Scalp Microbiome Dynamics Can Contribute to the Clinical Effect of a Novel Antiseborrheic Dermatitis Shampoo Containing Patented Antifungal Actives: A Randomized Controlled Study

Abstract

Introduction

Scalp seborrheic dermatitis (SD) can cause physical discomfort and social embarrassment in affected individuals. Mild-to-moderate scalp SD can be managed using topical products with antifungal, antiinflammatory, and keratolytic properties.

Methods

A two-phase, randomized, controlled study was conducted to evaluate the clinical efficacy of a newly formulated anti-SD shampoo containing two patented antifungal actives and to investigate the associated changes in the scalp microbiota. The intervention involved a 2-week intensive phase for the 42 subjects included in the study, consisting of the application of the anti-SD shampoo three times a week; a randomized [1:1], controlled, parallel-group 8-week maintenance phase consisting of the test group applying the study shampoo once a week alternately with a neutral shampoo twice a week; and the control group applying the neutral shampoo alone three times a week.

Results

Following the intensive phase, the scalp condition improved substantially, as evidenced by a significant decrease in the severity of dandruff, erythema, and pruritus, associated with an improvement of SD dysbiosis. These improvements were more sustained in the test group than in the control group during the maintenance phase. The rediversification of the scalp microbiota involved a significant increase in fungal and bacterial richness along with a decrease in the level of SD-predominant Malassezia fungi and Staphylococcus bacteria and an increase in the level of low-abundant fungi genera belonging to the Ascomycota phylum.

Conclusions

The synergistic effects of antimycotic and antiinflammatory agents in the study shampoo likely contributed to rebalancing the fungal and bacterial ecosystem, thus improving scalp symptoms.

ClinicalTrials.gov Identifier

NCT06578962 (retrospectively registered on 28 August 2024).

Supplementary Information

The online version contains supplementary material available at 10.1007/s13555-025-01408-z.

Key Summary Points

Why carry out this study?

Seborrheic dermatitis (SD) involves an imbalance in scalp microbiota, with an overgrowth of Malassezia fungi and certain bacteria, leading to symptoms that affect many individuals.

The study aimed to test if an anti-dandruff shampoo could restore microbial balance on the scalp, reducing SD symptoms.

What was learned from the study?

Clinical efficacy: The study demonstrated the good clinical efficacy of a newly formulated anti-dandruff shampoo on mild-to-moderate scalp SD, showing significant improvements in symptoms such as dandruff, erythema, and pruritus which were more sustained in the test group than in the control group during the maintenance phase.

Microbiota rebalancing: The shampoo effectively reduced Malassezia fungi and Staphylococcus species, while increasing the abundance of Cutibacterium, Penicillium, and Cladosporium, suggesting a rebalanced scalp ecosystem linked to improved scalp health and symptom relief.

Introduction

Seborrheic dermatitis (SD) is a common and recurrent chronic inflammatory skin condition affecting areas rich in sebaceous glands, such as the scalp, face, upper chest, and back [1]. It affects approximately 1–5% of adults worldwide [2,3] and may considerably impact quality of life by contributing to physical discomfort with persistent itching, causing social embarrassment and a potential decline in self-esteem [4].

SD has a multifactorial etiology that involves both intrinsic and extrinsic susceptibility factors, including genetic predisposition [5], immunological factors [6,7], neurogenic factors, emotional stress, as well as nutritional [8] and environmental (humidity and ambient temperature) factors [3,7]. In scalp SD, these factors contribute to a compromised epidermal barrier function, altered sebum composition, and increased local inflammation [9]. This is reflected by flaking or scaling, leading to white-to-yellowish oily or dry dandruff, with erythematous and often pruritic plaques [3,7]. Histological changes involve acanthosis, hyperkeratosis, focal spongiosis, and parakeratosis, demonstrating keratinocyte dysregulation with an altered “proliferation-to-differentiation” balance [1,3,10]. Perivascular and perifollicular immune infiltration may be also associated [3]. Excessive and altered sebum composition arises in a context of microbiota dysbiosis [11]. During SD, an unbalanced microbiota is observed [12], with the epidermal structural alteration contributing to colonization by specific microorganisms. Indeed, increased populations of certain fungal and bacterial species, such as members of the Malassezia [12–16], Staphylococcus [12,17,18], and Streptococcus [19] genera, have been reported in patients exhibiting SD, while Cutibacterium acnes (C. acnes), a component of the healthy scalp microbiota, was shown to be underrepresented [18].

For mild-to-moderate scalp SD, presenting with dandruff but without visible inflammation, first-line therapies rely on antifungal, antiinflammatory, and keratolytic topical agents [9,20–22]. Current research seeks to enhance the efficacy of topical products by addressing multiple contributing factors and improving long-term management, notably with regard to relapse. With this aim, a newly formulated anti-SD shampoo, containing two patented antifungal compounds (piroctone olamine [23] and ciclopirox olamine [24]; patent nos. WO2021/064316) that work synergistically, a keratolytic compound (keluamid), and soothing antioxidant agents (β-glycyrrhetinic acid [24]), has been developed.

