Subtype determination of Blastocystis isolates by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)

Abstract

The pathogenic role of the enteric parasite Blastocystis remains controversial. Recent studies have suggested that various subtypes (STs) found in human samples could be correlated to the presence or absence and variability of clinical manifestations, and that STs can differ with respect to drug sensitivity. Polymerase chain reaction (PCR) techniques used to determine these STs are expensive and are usually restricted to research laboratory settings. This study evaluates the potential application of the inexpensive matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) technique to discriminate Blastocystis STs. A database of parasitic protein signatures was constructed for five Blastocystis STs, and the reference spectra were challenged with those from 19 axenic cultures of ST1, ST2, ST3, ST4 and ST8 and those from nine xenic liquid cultures of ST3 and ST4. Samples from axenic cultures were prepared using standard formic acid extraction and direct deposition procedures. The reference spectra revealed five distinct spectral profiles, and the database library allowed for discrimination between all of the cultures with reliability indices ranging from 2.038 to greater than 2.8 when an extraction was performed. The direct deposition procedure resulted in greater variability in the discrimination and direct MALDI-TOF MS identification from xenic liquid cultures was effective in 3 out of 9 samples. MALDI-TOF MS proved to be an effective technology for efficiently discriminating Blastocystis STs in axenic cultures.

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

“…However, in human parasitology mass spectrometry has had limited application, such as the detection of malarial hemozoin in blood, using technologies different from MALDI-TOF [ 19 ], or parasite identification or detection of their biomarkers by MALDI-TOF MS (eg. [ 1 , 3 , 5 – 7 , 20 ]).…”

“…Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) has recently revolutionized clinical microbiology [ 1 ], proving to be a reliable approach for solving certain problems linked to the identification and strain differentiation of microorganisms, and overcoming limitations of conventional methods [ 2 ]. This technique allows the rapid, accurate, and inexpensive identification of microorganisms from both cultures and biological samples.…”

“…As far as human intestinal parasites as concerned, the application of MALDI-TOF MS has been limited to obtaining general parasitic proteome data [ 4 , 5 ], to characterizing specific biomarkers for discriminating between Cryptosporidium and Giardia species [ 6 , 7 ], and to determining the subtype of Blastocystis sp. isolates from liquid xenic cultures [ 1 ].…”

Update on the Molecular Epidemiology and Diagnostic Tools for Blastocystis sp

“…A recent study showed the accuracy of MALDI-TOF MS for the speciation of five different Blastocystis STs [28]. Axenic cultures speciated by SSU-rDNA analysis were used to create profiles for the different Blastocystis subtypes.…”

MALDI-TOF MS Profiling-Advances in Species Identification of Pests, Parasites, and Vectors

“…In a similar study, a database was constructed from four reference strains and the two clinical isolate were identified as L. infantum (Culha et al, 2014). In another study, using 19 enteric parasite Blastocystis isolates from 19 patients, a database for five subtypes was created and the remaining specimens were identified by matching (Martiny et al, 2014). Likewise, the differentiation of E. histolytica and E. dispar was demonstrated after establishment of the reference spectra and discriminating peaks were matched with the proteins identified through SDS-PAGE MALDI TOF MS based protein identification approach (Calderaro et al, 2015).…”