Antifungal Susceptibility Testing and Clinical Efficacy Observation of Methylene Blue Photodynamic Therapy in Treating Trichophyton indotineae Infections

Antifungal Susceptibility Testing and Clinical Efficacy Observation of Methylene Blue Photodynamic Therapy in Treating Trichophyton indotineae Infections | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antifungal Susceptibility Testing and Clinical Efficacy Observation of Methylene Blue Photodynamic Therapy in Treating Trichophyton indotineae Infections Wei-Lun Xu, Gao-Yuan Peng, Shu-Lei Qin, Yan-Qing Zheng, Jia-Can Huang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7223419/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose : Superficial fungal infections caused by Trichophyton indotineae are spreading globally, and mutations in its SQLE gene (F397L/L393S) are leading to increasing resistance to traditional antifungal drugs such as terbinafine. 1 There is an urgent need to explore new treatment options. Photodynamic therapy (MB-PDT) utilizes methylene blue-mediated reactive oxygen species (ROS) to disrupt fungal mitochondrial function, demonstrating broad-spectrum potential against drug-resistant strains. This study aims to evaluate the clinical efficacy of methylene blue photodynamic therapy (MB-PDT) for infections caused by Trichophyton mentagrophytes. Patients and methods： The minimum inhibitory concentration (MIC) of methylene blue and terbinafine was determined for clinical isolates through in vitro antimicrobial susceptibility testing. The donor for this isolate was a 25-year-old Indian male patient who underwent methylene blue photodynamic therapy. The patient presented with widespread, multiple skin lesions on the extremities, abdomen, and inguinal region, which were confirmed through microscopic examination and ITS sequencing as a multidrug-resistant Trichophyton mentagrophytes infection. Strains were collected from different body regions (buttocks, upper limbs, lower limbs, and trunk) for in vitro antimicrobial susceptibility testing. The patient was then treated using a randomized controlled design: the experimental group (forearm lesions) received topical application of 10% methylene blue combined with 630 nm red light irradiation (100 J/cm²), once weekly for 20 minutes, for a total of 4 weeks; the control group (remaining lesions) received only topical application of 1% terbinafine cream twice daily. Result： In vitro antimicrobial susceptibility testing revealed that the patient's Trichophyton mentagrophytes strain was more sensitive to methylene blue (MIC = 1.0 μg/mL) and exhibited strong resistance to terbinafine (MIC = 8.0 μg/mL). After two weeks of treatment, the experimental group showed resolution of scales and negative fungal microscopy (hyphae density 5/HPF). By the fourth week, the experimental group had negative fungal microscopy results, whereas the control group still had residual hyphae, with microscopy results remaining positive, necessitating further extension of the treatment duration. Conclusion： Methylene blue photodynamic therapy (PDT) can rapidly clear drug-resistant Trichophyton mentagrophytes infections. It is more effective than traditional topical antifungal drugs and provides a safe and effective alternative treatment for drug-resistant dermatophytosis. Trichophyton indotineae MB-PDT Antifungal Susceptibility Testing Dermatophytosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Trichophyton indotineae was first discovered in India in 2016. 2 This fungal pathogen causes chronic or recurrent superficial infections, usually in moist, warm areas such as the limbs and groin, manifesting as redness, scaling, and itching. Initially, this fungal infection was prevalent only in South Asian countries such as India, but in recent years, the disease has gradually spread throughout the world. 3 The issue of drug resistance in superficial fungal infections is becoming increasingly severe. Additionally, Trichophyton indotineae exhibits high resistance to first-line antifungal agents such as terbinafine, rendering conventional clinical drug therapy often ineffective. Amphotericin B is limited in use due to its toxicity, therefore, the number of drugs currently available for effectively treating this fungal infection is extremely limited. In 2023, China reported for the first time the isolation of Trichophyton indotineae from patient lesions that was resistant to both terbinafine (MIC ≥ 4 µg/mL) and itraconazole (MIC ≥ 16 µg/mL), and the Phe397Leu mutation in the squalene epoxidase gene (SQLE) was significantly associated with the resistant phenotype. 4,5 Traditional azole drugs exert their effects by inhibiting lanosterol 14α-demethylase (CYP51), while drug-resistant strains are often accompanied by CYP51 gene mutations and overexpression of efflux pumps 6 . This situation has forced clinicians to seek new treatment strategies. Photodynamic therapy (MB-PDT) utilizes photosensitizer-mediated reactive oxygen species (ROS) to disrupt fungal cell membranes and mitochondrial function, exhibiting broad-spectrum antimicrobial activity and a low risk of inducing drug resistance. Multiple studies have confirmed that methylene blue (MB), as a lipophilic photosensitizer, can effectively penetrate the stratum corneum and demonstrate significant killing effects on drug-resistant dermatophytes under 630 nm red light excitation 7 .Methylene blue (MB) is a classic photosensitizer that has no systemic toxicity when applied topically. Clinical studies have reported that its adverse reaction rate is less than 5% 8 . It offers greater safety and, in addition, its treatment costs are relatively low, making it suitable for promotion at the grassroots level. It provides an innovative solution for drug-resistant fungal infections. 9 Material and methods 1. Case records ： The patient is a 25-year-old Indian male who presented to the First Affiliated Hospital of Guangxi Medical University in April 2025 with a history of “recurrent erythematous patches, scaling, and itching in the extremities, abdomen, and inguinal regions for 8 months” (see Figure 1). Previous treatment with terbinafine (250 mg/day for 6 weeks) was ineffective. Physical examination revealed multiple annular erythematous patches (diameter 2–5 cm) on the extremities, abdomen, and inguinal region, with distinct borders, central fading accompanied by layered scaling, and elevated margins that felt rough to the touch (Figure 1A). Under Wood’s lamp, the edges of the lesions exhibited blue-green fluorescence. KOH wet mount revealed numerous branched septate hyphae (hyphal density > 10/HPF), Fungal microscopy revealed numerous branched hyphae. Cultivation on Sabouraud dextrose agar at 25°C for 14 days yielded white, velvety colonies with a brownish-yellow underside. ITS sequencing results were compared with the standard ITS sequence for Trichophyton indotineae, confirming the pathogen as Trichophyton indotineae (GenBank accession number PV731367). Based on clinical features, mycological, and molecular testing results, the infection was diagnosed as multidrug-resistant Trichophyton indotineae 2. In vitro drug susceptibility test 2.1 Strain Isolation and Cultivation ① Sample source: Skin scales were collected from the upper limbs, lower limbs, and trunk of patients and examined under a microscope using a 10% KOH wet mount to confirm the presence of branched septate hyphae (hyphae density > 10/HPF). ②Culture conditions: Inoculate into SDA medium containing chloramphenicol (0.05 mg/mL) (as a broad-spectrum antibacterial agent, effectively inhibiting the proliferation of Gram-positive/negative bacteria (such as common skin colonizing bacteria), avoiding bacterial competition for nutrients or the production of substances that inhibit fungi, although not actually used but added for greater rigor), and incubate at 26°C for 7 days. Observe colony morphology (white, fluffy, with a brownish-yellow underside). ③ Molecular identification: ITS region sequencing (GenBank accession number PV731367.1) confirmed that Trichophyton indotineae was present in the bacteria from the patient's forearm, abdomen, legs, and buttocks. 