Identification of biomarker candidates for filarial parasite infections by analysis of extracellular vesicles.

Background: Improved diagnostic tools are needed for detecting active filarial infections in humans. Tests are available that detect adult circulating filarial antigen, but there are no sensitive and specific biomarker tests for brugian filariasis or loiasis. Here we explored whether extracellular vesicles released by filarial parasites contain diagnostic biomarker candidates.

Methods: Vesicles were isolated using VN96-affinity purification from supernatants of short-term cultured microfilariae (Mf) and analyzed by mass spectrometry (Bruker timsTOF). Parasite-specific proteins were identified by bioinformatic analysis and a protein was called present if supported by ≥ 2 spectra. After validation with cultures parasites, this approach was then used to analyze vesicles isolated from plasma of animals infected with and from humans with heavy infections.

Results: Vesicles from Mf cultures contained more than 300 proteins with high consistency across biological replicates. These included the known Mf excretory antigen BmR1 (AF225296). Over 150 proteins were detected in vesicles isolated from plasma samples from two infected animals. Vesicles isolated from plasma from 10 persons with high Mf densities contained consistently 21 proteins, 9 of them were supported by at least 5 unique peptides and 7 with spectral counts above 10. The protein EN70_10600 (an orthologue of the antigen BmR1, GenBank AF225296) was detected in all 10 samples with a total count of 91 spectra and a paralogue (EN70_10598) was detected in 6 samples with a total of 44 spectra.

Discussion: Extracellular vesicles released by filarial parasites and contain parasite proteins which can be reliably detected by mass spectrometry. This research provides the foundation to develop antigen detection assays to improve diagnosis of active filarial infections in humans.