Evaluation of pathogen specific urinary peptides in tick-borne illnesses

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-R2 = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

. Experimental design for pathogen derived, urinary peptide identification and authentication. Experimental analysis and bioinformatics pipeline was applied to a first set of n = 110 individuals, n = 10 acute Lyme borreliosis patients according to CDC criteria and n = 100 controls, comprising healthy and diseased non-Lyme participants. Parameters for peptide identification and authentications were established in this first phase. The method was verified in an independent set of n = 298 participants, including n = 148 non acute patients suspected of tick-borne illness and n = 150 healthy and diseased controls. Urinary peptides were validated by means of orthogonal methods including western blot analysis, parallel reaction monitoring, and a Babesia animal model. PTLDS post treatment Lyme disease syndrome.
Tick-borne pathogen peptides are present in the urine of 40% of nonacute patients with clinical suspicion of tick-borne illnesses. In the validation phase of the study, which was conducted blinded to the patient clinical category, urine samples from 148 non acute patients (n = 36 PTLDS; n = 112 clinically suspected of tick-borne illnesses) and 150 new healthy and diseased controls were analyzed. Patients (M/F = 0.45, median age = 48, IQR = 28.1) reported with symptoms including fatigue, musculoskeletal pain, cognitive impairment and a history of tick bite, fever, and/or EM rash. The control set (M/F = 1.75, median age = 35, IQR = 22) included healthy controls and disease control patients with clinical history of Chagas disease, tuberculosis and (B,C) urine was subjected to pre-analytical concentration using affinity particles, (D) affinity particle extracts were digested with trypsin and analyzed by means of non-targeted LC-MS/MS. This figure was prepared using CorelDRAW X7. Table 1. Demographic information of patients enrolled in this study. Demographic and clinical characteristics of sample cohort. Acute LB acute Lyme Borreliosis, PTLDS post treatment Lyme disease syndrome, NA-TBI suspected non-acute tick borne infection. a A history of Erythema migrans, fever, and facial palsy were recorded for PTLDS and TBI patients. b The urine was collected before treatment regimen for all patients. All 10 acute patients were verified to be two tier serology positive 3-6 weeks later.  Table 2. Borrelia peptides identified in the urine of acute Lyme borreliosis cases. The urine was collected before treatment regimen for all patients. All 10 acute patients were verified to be two tier serology positive 3-6 weeks later. Acute Lyme Borreliosis symptoms = joint pain, fatigue, facial palsy, neurological symptoms. a Developed after collection. www.nature.com/scientificreports/ controls were negative. n = 48 were positive for Borrelia, n = 17 positive for Babesia, n = 4 were positive for Bartonella, n = 2 were positive for Ehrlichia, n = 8 were positive for Borrelia and Babesia, n = 1 was positive for Borrelia and Bartonella, n = 1 was positive for Babesia and Bartonella, n = 1 was positive for Babesia, Bartonella and Anaplasma. Therefore, 48/148 patients (32%, Fig. 4A,B) were positive for only one pathogen and 10/148 samples (7%) were positive for 2 pathogens, and 1/148 (0.7%) was positive for 3 pathogens suggesting the co-existence of multiple infections (Fig. 4A, Fig. S5). Seven unique peptides belonging to the genus Francisella and common to the species tularensis, novicida, and hispaniensis, persica were also identified. Urinary peptides from species known to be non-pathogenic suggest a commensal host-microbe interaction (Table S7).
