute and recurrent VVC, it was found that
C. albicans strains isolated from acute VVC showed a higher average inhibition halo than those from recurrent infection when exposed to propolis extract and fluconazole, and this difference was statistically significant. This finding is very similar to that reported by Adjapong
et al.30 who compared the susceptibility of
C. albicans strains isolated from acute VVC and recurrent VVC, where the former showed greater sensitivity to fluconazole compared to those with recurrent infection, applying three doses of the antifungal (2 μg/mL, 4 μg/mL and 8 μg/mL).
30 In the case of propolis, precise studies comparing its effect on both types of strains could not be found; however, it can be suggested that, in recurrent candidiasis,
C. albicans expresses resistance to antifungal agents such as azoles and, therefore, to the metabolites present in propolis extract that induce their inhibition as well.
32
Finally, taking into account the specific objective of comparing the antifungal activity of propolis extract and fluconazole against strains of
C. albicans isolated from patients diagnosed with VVC, it was possible to establish significant differences between the antifungal activity of propolis extract and fluconazole, the latter showing the highest average inhibition halo; this finding was very similar to that found by Capoci
et al.
7 who used propolis and fluconazole as a positive control for the inhibition of biofilms caused by
C. albicans, the latter showing the greatest inhibitory effect against the strains of the microorganism in question.
7 This suggests that, although the metabolites of the product studied may be involved in the growth and development of the infectious agent, antifungal agents have been shown to be more effective, as they not only affect the external component of the fungi, but also the cellular interior, causing a greater and faster inhibition. It should be noted that the metabolites described above, in most cases, stimulate the inhibition of biofilm formation, however, the antifungal agents par excellence, such as fluconazole, can pass through the cytoplasmic membrane of the fungus and affect cellular respiration and therefore cause its death more rapidly.
32,33
It was concluded that:
Propolis extract showed antifungal activity against strains of
C. albicans isolated from patients diagnosed with both water-borne and recurrent vulvovaginal candidiasis.
There was a significant difference between the antifungal activity of propolis extract and fluconazole against
C. albicans strains isolated from patients diagnosed with vulvovaginal candidiasis.
There was a significant difference between the inhibition halos of
C. albicans strains isolated from patients diagnosed with acute and recurrent vulvovaginal candidiasis.
Data availability
Zenodo: Antifungal activity of propolis extract against
Candida albicans in patients with vulvovaginal candidiasis,
https://doi.org/10.5281/zenodo.6981744.
34
Data are available under the terms of the
Creative Commons Attribution 4.0 International license (CC-BY 4.0).
ReferencesMaureiraNVieraPFernandezA:
Susceptibilidad de Cepas de Candida Oral a Extracto Etanólico del Propóleo Chileno de Olmué.Int J Odontostomatol.2017;11(3):295–303.
10.4067/S0718-381X2017000300295ParkerMVaginitisSCVulvovaginitis:
Textbook of Natural Medicine.5° Edición.Missouri, Estados Unidos:
Elsevier;2021; pp.1840–1849.
10.1016/B978-0-323-43044-9.00224-7JoyaMGilMBastidas-PachecoG:
Actividad fungistática y fungicida de extractos etanólicos de propóleos sobre el crecimiento in vitro de cepas del género Candida.Tecnol en Marcha.2017;30(3):3–11.
10.18845/tm.v30i3.3268TulikangasPKSchimpfMO:
Genital and urinary tract infections.
General Gynecology.1° Edición.Philadelphia, Estados Unidos:
Elsevier Inc.;2007; pp.523–542.
10.1016/B978-032303247-6.10022-XPrieto-GranadaCLoboAMinhmM:
Skin Infections.Diagnostic Pathology of Infectious Disease.2° Edición.Philadelphia, Estados Unidos:
Elsevier Inc.;2018; pp.542–647.
10.1016/B978-0-323-44585-6.00020-5GavanjiSLarkiB:
Comparative effect of propolis of honey bee and some herbal extracts on
Candida Albicans.Chin J Integr Med.2015;23(3):201–207.
2614908310.1007/s11655-015-2074-9CapociIRGBonfim-MendonçaPDSAritaGS:
Propolis is an efficient fungicide and inhibitor of biofilm production by vaginal
Candida Albicans.Evidence-based Complement Altern Med.2015;2015(1):1–9.
