Urinary Borrelia Peptides Correlate with the General Symptom Questionnaire (GSQ-30) Scores in Symptomatic Patients with Suspicion of Tick-borne Illness

Background: Borreliosis infection and post-treatment Lyme disease syndrome are associated with nonspecific, multisystem symptoms, and there is currently no effective direct test for Lyme disease. The seroprevalence of Borrelia miyamotoi among individuals with suspected tick-borne illnesses and persistent symptoms is not well documented. Although bacterial extracellular vesicles (BEVs) have been suggested as potential sources of disease biomarkers, evidence supporting their role in Borrelia infection is lacking. Methods: Patients (N=66) with symptoms and with clinical suspicion of a tick-borne illness were enrolled in the study and followed longitudinally over the course of 1 year (min 1 visit, max 4 visits). The General Symptom Questionnaire (GSQ-30) was administered to the patients at each visit to measure symptom burden. Lyme and Borrelia miyamotoi serologies were conducted at LabCorp or Quest, under the standard of care. A mass spectrometry proteomic assay measuring Borrelia-specific peptides was conducted in the urine of the patients. Results: Among the analyzed patients, 65% had positive Lyme serology and 33% had positive B. miyamotoi serology with partial overlap (20%). Urinary Borrelia-specific peptides correlated with the GSQ-30 score after correcting for age, Lyme serology, and B. miyamotoi serology (coefficient = 0.57, t-test p-value < 0.00001). Urinary extracellular vesicles had positive reactivity in a p66 immunoassay, a known marker of Borrelia BEVs. Urinary peptides included glycerophosphodiester phosphodiesterase, variable small and large outer membrane proteins, the multicopy lipoprotein family, and Borrelia direct repeat proteins, which are also known markers of Borrelia BEVs. Conclusions: The B. miyamotoi seroprevalence (33%) in a symptomatic population with clinical suspicion of a tick-borne illness was higher than the seroprevalence estimated from previous studies. As it provides an objective measurement correlated with a standardized symptom burden score, urinary Borrelia peptide detection offers a promising avenue for future assay development. This proteomic analysis supported the presence of Borrelia BEVs in the urine of symptomatic patients with suspected tick-borne illnesses.