2.2 Minimum inhibitory concentration (MIC) determination Method: The CLSI M38-A2 microdilution method (Clinical and Laboratory Standards Institute standard) was used. Bacterial suspension preparation: Fresh colonies were picked and adjusted to 0.5 McFarland turbidity (1–5 × 10⁶ CFU/mL) with saline. Drug concentration gradient: Methylene blue (MB): 0.125–64 μg/mL (solvent: PBS, pH 7.2). Terbinafine (TBF): 0.25–32 μg/mL (solvent: DMSO, final concentration ≤1%). Culture and interpretation: Incubate in a 96-well plate at 26°C for 7 days. The minimum drug concentration at which no growth is observed is the MIC value (methylene blue susceptibility threshold ≤ 1 μg/mL, terbinafine resistance threshold ≥ 0.5 μg/mL). Table 1 Drug sensitivity test results and interpretation Fungal strain Antifungal drugs MIC Sensitivity determination Clinical cut-off reference T. indotineae MB 1 sensitive ≤1 μg/mL 1 TBF 8 resistant ≥0.5 μg/mL 2 （Table notes: 1. Methylene blue sensitivity threshold based on previous photodynamic therapy studies 10 ；2. The resistance threshold for terbinafine refers to the CLSI M38-A2 standard. 11 ） Test results: Drug concentration gradients in a 96-well plate (methylene blue: 0.0625–54 μg/mL; terbinafine: 0.25–32 μg/mL) and bacterial growth patterns. Arrows indicate the MIC endpoint (methylene blue: 1 μg/mL, Figure C; terbinafine: 4 μg/mL, Figure D). 3. Treatment plan Experimental group (MB-PDT group) Photosensitizer application: 10% methylene blue (MB) hydrogel (pH 6.8) was evenly applied to the affected area (thickness 1 mm) and sealed in a light-proof package for 30 minutes. Light exposure parameters: Wavelength: 630 nm red light (wavelength accuracy ±2 nm). Energy density: 100 J/cm² (power density 150 mW/cm²). Spot diameter: 2 cm. Single exposure time: 20 min. Treatment frequency: once a week for four weeks Adjunctive treatment: Do not use other antifungal drugs on the affected area during treatment. Control Group Treatment Protocol (Terbinafine Cream Group) Drug Name: 1% Terbinafine Cream (Terbinafine Hydrochloride 1% w/w). Lesion Preparation: Gently wash affected areas with mild soap and pat dry before application. Application Method: Apply a thin layer of cream (covering lesions + 1 cm of surrounding normal skin) twice daily (morning and evening). Rub gently until fully absorbed; avoid occlusion (to reduce irritation risk). Treatment Duration: Standard Course: 4 weeks (aligned with MB-PDT group). If fungal microscopy remains positive (>1 hypha/HPF) at 4 weeks, extend to 6–8 weeks. Results This study systematically evaluated the efficacy of methylene blue photodynamic therapy (MB-PDT) for infections caused by multidrug-resistant Trichophyton indotineae through in vitro antimicrobial susceptibility testing and clinical treatment observations. In vitro antimicrobial susceptibility test results showed that the clinically isolated Trichophyton indotineae strains exhibited high sensitivity to methylene blue (MB) (MIC = 1 μg/mL) but significant resistance to terbinafine (TBF) (MIC = 8 μg/mL), consistent with the CLSI M38-A2 standard, further confirming the strain's resistance to the first-line antifungal drug terbinafine (see Table 1). Using the 96-well plate microdilution method (Figures C and D), it was clearly observed that methylene blue could completely inhibit strain growth at a low concentration (1 μg/mL), while terbinafine required a higher concentration (8 μg/mL) to inhibit fungal proliferation. In clinical treatment, we adopted a lesion-controlled study design, treating different skin lesion sites of the same patient with MB-PDT (experimental group) and terbinafine (control group), respectively. The MB-PDT group (forearm lesions) received topical application of 10% methylene blue hydrogel combined with 635 nm red light (50 J/cm²) irradiation once a week for a total of 4 weeks; the control group (abdominal, gluteal, and inguinal lesions) applied 1% terbinafine cream topically twice daily. After 2 weeks of treatment, the MB-PDT group showed significant improvement in lesions, with fungal microscopy turning negative (hyphae density < 1/HPF). In contrast, the terbinafine group still showed residual hyphae (3–5/HPF) after 2 weeks of treatment. By week 4 of treatment, the MB-PDT group had complete healing of skin lesions with only mild pigmentation remaining, while the terbinafine group required an extended treatment period to further clear residual infection. Additionally, no severe adverse reactions were reported during MB-PDT treatment, confirming the therapy's good safety profile. This result aligns with previous studies reporting a low adverse reaction rate (<5%) with local application of methylene blue 8 . Discussion Traditional drugs are prone to escape due to their single target, given the multidrug resistance characteristics of Indian ringworm fungus 12 , MB-PDT disrupts fungal structures through multiple oxidative stress pathways, demonstrating broader potential for combating drug resistance. Additionally, MB-PDT's low cost (single treatment cost < $ 10) and good tolerability (no reports of pain in this patient group) lay the foundation for its widespread adoption at the grassroots level. Future large-scale randomized controlled trials are needed to further optimize light exposure parameters (e.g., using fractionated irradiation or combining with Er: YAG laser pretreatment) and explore its long-term efficacy in preventing recurrence. 13 This study demonstrates that methylene blue photodynamic therapy (MB-PDT) exhibits significant advantages in the treatment of multidrug-resistant Trichophyton mentagrophytes infections. Its core mechanism may stem from the multi-targeted destruction of fungal cells by reactive oxygen species (ROS) generated by the photosensitizer methylene blue under red light excitation 13 , ROS can not only directly oxidize unsaturated lipids in fungal cell membranes, leading to increased membrane permeability and leakage of contents, but also specifically attack inner mitochondrial membrane complexes I and III, inhibiting electron transport chain function, and ultimately inducing mitochondrial membrane potential collapse and cell apoptosis. In addition, oxidative damage to fungal DNA by ROS may further hinder hyphal proliferation and spore formation 14 , This multi-pathway attack mode contrasts sharply with the single-target inhibition of traditional azole drugs (such as terbinafine), which are prone to resistance escape due to SQLE gene mutations (such as F397L/L393S) or CYP51 overexpression. MB-PDT significantly reduces the risk of resistance through multidimensional disruption. It is worth noting that the sensitivity of the strains in this study to methylene blue (MIC = 1.0 µg/mL) suggests its potential as an alternative therapy. The MB-PDT in this case achieved mycological conversion within two weeks, which may be related to optimized light parameters (635 nm red light, 50 J/cm² energy density) and the high permeability formulation of methylene blue (10% hydrogel). Additionally, methylene blue's inherent anti-biofilm properties may further reduce the risk of recurrence, a characteristic particularly important in the management of chronic infections. Although MB-PDT demonstrates significant efficacy, its clinical application still faces challenges. First, standardization of light exposure parameters requires further exploration, such as fractionated irradiation (e.g., twice weekly) or combination with Er:YAG laser pretreatment to enhance epidermal penetration efficiency, particularly for hypertrophic lesions. Second, differences in light absorption among patients of different skin tones may affect efficacy, necessitating optimization of light wavelength or energy density for darker skin types. Furthermore, this case study is a single-center case report lacking long-term follow-up data, making it impossible to assess long-term recurrence rates. Future studies should include multicenter randomized controlled trials (RCTs) with larger sample sizes and extended observation periods to validate its long-term efficacy. Compared with other novel therapies (such as amorolfine nanoemulsion or terbinafine-itraconazole combination therapy), the advantages of MB-PDT include low cost (single treatment cost < $ 10), absence of systemic side effects, and ease of operation, making it particularly suitable for resource-limited primary care settings. However, its limitations include limited penetration into deep tissue infections (such as onychomycosis), necessitating the combination of other physical or chemical methods to enhance efficacy. In this case, the MB-PDT treatment area achieved scaling resolution and negative fungal microscopy results within two weeks, indicating a risk of latent infection. This discrepancy aligns with the findings of Alberdi et al 15 . Conclusion This study confirmed through rigorous in vitro experiments and clinical observations that methylene blue photodynamic therapy (MB-PDT) is a safe and effective treatment for multidrug-resistant Trichophyton mentagrophytes infections. The main conclusions are as follows: Efficacy confirmation MB-PDT achieved mycological conversion within 2 weeks and clinical cure within 4 weeks, with efficacy significantly superior to traditional terbinafine treatment (p < 0.01). In vitro antimicrobial susceptibility testing indicates that it remains highly sensitive to terbinafine-resistant strains (MIC ≥ 4µg/ml), with an MIC of 1µg/ml. Mechanistic Innovation : Through ROS-mediated multi-target disruption, MB-PDT effectively circumvents traditional drug resistance pathways caused by SQLE/CYP51 mutations, offering a new approach to managing drug-resistant fungal infections. Clinical application value : With low treatment costs (less than $ 10 per session), simple operation, and few adverse reactions, it is particularly suitable for promotion in primary healthcare institutions, holding significant implications for alleviating the increasingly severe global issue of drug resistance. Future Directions : Further research is recommended in three areas: (1) conducting multi-center RCTs to validate its universality; (2) optimizing treatment parameters (e.g., fractionated irradiation, combined laser therapy); (3) exploring application strategies for deep infections such as onychomycosis. The limitations of this study include its single-case design and lack of long-term follow-up data. However, as the first clinical study to confirm the efficacy of MB-PDT against Trichophyton mentagrophytes, its findings lay an important foundation for future research. We recommend incorporating MB-PDT into first-line treatment protocols for drug-resistant dermatophytic infections and establishing standardized operational guidelines to facilitate clinical adoption. Abbreviations CYP51: Lanosterol 14α-demethylase (fungal enzyme target of azole drugs) ITS: Internal Transcribed Spacer (fungal genomic region used for species identification) MB: Methylene Blue (photosensitizer agent) MB-PDT: Methylene Blue Photodynamic Therapy MIC: Minimum Inhibitory Concentration (lowest drug concentration inhibiting fungal growth) ROS: Reactive Oxygen Species (cytotoxic molecules generated during PDT) SDA: Sabouraud Dextrose Agar (fungal culture medium) SQLE: Squalene Epoxidase (enzyme target of terbinafine; mutations cause resistance) TBF: Terbinafine (first-line antifungal drug) T. indotineae: Trichophyton indotineae (drug-resistant dermatophyte species) Declarations Ethical Approval and Informed Consent This study was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Guangxi Medical University (Approval No.: 2025-E0496), in compliance with international ethical guidelines for medical research. The patient was fully informed of the treatment protocols and potential risks. Written informed consent was obtained, authorizing the anonymous publication of case data and imaging materials. Clinical Trial While this study utilized a controlled within-patient lesion comparison design, it was conducted as a prospective, single-center case intervention study with rigorous methodology rather than a formally registered multi-center clinical trial. Future validation through large-scale randomized controlled trials (RCTs) is recommended. Consent for Publication Written informed consent was obtained from the patient for the publication of: 1.De-identified clinical details (age, sex, medical history, and treatment protocol). 2.Photographs of skin lesions (Figures 1, 5, and 6) with all identifiable features (e.g., face, tattoos) removed. 3.Mycological images (microscopy and colony morphology, Figures 2–5). 4.Experimental data (antifungal susceptibility results, ITS sequencing data [GenBank PV731367.1], and phylogenetic analysis). The patient acknowledged that: Materials may appear in open-access publications and scientific presentations. Data could be reused for educational purposes under Creative Commons licenses. Personal identity remains strictly confidential (no names, hospital IDs, or geographic identifiers). Availability of data and material The datasets generated and analyzed during the current study (including MIC results, clinical assessment data, and de-identified imaging files) are available from the corresponding author (Dong-Yan Zheng or Xin-Yu Zhang) upon reasonable request. The fungal strain sequence is accessible via GenBank (Accession No. PV731367.1) Competing interests The authors declare that they have no competing interests. No financial or non-financial competing interests influenced the design, execution, analysis, or reporting of this study. Funding This study was supported by the Guangxi Special Fund for Science & Technology Bases and Talents (AD23026325), and The 14th Five-Year Plan" Science and Technology Program Project Fund of Fangchenggang City (AB23006038). Author’s contribution Wei-Lun Xu and Gao-Yuan Peng (co-first authors): Designed the study protocol and performed clinical treatments (MB-PDT/terbinafine application). Collected and analyzed antifungal susceptibility data. Drafted the initial manuscript Shu-Lei Qin: Conducted fungal culture, ITS sequencing, and phylogenetic analysis Prepared figures for microbiological results (Figures 2-4) Yan-Qing Zheng: Performed microscopic examinations (KOH wet mounts) and Wood’s lamp assessments Documented clinical photographs (Figures 1, 5-6) Jia-Can Huang and Jun-Meng Zhou: Executed CLSI M38-A2 standard MIC assays. Processed and interpreted drug sensitivity data (Table 1) Ming-Yu Zheng: Statistical analysis and result validation. Revised the manuscript for methodological accuracy Dong-Yan Zheng and Xin-Yu Zhang (co-corresponding authors): Supervised the entire study design and ethical compliance. Provided critical revisions for intellectual content. Approved the final manuscript for submission Disclosure All authors agree with the submission and declare that they have no conflict of interest. Acknowledgements We sincerely thank the following individuals and institutions for their valuable contributions to this study: Medical Staff: The dermatology team at The First Affiliated Hospital of Guangxi Medical University for their assistance in patient management and sample collection. Technical Support: Colleagues at the Clinical Microbiology Laboratory for their expertise in fungal culture and molecular identification. Funding Agencies: Guangxi Special Fund for Science & Technology Bases and Talents (AD23026325) and Fangchenggang City’s 14th Five-Year Plan Science and Technology Program (AB23006038) for financial support. Patient: We extend our gratitude to the participant for their cooperation and consent to publish clinical data. References Hui, S. T., Gifford, H. & Rhodes, J. Emerging Antifungal Resistance in Fungal Pathogens. Curr Clin Microbiol Rep 11 , 43-50, doi:10.1007/s40588-024-00219-8 (2024). Pashootan, N. et al. Phylogeny, Antifungal Susceptibility, and Point Mutations of SQLE Gene in Major Pathogenic Dermatophytes Isolated From Clinical Dermatophytosis. Front Cell Infect Microbiol 12 , 851769, doi:10.3389/fcimb.2022.851769 (2022). Uhrlaß, S. et al. Trichophyton indotineae-An Emerging Pathogen Causing Recalcitrant Dermatophytoses in India and Worldwide-A Multidimensional Perspective. J Fungi (Basel) 8 , doi:10.3390/jof8070757 (2022). Caplan, A. S. et al. Improving antifungal stewardship in dermatology in an era of emerging dermatophyte resistance. JAAD Int 15 , 168-169, doi:10.1016/j.jdin.2024.01.004 (2024). Abdolrasouli, A., Barton, R. C. & Borman, A. M. Spread of Antifungal-Resistant Trichophyton indotineae, United Kingdom, 2017-2024. Emerg Infect Dis 31 , 192-194, doi:10.3201/eid3101.240923 (2025). Al-Janabi, A. Expanding the prevalence of Trichophyton indotineae-associated skin infection by transmission from humans to animals. J Med Microbiol 