Species Distribution, Antifungal Susceptibility, and Factors Associated with Recurrence in Vulvovaginal Candidiasis
Round 1
Reviewer 1 Report (Previous Reviewer 1)
Comments and Suggestions for AuthorsI greatly appreciate the authors for revising the manuscript in response to my comments, and these revisions have addressed my prior concerns.
Author Response
Response:
We thank the reviewer for the positive evaluation of the revised manuscript and for the constructive comments that helped us improve our work.
Reviewer 2 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsThe manuscript addresses a clinically relevant question and has the potential to provide useful regional data on Candida species distribution and antifungal susceptibility in VVC. The sample size is good, and the authors addressed several methodological limitations. Here are my comments:
1) The title : “Factors Associated with Recurrence” is reasonable, but the manuscript should consistently use “association” rather than language implying causation. Please clarify.
2) Table 1 lists antibiotic use as 9.3% under common risk factors, but separately reports antibiotic use in 22.2% of patients. Please clarify
3) n= 345 used; however, in Table 1 under risk factors, total n is 343. Please clarify.
4)For better clarity, the epidemiological cutoff for each drug used should be discussed for classifying isolates as resistant or susceptible to that particular drug.
5) Data discrepancies that need clarification:
- Voriconazole: The overall susceptibility numbers differ between Table 1 and Table 2 (41.9/42.3% versus 42.6/43.0%). The authors should reconcile these discrepancies.
- Amphotericin B: Similarly, Table 1 reports 92.1% susceptible, whereas Table 2 reports 93.3%. This should be corrected
6) Why are some texts highlighted in yellow?
7) Several paragraphs repeat the same findings regarding C. albicans predominance, susceptibility, and clinical risk factors. Please shorten the discussion section.
8)Table 3: The recurrence groups total 343 rather than 345 when you add all the isolates under (<3 and >3) columns. Please clarify.
9) The authors should provide more information regarding the following in the methods section:
- the exact VITEK 2 card/system used;
- the testing period;
- MIC values, if available;
- quality-control procedures;
- how breakpoint changes during 2022–2025 affected classification;
- whether the same species-specific breakpoints were actually applied across all years.
10) The authors define recurrent VVC as ≥3 symptomatic episodes within 12 months, but it is unclear how previous episodes were documented. For better clarity please provide these.
Author Response
Dear Reviewer,
We sincerely thank you for your careful and constructive evaluation of our manuscript. We acknowledge that the concerns you raised were valid and important. We have addressed them as comprehensively as possible within the limitations of our retrospective study design and the available clinical and laboratory data. We believe that these revisions have improved the clarity and methodological transparency of the manuscript.
Manuscript title: Species Distribution, Antifungal Susceptibility, and Factors Associated with Recurrence in Vulvovaginal Candidiasis
We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We have carefully revised the manuscript in response to these comments. Our point-by-point responses and the corresponding changes are provided below.
Reviewer Comment 1
Comment: The title “Factors Associated with Recurrence” would be more appropriate, and causal terminology should be avoided.
Response: Thank you for this important comment. We agree that the retrospective observational design of our study allows the identification of associations but does not establish causality. The title, “Species Distribution, Antifungal Susceptibility, and Factors Associated with Recurrence in Vulvovaginal Candidiasis,” already reflects this association-based interpretation. We additionally reviewed the manuscript to ensure that causal terminology was avoided.
Changes in the manuscript: The manuscript was reviewed throughout to ensure consistent use of association-based terminology. The Discussion and limitations sections explicitly state that the retrospective observational design does not permit causal inference and that the observed associations should not be interpreted as causal relationships.
Reviewer Comment 2
Comment: There is a discrepancy in the frequency of antibiotic use (9.3% vs. 22.2%).
Response: Thank you for identifying this discrepancy. We re-evaluated the dataset and clarified the definition of antibiotic exposure. For the revised analysis, antibiotic use was defined as documented systemic antibacterial therapy within the 3 months preceding diagnosis, and the dedicated antibiotic-use variable in the clinical records was used consistently for all reported analyses. Accordingly, antibiotic exposure was present in 76 of 345 episodes (22.0%).
Changes in the manuscript: The Methods section now states: “Antibiotic use was defined as documented administration of systemic antibacterial therapy within the 3 months preceding the diagnosis; antifungal agents were not included in this category. The dedicated antibiotic-use variable in the clinical records was used for all analyses reported in the manuscript.” Table 1 and the Results section were revised to consistently report antibiotic use as 76/345 (22.0%).
Reviewer Comment 3
Comment: Although the study includes 345 cases, the risk-factor data in Table 1 appear to include only 343 cases.
Response: Thank you for noting this issue. The dataset and Table 1 were rechecked and corrected. The revised analysis includes 345 VVC episodes, with risk-factor status available for all included episodes. At least one risk factor was present in 144 episodes and absent in 201 episodes, totaling 345.
Changes in the manuscript: Table 1 was corrected to include all 345 episodes. A footnote clarifies that individual risk factors were not mutually exclusive and therefore may overlap.
Reviewer Comment 4
Comment: The epidemiological cutoff/clinical breakpoint used for each antifungal agent should be discussed.
Response: Thank you for this important methodological comment. Antifungal susceptibility results were interpreted according to the EUCAST clinical breakpoint tables applicable at the time of testing. Because EUCAST breakpoint tables were updated during the 2022–2025 study period, the breakpoint version in use at the time of testing was applied to each result rather than retrospectively reclassifying all isolates according to a single current version. EUCAST version 11.0 was used from December 2, 2024. Categorical results were reported only when an applicable species–drug clinical breakpoint was available.
Changes in the manuscript: The Microbiological Procedures section was revised to clarify the EUCAST breakpoint approach used during the study period, including the use of the breakpoint version applicable at the time of testing and the implementation of EUCAST version 11.0 from December 2, 2024. The manuscript also clarifies that categorical results were reported only when an applicable species–drug breakpoint was available.
