The value of urine-PCR for the diagnosis of Lyme borreliosis

Research project P 13668 Urine-PCR for routine diagnosis of Lyme borreliosis Elisabeth ABERER 28.06.1999 Serological laboratory tests for the diagnosis of Lyme borreliosis (LB), a multisystem disease with considerable morbidity, are still of restricted value. They can at best confirm the clinical diagnosis of disease, but are unable to monitor treatment success. A novel investigation technique, the molecular detection of Borrelia burgdorferi-DNA in the urine has gained importance worldwide. However, usually these tests have been performed on a heterogenous group of patients with different clinical symptoms of Lyme borreliosis, and are not used rountinely. Differences in the use of oligonucleotidsequences to be amplified derived from chromosomal genes or plasmids and heterogenous extraction methods have also brought different results. In previous studies with flagellin primers we could show that during, borrelia infection borrelia-DNA is excreted in the urine 4 days after infection up to 5-8 months after treatment depending on whether only erythema migrans; (EM) or signs for disseminated disease were present. The aim of our study is to confirm these results, to improve the technique of urine PCR, to reduce the risk of contamination, and to see whether this method is of value for routine diagnosis of LB by using additional primers and different preparation methods. We plan to test the urine PCR with 4 primers on patients with erythema migrans. Further tests will then be performed with the 2 best primers in 20 patients with EM and 15 patients with ACA before and after treatment, and then, after 3, 6, and 12 months. The sensitivity of urine PCR will be compared with sensitivity of strain isolation from skin biopsies. Further investigations are planned to be done in 150 patients with different dermatological diagnoses, 50 patients with arthritis or arthralgia or neuroborrehosis, arising after tick bite and/or EM, and for control purposes, in 50 blood donors. At the same time these patients will be tested serologically by commercial ELISA- methods. If this new test method is of value for routine diagnosis the innovative aspects of this project lie in the establishment of a new non-invasive laboratory method. Considerable progress could then be expected in the diagnosis of Lyme borreliosis, and thus a better management of disease, and economically, financial savings by inhibiting unnecessary antibiotic treatments.

Lyme borreliosis (LB) is a multisystem disease with considerable morbidity. In this project, the molecular diagnosis of LB from urine was newly established and proved more sensitive than serological methods in erythema migrans, the early manifestation of LB. Serological laboratory tests only confirm the clinical diagnosis of erythema migrans in about 50%, but are unsuitable for monitoring treatment success. The molecular diagnosis of Borrelia burgdorferi infection from urine has been shown as a valuable diagnostic procedure. B. burgdorferi - PCR has been performed in various studies, on heterogenous groups of patients using different primers derived from chromosomal genes or plasmids, and heterogenous extraction methods. The technique of urine PCR, however, was difficult to be reproduced. The aim of this project, therefore, was to analyse the urine PCR technique in detail. It was recognized that presample preparation steps were essential for a successful urine PCR. We found that patients with erythema migrans showed a positive urine PCR in 80%. B. burgdorferi DNA was detected up to 6 months after antibiotic treatment. Freezing of urine samples for more than 3 months reduced sensitivity to 25-30%. Nested PCR was performed using flagellin primers BBSCH 1, 2 / Fl 7, 59. The amplicons were visualized on agarose gel und the specificity of bands was proven by a DNA enzyme immunoassay. Further important steps were centrifugation of urine at 36.000 x g, extraction with DNAzol , and avoiding investigation of morning urine since the lowest borrelia DNA content was observed in the morning urine in contrast to evening urine. Urine PCR was performed in serial investigations in 126 patients with erythema migrans and 21 with acrodermatitis chronica atrophicans, in patients with arthritis (here synovial fluid was investigated additionally), neuroborreliosis, patients with various dermatoses und healthy controls. Altogether 891 samples were investigated. A considerable amount of patients with various dermatological clinical pictures, patients with musculoskeletal or neurological symptoms, and patients with sclerotic skin lesions where an association with B. burgdorferi infection had repeatedly been reported, showed a positive urine PCR whereas healthy individuals were only positive in 3.5%. Our detailed laboratory analyses showed that urine PCR can be of diagostic value. However, the amount of borrelia DNA in urine is low. Therefore, urine PCR has to performed by a strict protocol. After antibiotic treatment, borrelia DNA is found in the urine up to 6 months. This provides evidence that noninfectious, non viable borreliae can persist for a limited duration after antibiotics and are, according to the clinical picture, not associated with disease. Urine PCR technique can be recommended not only for routine diagnosis of erythema migrans but rather to confirm LB in seropositive and seronegative patients with unclear clinical symptoms attributed to LB, especially in seropositive individuals with arthritis/arthralgia to evaluate the need for antibiotic treatment. In conclusion, urine PCR proved useful as a new non-invasive laboratory method. Considerable progress can be expected in the diagnosis of LB, and subsequently a better management of disease, medically and economically, with financial savings due to avoidance of unnecessary antibiotics.