Urinary pathogen peptides revert to undetectable levels after symptom resolution. Longitudinal study of three patients provided anectdotal evidence that urinary peptides revert to undetectable levels after symptom resolution. Pre and post treatment urine collection was obtained from two acute LB patients. In one case (patient # 108838), three Borrelia peptides were identified at the time of positive serology and EM rash. Complete clearing of Borrelia peptides was observed after symptoms resolution with two 14-day courses of doxycycline. In the second patient (# 453742), urine was collected at different time points: (1) after tick bite and  (160)  www.nature.com/scientificreports/ before EM rash, (2) after development of the characteristic EM rash and before antibiotic treatment, (3) after 2 days of doxycycline treatment while the patient was still symptomatic. Three Borrelia peptides were detected in the urine before development of the EM rash. Borrelia peptides were then confirmed in the urine of the untreated, symptomatic patient. Borrelia peptides were also detected after two days of doxycycline treatment while the patient was still symptomatic (Table 3). A decline in peptide numbers following treatment was found   www.nature.com/scientificreports/ for patient # 957477, positive for Erhlichia chaffeensis, whose urine was collected the first day of treatment as well as after two and four weeks (Table 3).
Borrelia-specific urinary peptides are associated with chemotaxis, transmembrane transport, immune evasion and metabolism. Peptides (N = 160) from Borrelia species were the most abundant among the tick-borne infection pathogens investigated. Gene Ontology (GO) analysis of biological functions indicated that a large number of proteins were associated with chemotaxis, biosynthesis, transmembrane transport, immune evasion and DNA metabolism (Table S8). Chemotaxis and motility are required for Borrelia to establish infection in the mammalian host 21 . In this study, we identified peptides specific for chemotaxis and motility associated proteins including flagellin, CheA, and MotA. Transmembrane transport plays a role in drug resistance, in parasite-host interaction, in cell signaling and virulence 22 . Urinary peptides associated with transmembrane transport proteins included ABC transporter permease, acriflavine resistance protein, and mechanosensitive ion channel. In response to mammalian host immunity, Borrelia modulates its transcriptional activity to facilitate dissemination and immune evasion (Table S8) 23 . Cell envelope proteins are involved in a number of processes required for Borrelia to establish infection in the mammalian host, including cell adhesion, cell invasion and immune escape 22  Borrelia peptides in the cerebrospinal fluid of a clinically suspected neuroborreliosis patient are also detectable in the urine (anectdotal). The experimental protocol described in Fig. 2 and the algorithm of Fig. S1 were applied to matched cerebrospinal fluid (CSF) and urine samples from a clinically suspected Neuroborreliosis patient. Ten months prior to sample collection, the patient experienced worsening of neurological symptoms, including fainting, ataxia, and tremors in the face, neck and hands. Two peptides were detected in the CSF: OspC peptide LKEKHQDLGVANGDTTDNNAK and Acriflavine resistance protein peptide VTSNLDVEK. The same OspC peptide was detected in the urine.
The number of urinary peptides correlates with presence or absence of symptoms in non acute tick borne disease patients. Symptoms reported by non acute patients (PTLDS and patients with clinical suspicion of tick-borne illnesses) included previous EM rash, joint pain, fatigue, fever, facial palsy, and other neurological symptoms. A score of a 0 and 1 was attributed in absence or presence of any symptom designated in Table 1. Using an ordinal regression model, we found that for those subjects where clinical data were available (N = 46), urinary peptide number was positively correlated with presence or absence of symptoms (ordinal regression pseudo-R 2 = 0.392, p-value = 0.010) (Table S10).

Alignment analysis informs verification of protein database annotation and unambiguous species attribution of urinary peptides. Alignment analysis within evolutionarily related organisms
in the clade was conducted to achieve two goals: (1) verification of the protein database annotation, and (2) attribution of the peptide to an organism at the species level. In order to achieve the former, full length sequence of the protein associated with each urinary peptide was retrieved from the highest-ranking species in FASTA format and compared to homologous proteins (data from Basic Local Alignment Search Tool (BLAST), run on databases downloaded on February 15th 2020). In the case of Borrelia, annotated species of Borrelia were used, including: Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia bissettii, Borrelia bavariensis, Borrelia mayonii, Borrelia miyamotoi, Borrelia hermsii, Borrelia turicatae, Borrelia chilensis, Borrelia duttonii (Fig. S7). If the protein demonstrated greater than 60% homology, over the full query length, with other species in the query, then the database annotation was validated. In this study, protein database annotation was validated in all the instances and no rejection was necessary. In order to attribute peptides to an organism at the species level, the peptide sequence was studied in the context of homologous proteins in the clade. A peptide was unambiguously attributed to a species if the peptide sequence had 100% match with the given species and less than 90% sequence identity to any other species investigated (Fig. S7). Species variation can be an important cause of diagnostic inaccuracy due to lack of reactivity of detection reagents 22 .