Reviewer Comment 5
Comment: There are discrepancies between Tables 1 and 2 regarding voriconazole and amphotericin B susceptibility results.
Response: Thank you for identifying these inconsistencies. We carefully rechecked the susceptibility dataset and harmonized the denominators and reporting across the manuscript and tables. Percentages are now calculated only from isolates with categorically interpretable susceptibility results. Technically unreportable (TRM) results and isolates for which only numerical MIC values without categorical interpretation were available were excluded from percentage calculations and comparative analyses and are reported separately where applicable.
Changes in the manuscript: The revised overall voriconazole results are 42.7% susceptible, 43.0% resistant, and 14.3% susceptible at increased exposure, with five TRM results. Amphotericin B results are 93.3% susceptible and 6.7% resistant, with four TRM results. Table 2 and its footnote were revised accordingly. We also clarified that 32 C. glabrata isolates had only numerical voriconazole MIC values without categorical interpretation and were therefore excluded from categorical percentage calculations.
Reviewer Comment 6
Comment: Please address/remove the yellow-highlighted sections.
Response: Thank you. The highlighted sections were reviewed during revision.
Changes in the manuscript: The highlighted formatting was removed/corrected in the revised manuscript.
Reviewer Comment 7
Comment: The Discussion is repetitive and should be shortened/reorganized.
Response: We agree with the reviewer. The Discussion was revised to reduce repetition and improve its focus.
Changes in the manuscript: Repetitive descriptions of species distribution, antifungal susceptibility, and risk factors were removed or consolidated. The revised Discussion focuses on interpretation of the principal findings, comparison with previous studies, clinical implications, and study limitations.
Reviewer Comment 8
Comment: The totals in Table 3 appear to be 343 rather than 345.
Response: Thank you for identifying this discrepancy. Table 3 was rechecked against the final dataset and corrected. The recurrence analysis now includes all 345 episodes, comprising 199 episodes in the ≥3 episodes/year group and 146 episodes in the <3 episodes/year group.
Changes in the manuscript: Table 3 was corrected using the final dataset. The table footnote also clarifies that susceptibility percentages were calculated using evaluable susceptibility results within each recurrence group.
Reviewer Comment 9
Comment: Please provide more detailed information regarding the VITEK system/cards, study period, MIC interpretation, quality control, breakpoint changes during 2022–2025, and whether the same breakpoints were applied throughout the study.
Response: Thank you for this detailed methodological comment. We expanded the Microbiological Procedures section to provide the available methodological details. Species identification was performed using the VITEK® 2 automated system with the VITEK® 2 YST identification card, and antifungal susceptibility testing was performed using the VITEK® 2 AST-YS08 susceptibility testing card. The study period was January 1, 2022 to December 31, 2025. Antifungal susceptibility results were interpreted according to the EUCAST clinical breakpoint tables applicable at the time of testing. Because the breakpoint tables were updated during the study period, the version in use at the time of testing was applied rather than retrospectively applying a single current version to all isolates; EUCAST version 11.0 was used from December 2, 2024. Numerical MIC-only results without categorical interpretation were excluded from categorical percentage calculations and comparative analyses. The laboratory participated in an external quality assessment program (LabPT, Türkiye) for microbiological identification during the study period.
Changes in the manuscript: The revised Methods section now specifies the study period, the VITEK® 2 YST and AST-YS08 cards, the handling of numerical MIC-only results, and the EUCAST breakpoint approach used throughout the study period. External quality assessment participation for microbiological identification is also stated.
Reviewer Comment 10
Comment: RVVC was defined as ≥3 symptomatic episodes within 12 months, but it is unclear how previous episodes were documented or confirmed.
Response: Thank you for raising this point. We clarified the definition and ascertainment of recurrent episodes in the Methods section. Previous episodes were retrospectively identified from the patients’ medical records.
Changes in the manuscript: The revised Methods section states that each recurrent episode was required to have clinical symptoms compatible with vulvovaginal candidiasis and isolation of Candida spp. by positive vaginal culture. Episodes caused by different Candida species were also considered recurrent VVC when each episode fulfilled the predefined clinical and microbiological diagnostic criteria.
Closing statement
We sincerely thank the reviewer for these constructive comments. We believe that the revisions, including clarification of the study population and recurrence definition, re-evaluation of risk-factor and antifungal susceptibility data, expansion of the microbiological methodology, and revision of the statistical analyses and Discussion, have improved the clarity and methodological transparency of the manuscript.
Round 2
Reviewer 2 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsThe authors have answered to my queries satisfactorily and the manuscript has improved.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors(1) The study enrolled patients presenting between January 1, 2022 and December 31, 2025, while ethical approval was only granted on September 22, 2025. Since the patient recruitment period was still ongoing at the time of approval, the research was initiated prior to obtaining ethical clearance. This practice violates the fundamental ethical guidelines for clinical research.
(2) The study enrolled female participants aged 18 years and above, yet no age stratification was performed, nor was the distribution of participants across reproductive and menopausal subgroups reported. Given that the onset and recurrence of vulvovaginal candidiasis (VVC) are closely correlated with age and hormonal status, the absence of such stratification constitutes a major variable deficiency.
(3) The exclusion criteria are ambiguously defined. The study excluded immunocompromised individuals, including people living with HIV, organ transplant recipients, patients receiving chemotherapy, long-term users of corticosteroids or immunosuppressive agents, and those with primary immunodeficiencies. However, detailed screening procedures were not described, such as routine HIV testing protocols or verification criteria for the dosage and duration of corticosteroid administration. It is therefore impossible to verify whether the exclusion criteria were strictly implemented.
