# Emerging Optical Techniques for the Diagnosis of Onychomycosis
## 2 Spectroscopy
### Raman Spectroscopy (RS)
Raman spectra were recorded using the i-Raman Plus portable Raman spectrometer (B&W Tek, Inc., Newark, DE, USA) with an excitation wavelength of 785 nm. included additional contributions from the less stable gauche-gauche-trans conformation. It was also proposed that specimens from different patients had different S-S conformations, and the authors suggested that disulphide bond Raman density and shift might reflect the pathological level of severity and/or infection phase. However, since no identification of the causative organism at the species level was performed, we argue that variations of the S-S bond conformation can be pathogen-specific. To that end, [PERSON] et al., [24] performed RS measurements on 52 mycologically-characterized nail clippings in order to differentiate between healthy and either _T. rubrum_ or _Candidia_ infected nails. In uninfected nails, the energetically stable gauche-gauche-gauche conformation was observed at 513-515 cm\({}^{-1}\). However, in samples infected with _Candidia_ species, a secondary shoulder at 519 cm\({}^{-1}\) was observed, indicating the presence of the less stable gauche-gauche-trans conformation as well as the heterogeneity of the S-S bonds conformation among nails infected by different species. Likewise, Raman spectra of nails infected by _T. rubrum_ featured two weak but well-defined bands at 619 cm\({}^{-1}\) and 648 cm\({}^{-1}\), attributed to the C-S stretching vibration, that were not observed in either healthy or _Candidia_-infected nails. Principal component analysis (PCA) successfully clustered specimens by species, and the corresponding loadings indicated that the bands responsible for the separation were assigned to the disulphide bridges. Subsequent classification of the RS spectra test set, using the soft independent modeling of class analogy (SIMCA) method, yielded 92.8% accuracy in discriminating between the three classes (healthy, _T. rubrum_- and _Candidia_-infected nails) and 100% accuracy in classifying healthy vs onychomycotic nails. These findings indicate that species-specific differences in fungal invasion mechanisms might trigger diverse chemical alterations of the nail plate, which can be reflected in the acquired Raman spectra and could be explored to differentiate among the culprit species of onychomycosis at patient-level in vivo. Dermatophytes, like _T. rubrum_, employ extracellular proteases (keratinases) which, together with the secretion of sulphite as a reducing agent [25], digest S-S bonds in order to assimilate degraded keratin moieties as a protein source [26]. Similarly, _Candidia_ species make use of extracellular enzymes with keratinocyte activity [27] to disrupt the S-S cross-linking of keratin, and in addition they excrete sulfite when confronted with elevated cysteine concentrations [28]. Moreover, in an ex vivo onychomycosis model (infection of nail clippings), RS was explored to differentiate between _Trichophyta_ dermatophytic fungi (_T. rubrum_, _T. mentagrophytes_ and _T. tonsurans_) and the non-dermatophytic species _S. brevicaulis_ and _C. albicans_[29]. A correlation matrix approach was used to generate a dendrogram which visually illustrated the cluster arrangements, representing the dermatophytic in one cluster and the non-dermatophytic/yeast nail infections in a separate one. The 500-640 cm\({}^{-1}\) spectral region was found to be the most promising discriminating factor since cysteine S-S and C-S bonds act as a correlate of nail rigidity and hardness. These bonds imply a densely-packed, folded structure of keratin with multiple S-S cross-linking and gauche-gauche-gauche conformation.