Discrimination among Cryptococcus deneoformans, C. neoformans and interspecies hybrids using MALDI-TOF Mass Spectrometry

Abstract

ABSTRACTBackgroundDifferentiation of the species within the Cryptococcus neoformans complex (C. deneoformans, C. neoformans and C. neoformans interspecies hybrids –C. deneoformans x C. neoformans-) is important to define the epidemiology of the infection.ObjectivesIn this study we attempted the discrimination of three C. neoformans species using MALDI-TOF MS coupled with an in-house library.MethodsAll Cryptococcus spp. isolates were identified by AFLP markers. An in-house database was constructed 26 well characterized C. deneoformans, C. neoformans and interspecies hybrids. Forty-four Cryptococcus spp. isolates were blindly identified using MALDI-TOF MS (Bruker Daltonics) and the expanded library. Their protein spectra were also submitted to hierarchical clustering and the resulting species were verified via Partial Least Squares Differential Analysis (PLS-DA) and Support-Vector Machine (SVM).ResultsMALDI-TOF MS coupled with the in-house library allowed 100% correct identification of C. deneoformans and C. neoformans but misidentified the interspecies hybrids. The same level of discrimination among C. deneoformans and C. neoformans was achieved applying SVM. The application of the PLS-DA and SVM algorithms in a two-step analysis allowed 96.95% and 96.55% correct discrimination of C. neoformans from the interspecies hybrids, respectively. Besides, PCA analysis prior to SVM provided 98.45% correct discrimination of the 3 species analysed in a one-step analysis.ConclusionsOur results indicate that MALDI-TOF MS could be a rapid and reliable tool for the correct discrimination of C. deneoformans and C. neoformans. The correct identification of the interspecies hybrids could only be achieved by hierarchical clustering with other protein spectra from the same species.