(4) Key information regarding participants’ lifestyle and medication history is incomplete. The authors only reported the mean age and prevalence of partial risk factors, while omitting several well-established contributors to VVC recurrence: sexual history, personal hygiene habits, vaginal douching, oral contraceptive use, detailed blood glucose profiles and vaginal microbiota status. The lack of these critical indicators renders the risk factor analysis inadequate.
(5) Recurrent VVC was defined as cases satisfying three criteria simultaneously: typical clinical symptoms, isolation of the identical Candida species, and positive vaginal culture. Nevertheless, several key details were not specified: the time interval between consecutive episodes, the differentiation between asymptomatic fungal colonization and symptomatic infection, and the classification rules for cases with species switching during recurrent episodes.
(6) Substandard approaches were applied to antifungal susceptibility testing and data analysis. Although MIC interpretations followed EUCAST guidelines, the specific MIC breakpoints were not provided, which undermines the methodological reproducibility of this study. Isolates marked as Technically Unreportable (TRM) were directly excluded from analysis, with no description of their proportion or distribution across study groups. If TRM isolates were concentrated in the recurrence group or specific Candida species, the susceptibility results would be biased. Additionally, the authors only compared overall susceptibility rates between groups, without conducting stratified analyses by fungal species, leading to insufficient analytical depth.
(7) The protocols for Candida species identification and laboratory testing are overly briefly described. The study only stated the use of the VITEK 2 system and Sabouraud Dextrose Agar, but omitted details about specimen collection, transportation, incubation conditions and strain reconfirmation procedures. For rare species such as Candida krusei and Candida lusitaniae, it was not clarified whether supplementary identification methods were adopted.
(8) Multiple flaws exist in the design and definition of risk factors. First, core risk factors including smoking, antibiotic exposure and intrauterine device use lack standardized operational definitions, greatly reducing study reproducibility. Second, the composite variable "presence of clinical risk factors" was established without clear assignment and grouping rules, resulting in flawed statistical logic. Third, major confounding factors such as vaginal douching and oral contraceptive use were not included or adjusted for, which may bias the associations between risk factors and VVC recurrence. Fourth, protocols for retrospective data extraction and quality control were not elaborated, bringing a high risk of information bias. It is suggested that the authors formulate standardized definitions and assignment rules for all risk factors, incorporate key confounding variables, and re-run statistical analyses accordingly.
(9) Deficiencies were found in statistical model design and variable selection. A threshold of p < 0.10 was used to select variables for the multivariate regression model, but the rationale for this cutoff was not explained. The forward stepwise regression method adopted in this study is vulnerable to confounding variables and may produce unstable results. Moreover, the authors only performed univariate and multivariate logistic regression without adjusting for potential interactions between age, disease duration and comorbidities. The Nagelkerke R² of 0.216 and classification accuracy of 65.9% indicate that the model has limited explanatory power, implying that multiple influential factors remain unaddressed.
(10) Multiple tables (Table 1-Table 4) cited throughout the article are absent.
Author Response
Dear Reviewers,
We sincerely thank the reviewers for their careful evaluation of our manuscript and for their detailed and constructive comments. We have revised the manuscript accordingly. For clarity, each reviewer comment is followed by our response and a summary of the corresponding changes made in the revised manuscript.
Reviewer 1
Reviewer Comment 1
The study enrolled patients presenting between January 1, 2022 and December 31, 2025, while ethical approval was only granted on September 22, 2025. Since the patient recruitment period was still ongoing at the time of approval, the research was initiated prior to obtaining ethical clearance. This practice violates the fundamental ethical guidelines for clinical research.
Response
Thank you for raising this important ethical concern. We agree that the wording in the previous version may have created the incorrect impression that patients were prospectively recruited before ethical approval. We would like to clarify that this was a retrospective observational study based exclusively on anonymized routinely collected clinical records, and no patients were prospectively recruited or subjected to any study-specific intervention. Ethical approval was obtained from the Toros University Scientific Research and Publication Ethics Committee on September 22, 2025 (Decision No. 2025-07/141). Data extraction for research purposes and statistical analyses were initiated only after ethical approval had been obtained. The study period (January 1, 2022–December 31, 2025) refers to the period covered by the routinely collected clinical records and does not represent a prospective patient recruitment period. To avoid any misunderstanding, we have revised the Methods section to explicitly clarify the retrospective nature of the study, the absence of prospective recruitment or research-related interventions, and the fact that data extraction and statistical analyses commenced only after ethics committee approval. The requirement for informed consent was waived by the ethics committee because the study used anonymized routinely collected clinical data.
Changes in the Manuscript
The Methods section now states:
“This retrospective study was based exclusively on anonymized routinely collected clinical records. No patients were recruited prospectively, and no interventions were performed for research purposes. Data extraction and statistical analyses were initiated only after ethical approval had been obtained.”
——————————————————————————————————————————
Reviewer Comment 2
The study enrolled female participants aged 18 years and above, yet no age stratification was performed, nor was the distribution of participants across reproductive and menopausal subgroups reported. Given that the onset and recurrence of vulvovaginal candidiasis (VVC) are closely correlated with age and hormonal status, the absence of such stratification constitutes a major variable deficiency.
Response
Thank you for this important comment. We agree that age and hormonal status may influence the occurrence and recurrence of vulvovaginal candidiasis. The mean age of the study population was 33.7 ± 7.2 years (range, 18–52 years). Because of the retrospective design of the study, menopausal status and other detailed hormonal variables were not consistently documented in the medical records and therefore could not be reliably incorporated into the subgroup or multivariable analyses. We considered retrospective assignment of menopausal status based solely on chronological age to be methodologically inappropriate and potentially subject to misclassification. We have therefore explicitly acknowledged the absence of menopausal status and related variables as a limitation of the study. Future prospective studies incorporating predefined age and reproductive/hormonal status categories are warranted to determine their potential influence on recurrent VVC.