Orthogonal technologies western blot analysis and parallel reaction monitoring confirm urinary peptide identification. Pathogen-specific, urinary peptides were confirmed using affinity particle

Babesia microti derived peptides are detected in the urine and blood of an animal model of persistent infection and correlate with parasitemia.
In order to substantiate the hypothesis that peptides derived from a tick-borne pathogen in later phases of infection can be detected in peripheral body fluids such as urine, we analyzed bodily fluids derived from golden hamsters (Mesocricetus auratus) infected with Babesia microti. Six golden hamsters with parasitemia ranging from 0 to 42% and one uninfected control were studied at different times after infection, ranging from 3 to 6 months to mimic chronic infection. PCR to Babesia ITS regions 25 Table S12), members of the BMN2 family were detected in RBCs (BMR1_03g00020, BMR1_02g04265; BmR1_04g09980) and in urine of chronic hamsters (BMR1_02g04265). BMN2 family members are considered to be relevant in the immune evasion as suggested by their high mutation frequency and low immunogenicity 25 , and were previously reported to be highly expressed antigens in Babesia microti. 3/62 (5%) Babesia peptides found in urine of non acute patients (Tables S4 and S5) were also identified in body fluids of infected hamsters (Table S12, Magni et al. 26 ). These include importin-5 (BMR1_02g00750), guanine nucleotide-binding protein subunit beta-2-like 1 protein (BmR1_04g08285), hypothetical protein (BMR1_03g04255).

Discussion
The goal of this study is to introduce a method for investigating candidate pathogen specific peptides in patients diagnosed with acute Borreliosis or suspected of tick-borne illnesses including Borreliosis, Babesiosis Anaplasmosis, Ehrlichiosis, Tick-borne encephalitis virus, Powassan Virus disease, Rickettsiosis, TBRF, and Tularemia 27 .
Past studies have focused on serology, culture, or erythrocyte microscopy (Babesiosis) to study single tick-borne infections, and little is known the incidence of co-infections 7,10,12,28 . The analytical sensitivity of MS analysis is currently in the range of 10-100 ng/mL when analyzing complex matrices without pre-analytical processing 11,29 , hence mass spectrometry analysis applied directly to body fluid samples lacks the sensitivity needed for low abundance pathogen derived protein detection. We show that preprocessing the sample with affinity hydrogel particles 18,19,[30][31][32][33][34] concentrates the low abundance biomarkers to achieve mass spectrometry sensitivity in the low picogram/mL range 32 . Additionally, the present method ensures linearity and precision of the assay in physiologically relevant protein concentration ranges (Table S1). Affinity hydrogel particles 18,19,[30][31][32][33][34] consist of polymeric networks functionalized with high affinity chemical baits that capture, concentrate, and preserve solution phase analytes in one step, while excluding interfering high abundance proteins (Supplementary Methods) that would otherwise negatively affect the analytical sensitivity of  Figure S16. (F,G) Western blot analysis detected the presence of Babesia BmSA1 and BMR1_03g00947 antigens in patients 542019, 413743, and 908230 who had at least one Babesia peptide in their urinary peptidome analysis conducted with mass spectrometry (2, 3, and 1, respectively). Patients 891284, 991873, and 243325 who had 2, 1, and 1 Babesia mass spectrometry peptide, respectively, did not show clear reactivity towards these two antibodies. Bands at a molecular weigth lower than the full length protein (48.7 kDa and 35.4 kDa for BMR1_03g00947 and BmSA1, respectively) are indicative of degradation products that pass glomerular filtration. PC is Babesia infected hamster red blood cells (RBC) diluted in urine. NC is healthy donor urine. PC − NP and PC + NP is positive control in absence and presence of nanoparticle processing, respectively. (H) Babesia