Detection of Borrelia burgdorferi DNA in urine samples and cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by polymerase chain reaction

Abstract

A polymerase chain reaction (PCR) was developed for use in the identification of a 248-bp fragment of the Borrelia burgdorferi flagellin gene in urine and cerebrospinal fluid (CSF) from patients with Lyme neuroborreliosis. The specificities of the PCR products were confirmed by DNA-DNA hybridization with an internal probe. The assay had a detection limit of 10 in vitro-cultivated B. burgdorferi. The PCR assay seemed to be species wide as well as species specific, since DNA from all 21 B. burgdorferi isolates from humans tested but not from Borrelia hermsii or Treponema pallidum could be amplified. We tested 10 consecutively diagnosed patients with untreated neuroborreliosis. There was lymphocytic pleocytosis and intrathecal B. burgdorferispecific antibody synthesis in the CSF of all patients. Urine and CSF samples were investigated by PCR before, during, and up to 8.5 months after therapy. B. burgdorferi DNA was detected in urine samples from nine patients; five patients, including two patients with chronic neuroborreliosis, were PCR positive prior to treatment, whereas urine samples from the remaining four patients obtained 3 to 6 days after the onset of therapy became PCR positive. All urine samples obtained >4 weeks after therapy were negative by PCR. PCR of CSF was less sensitive, and samples from only four patients, including one with chronic neuroborreliosis, were positive. We conclude that urine is a more suitable sample source than CSF for use in B. burgdorferi DNA detection by PCR. Normalization of inflammatory CSF changes and the negative PCR results during follow-up even in patients with chronic neuroborreliosis do not point to a persistent infection. The future role of PCR as a diagnostic tool for Lyme neuroborreliosis is still uncertain.

Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay

“…The significance of PCR-based assays to complement the clinical or serologic diagnosis of Lyme disease has so far been widely documented [1,[10][11][12][13][14][15][16][17][18][19][20][21]. However, the majority of the published PCR protocols are not suitable for day-to-day testing in a routine laboratory setting.…”

“…During the last decade, the PCR technology has been extensively used as an alternative diagnostic instrument for the detection of Borrelia burgdorferi DNA in clinical specimens [1,[10][11][12][13][14][15][16][17][18][19][20]. In these studies, the PCR assay allowed the successful detection of Borrelia burgdorferi DNA in blood, CSF, urine, skin biopsy, synovial membrane and synovial fluid specimens obtained from selected categories of patients with Lyme disease.…”

Polymerase Chain Reaction Analysis of Borrelia Species Isolated in Japan

“…Serodiagnosis is generally used for the diagnosis of Lyme disease in Japan. Recently, polymerase chain reaction (PCR) (31) has been applied for the diagnosis of Lyme disease in Europe (21) and North America (16). However, it is not shown whether these primers are able to react with Japanese isolates.…”

A road map for PCR from clinical material

“…Results like these are not entirely unexpected when it is kept in mind that urine is the main drainage point for metabolite and drug excretion from the body, such that its composition is therefore highly variab1e.A more subtle demonstration of the extent of this inhibition is shown by a study that involved "seeding" urine samples with the spirochaete Borrelia burgdorferi, whereby the sensitivity of the PCR decreased ten-fold compared with the sensitivity when a B . burgdorferi suspension in PBS was used (60).…”

“…The majority of pubIished articles describing the PCR detection of bacteria, including Mycobacteuiurn sp., within urine involve proteinase Wdetergent treatment of bacterial pellets (69,70) followed by either boiling (71) or, in some cases, by phenol/chloroform extraction and DNA precipitation (62,72). Simpler variations on these themes that appear to be equally effective, at least in some instances, include the use of boiling and washing steps prior to PCR (70,73), sonication (tends to produce erratic results, 70), and the addition of a Chelex-100 resin solution (60). It should be remembered that in a few of these studies artificially "seeded" urine samples are used, such that the usual variability in terms of the inhibitory Other methods initially described for preparation of urine samples containing CMV that appear to work well and that are likely to have general application for the detection of a broader range of infectious agents include dialysis (effective in some instances, although considered too cumbersome as a routine method for treating urine samples) (63), PEG 6000 precipitation of viral particles (63,67), and glass-powder ("glass-milk") treatment (59,67).…”