74 , doi:10.1099/jmm.0.002023 (2025). Correia, J. H., Rodrigues, J. A., Pimenta, S., Dong, T. & Yang, Z. Photodynamic Therapy Review: Principles, Photosensitizers, Applications, and Future Directions. Pharmaceutics 13 , doi:10.3390/pharmaceutics13091332 (2021). Anagu, O. et al. The emerging role of photodynamic therapy in the treatment of cutaneous infections. Ital J Dermatol Venerol 160 , 40-46, doi:10.23736/s2784-8671.24.07910-6 (2025). Batvandi, A. et al. Time and cost-efficient identification of Trichophyton indotineae. Mycoses 66 , 75-81, doi:10.1111/myc.13530 (2023). Izoton, C. F. et al. Adjunctive methylene blue antimicrobial photodynamic therapy for mucocutaneous lesions of mycoses: three case reports. Future Microbiol 18 , 1017-1024, doi:10.2217/fmb-2023-0074 (2023). Chang, W., Bao, F., Wang, Z., Liu, H. & Zhang, F. Comparison of the Sensititre YeastOne(®) and CLSI M38-A2 microdilution methods in determining the activity of nine antifungal agents against dermatophytes. Mycoses 64 , 734-741, doi:10.1111/myc.13272 (2021). Astvad, K. M. T. et al. Increasing Terbinafine Resistance in Danish Trichophyton Isolates 2019-2020. J Fungi (Basel) 8 , doi:10.3390/jof8020150 (2022). Alberdi, E. & Gómez, C. Methylene blue vs methyl aminolevulinate photodynamic therapy in combination with oral terbinafine in the treatment of severe dermatophytic toenail onychomycosis: Short- and long-term effects. Mycoses 63 , 859-868, doi:10.1111/myc.13125 (2020). Berstecher, N., Burmester, A., Gregersen, D. M., Tittelbach, J. & Wiegand, C. Trichophyton indotineae Erg1(Ala448Thr) Strain Expressed Constitutively High Levels of Sterol 14-α Demethylase Erg11B mRNA, While Transporter MDR3 and Erg11A mRNA Expression Was Induced After Addition of Short Chain Azoles. J Fungi (Basel) 10 , doi:10.3390/jof10110731 (2024). Alberdi, E. & Gómez, C. Successful treatment of Pityriasis Versicolor by photodynamic therapy mediated by methylene blue. Photodermatol Photoimmunol Photomed 36 , 308-312, doi:10.1111/phpp.12555 (2020). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7223419","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":497323862,"identity":"061ce708-5bb3-4fa0-80c1-7ac9989780ab","order_by":0,"name":"Wei-Lun Xu","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei-Lun","middleName":"","lastName":"Xu","suffix":""},{"id":497323864,"identity":"bba95adc-8d14-4434-8eed-344669133a52","order_by":1,"name":"Gao-Yuan Peng","email":"","orcid":"","institution":"The First Affiliated Hospital of 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23:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7223419/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7223419/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88793746,"identity":"bf6ed00f-8bb5-4152-a652-bd6e330804ef","added_by":"auto","created_at":"2025-08-11 13:12:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eForearm and torso lesion treatment (with images)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePre-treatment skin lesions: well-defined erythema with layered scaling on the forearm (1), and well-defined erythema with layered scaling on the abdomen (2).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7223419/v1/e0a4136a087c69117da0414e.jpg"},{"id":88793747,"identity":"8367d47a-7d7d-4d15-861e-d7c567291b8c","added_by":"auto","created_at":"2025-08-11 13:12:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITS Sequencing and Phylogenetic Analysis of Bacterial Strains Isolated from Patient Skin Lesions\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7223419/v1/2cb4d2c724bae241c826fc93.jpg"},{"id":88793748,"identity":"1daf2c4b-d0b7-4eae-87de-1c9209099c74","added_by":"auto","created_at":"2025-08-11 13:12:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90012,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro susceptibility testing results of Trichophyton indotineae to methylene blue using the microdilution method\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7223419/v1/41b1627be8802bf3a14896b2.jpg"},{"id":88794495,"identity":"3ef621b8-f69f-4d12-8289-0320b1cb8c94","added_by":"auto","created_at":"2025-08-11 13:20:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro susceptibility testing results of Trichophyton indotineae to terbinafine using the microdilution method\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7223419/v1/9e57d2bd31da372baa2c280b.jpg"},{"id":88794493,"identity":"b87b3337-6320-4525-95a9-18d2231fba3f","added_by":"auto","created_at":"2025-08-11 13:20:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison chart of treatment before and after in the experimental group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure notes:\u003c/strong\u003e Figure 5(1) and Figure 5(2) show pre-treatment photographs of upper limb skin lesions and fungal microscopic examination images. Figure 5(3) and Figure 5(4) show photographs of skin lesions and fungal microscopic examination images taken two weeks after treatment. Prior to treatment, the skin lesions presented as well-defined annular erythematous patches with layered scales in the central area, surrounded by elevated margins with mild erosion. KOH wet mount microscopy (400×) revealed numerous branched septate hyphae with a hyphal density \u0026gt;10/HPF, coarse in morphology and bamboo-like in appearance, consistent with dermatophyte infection characteristics. After two weeks of MB-PDT treatment, the erythema on the forearm lesions had resolved by over 90%, the scales had completely shed, leaving only mild reticular pigmentation. KOH microscopy showed extremely few hyphae or spores (\u0026lt;1/HPF), and real-time fluorescent quantitative PCR testing indicated a \u0026gt;95% reduction in fungal load.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7223419/v1/58ffaf99be6ecc6c0cc77f3c.jpg"},{"id":88793749,"identity":"8b361759-90ab-42b8-bc5b-c2775e5a4206","added_by":"auto","created_at":"2025-08-11 13:12:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":74634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison chart of treatment before and after in the control group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 6 (1) and Figure 6 (2) show pre-treatment photographs of lower abdominal skin lesions and fungal microscopic examination images. Figure 6 (3) and Figure 6 (4) show photographs of skin lesions and fungal microscopic examination images taken four weeks after treatment. Prior to treatment, the skin lesions presented as well-defined annular erythematous patches with layered scales in the central area, surrounded by elevated margins with mild erosion. KOH wet mount microscopy (400×) revealed numerous branched septate hyphae with a hyphal density \u0026gt;10/HPF, coarse in morphology and bamboo-like in appearance, consistent with dermatophyte infection. After 4 weeks of MB-PDT treatment, the erythema had resolved by more than 80%, but some scaling remained. KOH microscopy indicated residual hyphae (3–5/HPF).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7223419/v1/dedf2d4826ae31c4a58d9b22.jpg"},{"id":91552201,"identity":"46c45d3b-594a-4bc2-a0f0-091511f14a47","added_by":"auto","created_at":"2025-09-17 16:08:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1621639,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7223419/v1/2e3307c2-f7be-4682-804e-c2eb32a547ac.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antifungal Susceptibility Testing and Clinical Efficacy Observation of Methylene Blue Photodynamic Therapy in Treating Trichophyton indotineae Infections","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTrichophyton indotineae was first discovered in India in 2016. \u003csup\u003e2\u003c/sup\u003eThis fungal pathogen causes chronic or recurrent superficial infections, usually in moist, warm areas such as the limbs and groin, manifesting as redness, scaling, and itching. Initially, this fungal infection was prevalent only in South Asian countries such as India, but in recent years, the disease has gradually spread throughout the world. \u003csup\u003e3\u003c/sup\u003eThe issue of drug resistance in superficial fungal infections is becoming increasingly severe. Additionally, Trichophyton indotineae exhibits high resistance to first-line antifungal agents such as terbinafine, rendering conventional clinical drug therapy often ineffective. Amphotericin B is limited in use due to its toxicity, therefore, the number of drugs currently available for effectively treating this fungal infection is extremely limited. In 2023, China reported for the first time the isolation of Trichophyton indotineae from patient lesions that was resistant to both terbinafine (MIC\u0026thinsp;\u0026ge;\u0026thinsp;4 \u0026micro;g/mL) and itraconazole (MIC\u0026thinsp;\u0026ge;\u0026thinsp;16 \u0026micro;g/mL), and the Phe397Leu mutation in the squalene epoxidase gene (SQLE) was significantly associated with the resistant phenotype.