peptides were identified in the blood and urine of a hamster animal model at early 36   www.nature.com/scientificreports/ mass spectrometry analysis 35 . In previous studies we successfully pre-processed biofluids with hydrogel particles dramatically increase the sensitivity of the analytical technique used downstream and allowing the detection of previously undetectable markers of Mycobacterium tuberculosis 18 , Toxoplasma gondii 20 , and Trypanosoma cruzi 19 , and Babesia microti 26,36 . We also combined hydrogel particle pre-processing with a highly sensitive immunoassay 30 to detect OspA, a relevant biomarker for Lyme borreliosis. Our 3-tier authentication algorithm, which requires 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and lack of identity with human or any other organism (Fig. S1), dramatically reduces the number of false positives that would have been otherwise called using direct MS sequencing by conventional MS software (Fig. S4). Biologic and technical validation of the algorithm employs CDC criteria, serology positive acute Lyme patients, concomitant urine western blot immunoassays, orthogonal targeted identification using PRM, and an animal model of persistent Babesiosis. PRM is a targeted proteomic approach able to simultaneously monitor all fragment ions derived from selected peptides with high resolution and accuracy 37 . Orthogonality between discovery phase and PRM can be obtained through a different combination of fragmentation strategies and mass analyzers. In discovery, precursors were fragmented with collision induced dissociation (CID), and product ions measured in the ion trap analyzer; in PRM, fragmentation was obtained with high energy collision induced dissociation (HCD) and product ions were measured in the Orbitrap mass spectrometer. Stringent mass tolerance filters (≤ 1 ppm) were applied to the product ions in the spectra, allowing for a highly confident peptide identification.
The method yielded zero false positives in 250 diseased and healthy controls and identified up to five specific urinary Borrelia peptides in 10 acute LB patients, including proteins that are part of the panel for the standard Lyme serological test ( Table 2). For 2 of the acute LB patients as well as for a PTLDS patient we were able to obtain urine samples at different time points (Table 3). As a result, we were able to anectdotically observe a decrease in the number of peptides during antibiotic treatment and absence of tick-borne pathogen peptides after successful treatment and symptom remission.
Addressing the question of persistent infection, 279 different urinary peptides, derived from the surface or subcellular compartments of pathogenic strains of tick-borne pathogens, were identified in non acute patients (PTLDS and patients with clinical suspicion of tick-borne illnesses). In 40% (n = 59/148) of them we identified two or more peptides unique for at least one tick-borne pathogen and the number of urinary pathogen derived peptides correlated with the presence or absence of symptoms (p < 0.011) reported by the treating physician when available. 32% (n = 48/155) of patients presented peptides derived from one pathogen, while 7% presented (n = 10/148) peptides from two pathogens, and less than 1% (n = 1/148) presented 3 pathogens.
Borrelia was the most frequently represented organism. A large number of identified proteins are located on the membrane surface and several are known to be antigenic [38][39][40][41] . It is important to note that Borrelia undergoes several changes during host infections which require the production of new membrane proteins that could be used for immune evasion or adaptation to the new environment 38,42,43 . Multiple proteins identified herein are currently recognized as antigens in the standard serological test: OspC, Flagellin 44 . Among the Borrelia genus, the highest number of peptides were derived from species related to Lyme borreliosis.
