Extracellular vesicle microRNA signature as a highly accurate diagnostic biomarker for human brucellosis

Background Brucellosis remains an important public health problem globally. Rapid and accurate diagnosis is crucial for the treatment of brucellosis. However, current diagnostic methods for brucellosis are limited, posing significant clinical challenges. Extracellular vesicles (EVs)-derived microRNAs (miRNAs) potentially offer a novel, non-invasive approach for accurate diagnosis of brucellosis. Methods Small RNA sequencing was conducted to identify candidate miRNAs as potential diagnostic biomarkers in serum-derived EVs from patients with brucellosis and healthy individuals. These miRNA candidates were further validated in serum-derived EVs, serum free of EVs (EVs-free), and serum within an exploratory set and a preliminary validation set using a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and a logistic regression model to establish and validate the diagnostic signature. Results The sequencing analysis initially screened and identified a panel of 52 overexpressed miRNAs in serum-derived EVs from patients with brucellosis. Subsequently, qRT-PCR demonstrated that 10 of the top 12 most differentially expressed miRNAs (miR-20a-5p, miR-320a-3p, let-7b-5p, miR-374a-5p, miR-93-5p, miR-186-5p, let-7d-5p, miR-151a-3p, miR-361-5p, and miR-98-5p), effectively distinguished patients with brucellosis from healthy individuals. Among single miRNAs, miR-361-5p (area under the curve [AUC], 0.917 [95% CI, 0.760 -1.000]) has the largest AUC. After logistic regression analysis and qRT-PCR, it was found that a miRNA signature (miR-20a-5p and miR-93-5p) in serum-derived EVs performed more robust in differentiating between patients with brucellosis and healthy individuals (exploratory set: AUC = 1.000; preliminary validation set: AUC = 0.988, 95% CI: 0.949 -1.000). Moreover, there was no significant difference in the expression levels of this miRNA signature in EVs-free and serum. EVs were enriched with miRNAs that are closely associated with Brucella infection. Conclusion MiR-20a-5p and miR-93-5p in serum-derived EVs serve as highly accurate diagnostic biomarkers for brucellosis, offering a rapid and reliable alternative to conventional diagnostic methods.