Detection and quantification of Plasmodium falciparum in human blood by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a proof of concept study

Abstract

Background: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has revolutionized identification of bacteria and is becoming available in an increasing number of laboratories in malaria-endemic countries. The purpose of this proof-of-concept study was to explore the potential of MALDI-TOF as a diagnostic tool for direct detection and quantification of Plasmodium falciparum in human blood.

Methods: Three different P. falciparum strains (3D7, HB3 and IT4) were cultured and synchronized following standard protocols. Ring-stages were diluted in fresh blood group 0 blood drawn in EDTA from healthy subjects to mimic clinical samples. Samples were treated with saponin and washed in PBS to concentrate protein material. Samples were analysed using a Microflex LT MALDI-TOF and resulting mass spectra were compared using FlexAnalysis software.

Results: More than 10 peaks specific for P. falciparum were identified. The identified peaks were consistent among the three genetically unrelated strains. Identification was possible in clinically relevant concentrations of 0.1% infected red blood cells, and a close relationship between peak intensity and the percentage of infected red blood cells was seen.

Conclusion: The findings indicate that the method has the potential to detect and quantify P. falciparum at clinically relevant infection intensities and provides proof-of-concept for MALDI-TOF-based diagnosis of human malaria. Further research is needed to include other Plasmodium spp., wildtype parasite isolates and to increase sensitivity. MALDI-TOF may be a useful tool for mass-screening purposes and for diagnosis of malaria in settings where it is readily available.

Fig. 1

MALDI-TOF spectra of Plasmodium falciparum and negative controls. An example of mass spectra in the sample range 1000–20.000 m/z. A = Negative control of uninfected erythrocytes. B = Positive sample with 3,1% 3D7 Plasmodium falciparum-infected erythrocytes. Each peak in the spectra represents proteins of a specific charge and size. A full description of peak differences can be found in Table 1. Plasmodium cultures and negative controls were prepared using the same protocols. See Methods section for details

Fig. 2

Plasmodium falciparum specific peak at 6760 m/z at different concentrations. The Plasmodium falciparum-specific peak at 6760 m/z in samples with different concentrations of infected red blood cells (IRBC). Each panel corresponds to a different concentration as indicated by the IRBC %. Traces from three independent sample preparations are shown for each concentration. Please note change in vertical scale between panels

Fig. 3

Correlation between peak intensity and % IRBC for different Plasmodium falciparum specific peaks. Correlation between signal intensity of the Plasmodium falciparum specific peaks at 1130, 4332, 6500, 6760, 8568 and 129,993 m/z and the concentration of P. falciparum-infected red blood cells. Each panel represents a specific peak as indicated in the panel headline. Blue dots represent individual sample preparations at each concentration. Orange dots represent the mean peak intensity of the given concentration. The orange line represents a linear fit of mean peak intensity and concentration of P. falciparum-infected red blood cells