In this study, at least two different peptides associated with Borrelia were found in n = 48/148 non acute patients suspected of tick-borne illnesses. Represented Borrelia species included Lyme-associated as well as TBRF-associated species. In many subjects both Lyme and TBRF associated species were found (Tables S4 and  S5). We identified 42 unique peptides specific for TBRF Borrelia species (Tables S4, S5), and 24 peptides from Borrelia miyamotoi, which is being diagnosed in the United States in an increasing number of patients 45 . Recent evidence shows that TBRF Borrelia species can also be carried by Ixodes ticks, the same vector that transmits Lyme borreliosis [46][47][48] . TBRF is an often-neglected disease and may go underdiagnosed in many patients. In fact, TBRF patients can yield a positive serology for Lyme borreliosis because of proteins with overlapping antigenic similarities with Lyme Borrelia species 49,50 , thus TBRF true prevalence can be underestimated.
We chose to build the mass spectrometry proteomics database to comprise as many pathogenic species as possible, including pathogen species that are currently thought to be limited to narrow geographic location (e.g. Borrelia mayonii detected only in the US upper Midwest, Wisconsin and Minnesota). The reason of this choice was two fold: (1) a larger database increases the algorithm sensitivity and specificity; (2) we wanted to devise an analytical tool that is robust across all geographical locations, in face of the possibility that climate change may induce unexpected relocations of ticks and vertebrate reservoirs.
We hypothesize that Borrelia peptides present in the urine can be derived from two distinct sources: (a) pathogen peptides previously processed by immune cells, such as antigen presenting cells, and (b) peptides that are released directly by viable or non viable spirochetes (Fig. 6). Bacterial cell wall fragments have been previously detected in rats months after inoculation 51 and a recent study shows the persistence of Borrelia peptidoglycan in the synovial environment for several weeks after antibiotic treatment 17 . It is also known that antigens from several pathogens, including Borrelia, can persist in the lymph nodes in association with antigen presenting cells such as dendritic cells (DC) 52 and also with non-hematopoietic cells such as lymph node stromal cells 53,54 for at least several weeks after infection resolution 53 . DCs play a crucial role in the development of a specific T-cell mediated response to Borrelia after phagocytosis 55,56 . Published studies support the evidence that Borrelia peptides are displayed in conjunction with MHC class II molecules 52 and with CD1 proteins 57-59 on the surface of professional antigen presenting cells. Additionally, endosomal Toll Like Receptors bind to Borrelia processed antigens and initiate a MyD88 mediated signaling response that leads to expression of IL-1beta and inflammatory cytokines 60 . As a result of their persistence in the lymph nodes, peptides can be shed over time due to apoptosis of the antigen presenting cell or to secretion of soluble molecules or extracellular vesicles 61 . While the concentration of these peptides is unknown, we cannot exclude that it may still fall within the analytical range of detection. In support of this hypothesis, we have found that the OspC peptide AILKTNGDKTLGAAELEK, frequently recurring in this study, is compatible with MHC class II binding using the algorithm RankPep 62 www.nature.com/scientificreports/ computational method predicts the likelihood of peptide-MHC class II binding based on primary and secondary structure parameters. Predictions were conducted using HLA-DR haplotypes, based on the published evidence that HLA-DR frequency is significantly increased in patients that suffer from antibiotic refractory Lyme rheumatoid arthritis 64 and Neuroborreliosis 65 . Alternatively, Borrelia peptides can derive directly from spirochetes present in different tissues (e.g. synovial connective tissue 66 , brain 67 , and heart 67 ). Borrelia is known to secrete soluble proteins 68 and to produce extracellular vesicles 76 that contain a variety of molecules such as nucleic acids, intracellular and membrane proteins, lipopolysaccharides, phospholipids, and metabolites. Additionally, peptide molecules can be released by degenerated, non viable bacterial fragments (Fig. 6B). Peptides can then reach the blood circulation by permeating the blood vessel wall to enter the blood circulation. They are then filtered in the kidneys by the glomeruli and accumulate over time in the urine (Fig. 6C-E) 69 . This momentary shedding of pathogen molecules from living or non viable organisms in the blood is integrated over time as they accumulate in the urine. The number of pathogen peptides in the urine of untreated individuals were not higher than in PTLDS and NA-TBI patients. The scientific premise is that the number of urinary pathogen peptides correlates with pathogen burden, as demonstrated in other infections including tuberculosis 18 , Babesiosis 26,36 and Chagas disease 19 . In the acute, untreated phase, the total body burden of Borrelia organisms is low and there may have not been sufficient time for immune mediated attack and break down of pathogen organisms into antigenic peptides that are small enough to pass through glomeruli filtration (Fig. 6). We expect that, due to low body burden of disease in the early stages and late stages of Borrelia infection, we would find similar number of peptides.