The clinical efficacy of this novel shampoo formulation was evaluated in subjects with mild-to-moderate SD in a two-phase, randomized, controlled study. Specific changes in scalp microbiota composition and abundance associated with the product use were investigated to further characterize the effects of the shampoo on SD scalp dysbiosis. Both assessments were performed (1) after 2 weeks of intensive use of the study shampoo and (2) after 8 weeks of a maintenance phase where participants used the study shampoo in conjunction with a neutral shampoo (test group) or the neutral shampoo alone (control group). The primary efficacy endpoint of the study was the change in dandruff severity during the maintenance phase. The overall tolerability of the products was also assessed.

Methods

Study Design

This prospective, two-center trial was conducted from November 2020 to May 2021 at the Centre de Recherche sur la Peau (Hôtel Dieu, Toulouse, France) and the Centre de Santé Sabouraud (Hôpital Saint-Louis, Paris, France). This study comprised two phases: an open-label intensive phase of the product use and a randomized [1:1], controlled, parallel-group maintenance phase (Fig. 1A).

Fig. 1.

Study design (A) and flow chart (B). Description of the intensive phase and maintenance phase of the intervention scheme, and parameters evaluated in the subjects during the 4 study visits: week 0 (W0, baseline), W2 (end of intensive phase/randomization), W6, and W10 (end of maintenance phase). IGA investigator global assessment, PGA patient global assessment

Ethical Approval

The study was performed in compliance with the Declaration of Helsinki and its subsequent amendments, as well as good clinical practice guidelines (CHMP/ICH/135/1995 and integrated addendum). It also conformed to Regulation (EC) no. 1223/2009 of the European Commission and the French Decree (no. 2017-884) pertaining to research on cosmetic products. As the registration of studies evaluating cosmetic products is not mandatory in the European Union, the study was retrospectively registered under trial number NCT06578962 (28 August 2024) for publication. Prior to enrollment, all subjects provided written informed consent.

Participants

Female or male subjects aged 18–60 years, with skin phototypes I–V (Fitzpatrick scale) and mild-to-moderate recurrent scalp SD, were eligible to participate in the study. More specifically, participants had to present clinically visible dandruff (score ≥ 20 on a scale from 0 to 80 [25]), with a squamous state intensity > 2 (on a scale from 0 [absent] to 3 [severe]) in at least one of four scalp areas, erythema (score ≥ 1 on a scale from 0 [absent] to 3 [severe]) in at least one desquamating scalp area, and pruritus (score ≥ 3 on a scale from 0 to 10) for the last 3 days as reported by the subjects, and recurrent episodes (at least twice a year). Other exclusion criteria are listed in the Supplementary Material.

Interventions

Products

The study product was an anti-dandruff shampoo (KELUAL DS, Ducray Laboratories) containing the following active ingredients: piroctone olamine and ciclopirox olamine (patent nos. WO2021/064316), keluamid, and β-glycyrrhetinic acid. The control product was a neutral shampoo (Extra Doux, Ducray Laboratories).

Intervention Scheme

During the 2-week intensive phase, all subjects applied the study shampoo on the whole scalp three times a week for 2 weeks, at home (Fig. 1A). Thereafter, for the 8-week maintenance phase, the subjects were randomized into two groups according to a computer-generated randomization list established by Pierre Fabre Laboratories:

• The test group had to apply the study shampoo once a week, alternating with the neutral shampoo twice a week.

• The control group had to apply the neutral shampoo alone three times a week.

The application procedure was identical for the two products: the participants had to apply the shampoo onto the whole scalp, lather, rinse, then apply again, and leave for few minutes before rinsing. The shampoo had to be applied at least three days prior to each assessment visit, with no application of other topical hair products or water in between.

Assessment Schedule

A total of four visits was scheduled over the 10-week study period: at inclusion (week 0, W0), 2 weeks (W2) later at the end of the intensive phase/start of the maintenance phase, at week 6 (W6), and at the end of the study, i.e., 10 weeks after the intervention initiation (W10) (Fig. 1A). At each visit, the investigator examined the scalp of the subjects to assess clinical parameter scores and global efficacy and performed a swab sampling on SD-affected areas identified at the W0 visit. Subject compliance was assessed by comparing the self-recorded number of product applications with the theoretical number expected at visits W2, W6, and W10.

Evaluation Criteria

Clinical Efficacy

The primary efficacy endpoint was the change in dandruff severity during the maintenance phase (between W2 and W10), as assessed by the investigator using a clinical dandruff score according to the method of Squire and Goode [25].

Secondary efficacy endpoints were:

• The change in dandruff severity during the intensive phase (between W0 and W2), the maintenance phase (between W2 and W6), and overall (between W0 and W6 and W0 and W10) as assessed by the investigator using the clinical dandruff score.

• The changes in erythema and pruritus severity experienced in the last 3 days during both study phases, as assessed by the investigator using a 4-point scale (from 0 [absent] to 3 [severe]) for erythema severity and by the subject using a 10-point scale (from 0 to 10) for pruritus severity.

