Detection and differentiation of causative organisms of onychomycosis in an ex vivo nail model by means of Raman spectroscopy.

BACKGROUND: Onychomycosis is worldwide the most prevalent infection of the nail. It is mainly caused by the dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes and to a lesser extent Trichophyton tonsurans. The yeast Candida albicans and the mould Scopulariopsis brevicaulis can also cause onychomycosis. Management of these nail conditions may require appropriate treatment methods and therefore the identification of the causative species can be of importance. However, the determination of agents causing onychomycosis is still not optimal. OBJECTIVES: To detect and differentiate causative organisms of onychomycosis in an ex vivo nail model by means of Raman spectroscopy. The work focusses is on the discriminative power of Raman spectroscopy for detection of differences between T. rubrum, T. mentagrophytus and T. tonsurans on human nail and distinguishing these dermatophytic from the non-dermatophytic species S. brevicaulis and C. albicans. METHODS: Raman spectra (200/sample) were taken from 50-µm human nail slices infected with T. rubrum, T. mentagrophytus, T. tonsurans, S. brevicaulis or C. albicans using a 2500 High-Performance Raman Module and 785-nm diode laser. Processed spectra were analysed by sorting the correlation matrix and presented as dendrogram and heat map. Raman spectra from suspended dermatophytic microconidia were taken for mutual comparisons. RESULTS: Spectral differences between the dermatophytes T. rubrum, T. mentagrophytus and T. tonsurans (635-795, 840-894, 1018-1112, 1206-1372, 1566-1700/cm) and the non-dermatophytes S. brevicaulis and C. albicans (442-610, 692-758, 866-914, 1020-1100, 1138-1380,1492-1602/cm) growing on nail were confirmed by clustering correlation showing two main clusters. Dissimilarities between tested dermatophytes were also found with T. rubrum being most different. Raman spectra of the dermatophytic microconidia varied over the whole tested 400-1800/cm range. CONCLUSION: Important dermatophytic and non-dermatophytic agents of onychomycosis growing on ex vivo human nail can be distinguished specifically and non-invasively by Raman spectroscopy.

The use of Raman spectroscopy in the epidemiology of methicillin-resistant Staphylococcus aureus of human- and animal-related clonal lineages.

In order to perform a cost-effective search and destroy policy for methicillin-resistant Staphylococcus aureus (MRSA), a quick and reliable typing method is essential. In an area with a high level of animal-related MRSA ST398, pulsed field gel electrophoresis (PFGE) typing and spa-typing are not sufficient to discriminate between co-incidental findings and true transmission of MRSA. This study is the first to retrospectively show the performance of Raman spectroscopy in 16 well-documented outbreaks. We analysed 525 isolates, 286 MRSA ST398 and 239 from other PFGE clusters with Raman spectroscopy. When epidemiologically linked isolates from the outbreaks were analysed with PFGE as the reference standard, Raman spectroscopy correctly identified 97% of cases that were indistinguishable from the index case. With Raman cluster analysis, the most dominant distinction was between MRSA ST398 and other MRSA of human clonal lineages. Within MRSA ST398, 22 different Raman clusters were identified. Raman typing correctly identified an ST398 (spa type t567) outbreak in a hospital setting. No direct correlation was observed between Raman clusters and spa types. We conclude that Raman spectroscopy is a quick and reliable method of MRSA typing, which can be used in outbreak settings and it is comparable to PFGE, with the added advantage that PFGE non-typeable isolates can also be readily typed using the same sample preparation protocol.