This study has several limitations. Only 10 acute samples were analyzed in the training phase of the algorithm. Due to the fact that we have high precision and large effect size (3.35), even using a very low number of observations in the Lyme group, we achieved a power of 0.9999 (alpha = 0.05, Wilcoxon Mann Whitney).
We lack any independent means (e.g. culture or PCR) to verify a tick-borne pathogen infection in non acute patients with pathogen peptides in the urine. Moreover, while symptomatic non-TBI controls used in this study www.nature.com/scientificreports/ were collected from individuals living in geographical areas wth very low incidence of Borreliosis and healthy non-TBI controls had no history of a clinical diagnosis of tick borne illnesses, participants were not tested for Lyme and other TBI serology. Under standard of care, it is not justified to conduct a serology test on a patient in absence of clinical suspicion of Lyme disease or tick borne illness. A binary scale was used to assess presence or absence of symptoms. This choice was motivated by the fact that the location of treating physicians contributing to this study ranges from Europe to the USA east coast, the Baltimore Washington area, and the USA west coast. Thus we judged that there is no practical way to normalize a quantitative symptom level severity score across the physicians. Nevertheless, as has been previously shown a yes/no binary recording of a subjective symptom, such as level of pain, can be just as accurate across physicians as a ranking score 70,71 Although a binary approach provides less information and granularity than a ranking score, in the interest of increasing accuracy and rigor we chose to use presence or absence of symptoms that fall under the IDSA guidelines 13 . We report the correlation as merely descriptive and we cannot consider it clinically for a treatment decision. It would be interesting to investigate the findings further on a larger dataset, and possibly grade the severity of each symptom, after using some means to normalize the scoring across treating physicians.
We did not use a Borrelia animal model for this study; nevertheless, we attempted to study the concept of chronic disease shedding in the well-established Babesia animal model. In previous papers 26,36 we showed the ability to identify Babesia peptides in urine of acutely infected hamsters; in the current paper we are showing the persistence of peptides in chronically infected hamsters even in presence of very low or undetectable parasitemia.
A further limitation of the study relates to the unknown temporal variation in shedding of tick-borne pathogen proteins in a patient who harbors an active infection. Thus, if a symptomatic patient is negative for urinary tick-borne pathogen specific peptides, even though they are actively infected, they might not be shedding the pathogen protein at the time the urine was collected. On the other hand, pathogen specific peptides shed in the urine may be leftover from a prior active infection and may reflect discharge of intracellular processed proteins following immune cell phagocytosis (Fig. 6). These limitations may be addressed in future studies that sample urine longitudinally over time before and after antibiotic therapy in patients with acute Lyme borreliosis who successfully clear the symptoms and in those who do not.
Despite these limitations, the method is cost-effective, is not restricted to specialized centers, and has potential for routine diagnosis of TBI following thorough independent validation.

Study design.