• The overall efficacy on SD, as evaluated by the investigator at each visit (starting from W2) using the investigator global assessment (IGA) score (from 1 [worsening] to 5 [complete resolution]).

• The global efficacy on SD, as evaluated by the patient using the patient global assessment (PGA) score (from 1 [worsening] to 5 [complete resolution]), and changes in squamous state and scalp discomfort (both scores from 0 [absent] to 3 [severe]) were assessed through weekly self-assessment questionnaires.

Microbiota Analyses

Changes in the composition and abundance of scalp microbiota between W0 and W2 and W2 and W10 were another secondary endpoint of the study. For this assessment, total genomic DNA was extracted from swab samples from a subset of subjects, following a two-step lysis/purification protocol on a QIAcube^® instrument (Qiagen).

Microbiome Analyses

Amplicon sequencing microbiome analyses of scalp bacterial and fungal populations were performed at the Plateforme Génomique GeT-IT (INRAE transfert METYS in Castanet-Tolosan, France), as previously described in Maître et al. [26]. Sequencing of the V1–V3 region of the 16S ribosomal RNA (16SrRNA) for bacteria, and the internal transcribed spacer 1 (ITS1) region for fungi was done using Illumina Miseq^® technology. DNA sequences underwent preprocessing to eliminate barcodes, primers, and DNA chimeras. Sequences exhibiting 100% homology were consolidated into unique sequences, followed by the creation of operational taxonomic units (OTUs) at a 97% threshold. Bioinformatics analyses were conducted to identify bacteria and fungi at both the phylum and genus taxonomic levels, using Mothur and the Greengenes database (https://mothur.org/wiki/greengenes-formatted_databases) for bacteria or Mothur and the Findley ITS database for fungi [27].

Droplet Digital PCR of Microbial Species

The absolute quantification of four microorganism species (Staphylococcus epidermidis, Cutibacterium acnes, Malassezia restricta, and Malassezia globosa) was performed using droplet digital PCR (ddPCR™) with the QX200™ and AutoDG™ ddPCR system (Bio-Rad Laboratories). Strain references for normalization and quantification and primers are reported in Supplementary Table S1.

Tolerability

Product tolerability was evaluated by the investigator in all subjects at each visit, by reporting dermatological adverse events (DAEs) or nondermatological adverse events (AEs) in the case report form. The relationship with the investigational product was also assessed.

Statistical Methods

Sample Size

On the basis of preliminary data, a mean change in dandruff severity score of −2.1 ± 5.6 points was expected in the test group versus 7.1 ± 9.2 points in the control group. Therefore, considering a type I error (α) of 5%, a type II error (β) of 10%, and 10% of lost-to-follow-up subjects or premature withdrawals, we calculated that a total number of 24 subjects per group was needed to demonstrate a significant difference between the two study groups.

Analysis Sets

The safety and efficacy analyses were carried out on the full analysis set (FAS), consisting of all subjects included in the study.

Analysis of the Primary Endpoint

A mixed model for repeated measures (MMRM) was used to analyze changes in the dandruff clinical score between visits W2 and W10. Fixed factors included group, visit, and the interaction term visit × group. The “subject” factor was included as a repeated factor, and W2 value was included as a covariate. Intragroup and intergroup analyses were conducted by computing and comparing adjusted means derived from the mixed model.

Analyses of the Secondary Endpoints

For quantitative variables, a MMRM was also used to analyze changes between the baseline visit W0 and visits W2, W6, and W10, respectively, and the group, visit, and the interaction term group × visit were included as fixed factors. The subject was considered as a random factor, and the baseline value W0 was considered as a covariate. Intragroup and intergroup analyses at each visit, as well as a global analysis across all visits, were performed by calculating and comparing adjusted means derived from the mixed model.

For qualitative variables, intragroup comparisons were conducted using Kruskal–Wallis tests to assess changes between either visit W0 or visit W2, and subsequent visits (W2, W6, and W10). If a significant result was obtained, the Wilcoxon signed-rank test was used to compare changes between the timepoints within each group. For intergroup comparisons, the Wilcoxon rank-sum test was used to compare groups at each visit.

For microbiome analyses, alpha- and beta-diversity analyses were conducted for each microbial population (bacteria and fungi) after the normalization by rarefaction of the OTUs [28]. A total of four alpha-diversity indices, characterizing intracommunity diversity, was computed: Observed (number of observed OTUs, i.e., observed richness), Chao1 (observed OTUs added to the estimated number of unobserved OTUs, i.e., richness), Shannon (which considers both observed richness and evenness of OTU abundance distribution, i.e., richness and evenness), and Inverse (Inv)-Simpson (similar to Shannon but with higher penalization for lack of evenness). In total, four beta-diversity indices, aiming to assess intercommunity diversity, were also used: Jaccard (proportion of OTUs not shared), Bray–Curtis (proportion of abundance not shared), unweighted UniFrac (proportion of branch lengths in the phylogenetic tree not shared), and weighted UniFrac (quantitative version of the UniFrac index that weights each branch length by the difference in relative abundance). For each alpha-diversity index, a mixed linear model was used to assess changes from W0. Fixed factors included group, visit and the interaction term visit × group. The “subject” factor was included as a random factor, and the baseline value W0 as a covariate. A linear model was also used to assess changes from W2, with group as a fixed factor and W2 value as a covariate. Intragroup and intergroup analyses were conducted by computing and comparing adjusted means derived from the models. For each beta-diversity index, statistical intragroup differences were determined using a permutational multivariate analysis of variance (PERMANOVA) test [29], considering sample pairing.

