MALDI-TOF Mass Spectrometry for the Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar

Abstract

Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson’s medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples.

Sensing parasites: Proteomic and advanced bio-detection alternatives

“…The above mentioned technology has also been applied in parasitic diseases by sensing unusually truncated host proteins in response to Trypanosoma cruzi infection with high sensitivity and specificity [33]. Moreover, matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) is a valuable diagnostic technique already applied for detecting and differentiating Entamoeba Spp or Babesia canis canis infections, and becoming a miniaturised bioanalytical tool for detecting and discerning proteins from bio-complex samples [34][35][36]. MALDI mass spectrometry has applications in biomarker discovery, pathogen identification and has great potential for lipid-based biomarker sensing [37,38].…”

Pathogenic Entamoeba histolytica, but not Entamoeba dispar, induce neutrophil extracellular trap (NET) formation

“…Because both species cannot be distinguished microscopically, alternative detection methods capable of discriminating between the 2 microorganisms have been developed. They include PCR assay, real‐time PCR assay, ELISA assay, and even a MALDI‐TOF mass spectrometry analysis …”

“…real-time PCR assay, 16 ELISA assay, 17 and even a MALDI-TOF mass spectrometry analysis. 18 An infection by E. histolytica is characterized by intense inflammation with many infiltrating neutrophils. 19 Neutrophils migrate from the circulation to sites of infection, where they display several antimicrobial functions.…”

MALDI-TOF mass spectrometry as a potential tool for Trichomonas vaginalis identification

“…[ 22 – 25 ]). Recently, MALDI-TOF MS was applied for the first time to the differentiation of E. histolytica and E. dispar strains isolated from clinical samples [ 26 ].…”