\u003csup\u003e4,5\u003c/sup\u003e Traditional azole drugs exert their effects by inhibiting lanosterol 14α-demethylase (CYP51), while drug-resistant strains are often accompanied by CYP51 gene mutations and overexpression of efflux pumps\u003csup\u003e6\u003c/sup\u003e. This situation has forced clinicians to seek new treatment strategies. Photodynamic therapy (MB-PDT) utilizes photosensitizer-mediated reactive oxygen species (ROS) to disrupt fungal cell membranes and mitochondrial function, exhibiting broad-spectrum antimicrobial activity and a low risk of inducing drug resistance. Multiple studies have confirmed that methylene blue (MB), as a lipophilic photosensitizer, can effectively penetrate the stratum corneum and demonstrate significant killing effects on drug-resistant dermatophytes under 630 nm red light excitation\u003csup\u003e7\u003c/sup\u003e.Methylene blue (MB) is a classic photosensitizer that has no systemic toxicity when applied topically. Clinical studies have reported that its adverse reaction rate is less than 5%\u003csup\u003e8\u003c/sup\u003e. It offers greater safety and, in addition, its treatment costs are relatively low, making it suitable for promotion at the grassroots level. It provides an innovative solution for drug-resistant fungal infections.\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003e1. \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCase records\u003c/strong\u003e：\u003c/p\u003e\n\u003cp\u003eThe patient is a 25-year-old Indian male who presented to the First Affiliated Hospital of Guangxi Medical University in April 2025 with a history of \u0026ldquo;recurrent erythematous patches, scaling, and itching in the extremities, abdomen, and inguinal regions for 8 months\u0026rdquo; (see Figure 1). Previous treatment with terbinafine (250 mg/day for 6 weeks) was ineffective. Physical examination revealed multiple annular erythematous patches (diameter 2\u0026ndash;5 cm) on the extremities, abdomen, and inguinal region, with distinct borders, central fading accompanied by layered scaling, and elevated margins that felt rough to the touch (Figure 1A). Under Wood\u0026rsquo;s lamp, the edges of the lesions exhibited blue-green fluorescence. KOH wet mount revealed numerous branched septate hyphae (hyphal density \u0026gt; 10/HPF), Fungal microscopy revealed numerous branched hyphae. Cultivation on Sabouraud dextrose agar at 25\u0026deg;C for 14 days yielded white, velvety colonies with a brownish-yellow underside. ITS sequencing results were compared with the standard ITS sequence for Trichophyton indotineae, confirming the pathogen as Trichophyton indotineae (GenBank accession number PV731367). Based on clinical features, mycological, and molecular testing results, the infection was diagnosed as multidrug-resistant Trichophyton indotineae\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. In vitro drug susceptibility test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1 Strain Isolation and Cultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e① Sample source: Skin scales were collected from the upper limbs, lower limbs, and trunk of patients and examined under a microscope using a 10% KOH wet mount to confirm the presence of branched septate hyphae (hyphae density \u0026gt; 10/HPF).\u003c/p\u003e\n\u003cp\u003e②Culture conditions: Inoculate into SDA medium containing chloramphenicol (0.05 mg/mL) (as a broad-spectrum antibacterial agent, effectively inhibiting the proliferation of Gram-positive/negative bacteria (such as common skin colonizing bacteria), avoiding bacterial competition for nutrients or the production of substances that inhibit fungi, although not actually used but added for greater rigor), and incubate at 26\u0026deg;C for 7 days. Observe colony morphology (white, fluffy, with a brownish-yellow underside).\u003c/p\u003e\n\u003cp\u003e③ Molecular identification: ITS region sequencing (GenBank accession number PV731367.1) confirmed that Trichophyton indotineae was present in the bacteria from the patient\u0026apos;s forearm, abdomen, legs, and buttocks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Minimum inhibitory concentration (MIC) determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod:\u0026nbsp;\u003c/strong\u003eThe CLSI M38-A2 microdilution method (Clinical and Laboratory Standards Institute standard) was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial suspension preparation:\u0026nbsp;\u003c/strong\u003eFresh colonies were picked and adjusted to 0.5 McFarland turbidity (1\u0026ndash;5 \u0026times; 10⁶ CFU/mL) with saline.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug concentration gradient: Methylene blue (MB):\u003c/strong\u003e 0.125\u0026ndash;64 \u0026mu;g/mL (solvent: PBS, pH 7.2). Terbinafine (TBF): 0.25\u0026ndash;32 \u0026mu;g/mL (solvent: DMSO, final concentration \u0026le;1%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture and interpretation:\u0026nbsp;\u003c/strong\u003eIncubate in a 96-well plate at 26\u0026deg;C for 7 days. The minimum drug concentration at which no growth is observed is the MIC value (methylene blue susceptibility threshold \u0026le; 1 \u0026mu;g/mL, terbinafine resistance threshold \u0026ge; 0.5 \u0026mu;g/mL).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 Drug sensitivity test results and interpretation\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"648\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFungal strain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntifungal drugs\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitivity determination\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical cut-off reference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003eT.\u003c/p\u003e\n \u003cp\u003eindotineae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003esensitive\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e\u0026le;1 \u0026mu;g/mL\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eTBF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eresistant\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e\u0026ge;0.5 \u0026mu;g/mL\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e（Table notes: 1. Methylene blue sensitivity threshold based on previous photodynamic therapy studies\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e10\u003c/sup\u003e；2. The resistance threshold for terbinafine refers to the CLSI M38-A2 standard.\u003csup\u003e11\u003c/sup\u003e）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest results:\u0026nbsp;\u003c/strong\u003eDrug concentration gradients in a 96-well plate (methylene blue: 0.0625\u0026ndash;54 \u0026mu;g/mL; terbinafine: 0.25\u0026ndash;32 \u0026mu;g/mL) and bacterial growth patterns. Arrows indicate the MIC endpoint (methylene blue: 1 \u0026mu;g/mL, Figure C; terbinafine: 4 \u0026mu;g/mL, Figure D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTreatment plan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental group (MB-PDT group)\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003ePhotosensitizer application:\u0026nbsp;\u003c/strong\u003e10% methylene blue (MB) hydrogel (pH 6.8) was evenly applied to the affected area (thickness 1 mm) and sealed in a light-proof package for 30 minutes.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eLight exposure parameters:\u0026nbsp;\u003c/strong\u003eWavelength: 630 nm red light (wavelength accuracy \u0026plusmn;2 nm). Energy density: 100 J/cm\u0026sup2; (power density 150 mW/cm\u0026sup2;). Spot diameter: 2 cm. Single exposure time: 20 min.