Moreover, differential analyses of the relative abundances of scalp bacteria and fungi were conducted to identify genera with significant changes within study groups over time, and between study groups. For these analyses, genera with low prevalence (less than ten strictly positive values in all the samples) and unclassified genera were filtered out. The Wilcoxon signed-rank test was then used for the comparison of relative abundances between W2 and W0 on the whole study population (intensive phase) and between W10 and W2 in each study group (maintenance phase). The Wilcoxon rank-sum test was used for comparing changes in relative abundances (W10–W2) between the two groups during the maintenance phase. A multiple-testing correction of Benjamini and Hochberg was applied to obtain adjusted p-values.

The statistical analyses were performed using SAS (version V9.4) and R (version 4.2.1), with a significance level set at 5%. Phylogenetic tree schemes were generated with the function heat tree of the R library Metacoder.

Results

Participants and Product Use

In total, 42 (N = 42) subjects were enrolled in the intensive phase (study population), then randomized into the test (N = 21) and the control (N = 21) groups for the maintenance phase (Fig. 1B). One subject in the control group prematurely withdrew from the study for personal reasons. The mean age of enrolled participants was 35.8 ± 11.4 years, and 81.0% were women and 19.0% men. There were no intergroup differences in baseline demographic or clinical characteristics (Table 1).

Table 1.

Demographic and clinical characteristics of the participants

Parameters	Test group N = 21	Control group N = 21	Overall N = 42
Age in years, mean ± SD	36.1 ± 12.3	35.6 ± 10.7	35.8 ± 11.4
Sex, n (%)
Female	17 (81.0)	17 (81.0)	34 (81.0)
Male	4 (19.0)	4 (19.0)	8 (19.0)
Skin phototypea, n (%)
I	0 (0.0)	1 (4.8)	1 (2.4)
II	2 (9.5)	6 (28.6)	8 (19.1)
III	14 (66.7)	12 (57.1)	26 (61.9)
IV	4 (19.1)	2 (9.5)	6 (14.3)
V	1 (4.7)	0 (0.0)	1 (2.4)
Dandruff severity score, mean ± SD	29.2 ± 10.0	30.1 ± 9.7	29.6 ± 9.8
Facial seborrheic dermatitis, n (%)	2 (9.5)	1 (4.8)	3 (7.1)

n number of subjects, SD standard deviation

^aAccording to the Fitzpatrick classification

All subjects (N = 42 during the intensive phase and N = 21 during the maintenance phase) reported 80.0–120.0% compliance with the study product use.

Clinical Efficacy

Change in Dandruff Severity During the Maintenance Phase

Between W2 and W10, the mean dandruff severity score significantly decreased by 56.0% (−4.2 points, p = 0.003) in the test group, whereas it significantly increased by 117.4% in the control group (11.2 points, p < 0.0001) (Fig. 2A and Supplementary Table S2). The intergroup difference was highly significant (p < 0.0001).

Fig. 2.

Clinical symptoms evaluated and global clinical efficacy. Changes in dandruff (A), erythema (B), and pruritus (C) severity mean scores during the intensive (N = 42) and the maintenance (N = 21 in each group) phases. During the maintenance phase, intergroup analyses are shown in black, and intragroup analyses are shown in green (control group) and blue (test group) at the selected timepoint versus week 2 (W2). *p < 0.05; **p < 0.001; ***p < 0.0001. D Investigator global assessment (IGA) and E patient global assessment (PGA) at W2 in the whole study population (N = 42) and at W6 and W10 in the control and test groups (N = 21 in each group). The percentages of subjects who had improvement (comprising complete resolution, significant improvement, and slight improvement), stabilization, or worsening of scalp condition are shown next to the bars at W6 and W10. The Wilcoxon rank-sum test was used to compare groups. **p < 0.001; ***p < 0.0001

Secondary Clinical Endpoints

Changes in Dandruff Severity Score Over the Whole Study Period

Compared with baseline (W0), the mean dandruff severity score significantly decreased by 70.9% in the whole study population at the end of the intensive phase (W2, Fig. 2A). Thereafter, during the maintenance phase, further statistically significant decreases in the mean dandruff severity score were observed in the test group at both W6 and W10 versus W0 (−87.3% and −88.6%, respectively) and at W6 versus W2 (−51.0%). Conversely, in the control group, the mean score increased back at W6 (57.0%) and W10 (117.4%) compared with W2, but scores at W6 and W10 remained significantly lower than that at W0 (Fig. 2A). Overall, the clinical improvement of dandruff severity was more pronounced in the test group than in the control group, with statistically significant differences between the two groups at both W6 and W10. p-values associated to changes in dandruff severity scores are presented in Supplementary Table S2.