Changes in the Manuscript
The limitation has been explicitly stated in the revised manuscript:
“Finally, several potentially relevant variables, including body mass index, menopausal status, sexual behavior, vaginal hygiene practices, vaginal douching, oral contraceptive use, probiotic use, glycemic control, and vaginal microbiota composition, were not routinely available in the medical records and therefore could not be included in the analyses.”
——————————————————————————————————————————
Reviewer Comment 3
The exclusion criteria are ambiguously defined. The study excluded immunocompromised individuals, including people living with HIV, organ transplant recipients, patients receiving chemotherapy, long-term users of corticosteroids or immunosuppressive agents, and those with primary immunodeficiencies. However, detailed screening procedures were not described, such as routine HIV testing protocols or verification criteria for the dosage and duration of corticosteroid administration. It is therefore impossible to verify whether the exclusion criteria were strictly implemented.
Response
Thank you for highlighting the need for greater clarity regarding the exclusion criteria. We agree that the previous version did not provide sufficient detail regarding the definition and ascertainment of immunocompromised status. In the revised manuscript, we have explicitly defined immunocompromised patients as those with documented HIV infection, solid organ or hematopoietic stem cell transplantation, active malignancy receiving chemotherapy, treatment with biological immunosuppressive agents, systemic corticosteroid therapy equivalent to ≥20 mg/day of prednisolone for ≥14 days, or primary immunodeficiency disorders. Because this was a retrospective study based on routinely collected clinical data, eligibility and exclusion criteria were determined from the patients' available medical records. No additional laboratory screening, including routine HIV testing, was performed solely for research purposes. We have clarified these procedures in the revised Methods section to ensure transparency regarding how the exclusion criteria were implemented.
Changes in the Manuscript
The following clarification has been added to the Exclusion Criteria subsection:
“Immunocompromised patients were defined as those with human immunodeficiency virus (HIV) infection, solid organ or hematopoietic stem cell transplantation, active malignancy receiving chemotherapy, treatment with biological immunosuppressive agents, systemic corticosteroid therapy equivalent to ≥20 mg/day of prednisolone for ≥14 days, or primary immunodeficiency disorders. Eligibility was determined from routinely available medical records. No additional laboratory screening (including routine HIV testing) was performed solely for research purposes.”
——————————————————————————————————————————
Reviewer Comment 4
Key information regarding participants’ lifestyle and medication history is incomplete. The authors only reported the mean age and prevalence of partial risk factors, while omitting several well-established contributors to VVC recurrence: sexual history, personal hygiene habits, vaginal douching, oral contraceptive use, detailed blood glucose profiles and vaginal microbiota status. The lack of these critical indicators renders the risk factor analysis inadequate.
Response
Thank you for this important observation. We agree that sexual behavior, personal hygiene practices, vaginal douching, oral contraceptive use, glycemic control, and vaginal microbiota composition are potentially relevant factors in the development and recurrence of vulvovaginal candidiasis. However, because this was a retrospective study based on routinely collected clinical records, these variables were not consistently documented and therefore could not be reliably included in the present analyses. We considered that attempting to retrospectively reconstruct these variables from incomplete records could introduce substantial misclassification and information bias. In the revised manuscript, we have therefore more clearly defined the clinical risk factors that were available and analyzed, including smoking, recent systemic antibiotic use, intrauterine device use, pregnancy, and other documented comorbidities. We have also explicitly acknowledged the absence of sexual behavior, vaginal hygiene practices, vaginal douching, oral contraceptive use, glycemic control, and vaginal microbiota composition as an important limitation of the study. Accordingly, the associations identified in this study should be interpreted within the context of the variables available in the retrospective dataset, and we have avoided implying that our risk-factor model comprehensively captures all established determinants of recurrent VVC. Prospective studies incorporating standardized assessment of these variables are warranted.
Changes in the Manuscript
The Limitations section has been expanded to state:
“Finally, several potentially relevant variables, including body mass index, menopausal status, sexual behavior, vaginal hygiene practices, vaginal douching, oral contraceptive use, probiotic use, glycemic control, and vaginal microbiota composition, were not routinely available in the medical records and therefore could not be included in the analyses.”
——————————————————————————————————————————
Reviewer Comment 5
Recurrent VVC was defined as cases satisfying three criteria simultaneously: typical clinical symptoms, isolation of the identical Candida species, and positive vaginal culture. Nevertheless, several key details were not specified: the time interval between consecutive episodes, the differentiation between asymptomatic fungal colonization and symptomatic infection, and the classification rules for cases with species switching during recurrent episodes.
Response
Thank you for this important comment. We agree that the definition of recurrent vulvovaginal candidiasis required further clarification, and the relevant section has been revised accordingly. In the revised manuscript, RVVC was defined as ≥3 clinically documented symptomatic episodes within a 12-month period. Each episode was required to include clinical symptoms compatible with VVC and a positive vaginal culture for Candida spp. Thus, asymptomatic culture positivity was not considered an episode of VVC; women with positive vaginal cultures in the absence of compatible clinical symptoms were considered to have Candida colonization rather than infection and were excluded. We also clarified that isolation of the same Candida species was not required for an episode to qualify as recurrent VVC. Episodes involving a change in Candida species were considered recurrent episodes provided that each episode independently fulfilled the predefined clinical and microbiological diagnostic criteria. Regarding the interval between individual episodes, no predefined minimum interval between consecutive episodes was applied; recurrence was determined according to the occurrence of ≥3 separately clinically documented symptomatic and culture-confirmed episodes within a 12-month period.
Changes in the Manuscript
The Definition of Recurrence section has been revised to state:
“For the purposes of this study, RVVC was defined as ≥3 clinically documented symptomatic episodes within 12 months... To ensure diagnostic accuracy, each recurrent episode was required to meet all of the following criteria: Clinical symptoms compatible with vulvovaginal candidiasis; Isolation of Candida spp. by positive vaginal culture. Episodes caused by different Candida species were also considered recurrent VVC, provided that each episode fulfilled the predefined clinical and microbiological diagnostic criteria.”