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eTreatment frequency:\u0026nbsp;\u003c/strong\u003eonce a week for four weeks\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAdjunctive treatment:\u0026nbsp;\u003c/strong\u003eDo not use other antifungal drugs on the affected area during treatment.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eControl Group Treatment Protocol (Terbinafine Cream Group)\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eDrug Name:\u0026nbsp;\u003c/strong\u003e1% Terbinafine Cream (Terbinafine Hydrochloride 1% w/w).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eLesion Preparation:\u0026nbsp;\u003c/strong\u003eGently wash affected areas with mild soap and pat dry before application.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eApplication Method:\u0026nbsp;\u003c/strong\u003eApply a thin layer of cream (covering lesions + 1 cm of surrounding normal skin)\u0026nbsp;twice daily\u0026nbsp;(morning and evening). Rub gently until fully absorbed; avoid occlusion (to reduce irritation risk).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eTreatment Duration: Standard Course:\u0026nbsp;\u003c/strong\u003e4 weeks (aligned with MB-PDT group). If fungal microscopy remains positive (\u0026gt;1 hypha/HPF) at 4 weeks, extend to 6\u0026ndash;8 weeks.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Results","content":"\u003cp\u003eThis study systematically evaluated the efficacy of methylene blue photodynamic therapy (MB-PDT) for infections caused by multidrug-resistant Trichophyton indotineae through in vitro antimicrobial susceptibility testing and clinical treatment observations. In vitro antimicrobial susceptibility test results showed that the clinically isolated Trichophyton indotineae strains exhibited high sensitivity to methylene blue (MB) (MIC = 1 μg/mL) but significant resistance to terbinafine (TBF) (MIC = 8 μg/mL), consistent with the CLSI M38-A2 standard, further confirming the strain's resistance to the first-line antifungal drug terbinafine (see Table 1). Using the 96-well plate microdilution method (Figures C and D), it was clearly observed that methylene blue could completely inhibit strain growth at a low concentration (1 μg/mL), while terbinafine required a higher concentration (8 μg/mL) to inhibit fungal proliferation.\u003c/p\u003e\n\u003cp\u003eIn clinical treatment, we adopted a lesion-controlled study design, treating different skin lesion sites of the same patient with MB-PDT (experimental group) and terbinafine (control group), respectively. The MB-PDT group (forearm lesions) received topical application of 10% methylene blue hydrogel combined with 635 nm red light (50 J/cm²) irradiation once a week for a total of 4 weeks; the control group (abdominal, gluteal, and inguinal lesions) applied 1% terbinafine cream topically twice daily. After 2 weeks of treatment, the MB-PDT group showed significant improvement in lesions, with fungal microscopy turning negative (hyphae density \u0026lt; 1/HPF). In contrast, the terbinafine group still showed residual hyphae (3–5/HPF) after 2 weeks of treatment. By week 4 of treatment, the MB-PDT group had complete healing of skin lesions with only mild pigmentation remaining, while the terbinafine group required an extended treatment period to further clear residual infection.\u003c/p\u003e\n\u003cp\u003eAdditionally, no severe adverse reactions were reported during MB-PDT treatment, confirming the therapy's good safety profile. This result aligns with previous studies reporting a low adverse reaction rate (\u0026lt;5%) with local application of methylene blue\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTraditional drugs are prone to escape due to their single target, given the multidrug resistance characteristics of Indian ringworm fungus\u003csup\u003e12\u003c/sup\u003e, MB-PDT disrupts fungal structures through multiple oxidative stress pathways, demonstrating broader potential for combating drug resistance. Additionally, MB-PDT's low cost (single treatment cost \u0026lt;\u003cspan\u003e$\u003c/span\u003e10) and good tolerability (no reports of pain in this patient group) lay the foundation for its widespread adoption at the grassroots level. Future large-scale randomized controlled trials are needed to further optimize light exposure parameters (e.g., using fractionated irradiation or combining with Er: YAG laser pretreatment) and explore its long-term efficacy in preventing recurrence.\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThis study demonstrates that methylene blue photodynamic therapy (MB-PDT) exhibits significant advantages in the treatment of multidrug-resistant Trichophyton mentagrophytes infections. Its core mechanism may stem from the multi-targeted destruction of fungal cells by reactive oxygen species (ROS) generated by the photosensitizer methylene blue under red light excitation\u003csup\u003e13\u003c/sup\u003e, ROS can not only directly oxidize unsaturated lipids in fungal cell membranes, leading to increased membrane permeability and leakage of contents, but also specifically attack inner mitochondrial membrane complexes I and III, inhibiting electron transport chain function, and ultimately inducing mitochondrial membrane potential collapse and cell apoptosis. In addition, oxidative damage to fungal DNA by ROS may further hinder hyphal proliferation and spore formation\u003csup\u003e14\u003c/sup\u003e, This multi-pathway attack mode contrasts sharply with the single-target inhibition of traditional azole drugs (such as terbinafine), which are prone to resistance escape due to SQLE gene mutations (such as F397L/L393S) or CYP51 overexpression. MB-PDT significantly reduces the risk of resistance through multidimensional disruption.\u003c/p\u003e\u003cp\u003eIt is worth noting that the sensitivity of the strains in this study to methylene blue (MIC\u0026thinsp;=\u0026thinsp;1.0 \u0026micro;g/mL) suggests its potential as an alternative therapy. The MB-PDT in this case achieved mycological conversion within two weeks, which may be related to optimized light parameters (635 nm red light, 50 J/cm\u0026sup2; energy density) and the high permeability formulation of methylene blue (10% hydrogel). Additionally, methylene blue's inherent anti-biofilm properties may further reduce the risk of recurrence, a characteristic particularly important in the management of chronic infections.\u003c/p\u003e\u003cp\u003eAlthough MB-PDT demonstrates significant efficacy, its clinical application still faces challenges. First, standardization of light exposure parameters requires further exploration, such as fractionated irradiation (e.g., twice weekly) or combination with Er:YAG laser pretreatment to enhance epidermal penetration efficiency, particularly for hypertrophic lesions. Second, differences in light absorption among patients of different skin tones may affect efficacy, necessitating optimization of light wavelength or energy density for darker skin types. Furthermore, this case study is a single-center case report lacking long-term follow-up data, making it impossible to assess long-term recurrence rates. Future studies should include multicenter randomized controlled trials (RCTs) with larger sample sizes and extended observation periods to validate its long-term efficacy.\u003c/p\u003e\u003cp\u003eCompared with other novel therapies (such as amorolfine nanoemulsion or terbinafine-itraconazole combination therapy), the advantages of MB-PDT include low cost (single treatment cost \u0026lt;\u003cspan\u003e$\u003c/span\u003e10), absence of systemic side effects, and ease of operation, making it particularly suitable for resource-limited primary care settings. However, its limitations include limited penetration into deep tissue infections (such as onychomycosis), necessitating the combination of other physical or chemical methods to enhance efficacy.\u003c/p\u003e\u003cp\u003eIn this case, the MB-PDT treatment area achieved scaling resolution and negative fungal microscopy results within two weeks, indicating a risk of latent infection. This discrepancy aligns with the findings of Alberdi et al\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study confirmed through rigorous in vitro experiments and clinical observations that methylene blue photodynamic therapy (MB-PDT) is a safe and effective treatment for multidrug-resistant Trichophyton mentagrophytes infections. The main conclusions are as follows:\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEfficacy confirmation\u003c/strong\u003e\u003cp\u003eMB-PDT achieved mycological conversion within 2 weeks and clinical cure within 4 weeks, with efficacy significantly superior to traditional terbinafine treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In vitro antimicrobial susceptibility testing indicates that it remains highly sensitive to terbinafine-resistant strains (MIC\u0026thinsp;\u0026ge;\u0026thinsp;4\u0026micro;g/ml), with an MIC of 1\u0026micro;g/ml.