Changes in Erythema and Pruritus Severity Scores

The changes in erythema and pruritus severity scores followed a profile similar to that observed for the dandruff severity score (Fig. 2B, C). Both scores significantly decreased after the intensive phase, by 51.7% and 49.6%, respectively, in the whole study population. Both scores continued to decrease during the maintenance phase in the test group, by 30.2% and 69.1%, respectively, between W2 and W10. Significant decreases were also observed between W0 and W6 (−71.8% and −80.3%, respectively) and between W0 and W10 (−71.8% and −88.1%, respectively). In contrast, in the control group, both scores increased between W2 and W10 (50.0% and 41.9%, respectively), but the scores remained significantly lower at W6 and W10 compared with W0 (erythema: −23.4% and −10.4%; pruritus: −27.50% and −20.3%, respectively). Clinical improvement in erythema and pruritus was more pronounced in the test group than in the control group, with statistically significant differences between the two groups at both W6 and W10. p-values associated to changes in severity scores are presented in Supplementary Table S2.

Global Clinical Efficacy Assessment

According to the IGA (Fig. 2D) and PGA (Fig. 2E), the general scalp condition of the subjects further improved at W6 in the test group compared with the control group, with 81% (IGA) and 71% (PGA) of the subjects with SD improvement at W6 (including slight and significant improvements and complete resolution) in the test group versus 24% and 30%, respectively, in the control group. Global clinical efficacy was also better in the test group at the end of the study, with a significant intergroup difference at W10 (p < 0.001).

Assessment of Squamous State and Scalp Discomfort

Squamous state and scalp discomfort assessed by the subjects showed significant improvements during the intensive phase of the study product use (Supplementary Table S3). At the end of the maintenance phase, both parameters were improved in the test group, but not in the control group. A significant intergroup difference was observed for these parameters at W6 and W10 (Supplementary Table S4).

Scalp Microbiota Analysis during the Intensive Phase

Fungi

Fungal amplicon sequencing analysis was performed on scalp samples of a subset of 31 patients. On the basis of the analysis of a total of 868 OTUs, we found that Malassezia and Malasseziales were the most abundant fungi at baseline (Fig. 3A, D and Supplementary Fig. S1A). During the intensive phase, the abundance of this genus significantly decreased, whereas the relative abundances of the Cladosporium, Penicillium, Saccharomyces, and Debaryomyces genera (belonging to the Ascomycota phylum) significantly increased, as shown by differential analyses (Supplementary Table S5). More specifically, at the species level, ddPCR results showed that the abundances of Malassezia globosa (M. globosa) and Malassezia restricta (M. restricta) significantly decreased at W2 versus W0 (Fig. 3B and Supplementary Table S9, p < 0.001).

Fig. 3.

Scalp fungal analysis. Intensive phase: Subfigures A–D. Maintenance Phase: Subfigures E–F. A Mean relative abundances (%) of the top ten fungal genera at baseline week 0 (W0) and after the intensive phase (W2) in a subset of subjects (N = 31). B Absolute quantification of Malassezia globosa and Malassezia restricta at W0 and W2 (mean ± SD Log10 values), as determined by droplet digital PCR™ (N = 42). Differences between time points: **p < 0.001. C Boxplots for the four alpha-diversity indices analyzed during the intensive phase (median [min–max], N = 31). Differences between time points: **p < 0.001; ***p < 0.0001 D Phylogenetic trees of the scalp fungal population at baseline (W0) and after the intensive phase (W2). Colors and thickness of the branches are proportional to the abundance of each phylum, class, family, or genus. E Changes in the mean relative abundances of the top ten fungal genera during the maintenance phase (W2 to W10) in the control (N = 16) and test (N = 15) groups. F Boxplots for the four alpha-diversity indices analyzed during the maintenance phase (median [min–max]). Differences between time points: ***p ≤ 0.0001

At the end of the intensive phase, the four alpha-diversity indices analyzed had significantly increased compared with baseline (Fig. 3C and Supplementary Table S7), indicating an increase in fungal richness and evenness. The composition of the scalp fungal population was also substantially changed compared with baseline, as reflected by significant differences in the four beta-diversity indices (all p < 0.001).

Bacteria

As for the fungal population, the relative abundances of bacteria were determined from samples of a subset of 31 patients with a total of 3285 OTUs. At baseline, the most abundant genera were Cutibacterium and Staphylococcus (Fig. 4A, D and Supplementary Fig. S1B). After the 2-week intensive phase, the relative abundances of Cutibacterium, Anaerococcus, and Peptoniphilus genera had significantly increased, whereas that of Staphylococcus and Stenotrophomonas had significantly decreased (Supplementary Table S11). As determined by the ddPCR quantification, at the species level, a lower mean level of Staphylococcus epidermidis (S. epidermidis) was observed at W2 compared with W0 (p < 0.001), whereas the level of C. acnes remained relatively stable (Fig. 4B and Supplementary Table S15).

