(A) A schematic workflow of the EV isolation protocol from i.v. catheters of rats infected with Candida. Following blood collection, fungal EVs were isolated and enriched via IA using Rb-CA-EV-pAbs. (B) Western blot titration of C. albicans biofilm EVs by Rb-CA-EV-pAbs. (C) IAC of C. albicans EVs with Rb-CA-EV-pAbs visualized by super-resolution microscopy. Inset: no EV signal was detected in the absence of Rb-CA-EV-pAbs. Scale bar: 1.5 µm. (D) Western blot analysis of in vivo C. albicans EVs in uninfected control and C. albicans–infected rat serum EVs by Rb-CA-EV-pAbs. No EV signal was detected in uninfected rat sera. (E) NTA-based concentrations of EVs in unprocessed (input) and processed (output) rat serum samples collected from intravenous catheters (n = 3, P < 0.001). (F) Mass spectrometry–based analysis of fungal-unique biomarker cerebrosides. (G) NTA-based sizing of EVs in unprocessed (input) and processed (output) rat blood samples from Candida biofilm i.v. catheters. (H) Representative cryoEM images showing in vivo EVs isolated from Candida-infected rat catheter. Scale bar: 100 nm. (I) Scanning electron micrographs of C. albicans biofilms of WT and ESCRT-associated hse1ΔΔ mutant strains from rat catheters. Scale bar: 400 μm. (J) Imaging flow cytometry quantitative analysis of in vivo EVs in the C. albicans of WT and ESCRT-associated Hse1ΔΔ strains from rat venous catheters (n = 4, 6 technical each). Kruskal-Wallis 1-way ANOVA, followed by uncorrected Dunn’s multiple-comparison test. ****P < 0.001. (K) Venn diagram depicting the qualitative profiling of in vitro and in vivo C. albicans proteomes.