A method consisting of sample pre-analytical concentration, mass spectrometry analysis and a novel peptide authentication algorithm was applied to 408 urine patient specimens (Table 1) in order to discern the presence of peptides belonging to the proteome of selected tick-borne pathogens. Urine samples were subjected to pre-analytical concentration and mass spectrometry analysis. Urine specimens were divided in a training (N = 110 patients, 10 cases and 100 controls) and a non-overlapping validation (N = 298 patients, 148 cases and 150 controls) set that were used to establish the parameters of the peptide authentication algorithm to ensure that identified peptide sequences were uniquely attributable to tick-borne pathogens. Validation was conducted blinded to clinical category. The algorithm included three steps: (1) determination of physical and statistical parameters for mass spectrum matching, (2) BLAST searches of peptides longer 7 amino acids to ensure that the selected peptide sequence has percentage identity lower than 100% with proteins of non-tick pathogen organisms, and (3) validation of protein database annotation via alignment with homologous proteins of evolutionary related organisms in the clade (Fig. S1). At the conclusion of the analysis, we performed manual quality check of spectra and we did not find any discrepancy or incorrect attributions. Peptides identified with the method were verified using Western Blot analysis 30  . Acceptance criteria for (1) acute LB patients (N = 10) included the characteristic erythema migrans (EM) rash and positive two-tier LB serology according to CDC criteria The urine was collected before treatment regimen for all patients. All 10 acute patients were verified to be two tier serology positive 3 to 6 weeks later. (2) PTLDS patients were identified according to the Infectious Disease Society of America guidelines 13 . Patients were considered PTLDS if (a) they were previously diagnosed for Lyme disease (based on EM rash and positive two-tier serology) more than 6 months before the urine sample was collected, (b) if Lyme Borreliosis symptoms ameliorated following antibiotic treatment, and (c) if patients presented with fatigue, musculoskeletal pain, or cognitive impairment according to the IDSA criteria at the time of urine collection. Symptoms were judged by the physician to be functionally disabling. (3) Other non acute patients suspected of tick-borne disease (n = 112): patients had a history of tick bite and a history of clinical diagnosis of a tick borne illness. For patients suspected of a prior diagnosis of Lyme Borreliosis, clinical information, or IDSA categorized symptoms were not sufficient to fit the requirement of PTLDS according to the standards specified above. The ticks that bit the patients were not formally identified. This study met the requirements for IRB approval (Pro00008518, Chesapeake IRB) and followed principles of the Declaration of Helsinki. All participants provided written informed consent prior to the study. For minors, written informed consent from a parent or legal guardian was obtained. All methods were performed in accordance with relevant guidelines and regulations. Clinical and demographic data included age, Three-tier peptide identification and authentication algorithm. We developed an algorithm to perform peptide authentication, which incorporates stringent filtering criteria in order to minimize the false positive rate. The algorithm includes the following steps: (A) Statistical and physical parameters for spectrum matching. (1) Peptide false discovery rate (FDR) based on forward-reverse decoy < 1%, (2) Xcorr > 2.0, > 3.0 and > 4.0 for 2+, 3+, 4+ precursor ions, (3) q-value < 0.05, (4) precursor ion mass tolerance < 2 ppm and fragment ion mass tolerance < 0.5 Da, (5) If precursor is triply-charged: www.nature.com/scientificreports/ (5.1) Presence of a basic residue (K, R, H) within the sequence (excluding N-terminus and C-terminus residues), and (5.2) Presence of corresponding doubly charged precursor ion in the full mass chromatogram (MS1). (B) Unambiguous peptide attribution to one microorganism. In order to exclude peptides that share amino acid sequence with other organisms, each peptide attributed to a tick-borne pathogen was subjected to BLAST searches against the NCBI Reference Sequence database (RefSeq) 73 , a comprehensive dataset containing the available protein sequence information for any given species. A peptide showing 100% identity to any additional species included in the RefSeq database beyond the intended tick-borne pathogen was discarded. Peptide sequences shorter than 7 amino acids were discarded in order to minimize random error of attribution when searching for short-peptide sequences 74 . Date and time of BLAST search and database download were recorded.
(C) Validation of protein database annotation. The full-length protein, to which every peptide was attributed, was aligned with homologous proteins of evolutionary related organisms in the clade. If the full-length protein had greater than 60% identity with proteins in the query, the database annotation was considered valid.