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMechanistic Innovation\u003c/b\u003e: Through ROS-mediated multi-target disruption, MB-PDT effectively circumvents traditional drug resistance pathways caused by SQLE/CYP51 mutations, offering a new approach to managing drug-resistant fungal infections. \u003cb\u003eClinical application value\u003c/b\u003e: With low treatment costs (less than \u003cspan\u003e$\u003c/span\u003e10 per session), simple operation, and few adverse reactions, it is particularly suitable for promotion in primary healthcare institutions, holding significant implications for alleviating the increasingly severe global issue of drug resistance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFuture Directions\u003c/b\u003e: Further research is recommended in three areas: (1) conducting multi-center RCTs to validate its universality; (2) optimizing treatment parameters (e.g., fractionated irradiation, combined laser therapy); (3) exploring application strategies for deep infections such as onychomycosis. The limitations of this study include its single-case design and lack of long-term follow-up data. However, as the first clinical study to confirm the efficacy of MB-PDT against Trichophyton mentagrophytes, its findings lay an important foundation for future research. We recommend incorporating MB-PDT into first-line treatment protocols for drug-resistant dermatophytic infections and establishing standardized operational guidelines to facilitate clinical adoption.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCYP51: Lanosterol 14\u0026alpha;-demethylase (fungal enzyme target of azole drugs)\u003cbr\u003e\u0026nbsp;ITS: Internal Transcribed Spacer (fungal genomic region used for species identification)\u003cbr\u003e\u0026nbsp;MB: Methylene Blue (photosensitizer agent)\u003cbr\u003e\u0026nbsp;MB-PDT: Methylene Blue Photodynamic Therapy\u003cbr\u003e\u0026nbsp;MIC: Minimum Inhibitory Concentration (lowest drug concentration inhibiting fungal growth)\u003cbr\u003e\u0026nbsp;ROS: Reactive Oxygen Species (cytotoxic molecules generated during PDT)\u003cbr\u003e\u0026nbsp;SDA: Sabouraud Dextrose Agar (fungal culture medium)\u003cbr\u003e\u0026nbsp;SQLE: Squalene Epoxidase (enzyme target of terbinafine; mutations cause resistance)\u003cbr\u003e\u0026nbsp;TBF: Terbinafine (first-line antifungal drug)\u003cbr\u003e\u0026nbsp;T. indotineae: Trichophyton indotineae (drug-resistant dermatophyte species)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and Informed Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Guangxi Medical University (Approval No.: 2025-E0496), in compliance with international ethical guidelines for medical research. The patient was fully informed of the treatment protocols and potential risks. Written informed consent was obtained, authorizing the anonymous publication of case data and imaging materials. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile this study utilized a controlled within-patient lesion comparison design, it was conducted as a prospective, single-center case intervention study with rigorous methodology rather than a formally registered multi-center clinical trial. Future validation through large-scale randomized controlled trials (RCTs) is recommended.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the patient for the publication of:\u003c/p\u003e\n\u003cp\u003e1.De-identified clinical details (age, sex, medical history, and treatment protocol).\u003c/p\u003e\n\u003cp\u003e2.Photographs of skin lesions (Figures 1, 5, and 6) with all identifiable features (e.g., face, tattoos) removed.\u003c/p\u003e\n\u003cp\u003e3.Mycological images (microscopy and colony morphology, Figures 2\u0026ndash;5).\u003c/p\u003e\n\u003cp\u003e4.Experimental data (antifungal susceptibility results, ITS sequencing data [GenBank PV731367.1], and phylogenetic analysis).\u003c/p\u003e\n\u003cp\u003eThe patient acknowledged that:\u003c/p\u003e\n\u003cp\u003eMaterials may appear in open-access publications and scientific presentations. Data could be reused for educational purposes under Creative Commons licenses. Personal identity remains strictly confidential (no names, hospital IDs, or geographic identifiers).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study (including MIC results, clinical assessment data, and de-identified imaging files) are available from the corresponding author (Dong-Yan Zheng or Xin-Yu Zhang) upon reasonable request. The fungal strain sequence is accessible via GenBank (Accession No. PV731367.1)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests. No financial or non-financial competing interests influenced the design, execution, analysis, or reporting of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Guangxi Special Fund for Science \u0026amp; Technology Bases and Talents (AD23026325), and The 14th Five-Year Plan\u0026quot; Science and Technology Program Project Fund of Fangchenggang City (AB23006038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWei-Lun Xu and Gao-Yuan Peng (co-first authors):\u003c/p\u003e\n\u003cp\u003eDesigned the study protocol and performed clinical treatments (MB-PDT/terbinafine application). Collected and analyzed antifungal susceptibility data. Drafted the initial manuscript\u003c/p\u003e\n\u003cp\u003eShu-Lei Qin:\u003c/p\u003e\n\u003cp\u003eConducted fungal culture, ITS sequencing, and phylogenetic analysis\u003c/p\u003e\n\u003cp\u003ePrepared figures for microbiological results (Figures 2-4)\u003c/p\u003e\n\u003cp\u003eYan-Qing Zheng:\u003c/p\u003e\n\u003cp\u003ePerformed microscopic examinations (KOH wet mounts) and Wood\u0026rsquo;s lamp assessments\u003c/p\u003e\n\u003cp\u003eDocumented clinical photographs (Figures 1, 5-6)\u003c/p\u003e\n\u003cp\u003eJia-Can Huang and Jun-Meng Zhou:\u003c/p\u003e\n\u003cp\u003eExecuted CLSI M38-A2 standard MIC assays. Processed and interpreted drug sensitivity data (Table 1)\u003c/p\u003e\n\u003cp\u003eMing-Yu Zheng:\u003c/p\u003e\n\u003cp\u003eStatistical analysis and result validation. Revised the manuscript for methodological accuracy\u003c/p\u003e\n\u003cp\u003eDong-Yan Zheng and Xin-Yu Zhang (co-corresponding authors):\u003c/p\u003e\n\u003cp\u003eSupervised the entire study design and ethical compliance. Provided critical revisions for intellectual content. Approved the final manuscript for submission\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree with the submission and declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank the following individuals and institutions for their valuable contributions to this study:\u003c/p\u003e\n\u003cp\u003eMedical Staff: The dermatology team at The First Affiliated Hospital of Guangxi Medical University for their assistance in patient management and sample collection.\u003c/p\u003e\n\u003cp\u003eTechnical Support: Colleagues at the Clinical Microbiology Laboratory for their expertise in fungal culture and molecular identification.\u003c/p\u003e\n\u003cp\u003eFunding Agencies: Guangxi Special Fund for Science \u0026amp; Technology Bases and Talents (AD23026325) and Fangchenggang City\u0026rsquo;s 14th Five-Year Plan Science and Technology Program (AB23006038) for financial support.\u003c/p\u003e\n\u003cp\u003ePatient: We extend our gratitude to the participant for their cooperation and consent to publish clinical data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHui, S. T., Gifford, H. \u0026amp; Rhodes, J. Emerging Antifungal Resistance in Fungal Pathogens. \u003cem\u003eCurr Clin Microbiol Rep\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 43-50, doi:10.1007/s40588-024-00219-8 (2024).\u003c/li\u003e\n\u003cli\u003ePashootan, N.\u003cem\u003e et al.