Evaluation of the detection ofBorrelia burgdorferi DNA in urine samples by polymerase chain reaction

Abstract

It is difficult in some cases to identify an infection caused by Borrelia burgdorferi and to monitor the effect of therapy. Seropositivity will persist even after successful treatment and therefore may suggest ongoing infection. For direct detection of B. burgdorferi DNA in human urine samples, the polymerase chain reaction (PCR) was evaluated. A published primer system was selected, which amplifies a 259 bp fragment from the gene encoding the 23S rRNA. The lower detection limit of the primer system was 10 fg of extracted B. burgdorferi DNA. Several methods for the pretreatment of urine samples were tested. Of these, the Geneclean kit (Bio 101, USA) showed the best results. A total of 114 urine samples from 74 patients belonging to three clinical groups was investigated: (i) 51 samples from 26 patients with active Lyme disease, (ii) 36 samples from 27 patients with previous infection but no symptoms at the time the urine was collected, and (iii) 27 samples from 21 seronegative control patients without Lyme disease. B. burgdorferi DNA was detected in 25 urine samples of 17 patients with active disease, whereas 26 samples from this group of patients were negative. Only one asymptomatic case with previous infection showed a positive result, and the urine samples of the patients without Lyme disease were uniformly negative. Two of four patients from whom samples before and directly after onset of therapy were available converted from negative to positive PCR results after initiation of therapy, accompanied by the symptoms of a Jarisch-Herxheimer reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Methodological Aspects

“…In addition to reports summarized in Table 7, B. burgdorferi DNA was detected by PCR in blood (189), SF (140,165), CSF (101), and urine (52,79,105,155,189) of patients with LA. Maiwald et al (105) found that 11 of 15 patients with LA (1 with arthralgia and a typical history of a tick bite and EM, 9 with arthritis as a major symptom with other etiologies ruled out, and 5 with arthritis in combination with other manifestations) were reactive on tests of urine by a PCR that amplified a 259-bp fragment of the 23S rRNA gene (161).…”

“…Not surprisingly, procedures involving simpler methods have been developed; these include simple boiling of the specimen (88), centrifugation and boiling (31), alkali lysis (111), adsorption to coated or uncoated silica in the presence of chaotropic salts (7,21,51,68,105), boiling and concentration by centrifugation and ultrafiltration (128), and boiling in the presence of a cation exchanger (Chelex 100; Bio-Rad, Richmond, Calif.) (93). Although the mechanism by which Chelex improves the DNA extraction, is not known, it is thought to aid in stabilizing the DNA double helix (182).…”

“…DNA extraction procedures have been compared in a dilution study of B. burgdorferi with sterile water and CSF (97) and a few clinical studies with urine (68,93,105), CSF (68,128), and synovial fluid (SF) (123) as specimens for PCR.…”

“…Urine can be a suitable specimen for diagnosis; however, little is known about how spirochetes invade the urine or if whole borreliae are present in human urine, over though borreliae have been previously demonstrated in the urine of infected mice. By using a method in which soluble DNA is adsorbed to glass beads without previous enzymatic digestion or boiling, it was recently shown that soluble DNA or antigen-associated DNA is excreted into the urine (105).…”

“…Maiwald et al (105) found that 11 of 15 patients with LA (1 with arthralgia and a typical history of a tick bite and EM, 9 with arthritis as a major symptom with other etiologies ruled out, and 5 with arthritis in combination with other manifestations) were reactive on tests of urine by a PCR that amplified a 259-bp fragment of the 23S rRNA gene (161). A flagellinspecific DNA could be amplified in urine from 9 of 24 (37.5%) patients with LA, but no further details of the patients are given (79).…”