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.

Course of Borrelia burgdorferi DNA shedding in urine after treatment.

Diagnosis of Lyme borreliosis by urine polymerase chain reaction (PCR) has been recognized as having better diagnostic sensitivity in patients with erythema migrans than serological methods. We made serial tests with 192 urine specimens from 70 patients with erythema migrans and 60 urine specimens from 21 patients with acrodermatitis chronica atrophicans to evaluate the course of positive urine PCR after antibiotic treatment. Before treatment, urine samples from patients with erythema migrans showed a positive PCR in 27/34 samples (79%), and those from patients with acrodermatitis chronica atrophicans in 7/11 (63%). The specificity of bands was proven by hybridization with GEN-ETI-KTM-DEIA kit in 40/41 samples. Borrelia DNA in urine decreased gradually within the observation period of one year in both patients with erythema migrans and acrodermatitis chronica atrophicans, and persisted without clinical symptoms in 4/45 patients with erythema migrans (8%) after 12 months. Urine PCR can serve as a diagnostic method in early Lyme borreliosis and also in seropositive patients with unclear clinical symptoms.

Molecular and serological diagnosis of Borrelia burgdorferi infection among patients with diagnosed Erythema migrans.

The aim of the study was to assess the frequency of Borrelia burgdorferi DNA detection in the blood and urine of patients diagnosed with erythema migrans, and compare the results of PCR-based methods with ELISA methodology. The latter was used to detect serum antibodies against Borrelia burgdorferi of the IgM and IgG classes, before and after antibiotic therapy. The study included 86 patients hospitalized in the Department of Infectious Diseases and Neuroinfections in the Medical Academy in Bialystok, diagnosed with the erythema migrans phase of Lyme borreliosis. Examinations were carried out twice: the first at the moment of diagnosis (Trial 1), the second after 4 weeks of antibiotic therapy. The study showed that antibiotic therapy in the early phase of borreliosis does not decrease the sensitivity of PCR and that after 4 weeks of therapy (Trial 2), spirochete DNA is still detectable in most patients (45/86). There was no correlation between detectability of spirochete DNA and the presence of antibodies against B. burgdorferi s.l. (assessed by ELISA) during the course of erythema migrans. The largest percentage of positive results in the detection of B. burgdorferi s.l. DNA was observed in patients who simultaneously possessed IgM and IgG antibodies against B. burgdorferi, while the lowest percentage of PCR positive results was among patients with only IgM antibodies.

Detection of spirochaetal DNA simultaneously in skin biopsies, peripheral blood and urine from patients with erythema migrans.

Lyme borreliosis is an emerging zoonosis transmitted by infected hard-bodied ticks. The disease is multisystemic. In the initial stage its typical manifestation is the erythema migrans, a cutaneous lesion that occurs in up to 90% of patients. In order to investigate the presence of the specific agent, Borrelia burgdorferi, in the early stages of the disease, DNA from skin biopsies, urine and peripheral blood of 30 patients with clinically documented erythema migrans and without apparent systemic involvement was analysed by polymerase chain reaction. Borrelia DNA in both blood and skin biopsies was detected in 23 patients, while in 9 patients it was discovered in urine and skin biopsies. These results demonstrate that Borrelia DNA is detectable systemically also in patients with early Lyme borreliosis and strongly suggest a possible dissemination of the causative agents even when only a local infection is assumed.

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in urine specimens of patients with erythema migrans lesions.

A polymerase chain reaction (PCR) assay was developed for the detection of Borrelia burgdorferi-specific DNA in the urine of patients with erythema migrans (EM). The target for the PCR was a specific region of the flagellin gene, and DNA was extracted from urine by Chelex resin. The detection limit was 1-10 genomes of B. burgdorferi, B. garinii or B. afzelii. A prospective study was performed with 12 consecutively diagnosed patients with EM, to evaluate the PCR assay on clinical samples. Borrelia burgdorferi-specific DNA could be detected in urine specimens from the 12 patients with EM before antibiotic therapy. Five weeks after therapy all the patients were negative by PCR of urine. Results of the present study confirm that the described PCR assay is sensitive and that this sort of test allows monitoring of the efficacy of therapy in patients with early Lyme borreliosis.