——————————————————————————————————————————
Reviewer Comment 6
Substandard approaches were applied to antifungal susceptibility testing and data analysis. Although MIC interpretations followed EUCAST guidelines, the specific MIC breakpoints were not provided, which undermines the methodological reproducibility of this study. Isolates marked as Technically Unreportable (TRM) were directly excluded from analysis, with no description of their proportion or distribution across study groups. If TRM isolates were concentrated in the recurrence group or specific Candida species, the susceptibility results would be biased. Additionally, the authors only compared overall susceptibility rates between groups, without conducting stratified analyses by fungal species, leading to insufficient analytical depth.
Response
Thank you for this detailed and important comment. We agree that the methodology and presentation of the antifungal susceptibility results required greater clarification, and we have substantially revised these sections. First, we have clarified that species identification and antifungal susceptibility testing were performed using the VITEK® 2 automated system and that MIC results were interpreted according to the EUCAST Version 12.1 clinical breakpoints. Rather than applying locally defined susceptibility thresholds, interpretations were based on the species- and antifungal-specific EUCAST criteria applicable to the tested isolates. Second, TRM results are no longer implicitly excluded without description. In the revised manuscript, TRM results are reported separately, and susceptibility percentages are calculated using isolates with interpretable susceptibility results as the denominator. The numbers and proportions of TRM results are now explicitly presented in Table 2, including their distribution according to Candida species. Third, we have expanded the analysis to provide species-specific descriptive susceptibility data. Table 2 now presents fluconazole, voriconazole, caspofungin, amphotericin B, and micafungin susceptibility results separately according to Candida species. This allows the marked differences in antifungal susceptibility between C. albicans and non-albicans Candida species to be evaluated rather than relying solely on pooled overall susceptibility estimates. We acknowledge that further statistical stratification of susceptibility according to both Candida species and recurrence status would result in very small cell sizes for several non-albicans Candida species and would therefore produce unstable and potentially misleading statistical estimates. We have consequently presented species-specific susceptibility descriptively while retaining the recurrence-group comparisons for the overall susceptibility analysis.
Changes in the Manuscript
The Microbiological Procedures section has been revised to clarify:
“Candida species identification and antifungal susceptibility testing were performed using the VITEK® 2 automated system... Antifungal susceptibility results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Version 12.1 clinical breakpoints, based on minimum inhibitory concentration (MIC) values... Results categorized as technically unreportable (TRM)... were reported separately and excluded only from comparative susceptibility analyses.”
——————————————————————————————————————————
Reviewer Comment 7
The protocols for Candida species identification and laboratory testing are overly briefly described. The study only stated the use of the VITEK 2 system and Sabouraud Dextrose Agar, but omitted details about specimen collection, transportation, incubation conditions and strain reconfirmation procedures. For rare species such as Candida krusei and Candida lusitaniae, it was not clarified whether supplementary identification methods were adopted.
Response
Thank you for this important comment. We agree that the microbiological procedures required additional methodological detail. Accordingly, the Microbiological Procedures section has been expanded in the revised manuscript. We have clarified that vaginal swab specimens were transported immediately to the microbiology laboratory and inoculated onto Sabouraud Dextrose Agar (RTA, Türkiye). The inoculated media were incubated at 35°C under aerobic conditions according to routine laboratory procedures. Candida species identification and antifungal susceptibility testing were subsequently performed using the VITEK® 2 automated system (bioMérieux, Marcy-l'Étoile, France). As this was a retrospective study based on routine clinical microbiology records, the analysis relied on the species identification results generated during routine diagnostic practice. No supplementary molecular or MALDI-TOF-based reconfirmation of rare Candida species was performed specifically for the purposes of this study. We have therefore avoided implying that rare isolates were independently reconfirmed by an additional identification method. We acknowledge that the absence of supplementary confirmation for uncommon Candida species represents a methodological limitation, particularly given the small number of rare isolates, and these findings should therefore be interpreted cautiously.
Changes in the Manuscript
The Microbiological Procedures section has been expanded to state:
“Vaginal swab specimens were transported immediately to the microbiology laboratory and inoculated onto Sabouraud Dextrose Agar (RTA, Türkiye). The inoculated media were incubated at 35°C under aerobic conditions according to routine laboratory procedures. Candida species identification and antifungal susceptibility testing were performed using the VITEK® 2 automated system (bioMérieux, Marcy-l'Étoile, France).”
——————————————————————————————————————————
Reviewer Comment 8
Multiple flaws exist in the design and definition of risk factors...
Response
Thank you for this detailed and constructive comment. We agree that the definitions and ascertainment of clinical risk factors required greater methodological clarity. We have therefore substantially revised the relevant sections of the manuscript. First, standardized operational definitions have now been provided for the principal clinical risk factors. Smoking was defined as documented tobacco use recorded in the medical record at the time of diagnosis. Antibiotic exposure was defined as systemic antibacterial therapy administered within the 3 months preceding the diagnosis; antifungal agents were not included in this category. IUD use was defined as the presence of an intrauterine device at the time of diagnosis, and pregnancy was defined as a documented ongoing pregnancy at presentation. Second, we have clarified the construction of the composite variable “presence of clinical risk factors.” This variable was defined as the presence of at least one of the predefined clinical factors, including smoking, recent antibiotic exposure, IUD use, pregnancy, or other documented comorbidities. Third, we agree that additional potential confounders, including vaginal douching, oral contraceptive use, sexual behavior, vaginal hygiene practices, glycemic control, and vaginal microbiota composition, may influence VVC recurrence. However, because this was a retrospective study based on routinely collected clinical records, these variables were not consistently available and could not be reliably incorporated into the multivariable analysis. Retrospective reconstruction or imputation of undocumented exposures was considered inappropriate because it could introduce additional misclassification and information bias. These unavailable potential confounders have therefore been explicitly acknowledged in the revised Limitations section. Finally, we have clarified that eligibility and clinical variables were determined from routinely available medical records. We acknowledge that the retrospective nature of data ascertainment may result in incomplete documentation of certain exposures, particularly smoking and antibiotic use, and this potential information bias has also been explicitly acknowledged as a study limitation. Accordingly, the associations observed in the multivariable analysis should be interpreted as associations based on the clinical variables reliably available in the retrospective dataset rather than as a comprehensive model incorporating all established determinants of recurrent VVC.