2581502910.1155/2015/287693MendozaE:
Estudio comparativo del efecto antifúngico del extracto etánolico de propóleos de la sierra y costa liberteña sobre cepas de
Candida Albicans ATCC 10231, Trujillo - 2018. Tesis para Optar el Grado de Bachiller en Medicina, Universidad Católica los Ángeles de Chimbote.2019.
Reference SourceHuaytallaRMGálvezCMCarhuapoma-YanceM:
Efecto inhibidor in vitro del extracto etanólico de propóleo al 15% y 30% frente a cepas de Lactobacillus acidophilus.Rev Estomatológica Hered.2018;28(1):36–43.
10.20453/reh.v28i1.3281SobelJ:
Vulvovaginal candidosis.Lancet.2007;369(1):1961–1971.
10.1016/S0140-6736(07)60917-9CallaKQuispeD:
Evaluación del efecto antimicrobiano in vitro del propolis “Propóleos” de Tres Regiones del Sur del Perú sobre el crecimiento de
Candida Albicans,
Escherichia coli,
Staphylococcus aureus y
Pseudomona aeruginosa. Tesis Para optar el Título Profesional de Químico Farmacéutico, Universidad Católica de Santa María.2016.
Reference SourceLeónGSacsaquispeSZuritaS:
Efecto antifúngico in vitro sobre el crecimiento en
Candida Albicans ATCC 90028, Candida glabrata ATCC 90030 y Candida krusei ATCC 6258 expuestas al propóleos de Oxapampa a las 24, 48 y 120 horas.Rev Investig la Univ Norbert Wiener.2014;3(1):23–29.
10.37768/unw.rinv.03.01.002Reference SourceDe la CruzM:
Actividad Antimicotica Del Extracto Etanólico De Propóleo Sobre El Crecimiento in vitro De
Candida Albicans. Tesis para Optar el Grado Académico de Bachiller en Estomatología, Universidad Nacional de Trujillo.2013.
Reference SourceFirdausSHassanNAamirM:
FbD directed fabrication and investigation of luliconazole based SLN gel for the amelioration of candidal vulvovaginitis: a 2 T (thermosensitive & transvaginal) approach.Saudi J Biol Sci.2021;28(1):317–326.
3342431210.1016/j.sjbs.2020.10.005DovnikAGolleANovakD:
Treatment of vulvovaginal candidiasis: a review of the literature.Acta Dermatovenerologica.2015;24(9):5–7.
10.15570/actaapa.2015.2FaustinoCPinheiroL:
Lipid systems for the delivery of amphotericin B in antifungal therapy.Pharmaceutics.2020;12(1):1–47.
3190626810.3390/pharmaceutics12010029HuangKZhangBShenZY:
Enhanced amphotericin B production by genetically engineered Streptomyces nodosus.Microbiol Res.2021;242(1):126623–126636.
3318907310.1016/j.micres.2020.126623LírioJGiraldoPCAmaralRL:
Antifungal (oral and vaginal) therapy for recurrent vulvovaginal candidiasis: A systematic review protocol.BMJ Open.2019;9(5):1–6.
3112299110.1136/bmjopen-2018-027489World Health Organization (WHO):
Candida auris, un patógeno emergente. Acciones y prevención en Colombia.WHO;2021 [citado el 14 de noviembre de 2021].
Reference SourceCabezasEAndradeDTorresJ:
Introducción a la Metodología de la investigación científica com.ed. F.F.A.A. ESPE;. Ecuador.2018 [citado 21 noviembre 2021]. Prim. Edic.
Reference SourceÑaupasHValdiviaMPalaciosJ:
Metodología de la investigación cuantitativa – cualitativa y redacción de tesis.5ta ed.Colombia:
Ediciones de la U;2018.Hernández-SampieriRy MendozaC:
Metodología de la investigación. Las rutas cuantitativa, cualitativa y mixta.México:
Grupo editorial Mc Graw Hill Education;2018.BerndtAE:
Sampling Methods.J Hum Lact.2020;36(2):224–226.
10.1177/0890334420906850BauerAKirbyWMSherrisJ:
Antibiotic susceptibility testing by a standardized single disk method.Am J Clin Pathol.1966;45(4):493–496.