Fig. 4.

Scalp bacterial analysis. Intensive phase: Subfigures A–D. Maintenance Phase: Subfigures E–F. A Mean relative abundances (%) of the top ten bacterial genera at baseline week 0 (W0) and after the intensive phase (W2) in a subset of subjects (N = 31). B Absolute quantification of Cutibacterium acnes and Staphylococcus epidermidis at W0 and W2 (mean ± SD Log10 values), as determined by droplet digital PCR™ (N = 42). Differences between time points: **p < 0.001. C Boxplots for the four alpha-diversity indices analyzed during the intensive phase (median [min–max], N = 31). Differences between time points: *p < 0.05. D Phylogenetic trees of the scalp bacterial population at baseline (W0) and after the intensive phase (W2). Colors and thickness of the branches are proportional to the abundance of each phylum, class, family, or genus. E Changes in the mean relative abundances of the top ten bacterial genera during the maintenance phase (W2 to W10) in the control (N = 16) and test (N = 15) groups. F Boxplots for the four alpha-diversity indices analyzed during the maintenance phase (median [min–max]). Differences between time points: *p < 0.05

In addition, bacterial Observed and Chao1 alpha-diversity indices (indicative of population richness) were significantly increased at W2 (Fig. 4C and Supplementary Table S13), and the four beta-diversity indices were significantly modified: Jaccard (p = 0.017), Bray Curtis (p < 0.001), UniFrac (p = 0.024), and weighted UniFrac (p < 0.001) indices.

Scalp Microbiota Analysis During the Maintenance Phase

Fungi

During the maintenance phase of the intervention, the relative abundance of Malassezia fungi increased back in both the test and control groups (Fig. 3E). Compared with W2, this increase was significant at W10 in the control group only (p < 0.01, Supplementary Table S6). Moreover, the mean levels of both M. restricta and M. globosa species significantly increased in the control group at W10 (p < 0.001, Supplementary Table S10) reaching similar levels to those observed at baseline (Supplementary Table S9). Conversely, though increases were also observed, in the test group, the levels of these two species remained lower than at baseline. The differential analysis also showed that the relative levels of Cladosporium and Penicillium significantly decreased in the control group (Supplementary Table S6), whereas no significant changes were found in the test group.

Consistently, all alpha-diversity indices remained stable in the test group during the maintenance phase, whereas three of the four indices significantly decreased in the control group (Fig. 3F and Supplementary Table S8). The difference between both groups was statistically significant. Regarding beta-diversity indices, only the weighted UniFrac index was significantly different in the test group (W10 versus W2, p = 0.021), whereas in the control group, the four indices were significantly changed (Jaccard: p < 0.001, Bray Curtis: p = 0.005, UniFrac: p = 0.001, and weighted UniFrac: p < 0.001).

Bacteria

In the test group, no significant changes in bacterial relative abundances were found at W10 versus W2, with Cutibacterium remaining the predominant genera, whereas, in the control group, there was a significant increase in the Staphylococcus bacteria population (Fig. 4E and Supplementary Table S12). The differential analysis showed no other significant changes. However, at the species level, both C. acnes and S. epidermidis abundances significantly increased in the test group at W10 versus W2 (p < 0.05, Supplementary Table S16), whereas no changes in the levels of these species were observed in the control group.

Furthermore, over the course of the maintenance phase (W10 versus W2), the Observed and Chao1 alpha-diversity indices (richness) showed a trend of decrease in both groups, while the Shannon and Inv-Simpson indices (evenness) significantly decreased in the control group (Fig. 4F and Supplementary Table S14), but the intergroup difference was not significant. The beta-diversity indices were also significantly modified in both groups, for three of the four beta-diversity indices in the test group (Jaccard: p = 0.034, Bray Curtis: p = 0.002, UniFrac: p = 0.097 and weighted UniFrac: p = 0.004) and all four indices in the control group (Jaccard: p = 0.001, Bray Curtis: p = 0.001, UniFrac: p = 0.001, and weighted UniFrac: p = 0.001).

Dermatological Tolerability

The study product was overall well-tolerated over the study course. Only two DAEs in two patients (mild burning sensation and moderate pruritus during product application, respectively) were reported during the intensive phase and considered possibly related to the study product.

Discussion

This randomized, controlled study demonstrated the efficacy of an anti-SD shampoo used over a 10-week intervention consisting of an intensive phase followed by a maintenance phase as recommended for the management of mild-to-moderate scalp SD [9]. Following a thorough clinical evaluation, we found improvement in the scalp condition after the 2-week intensive phase, which was notably sustained during the 8-week maintenance phase in the test group. In this group, the objective symptoms, including dandruff, erythema, and pruritus severity, were significantly improved at the end of the maintenance phase. Similarly, the subjective symptoms, such as squamous state and scalp discomfort, also showed significant improvement. These improvements were significantly greater than in the control subjects who exclusively used the neutral shampoo during the maintenance phase.