Attribution of urinary peptides to an organism at the species level was conducted as follows. Full length homologous proteins in related microorganism were aligned using the JalView software. For Borrelia, the following species were taken into consideration: Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia bavariensis, Borrelia mayonii, Borrelia miyamotoi, Borrelia hermsii, Borrelia turicatae, Borrelia recurrentis, Borrelia chilensis, Borrelia crocidurae, Borrelia duttonii, Borrelia bissettii. A peptide showing 100% identity to a single species and < 90% to other species was attributed to the microorganism at the species level. A peptide showing 100% identity to one or more species and > 90% identity to different species was not attributed to the microorganism at the species level, and all the species with 100% identity were reported (Table S4).

Targeted peptide identification with parallel reaction monitoring. LC-MS/MS was performed on
an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Scientific) coupled with a nanospray EASY-nLC 1200 UHPLC. Reversed-phase chromatography was performed using PepMap RSLC 75 μm i.d. × 15 cm long with 2 μm, C18 resin LC column (ThermoFisher). Peptides were eluted using a linear gradient of 5% mobile phase B to 50% mobile phase B in 15 min at 300 nl/min, then to 100% mobile phase B for an additional 2 min. The Orbitrap Fusion was operated in data independent acquisition parallel reaction monitoring mode. A targeted list of precursor ions of the peptides of interest AVEIKTLDELK (m/z = 420.24; z = 3), LKNSHAELGVAGNGATTDE-NAQK (m/z = 775.72; z = 2), NDVSEEKPEIK (m/z = 644.32) were isolated and fragmented by Higher-energy C-trap Dissociation (HCD) with 35% normalized collision and detected at a mass resolution of 60,000. The data were then analyzed using Skyline v3.6 (University of Washington, MacCoss Lab) to determine the presence or absence of peptides of interest.

Propagation of Babesia microti in hamsters. Babesia microti GI (BEI Resources NR-44070; ATCC
PRA-398) was originally isolated from blood obtained from a human case of babesiosis in Nantucket, Massachusetts, USA, in 1983 74,75 . The isolate was maintained by in vivo propagation in Golden Syrian hamsters (Harlan Laboratories, stock: HsdHan:AURA) according to published protocols 76,77 and procedures approved by the ATCC IACUC. Ten hamsters were inoculated with ~ 10 8 parasitized erythrocytes in 0.5 ml of blood. Blood samples were collected by the peri-orbital route following inhalational anesthesia with isoflurane and parasitemia was determined by microscopic examination of Giemsa-stained blood films at different times of infection. A minimum of 500 erythrocytes were counted to calculate the percent parasitemia of each sample. This included all parasitized cells regardless of intraerythrocytic stage or number of parasites per cell. After 30 days of infection, four hamsters (acute group) were anesthetized by ketamine injection (50 mg/kg) and 0.5 ml of blood with and without heparin was collected from each animal. Urine samples (~ 0.1 ml) were collected directly from the bladders with a syringe during abdominal surgery and animals were subsequently euthanized using carbon dioxide inhalation. The six remaining hamsters (chronic group) were monitored for 6 months and blood and urine samples were collected as described above.
Statistical analysis. Ordinal regression analysis was performed to evaluate correlation between the number of urinary pathogen derived peptides and presence or absence of clinical symptoms in non acute symptomatic patients suspected of tick-borne diseases. Linear regression analysis was performed to evaluate the correlation of the number of Babesia derived peptides with parasitemia in the hamster animal model experiment. T-test was used to test the significance of regression. Statistical analyses were performed using SPSS v. 19.0 (IBM Corp.). Descriptive statistical analysis of data derived from LD and non acute patients, controls, and hamsters was performed using Python 3 Pandas library and MicrosoftExcel. Visualization were obtained using Python 3 Matplotlib 3.1.1, Seaborn 9.0 libraries and Excel.

Data availability
All peptide sequences and matching accession numbers reported in this study are made available in the Supplementary Information. Materials used in this study can be obtained by request to A.L.