MALDI-TOF mass spectrometry for the research of specific biomarkers of Leishmania infection by analysing strains isolated from biological samples.

Objectives.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) recently emerged as a first-line method for the accurateidentification of bacteria, but few data are available for protozoa.We investigated the potential role of MALDI-TOF MS for the rapid identification of specificbiomarkers of Leishmania spp. byanalysing strains isolated in our laboratory from clinical samples. Methods. In this study, we used a L. major strain 1174 tofind specific biomarkers able to identify Leishmaniaspecies directly in culture medium. This strain was isolated in our laboratory from the skin biopsy of apatient with suspected cutaneous old world leishmaniasis and it was characterized as L.major by analysis of isoenzymes. Leishmania spp. strain 1585, isolated in ourlaboratory from the skin biopsy of a patient with suspected cutaneous old worldleishmaniasis was identifiedas a Leishmania spp. by specific18S-rDNA Real-time PCR and showed the same melting peak as strain 1174; thisstrain was used to verify the presence/absence of the same biomarkers of strain1174. Aliquots of cultures of these strains in Evan’smodified Tobies’s medium were submitted to formic acid/acetonitrile protein extraction.The spectra obtained with the instrument Microflex LT massspectrometer (Bruker Daltonics, Germany) wereanalyzed and subsequently imported into the ClinProTools software version 2.2(Bruker Daltonics, Germany) to carry out a statistical analysis in order toverify the presence of specific peaks. Results. The 2 strains yielded a protein profilewhich was found to be reproducible over several, independent experiments and nodifferences were observed when strains were grown in different lots of media. Theprofiles obtained for each of the 2 strains analyzed in this study showed thepresence of 2 specific peaks (9,692 and 11,184 Da) that were not present in Evans’medium used for their cultivation. Conclusion. The detection of the same 2 peaks in the 2 strains(absent in the culture medium) may be usefulfor the identification of Leishmania strainsisolated from biological samples by MALDI-TOF MS. Our future goal will be to analyse severalother Leishmania spp. strains availablein our laboratory in order to detect the presence/absence of species-specific biomarkersfor a possible identification of isolated strains directly from biological samples.