Changes in the Manuscript
The Variables Assessed section now provides explicit operational definitions:
“Smoking was defined as documented tobacco use recorded in the medical record at the time of diagnosis. Antibiotic use was defined as the administration of systemic antibacterial therapy within the 3 months preceding the diagnosis; antifungal agents were not included in this category. Intrauterine device (IUD) use was defined as the presence of an IUD at the time of diagnosis. Pregnancy was defined as a documented ongoing pregnancy at the time of presentation.”
——————————————————————————————————————————
Reviewer Comment 9
Deficiencies were found in statistical model design and variable selection. A threshold of p < 0.10 was used to select variables for the multivariate regression model, but the rationale for this cutoff was not explained. The forward stepwise regression method adopted in this study is vulnerable to confounding variables and may produce unstable results. Moreover, the authors only performed univariate and multivariate logistic regression without adjusting for potential interactions between age, disease duration and comorbidities. The Nagelkerke R² of 0.216 and classification accuracy of 65.9% indicate that the model has limited explanatory power, implying that multiple influential factors remain unaddressed.
Response
Thank you for this important methodological comment. We agree that the multivariable model should be interpreted cautiously given the retrospective nature of the dataset and the limited availability of several potentially relevant covariates. In the revised manuscript, we have clarified that variable selection was not based exclusively on statistical significance. Variables with a univariate p value <0.10 were considered as candidate variables to avoid prematurely excluding potentially relevant predictors, while variables considered clinically relevant based on previous literature and clinical judgment were also considered for inclusion in the multivariable model. We acknowledge the limitations associated with forward stepwise selection, including potential model instability and residual confounding. Furthermore, several variables that could potentially contribute to recurrence or interact with the evaluated predictors, including menopausal status, detailed hormonal and metabolic characteristics, sexual behavior, vaginal hygiene practices, oral contraceptive use, glycemic control, and vaginal microbiota composition, were not consistently available in the retrospective medical records and therefore could not be incorporated into interaction or adjusted analyses. We also agree that the Nagelkerke R² of 0.216 and overall classification accuracy of 65.9% indicate modest explanatory and discriminatory performance. Accordingly, the model should not be interpreted as a comprehensive predictive model for recurrent VVC. Rather, it was used as an exploratory analysis to identify associations between the clinical variables available in our retrospective dataset and recurrence. We have therefore adopted a more cautious interpretation of the regression findings and explicitly acknowledged the possibility of residual confounding and unmeasured determinants. Prospective studies incorporating standardized collection of additional clinical, behavioral, hormonal, metabolic, and microbiological variables are required to develop and validate more robust predictive models for recurrent VVC.
Changes in the Manuscript
The Statistical Analysis section now clarifies:
“Variables with a p value <0.10 in the univariate analysis, together with variables considered clinically relevant based on previous literature and clinical judgment, were entered into the multivariable logistic regression model, which was constructed using a forward stepwise selection method.”
——————————————————————————————————————————
Reviewer Comment 10
Multiple tables (Table 1–Table 4) cited throughout the article are absent.
Response
Thank you for pointing this out. We apologize for the omission in the previous version of the manuscript. All four tables (Tables 1–4) have now been included in the revised manuscript and are appropriately cited in the corresponding sections of the text. The tables provide the following information: Table 1: Baseline demographics and clinical characteristics of the study population. Table 2: Distribution of antifungal susceptibility according to Candida species. Table 3: Candida species and antifungal susceptibility results according to recurrence status (≥3 episodes/year). Table 4: Univariate and multivariable logistic regression analyses for factors associated with recurrent VVC.
Changes in the Manuscript
Tables 1–4 have been included in full in the revised manuscript and appropriately referenced in the corresponding Results sections.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe definition of recurrent VVC should be reconciled with current IDSA and European guideline definitions, particularly the difference between three versus four episodes per year. The authors should also describe the diagnostic criteria and evaluation used to distinguish symptomatic infection from Candida colonization. In the methods section, additional details regarding antifungal susceptibility testing would be helpful, including the EUCAST version used and how species without established breakpoints were interpreted. Clarification of the terms "moderately susceptible" and "intermediately susceptible" is also warranted. . Finally, some concepts in the introduction are repetitive and could be condensed, and the section discussing antifungal vaccines could be shortened
Comments on the Quality of English LanguageMinor changes. The manuscript is generally well written and easy to follow. However, several concepts are repeated throughout the Introduction and Discussion, which could be condensed to improve readability. There are also occasional grammatical errors, repeated words, inconsistent use of articles, and minor sentence construction issues. In addition, species nomenclature, punctuation, and spacing are not entirely consistent throughout the manuscript.
Author Response
Dear Reviewers,
We sincerely thank the reviewers for their careful evaluation of our manuscript and for their detailed and constructive comments. We have revised the manuscript accordingly. For clarity, each reviewer comment is followed by our response and a summary of the corresponding changes made in the revised manuscript.
Reviewer 2
Comment 1
The definition of recurrent VVC should be reconciled with current IDSA and European guideline definitions, particularly the difference between three versus four episodes per year.