532570710.1093/AJCP/45.4_TS.493AnjumSIUllahAKhanKA:
Composition and functional properties of propolis (bee glue): A review.Saudi J Biol Sci.2019;26(7):1695–1703.
3176264610.1016/j.sjbs.2018.08.013DIFCO:
The Difco Manual.11° Edició ed.Maryland, Estados Unidos:
Difco Laboratories;1998;157.
Reference SourceZuritaSAusejoF:
Manual De Procedimientos Técnicos Para El Diagnóstico Micológico.1° Edición ed.Lima, Perú:
Repositorio Institucional - INS;2017;139.
Reference SourceDiario El Peruano:
Ley de protección de datos personales – Ley N° 29733.Lima:
Diario El Peruano;2011.
Reference SourceBoisardSLe RayAMGattoJ:
Chemical composition, antioxidant and anti-AGEs activities of a French poplar type propolis.J Agric Food Chem.2014;62(6):1344–1351.
2444399410.1021/jf4053397AdjapongGHaleMGarrillA:
A comparative investigation of azole susceptibility in Candida isolates from vulvovaginal candidiasis and recurrent vulvovaginal candidiasis patients in Ghana.Med Mycol.2017;55(1):686–689.
2783864210.1093/mmy/myw122SobelJD:
Recurrent vulvovaginal candidiasis.Am J Obstet Gynecol.2016;214(1):15–21.
10.1016/j.ajog.2015.06.067Vademecum:
Fluconazol.2013 [citado el 29 de noviembre de 2021].
Reference SourceSeleemDPardiVMurataRM:
Review of flavonoids: A diverse group of natural compounds with anti-
Candida Albicans activity in vitro.Arch Oral Biol.2017;76(1):76–83.
2765990210.1016/j.archoralbio.2016.08.030PairazamanEAmbrocioCPDanielJ:
Antifungal activity of propolis extract against Candida albicans in patients with vulvovaginal candidiasis [Data set]. Zenodo.2022.
10.5281/zenodo.698174410.5256/f1000research.137365.r262081Reviewer response for version 1Gómez-GaviriaManuela1Refereehttps://orcid.org/0009-0006-6836-913X
Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, University of Guanajuato, Guanajuato, Guanajuato, Mexico
Competing interests: No competing interests were disclosed.
I consider that the article is very incomplete, the methodology used is not the best, in addition, details need to be reviewed in the introduction, as well as in the results and discussion. It is necessary to organize some spelling errors and clarify some things within the text.
I consider that there is a lot of information on this topic, and taking this into account, the bibliography used in this article is very little.
Introduction:
Paragraph 1: I consider it necessary to add more information about Candidiasis
Paragraph 4 y 5: These paragraphs could be together
Paragraph 6: I consider that in this paragraph should be included more information about new natural therapies
Check the spelling of scientific names.
Methodology:
Reliability pg 4: The choice of the technique of direct observation of inhibition halos to evaluate resistance and susceptibility can generate some controversy. I consider that it is better to use the microplate technique, since it has more precise control of the experimental conditions and the possibility of quantifying the results in a more objective and reproducible way.
Preparation of the extract pg 4: Can the solvent used for the extract affect the interaction with the fungus?
Figure 1 pg 5: This photo is not clear, if they are identifying C. albicans, why do they put C. tropical is in the petri plate?
Figure 2 pg 5: The caption has nothing to do with what is seen in the photo.
Quality control of the growth of C. albicans strains pg 6: “obtaining 5 µ/ml” Check concentration
Antifungal susceptibility testing pg 6: I consider it necessary to use different extract concentrations
Bauer et al.,: that reference is very old
Results
Check the spelling of scientific names.
Is the work clearly and accurately presented and does it cite the current literature?
No
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Is the study design appropriate and is the work technically sound?
No
Are the conclusions drawn adequately supported by the results?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
No
Reviewer Expertise:
Taking into account the area of study in which I am currently working, which is microbiology, immunology and molecular biology, I consider that this article does not meet the microbiological aspects necessary to be published. First, the method they use to determine susceptibility and resistance is obsolete. Currently there are new tools such as microplates to do this type of experiments. Second, the figures are not of good quality, nor are they appropriate for an original article. And finally, the methodology is confusing and not written properly.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.