Easy identification of leishmania species by mass spectrometry

Abstract

Background: Cutaneous leishmaniasis is caused by several Leishmania species that are associated with variable outcomes before and after therapy. Optimal treatment decision is based on an accurate identification of the infecting species but current methods to type Leishmania isolates are relatively complex and/or slow. Therefore, the initial treatment decision is generally presumptive, the infecting species being suspected on epidemiological and clinical grounds. A simple method to type cultured isolates would facilitate disease management.

Methodology: We analyzed MALDI-TOF spectra of promastigote pellets from 46 strains cultured in monophasic medium, including 20 short-term cultured isolates from French travelers (19 with CL, 1 with VL). As per routine procedure, clinical isolates were analyzed in parallel with Multilocus Sequence Typing (MLST) at the National Reference Center for Leishmania.

Principal findings: Automatic dendrogram analysis generated a classification of isolates consistent with reference determination of species based on MLST or hsp70 sequencing. A minute analysis of spectra based on a very simple, database-independent analysis of spectra based on the algorithm showed that the mutually exclusive presence of two pairs of peaks discriminated isolates considered by reference methods to belong either to the Viannia or Leishmania subgenus, and that within each subgenus presence or absence of a few peaks allowed discrimination to species complexes level.

Conclusions/significance: Analysis of cultured Leishmania isolates using mass spectrometry allows a rapid and simple classification to the species complex level consistent with reference methods, a potentially useful method to guide treatment decision in patients with cutaneous leishmaniasis.

Figure 1. Cluster analysis of MALDI-TOF MS 184 spectra from 46 Leishmania isolates (A) with distances displayed in relative units , and algorithm for a computer-independent interpretation of MALDI-TOF MS (B) based on presence/absence of peaks as displayed on Table 1.

Figure 2. Mass spectra from isolates belonging to either L. (V) braziliensis, L. (V) guyanensis ((V) stands for Viannia subgenus), L. (L) major, L. (L) infantum ((L) stands for Leishmania subgenus).

The two pairs of peaks discriminating the Viannia subgenus from the Leishmania subgenus are labeled in green and blue, respectively and indicated by vertical dotted lines. The 11120+/−(7) peak that identifies the Viannia subgenus is shown in insert squares at the right side of the figure to improve readability. Peaks differentiating species complexes are labeled with their corresponding molecular weights in colored squares. The software automatically provides the molecular weights for all peaks above signal background (grey labels). Peaks that identify species in each subgenus are shown in Black. A few peaks above background were not labeled on the figure to improve readability, the complete spectra are provided as supplementary figure S1.