Response: Thank you for this important comment. We agree that the definition of recurrent vulvovaginal candidiasis (RVVC) varies among international guidelines. We have revised both the Methods and Discussion sections to explicitly acknowledge this discrepancy. In the revised manuscript, we clarify that RVVC may be defined as three or more symptomatic episodes within 12 months in some contemporary guidelines, whereas European guidance has traditionally used four or more episodes annually.
For consistency with the prespecified definition used in the present study, RVVC was defined as ≥3 clinically documented symptomatic episodes within 12 months. We have now clearly stated the definition used in our study and its relationship to the differing definitions in international guidelines.
Changes made in the revised manuscript: The “Definition of Recurrence” subsection of the Methods and the corresponding paragraph in the Discussion have been revised accordingly.
Comment 2
The authors should also describe the diagnostic criteria and evaluation used to distinguish symptomatic infection from Candida colonization.
Response: Thank you for this valuable suggestion. We have expanded the Methods section to clearly distinguish symptomatic VVC from Candida colonization. Women were eligible for inclusion only when clinical findings compatible with VVC, including vaginal discharge, pruritus, burning sensation, or vulvovaginal erythema, were accompanied by a positive vaginal culture for Candida spp.
Women with a positive vaginal culture in the absence of compatible clinical symptoms were considered to have Candida colonization rather than symptomatic infection and were therefore excluded from the study.
In addition, each episode included in the definition of recurrent VVC was required to fulfill both clinical and microbiological criteria. Episodes caused by different Candida species were also considered recurrent episodes provided that each episode independently met these predefined criteria.
Changes made in the revised manuscript: These diagnostic criteria have been added to the “Inclusion Criteria” and “Definition of Recurrence” subsections of the Methods.
Comment 3
In the Methods section, additional details regarding antifungal susceptibility testing would be helpful, including the EUCAST version used and how species without established breakpoints were interpreted.
Response: Thank you for this important observation. We have expanded the “Microbiological Procedures” subsection to provide additional methodological details regarding antifungal susceptibility testing.
Candida species identification and antifungal susceptibility testing were performed using the VITEK® 2 automated system (bioMérieux, Marcy-l'Étoile, France). Because the study covered a multiyear retrospective period during which EUCAST breakpoint tables were updated, we revised the previous wording referring to a single EUCAST version. Categorical interpretations are now described as being based on EUCAST clinical breakpoints applicable to the corresponding species–antifungal combination.
For species–antifungal combinations for which an established EUCAST clinical breakpoint was unavailable, or when a categorical MIC interpretation could not be assigned, no S/I/R classification was made. Such results were reported separately as technically unreportable (TRM) and were excluded from comparative susceptibility analyses.
Changes made in the revised manuscript: The “Microbiological Procedures” subsection has been revised to clarify the susceptibility testing methodology and the handling of isolates without an interpretable clinical breakpoint.
Comment 4
Clarification of the terms "moderately susceptible" and "intermediately susceptible" is also warranted.
Response: We agree with the Reviewer and thank them for highlighting this terminology issue. Antifungal susceptibility terminology has now been standardized throughout the manuscript. The terms “moderately susceptible” and “intermediately susceptible” have been removed.
The susceptibility categories are now consistently described as:
S: susceptible at standard exposure
I: susceptible at increased exposure
R: resistant
Accordingly, previous references to “intermediate susceptibility” or “intermediately susceptible” have been revised to “susceptible at increased exposure (I),” where applicable. The corresponding table footnote has also been revised to define the S, I, and R categories explicitly.
Changes made in the revised manuscript: The terminology has been standardized throughout the Methods, Results, Discussion, and relevant table footnotes.
Comment 5
Finally, some concepts in the Introduction are repetitive and could be condensed.
Response: Thank you for this suggestion. We have carefully revised and condensed the Introduction. Repetitive statements regarding the epidemiology of VVC, Candida species distribution, recurrence, and antifungal susceptibility have been removed or combined. The revised Introduction is now more concise and focuses more directly on the rationale and objectives of the present study.
Changes made in the revised manuscript: The Introduction has been substantially condensed and reorganized to improve clarity and readability.
Comment 6
The section discussing antifungal vaccines could be shortened.
Response: We agree with the Reviewer. As the discussion of antifungal vaccines was not directly related to the primary objectives of the present study, this section has been substantially shortened/removed. The revised Discussion now focuses more directly on Candida species distribution, antifungal susceptibility patterns, clinical factors associated with recurrence, and the clinical implications of our findings.
Changes made in the revised manuscript: The antifungal vaccine discussion has been substantially reduced to improve the focus of the Discussion.
Comments on the Quality of English Language
Minor changes. The manuscript is generally well written and easy to follow. However, several concepts are repeated throughout the Introduction and Discussion, which could be condensed to improve readability. There are also occasional grammatical errors, repeated words, inconsistent use of articles, and minor sentence construction issues. In addition, species nomenclature, punctuation, and spacing are not entirely consistent throughout the manuscript.
Response: We thank the Reviewer for these helpful comments. The manuscript has been carefully revised for English grammar, sentence structure, article usage, punctuation, and spacing. Repetitive statements in the Introduction and Discussion have been removed or condensed. Candida species nomenclature and antifungal susceptibility terminology have also been reviewed and standardized throughout the manuscript to improve consistency and readability.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe article by Ozmen et al, evaluates the distribution of Candida species, antifungal susceptibility, and clinical predictors in causing recurrent vulvovaginal candidiasis (RVVC). The topic is clinically important as recurrence remains challenging. Here are my comments:
1) for susceptibility testing, the CLSI/ EUCAST breakpoints for individual drugs need to be mentioned. Please clarify.
2)for VVC, are echinocandins commonly used clinically? Due to pharmacokinetic limitations, echinocandins are not the standard first-line therapeutics in treating VVC. The clinical relevance in the manuscript needs to be better justified.