Furthermore, this study provided new insights into the scalp microbiota associated with SD and its dynamic during the intervention scheme. At baseline, SD scalp microbiome was characterized in our cohort by the predominance of Malassezia fungi (75.4% of total fungi) and high levels of Staphylococcus bacteria (36.8% of total bacteria). This dysregulated SD microbiota started to rebalance during the intensive phase of the intervention, and the microbiota balance was more stable in the test group than in the control group during the maintenance phase. The intensive use of the product resulted in a significant modification of the composition of the fungal and bacterial populations, associated with an increase in microbial richness, indicating a global diversification of the scalp microbiota, which likely contributed to the observed improvements in scalp symptoms. During the maintenance phase, the fungal population remained stable in the test group, whereas significant changes in fungal composition and decreased richness of this population were observed in the control group. Similarly, fewer changes in the bacterial population were observed in the test group than in the control group. In this group, the richness and evenness of the bacterial microbiome were significantly decreased, indicating a loss of bacterial diversity after the use of the anti-dandruff shampoo was stopped. These results coincided with a recurrence of SD clinical symptoms in the control group, supporting the key role of microbial homeostasis in maintaining a healthy scalp.

Several studies have previously reported microbiome alterations in SD, with shifts in richness and evenness of the scalp microbial population, which may contribute to the onset and perpetuation of the disease [12]. Although inconsistencies exist among studies—likely linked to differences in SD severity, analyzed body sites, sebum levels or inflammatory response, environmental factors, and other analytical parameters—Staphylococcus, Corynebacterium, and Brevibacterium were found to be the major bacterial genera associated with SD [12]. The balance between C. acnes and S. epidermidis species also appears to be essential [30], with evidence of an antagonistic relationship between both species [12,31]. In SD, the reduced abundance of C. acnes [18] and the colonization of the scalp by some Staphylococcus species, such as S. epidermidis, have been associated with local inflammation and transepidermal water loss favoring scaling and itching [12,17,32]. In our study, the absolute quantification showed the rebalancing of these two bacterial species following the intensive use of the anti-dandruff shampoo, with the specific decrease of S. epidermidis bacteria likely contributing to SD symptom relief and scalp condition improvement. Differential analyses also showed significant changes in the overall scalp bacterial composition at the phylum and genus levels, with increased abundances of Actinobacteria, including Cutibacterium species, and decreased abundances of Firmicutes, including Staphylococcus species (see phylogenic trees in Fig. 4D). Alpha- and beta-diversity analyses also revealed an increase in bacterial diversity and richness following the regular use of the anti-dandruff shampoo. These results suggest that a diversification of scalp-colonizing bacteria occurs following the intervention, which is consistent with the results obtained for another piroctone olamine-based shampoo used for 3 weeks, showing a decrease in the relative abundance of Staphylococcus bacteria (mainly S. capitis) and an increase in the relative abundance of Cutibacterium bacteria (mainly C. acnes) which resulted in significant beta-diversity differences before and after the intervention schedule [33]. On the basis of these data and the evidence of an inhibitory interaction between these two bacterial genera, we can hypothesize that the overgrowth of Staphylococcus in SD may be limited by the presence or proliferation of Cutibacterium that can secrete antimicrobial thiopeptides [31].

Moreover, SD was also previously found to be associated with an altered fungal population composition. The proliferation of Malassezia species, more specifically M. globosa and M. restricta, has been shown in subjects with SD [7,12,17,34,35]. These lipid-dependent fungi metabolize sebum lipids and release irritating free fatty acids and lipid peroxides [36,37] that can impair keratinocyte functions and promote the secretion of proinflammatory mediators involved in leukocyte recruitment and local inflammation [38–41]. The subsequent inflammatory reaction can alter scalp barrier function and exacerbate dysbiosis. Following the intensive use of our product, we observed significant changes in the composition of the scalp fungal population, with changes in the presence and absence of some species, along with higher fungal diversity and richness. More specifically, we found that seven fungal genera were significantly more abundant (Cladosporium, Penicillium, Saccharomyces, Debaryomyces—all in the Ascomycota phylum—Itersonilia, Inonotus, and Sistotrema), whereas the abundance of Malassezia, belonging to the Basidiomycota phylum, was decreased (see phylogenic trees in Fig. 3D), suggesting a diversification and rebalancing of the scalp fungal microbiota following the intensive phase of our intervention. This rebalanced profile was sustained in the test group during the 8-week maintenance phase. Significant modifications in the microbial population composition, in particular, fewer Malassezia fungi and Staphylococcus bacteria, have also been reported following other anti-SD interventions, such as a combination treatment with 2% ketoconazole gel and a selenium disulfide-containing shampoo [42] or a piroctone olamine-based shampoo [33]. In addition, an increased abundance of Ascomycota fungi has been linked to a healthy scalp [35]. Some genera in this phylum, such as Cladosporium, Penicillium, Saccharomyces, and Debaryomyces, of which their abundances were increased following the use of our study product, can produce antifungal proteins [43,44] as well as antibiotic, antioxidant, and antiinflammatory compounds [44–47]. This suggests that these genera could be involved in the rebalancing of SD fungal population during the intensive phase. The exact contribution of these fungal compounds to the scalp ecosystem improvement remains to be explored. Moreover, piroctone olamine, known to effectively control Malassezia growth, can act synergistically with ciclopirox olamine to confer potent antimycotic and antiinflammatory properties to the product [23,24]. Piroctone olamine can also reduce oxidative stress markers associated with dandruff and scalp microbiome [33]. Thus, the combination of these effects likely contributed to the observed efficacy of our anti-SD shampoo.

