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Background: Improved diagnostic tools are needed for detecting active filarial infections in humans. Tests are available that detect adult W. bancrofti 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 in vitro cultured B. malayi 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 B. malayi and from humans with heavy Loa loa infections., Results: Vesicles from Mf cultures contained more than 300 B. malayi proteins with high consistency across biological replicates. These included the known Mf excretory antigen BmR1 (AF225296). Over 150 B. malayi proteins were detected in vesicles isolated from plasma samples from two infected animals. Vesicles isolated from plasma from 10 persons with high L. loa 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 B. malayi 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 in vitro and in vivo 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors MM and BR declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Yates, Di Maggio, Rosa, Sprung, Erdmann-Gilmore, Townsend, Budge, Kamgno, Mitreva, Weil and Fischer.)

Background and Methods: Circulating Loa loa antigens are often detected in individuals with heavy L. loa infections by diagnostic tests for lymphatic filariasis (LF) caused by Wuchereria bancrofti . This is a major challenge to LF mapping and elimination efforts in loiasis co-endemic areas. However, it also provides an opportunity to identify antigen biomarkers for loiasis. To determine which L. loa antigens might be promising biomarkers for distinguishing true LF from loiasis, we screened for L. loa antigens in a group of individuals with heavy L. loa infections living in the Okola Health District of Cameroon. In this longitudinal study, participants were tested for cross-reactive antigenemia by filariasis test strip (FTS), ELISA, and western blot, and were monitored for FTS status at 6, 9, 12, and 15 months post-enrollment. We then identified specific circulating L. loa antigens by liquid chromatography-tandem mass spectrometry (LC-MS/MS) from baseline and 15-month plasma samples., Principal Findings and Conclusions: Among 73 FTS-positive (FTS+) and 13 FTS-negative (FTS-) participants with high L. loa microfilarial loads, 83% maintained their FTS status over the course of the study, while 17% experienced at least one FTS conversion event (from FTS+ to FTS- or vice versa). Cross-reactive antigens were detected in both FTS+ and FTS- sera by western blot, and there was poor agreement in antigen detection by FTS, western blot, and ELISA methods. One protein family, a group of Nas-14 metalloproteases, was detected by LC MS/MS in >80% of tested samples, including FTS- samples. These data identify Nas-14 as a promising loiasis biomarker potentially capable of distinguishing loiasis from lymphatic filariasis., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. The author(s) HND declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.