3) The sample collection, culture conditions, and species identification methodologies need to be described for better clarity and reproducibility.
4)Line 258: "Glabrata" is spelled as "Glabratta". The authors used ",s" instead of decimal (.) in lines 312 and 315.
5) The authors referenced Tables 1-4, however, the manuscript doesn't contain Tables 1-4. Please clarify.
6)The study only evaluates a limited set of risk factors. Risk factors that were not considered were vaginal hygiene, antifungal therapy history, BMI, sexual activities, probiotic use etc. These need to be acknowledged in the discussion,
7)The manuscript repeatedly uses causal statements. This study is retrospective and can only demonstrate association. Please clarify
Author Response
Dear Reviewers,
We sincerely thank the reviewers for their careful evaluation of our manuscript and for their detailed and constructive comments. We have revised the manuscript accordingly. For clarity, each reviewer comment is followed by our response and a summary of the corresponding changes made in the revised manuscript.
Reviewer 3
Comment 1
For susceptibility testing, the CLSI/EUCAST breakpoints for individual drugs need to be mentioned. Please clarify.
Response: Thank you for this important comment. We revised the Microbiological Procedures section to clarify how antifungal susceptibility categories were handled. Because this retrospective study spans 2022–2025, during which EUCAST antifungal breakpoint tables were updated, we did not retrospectively impose a single current breakpoint table on all historical laboratory results. The analysis used the categorical susceptibility interpretations documented in the laboratory records for the corresponding species–antifungal combinations. We also clarified that EUCAST breakpoints are species–drug specific rather than universal values for an antifungal agent. When an established breakpoint was unavailable for a species–drug combination, or when a categorical MIC interpretation could not be assigned, no S/I/R category was retrospectively inferred; such results were reported separately as technically unreportable (TRM). This avoids assigning unsupported breakpoint values to historical results.
Changes in the Manuscript: The Microbiological Procedures section now explains the retrospective use of documented categorical susceptibility results, the species–drug-specific nature of EUCAST breakpoints, the S/I/R terminology, and the handling of combinations without an established categorical interpretation.
——————————————————————————————————————————
Comment 2
For VVC, are echinocandins commonly used clinically? Due to pharmacokinetic limitations, echinocandins are not the standard first-line therapeutics in treating VVC. The clinical relevance in the manuscript needs to be better justified.
Response: We agree. The Discussion and Conclusion have been revised to make clear that echinocandins are not standard first-line therapy for vulvovaginal candidiasis and that the echinocandin findings in this study should be interpreted as in vitro microbiological susceptibility data rather than as evidence supporting routine clinical use for VVC. Their inclusion provides a broader description of the susceptibility profile of the isolates, particularly in the context of azole nonsusceptibility.
Changes in the Manuscript: The Discussion and Conclusion now explicitly state that echinocandins are not standard first-line therapy for VVC and that the reported echinocandin susceptibility results do not constitute a treatment recommendation.
——————————————————————————————————————————
Comment 3
The sample collection, culture conditions, and species identification methodologies need to be described for better clarity and reproducibility.
Response: Thank you. The Microbiological Procedures section has been expanded. Vaginal swab specimens were transported immediately to the microbiology laboratory, inoculated onto Sabouraud Dextrose Agar, and incubated at 35°C under aerobic conditions according to routine laboratory procedures. Candida species identification and antifungal susceptibility testing were performed using the VITEK® 2 automated system.
Changes in the Manuscript: Detailed specimen transport, culture medium, incubation conditions, and VITEK® 2 species identification/susceptibility methodology are now provided in the Methods section.
——————————————————————————————————————————
Comment 4
Line 258: “Glabrata” is spelled as “Glabratta”. The authors used commas instead of decimal points in lines 312 and 315.
Response: Thank you for identifying these typographical and formatting errors. The species name has been corrected to C. glabrata, and decimal commas in the manuscript and tables have been standardized to decimal points in accordance with English-language scientific formatting.
Changes in the Manuscript: The spelling and decimal-formatting errors have been corrected throughout the revised manuscript.
——————————————————————————————————————————
Comment 5
The authors referenced Tables 1–4; however, the manuscript does not contain Tables 1–4. Please clarify.
Response: We apologize for the omission in the earlier file. All four tables are included in the revised manuscript and are appropriately cited in the corresponding sections of the text.
Changes in the Manuscript: Tables 1–4 are now included in full and appropriately referenced.
——————————————————————————————————————————
Comment 6
The study only evaluates a limited set of risk factors. Risk factors that were not considered were vaginal hygiene, antifungal therapy history, BMI, sexual activities, probiotic use, etc. These need to be acknowledged in the Discussion.
Response: We agree. The Limitations section has been expanded to acknowledge that prior antifungal treatment history was not consistently available and that several potentially relevant variables—including body mass index, menopausal status, sexual behavior, vaginal hygiene practices, vaginal douching, oral contraceptive use, probiotic use, glycemic control, and vaginal microbiota composition—were not routinely available in the retrospective medical records and therefore could not be included in the analyses.
Changes in the Manuscript: These unmeasured or incompletely documented risk factors are now explicitly acknowledged in the Limitations section.
——————————————————————————————————————————
Comment 7
The manuscript repeatedly uses causal statements. This study is retrospective and can only demonstrate association. Please clarify.
Response: We fully agree. We revised the title and relevant wording throughout the Abstract, Introduction, Methods, Results, Discussion, and Conclusion to avoid causal or overly predictive interpretation. Terms such as “predictors” and causal formulations were replaced, where appropriate, with “factors associated with recurrence” and “associations.” The revised manuscript also explicitly states that the observational findings do not establish causality.
Changes in the Manuscript: The title has been revised to “Species Distribution, Antifungal Susceptibility, and Factors Associated with Recurrence in Vulvovaginal Candidiasis,” and causal language has been replaced by association-based terminology throughout the manuscript.