Limitations

Although conducted with a relatively small group size, this study offers a comprehensive clinical assessment within a randomized, controlled setting and addresses the unmet need for improved maintenance treatments for SD, which is a critical aspect of dermatological care [9]. This study also provided new insights into the scalp microbiota associated with SD and its rebalancing following the regular use of the anti-dandruff shampoo. The role of the host and microbial metabolites and lipids in SD condition, and how the study product affects them will be evaluated in another dedicated analysis. An extension study with a comparator product and longer follow-up of the subjects, between 3 and 6 months as recommended for maintenance treatment duration⁹, could also be performed to provide long-term efficacy data and gather further insights into the microbiota-modulating effects of this anti-SD shampoo, offering potential avenues for clinical applications, including its integration with other treatment modalities for more severe forms of SD.

Conclusions

The results of this study that evaluated a newly formulated anti-dandruff shampoo on mild-to-moderate scalp SD showed good clinical efficacy of the product with good tolerability during the intensive and maintenance phases of the intervention. The antioxidant and antiinflammatory properties of the shampoo combined with its specific antifungal effect on SD-predominant Malassezia fungi may have contributed to the repopulation of the scalp microbiota by low-abundance fungi of the Ascomycota phylum, restoring scalp homeostasis and improving SD symptoms. These data overall support that scalp microbial diversity is pivotal to maintaining a healthy scalp.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. In addition, Sophie Baradat, Mélanie Froliger, Virginie Turlier, Eléonore Gravier, Camille Géniès, Hélène Duplan, Sandrine Bessou-Touya, and Martine Maître contributed to the conception and design of the study; Sophie Baradat, Mélanie Froliger, and Martine Maître managed the study; Rasvan Marinescu and Pascal Reygagne conducted the study; Céline Huyghe, Anais Noustens, and Sandrine Alvarez-Georges contributed to the logistic management and analysis of samples; Sophie Baradat, Mélanie Froliger, Christophe Lauze, Eléonore Gravier, Aimée Simcic-Mori, and Martine Maître participated in the analysis and interpretation of data; Christophe Lauze, Eléonore Gravier, and Aimée Simcic-Mori contributed to the statistical analysis and interpretation of data; and Christophe Lauze, Eléonore Gravier, Aimée Simcic-Mori, Camille Géniès, Hélène Duplan, Valérie Mengeaud, Sandrine Bessou-Touya, Sophie Baradat, Martine Maître, Rasvan Marinescu, and Pascal Reygagne contributed to the drafting and/or critical revision of the manuscript.

Funding

This study, medical writing, editorial assistance, language editing services, and the journal’s rapid service fee were funded by Pierre Fabre Dermo-Cosmétique and Personal Care.

Data Availability

Data that support the findings of this study are available in the article or its associated Supplementary Materials.

Declarations

Conflict of Interest

Martine Maître, Sophie Baradat, Mélanie Froliger, Virginie Turlier, Aimée Simcic-Mori, Eléonore Gravier, Camille Géniès, Christophe Lauze, Céline Huyghe, Anais Noustens, Sandrine Alvarez-Georges, Rasvan Marinescu, Sandrine Bessou-Touya, Valérie Mengeaud, and Hélène Duplan are or were employees of Pierre Fabre Dermo-Cosmétique and Personal Care, France, at the time of the preparation of this manuscript and received salaries, but they do not have any financial interest in the findings described in this manuscript. Pascal Reygagne has previously received honorarium fees Pierre Fabre Dermo-Cosmétique and Personal Care for his role as a speaker, advisory board member, and investigator but did not receive any financial compensation for the performance in this study.

Ethical Approval

The study was performed in compliance with the Declaration of Helsinki and its subsequent amendments, as well as good clinical practice guidelines (CHMP/ICH/135/1995 and integrated addendum). It also conformed to Regulation (EC) No. 1223/2009 of the European Commission and the French Decree (no. 2017-884) pertaining to research on cosmetic products. As registration of studies evaluating cosmetic products is not mandatory in the European Union, the study was retrospectively registered under trial number NCT06578962 (28 August 2024) for publication. Prior to enrollment, all subjects provided written informed consent.