# Emerging Optical Techniques for the Diagnosis of Onychomycosis  
[PERSON]  
1 Department of Medical Physics, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece; [EMAIL_ADDRESS] (C.P.); [EMAIL_ADDRESS] (M.Z.V.)1  
[PERSON]  
2 Department of Skin & Venereal Diseases, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece; [EMAIL_ADDRESS] (G.G.); [EMAIL_ADDRESS] (I.D.B.)2  
[PERSON]  
2 Department of Skin & Venereal Diseases, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece; [EMAIL_ADDRESS] (G.G.); [EMAIL_ADDRESS] (I.D.B.)2  
[PERSON]  
3 Myocology Research Laboratory & UOA/HCPF Culture Collection, 15772 Athens, Greece; [EMAIL_ADDRESS] 34 Biomedicine SA, 11526 Athens, Greece 45 CONACYT-Universidad Autonoma de San Luis Potosi, 78210 San Luis Potosi, Mexico; [EMAIL_ADDRESS] 4  
Footnote 4: email: {[[https://www.mdpi.com/journals/](https://www.mdpi.com/journals/)]([https://www.mdpi.com/journals/](https://www.mdpi.com/journals/))  
secreted proteolytic activity of dermatophytes is manifested by excreting large quantities of sulphite for cleaving those disulphide bridges. The modified proteins become accessible for further digestion by various endo- and exoproteases that reportedly act as virulence factors [6]. Non- dermatophytic moulds are also implicated in the pathogenesis in some cases. The global prevalence of onychomycosis is expected to increase further due to the ageing of the population and the rising numbers of predisposed individuals [7; 8]. The gold standard for the diagnosis of onychomycosis involves direct microscopic evaluation of nail clippings and culture of nail samples in specialized media. Culture of tissue probes permits the identification of the causative organism and confirms its viability in situ [9]. However, it is time consuming--requiring about 3-4 weeks of incubation--and has lower sensitivity than microscopy [10]. Although direct microscopy is an inexpensive and widely available diagnostic technique, its sensitivity is highly operator-dependent [11]. Fungal chitin staining with calcofluor white can increase the overall sensitivity of the method. Histological investigation of nail plate samples after PAS staining is an alternative microscopic approach to the diagnosis of onychomycosis. Typically, these methods are less effective in identifying the culprit fungal pathogen at species level. More elaborate diagnostic alternatives for onychomycosis include the detection of dermatophyte DNA by PCR-based amplification [12] and an antibody-based immunochromatography strip method [13]. Recently, [PERSON] and [PERSON] [14] reported that culture was three to four times more likely to report false-negative results compared to PCR, highlighting the major setback of false-negative cultures. Finally, an additional challenge concerns the evaluation of treatment outcome and the follow up of patients for relapses. There is a need for the evaluation of objective criteria in order to increase the concordance between the clinical mycological assessment of affected nails (i.e., clinical cure) and the laboratory one (i.e., mycological clearance) [15].  
Optical techniques play a central role in modern clinical diagnostics and optical instrumentations or prototypes are currently being used or tested in clinical settings to assess health conditions and treatment outcomes. Their main advantage is that they allow real-time visualization of tissues at high spatial resolution, being minimally or non-invasive, and optionally portable. Herein, the evidence from the application of three imaging and spectroscopic modalities, namely optical coherence tomography (OCT), confocal laser scanning microscopy (CLSM), and Raman spectroscopy (RS) for the evaluation of onychomycosis, is presented. While they have not been established as reference methods, these techniques offer several advantages, being rapid, non-destructive, non-invasive and with the capacity for in vivo application.