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Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R 2  = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

Lyme borreliosis (LB), caused by bacteria of the Borrelia burgdorferi sensu lato (Borrelia) species, is the most common tick-borne infection in the northern hemisphere. LB diagnostics is based on clinical evaluation of the patient and on laboratory testing, where the main method is the detection of Borrelia specific antibodies in patient samples. There are, however, shortcomings in the current serology based LB diagnostics, especially its inability to differentiate ongoing infection from a previously treated one. Identification of specific biomarkers of diseases is a growing application of metabolomics. One of the main methods of metabolomics is nuclear magnetic resonance (NMR) spectroscopy. In the present study, our aim was to analyze whether Borrelia growth in vitro and infection in vivo in mice causes specific metabolite differences, and whether NMR can be used to detect them. For this purpose, we performed NMR analyses of in vitro culture medium samples, and of serum and urine samples of Borrelia infected and control mice. The results show, that there were significant differences in the concentrations of several amino acids, energy metabolites and aromatic compounds between Borrelia culture and control media, and between infected and control mouse serum and urine samples. For example, the concentration of L-phenylalanine increases in the Borrelia growth medium and in serum of infected mice, whereas the concentrations of allantoin and trigonelline decrease in the urine of infected mice. Therefore, we conclude that Borrelia infection causes measurable metabolome differences in vitro and in Borrelia infected mouse serum and urine samples, and that these can be detected with NMR.