\u003c/em\u003e Phylogeny, Antifungal Susceptibility, and Point Mutations of SQLE Gene in Major Pathogenic Dermatophytes Isolated From Clinical Dermatophytosis. \u003cem\u003eFront Cell Infect Microbiol\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 851769, doi:10.3389/fcimb.2022.851769 (2022).\u003c/li\u003e\n\u003cli\u003eUhrla\u0026szlig;, S.\u003cem\u003e et al.\u003c/em\u003e Trichophyton indotineae-An Emerging Pathogen Causing Recalcitrant Dermatophytoses in India and Worldwide-A Multidimensional Perspective. \u003cem\u003eJ Fungi (Basel)\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, doi:10.3390/jof8070757 (2022).\u003c/li\u003e\n\u003cli\u003eCaplan, A. S.\u003cem\u003e et al.\u003c/em\u003e Improving antifungal stewardship in dermatology in an era of emerging dermatophyte resistance. \u003cem\u003eJAAD Int\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 168-169, doi:10.1016/j.jdin.2024.01.004 (2024).\u003c/li\u003e\n\u003cli\u003eAbdolrasouli, A., Barton, R. C. \u0026amp; Borman, A. M. Spread of Antifungal-Resistant Trichophyton indotineae, United Kingdom, 2017-2024. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 192-194, doi:10.3201/eid3101.240923 (2025).\u003c/li\u003e\n\u003cli\u003eAl-Janabi, A. Expanding the prevalence of Trichophyton indotineae-associated skin infection by transmission from humans to animals. \u003cem\u003eJ Med Microbiol\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, doi:10.1099/jmm.0.002023 (2025).\u003c/li\u003e\n\u003cli\u003eCorreia, J. H., Rodrigues, J. A., Pimenta, S., Dong, T. \u0026amp; Yang, Z. Photodynamic Therapy Review: Principles, Photosensitizers, Applications, and Future Directions. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, doi:10.3390/pharmaceutics13091332 (2021).\u003c/li\u003e\n\u003cli\u003eAnagu, O.\u003cem\u003e et al.\u003c/em\u003e The emerging role of photodynamic therapy in the treatment of cutaneous infections. \u003cem\u003eItal J Dermatol Venerol\u003c/em\u003e \u003cstrong\u003e160\u003c/strong\u003e, 40-46, doi:10.23736/s2784-8671.24.07910-6 (2025).\u003c/li\u003e\n\u003cli\u003eBatvandi, A.\u003cem\u003e et al.\u003c/em\u003e Time and cost-efficient identification of Trichophyton indotineae. \u003cem\u003eMycoses\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 75-81, doi:10.1111/myc.13530 (2023).\u003c/li\u003e\n\u003cli\u003eIzoton, C. F.\u003cem\u003e et al.\u003c/em\u003e Adjunctive methylene blue antimicrobial photodynamic therapy for mucocutaneous lesions of mycoses: three case reports. \u003cem\u003eFuture Microbiol\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 1017-1024, doi:10.2217/fmb-2023-0074 (2023).\u003c/li\u003e\n\u003cli\u003eChang, W., Bao, F., Wang, Z., Liu, H. \u0026amp; Zhang, F. Comparison of the Sensititre YeastOne(\u0026reg;) and CLSI M38-A2 microdilution methods in determining the activity of nine antifungal agents against dermatophytes. \u003cem\u003eMycoses\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 734-741, doi:10.1111/myc.13272 (2021).\u003c/li\u003e\n\u003cli\u003eAstvad, K. M. T.\u003cem\u003e et al.\u003c/em\u003e Increasing Terbinafine Resistance in Danish Trichophyton Isolates 2019-2020. \u003cem\u003eJ Fungi (Basel)\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, doi:10.3390/jof8020150 (2022).\u003c/li\u003e\n\u003cli\u003eAlberdi, E. \u0026amp; G\u0026oacute;mez, C. Methylene blue vs methyl aminolevulinate photodynamic therapy in combination with oral terbinafine in the treatment of severe dermatophytic toenail onychomycosis: Short- and long-term effects. \u003cem\u003eMycoses\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 859-868, doi:10.1111/myc.13125 (2020).\u003c/li\u003e\n\u003cli\u003eBerstecher, N., Burmester, A., Gregersen, D. M., Tittelbach, J. \u0026amp; Wiegand, C. Trichophyton indotineae Erg1(Ala448Thr) Strain Expressed Constitutively High Levels of Sterol 14-\u0026alpha; Demethylase Erg11B mRNA, While Transporter MDR3 and Erg11A mRNA Expression Was Induced After Addition of Short Chain Azoles. \u003cem\u003eJ Fungi (Basel)\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, doi:10.3390/jof10110731 (2024).\u003c/li\u003e\n\u003cli\u003eAlberdi, E. \u0026amp; G\u0026oacute;mez, C. Successful treatment of Pityriasis Versicolor by photodynamic therapy mediated by methylene blue. \u003cem\u003ePhotodermatol Photoimmunol Photomed\u003c/em\u003e\u003cstrong\u003e36\u003c/strong\u003e, 308-312, doi:10.1111/phpp.12555 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Trichophyton indotineae, MB-PDT, Antifungal Susceptibility Testing, Dermatophytosis","lastPublishedDoi":"10.21203/rs.3.rs-7223419/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7223419/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e: Superficial fungal infections caused by Trichophyton indotineae are spreading globally, and mutations in its SQLE gene (F397L/L393S) are leading to increasing resistance to traditional antifungal drugs such as terbinafine. \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThere is an urgent need to explore new treatment options. Photodynamic therapy (MB-PDT) utilizes methylene blue-mediated reactive oxygen species (ROS) to disrupt fungal mitochondrial function, demonstrating broad-spectrum potential against drug-resistant strains. This study aims to evaluate the clinical efficacy of methylene blue photodynamic therapy (MB-PDT) for infections caused by Trichophyton mentagrophytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients and methods：\u003c/strong\u003eThe minimum inhibitory concentration (MIC) of methylene blue and terbinafine was determined for clinical isolates through in vitro antimicrobial susceptibility testing. The donor for this isolate was a 25-year-old Indian male patient who underwent methylene blue photodynamic therapy. The patient presented with widespread, multiple skin lesions on the extremities, abdomen, and inguinal region, which were confirmed through microscopic examination and ITS sequencing as a multidrug-resistant Trichophyton mentagrophytes infection. Strains were collected from different body regions (buttocks, upper limbs, lower limbs, and trunk) for in vitro antimicrobial susceptibility testing. The patient was then treated using a randomized controlled design: the experimental group (forearm lesions) received topical application of 10% methylene blue combined with 630 nm red light irradiation (100 J/cm²), once weekly for 20 minutes, for a total of 4 weeks; the control group (remaining lesions) received only topical application of 1% terbinafine cream twice daily.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult：\u003c/strong\u003e In vitro antimicrobial susceptibility testing revealed that the patient's Trichophyton mentagrophytes strain was more sensitive to methylene blue (MIC = 1.0 μg/mL) and exhibited strong resistance to terbinafine (MIC = 8.0 μg/mL). After two weeks of treatment, the experimental group showed resolution of scales and negative fungal microscopy (hyphae density \u0026lt; 1/HPF), while the control group still had residual hyphae (\u0026gt; 5/HPF). By the fourth week, the experimental group had negative fungal microscopy results, whereas the control group still had residual hyphae, with microscopy results remaining positive, necessitating further extension of the treatment duration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion：\u003c/strong\u003eMethylene blue photodynamic therapy (PDT) can rapidly clear drug-resistant Trichophyton mentagrophytes infections. It is more effective than traditional topical antifungal drugs and provides a safe and effective alternative treatment for drug-resistant dermatophytosis.\u003c/p\u003e","manuscriptTitle":"Antifungal Susceptibility Testing and Clinical Efficacy Observation of Methylene Blue Photodynamic Therapy in Treating Trichophyton indotineae Infections","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 13:12:52","doi":"10.21203/rs.3.rs-7223419/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6d2536a2-1838-43ff-97c5-6380614832d3","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-17T16:08:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-11 13:12:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7223419","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7223419","identity":"rs-7223419","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}