The research team, led by Salma M. Alsayed and colleagues at King Abdulaziz University in Jeddah, Saudi Arabia, followed PRISMA guidelines and prospectively registered the review in PROSPERO. They searched PubMed, Web of Science, and Google Scholar for studies published between 2021 and 2025, screened 609 articles after removing duplicates, and applied Joanna Briggs Institute critical appraisal tools to assess methodological quality. Most included studies scored between six and eight out of eight, indicating moderate to high quality. The team deliberately restricted eligibility to original research using microbiological or molecular methods for pathogen identification, excluding review articles and studies focused exclusively on bacteria.

The microbiological picture that emerged is strikingly diverse. Yeasts constituted 72.3 percent of fungal isolates, with Candida species dominating. Candida albicans was the most frequently reported species at 28.8 percent, but non-albicans Candida collectively accounted for 66.7 percent of yeast isolates, led by Candida parapsilosis at 18.2 percent, Candida tropicalis at 15.2 percent, and Nakaseomyces glabrata, formerly known as Candida glabrata, at 10.6 percent. Even more alarmingly, the multidrug-resistant pathogen Candida auris appeared in 1.5 percent of yeast isolates, a finding the authors describe as clinically noteworthy given its growing recognition as a serious hospital threat. Rare yeasts including Rhodotorula mucilaginosa and Cutaneotrichosporon debeurmannianum were also detected.

Filamentous fungi, or molds, made up 21.3 percent of reported fungal cases. Aspergillus species were the most common molds at 60 percent of filamentous isolates, with Aspergillus niger leading at 20 percent, followed by Aspergillus fumigatus at 15 percent and Aspergillus flavus at 10 percent. Penicillium and Fusarium species followed, while dermatophytes such as Trichophyton and Microsporum accounted for 6.4 percent of fungal isolates. The authors note that dermatophytes, which typically cause superficial infections like tinea pedis, may serve as entry points for deeper tissue invasion when skin barriers are already compromised by diabetes and poor glycemic control.

Perhaps the most concerning technical finding concerns antifungal susceptibility. Only three of the 26 included studies, or 11.5 percent, performed standardized antifungal susceptibility testing, yet within this limited dataset, resistance rates among Candida species ranged from 7.7 to 23 percent for select agents. Candida tropicalis demonstrated the highest resistance, with fluconazole resistance reaching 23 percent and resistance to voriconazole and amphotericin B at 7.7 percent each. Candida albicans remained largely susceptible, with approximately 13.3 percent fluconazole resistance. Trichosporon species exhibited intrinsic resistance to echinocandins despite susceptibility to azoles. The scarcity of susceptibility data represents a significant gap that limits optimization of antifungal therapy, particularly for immunocompromised diabetic patients with chronic wounds and repeated antimicrobial exposure.

The diagnostic landscape for detecting fungi in diabetic foot ulcers proved highly heterogeneous. Culture-based methods using Sabouraud dextrose agar and chromogenic media were the most frequently applied approach in 13 studies, while advanced techniques such as MALDI-TOF mass spectrometry, VITEK automated systems, and BD Phoenix analyzers were used in an equal number. Direct microscopic examination with potassium hydroxide and Lactophenol Cotton Blue staining appeared in nine studies, but molecular methods including PCR amplification and internal transcribed spacer sequencing were employed in only five. Histopathological analysis, which provides direct evidence of tissue invasion, was limited to two case-based studies. This heavy reliance on culture rather than molecular identification may substantially underestimate fungal diversity, since culture is inherently limited by fastidious growth requirements, prior antimicrobial exposure, and overgrowth by faster-growing organisms.

A critical ambiguity pervades the entire evidence base: most included studies did not distinguish between fungal colonization and true infection. Only three of 26 studies, all case reports, used histological staining such as hematoxylin and eosin or Grocott methenamine silver to confirm invasive fungal disease. The remaining investigations reported fungal isolation based solely on culture or microscopy without corroborating clinical criteria. This distinction matters enormously, because various microorganisms can colonize ulcers without causing tissue damage, while true infections involve microbial invasion and activate a host immune response. In polymicrobial wounds, where 69.2 percent of studies reported co-isolation of bacteria and fungi, determining the pathogenic contribution of any single fungal species becomes particularly challenging.

The polymicrobial nature of these wounds raises deeper questions about fungal-bacterial interactions within biofilms. Experimental data show that Candida albicans secretes quorum-sensing molecules that promote bacterial biofilm formation, while certain bacterial metabolites can enhance fungal virulence and increase resistance to both antifungal and antibiotic agents. Mixed bacterial-fungal communities exhibit significantly higher antimicrobial tolerance than single-species biofilms and may serve as reservoirs for horizontal transfer of resistance genes. None of the included studies investigated these mechanistic interactions, which the authors identify as a crucial area for future research beyond simple co-isolation reporting.

Geographic patterns also emerged, though the authors caution against overinterpretation given the small number of studies per region. South Asian cohorts from India, Pakistan, and Iraq reported a predominance of non-albicans Candida species, with one Iraqi study finding Candida albicans at only 13.3 percent of Candida isolates compared to 58.4 percent for non-albicans species. In contrast, a Kenyan cohort reported Candida albicans as the predominant species at 75 percent, while Chinese cohorts showed more varied patterns with Candida parapsilosis dominating at 44.4 percent. Evidence was concentrated in a limited number of countries, with China contributing the most studies, while regions including Saudi Arabia, the Middle East, and Africa remain underrepresented despite carrying substantial burdens of diabetic foot complications.

The authors argue that current clinical guidelines, including the IWGDF and IDSA recommendations on diabetes-related foot infections, frame microbial identification broadly without specific guidance on when fungal-targeted investigations should be pursued in chronic or non-healing ulcers. This absence of fungus-specific diagnostic recommendations may partly explain why fungal diagnostics remain inconsistently applied in routine practice. Failure to recognize fungal involvement may contribute to prolonged and unnecessary antibacterial therapy, while empirical antifungal treatment without confirmatory testing may increase selection pressure for resistance. The team proposes integrating clinical assessment, appropriate specimen collection, and stepwise diagnostic testing into a standardized workflow, and calls for prospective multicenter studies using molecular identification, standardized antifungal susceptibility testing with explicit CLSI or EUCAST breakpoints, and coordinated surveillance to clarify whether fungi are truly driving delayed healing or merely accompanying it. Until that evidence arrives, the message is clear: there is more to diabetic foot ulcers than bacteria.

Subject of Research: Fungal detection, diagnostic approaches, and antifungal susceptibility in diabetic foot ulcers: a systematic review of recent evidence

Article Title: Fungal detection, diagnostic approaches, and antifungal susceptibility in diabetic foot ulcers: a systematic review of recent evidence

Article References: Alsayed, S. M., Khateb, A. M., Yasir, M., Bogary, B., Aljabri, A. M., Al Atrouni, A., Baabdullah, R., Afandi, A., Turkistani, S. A., Abduljabbar, W. A., Ardawi, M. S. M., & Azhar, E. I. (2026). Fungal detection, diagnostic approaches, and antifungal susceptibility in diabetic foot ulcers: a systematic review of recent evidence. Frontiers in Fungal Biology, 7, Article 1915109. https://doi.org/10.3389/ffunb.2026.1915109

Image Credits: AI Generated

DOI: 10.3389/ffunb.2026.1915109

Keywords: Fungal, detection, diagnostic, approaches, antifungal, susceptibility, diabetic, foot, ulcers, systematic, review, recent