PATIENTS

Four patients with treatment resistant Lyme arthritis were investigated. After referral to our clinic Lyme borreliosis was diagnosed based on the following criteria: all patients lived in an area that is highly endemic for Lyme disease, all patients had a typical monarthritis or oligoarthritis involving the knees, no clinical or serological evidence for other inflammatory rheumatic diseases including rheumatoid arthritis, reactive arthritis or collagen vascular disease could be found, and all patients had a positive serological test for Lyme arthritis according to the CDC criteria.8 Table 1 gives the patients’ characteristics.

Patient 1, a 50 year old male patient had first been seen by an orthopaedic specialist because of an acute onset arthritis of the left knee. Serological tests had revealed a high IgG antibody titre againstB burgdorferi and the diagnosis of Lyme arthritis was made. Doxycycline, 200 mg/day, and diclofenac were prescribed and an intraarticular injection of dexamethasone had been performed. Arthritis persisted with rapidly recurring joint effusions and the patient was referred to our clinic. Here the patient presented with a moderate left gonarthritis and pronounced synovial proliferation. Serological examination for Lyme arthritis was positive for IgG antibodies in both ELISA and immunoblot. B burgdorferi DNA was detected in SF by PCR. The patient was treated with ceftriaxone, 2 g daily for two weeks. The arthritis improved significantly, but then recurred after about six weeks. The patient was treated with ceftriaxone again. Although PCR in synovial fluid became negative, gonarthritis persisted. A popliteal cyst developed and a bursitis of the left elbow occurred. An arthroscopic synovectomy of the left knee and a bursectomy were performed.

The second patient, a 51 year old woman, presented to our outpatient clinic with a two month history of an acute onset bilateral gonarthritis. The patient remembered having had a tick bite and an erythema migrans-like lesion at the right thigh eight years ago. Clinical and ultrasound examinations revealed an active arthritis of the left knee with synovial thickening, a pronounced synovial effusion, and a popliteal cyst. The right knee was only slightly swollen with a pasty thickening of the joint capsule. IgG antibodies against B burgdorferi were detectable by ELISA and immunoblot, and B burgdorferi PCR in SF was positive. The patient was treated with doxycycline 200 mg/day for four weeks without any effect. Ceftriaxone, 2 g/day, was given for three weeks with only moderate success. Two months after the antibiotic treatment had been completed the gonarthritis persisted. An arthrocentesis of the left knee with aspiration synovial biopsy was performed.

In patient 3, a 28 year old female patient, a subtotal arthroscopic synovectomy of the right knee had been performed elsewhere because of a gonarthritis with pronounced synovial proliferation that had persisted for nine months. Upon subsequent diagnostic procedures, a positive Lyme serology was observed. Lyme arthritis was diagnosed and treated with ceftriaxone. Treatment had to be discontinued at day 11 because of an allergic rash. Gonarthritis persisted and the patient was seen by a rheumatologist. PCR for B burgdorferi DNA was positive in SF, and doxycycline, 200 mg/day, was given for 30 days. In addition, corticosteroids were injected intraarticularly. However, only a temporary improvement of the arthritis was seen and the patient was referred to our clinic. Here, the patient presented with a severe arthritis of the right knee joint with a synovial effusion and a popliteal cyst. Lyme serology was positive for both IgG and IgM antibodies in ELISA and immunoblot. Arthroscopy was performed again showing a pronounced proliferation of the newly formed synovial membrane. SF samples and SM biopsy samples were obtained and subjected to PCR.

A 43 year old woman, patient 4, presented in our outpatient clinic complaining of a bilateral gonarthritis of seven months duration. ELISA and immunoblot showed a high IgG antibody titre against B burgdorferi with several specific bands, and the B burgdorferi PCR in urine and SF gave positive results. The patient was treated with ceftriaxone, 2 g daily for two weeks without any improvement. A few weeks later the patient was seen in another hospital with an acute polyarthritis involving both wrists and several metacarpophalangeal joints and a deterioration of the right gonarthritis. Treatment with doxycycline, 200 mg daily for 30 days, and prednisolone 10–20 mg per day was started. Again, there was no sufficient response and the patient was referred to our clinic. A closed needle arthrocentesis with synovial biopsy of the right knee was performed.

PCR

PCR was performed as described previously.4 In brief, 1–10 ml of SF or urine were centrifuged at 350 g for 20 minutes at 10°C. The pellet obtained was washed with 0.9% saline, cells were pelleted again, and DNA was extracted from the pellet by alkaline lysis. SM samples were disrupted mechanically, followed by a freeze and thaw procedure. The pieces obtained were subjected to alkaline lysis. Boiled B burgdorferi sensu stricto (strain LW2)9 were used as positive control (external amplification control). Negative controls with water and SF were also run in every determination.

Nested PCR was performed with two primer sets (TIB Molbiol, Berlin, Germany) targeting B burgdorferi specific gene sequences. One primer set (ospA primer) was specific for a portion of the plasmid located ospA gene (5′ outer primer GGG AAT AGG TCT AAT ATT AGC C; 3′ outer primer CAC TAA TTG TTA AAG TGG AAG T; 5′ nested primer GCA AAA TGT TAG CAG CCT TGA T; 3′ nested primer CTG TGT ATT CAA GTC TGG C; length of amplicons: 665 and 392 bp; EMBL accession number X66065).10 The second primer set (p66primer) targeted a sequence of a chromosomal gene encoding a 66 kDa protein (5′ outer primer CGA AGA TAC TAA ATC TGT; 3′ outer primer GAT CAA ATA TTT CAG CTT; 5′ nested primer TGC AGA AAC ACC TTT TGA AT; 3′ nested primer AAT CAG TTC CCA TTT GCA; length of amplicons 371 and 236 bp; EMBL accession number M58429).11 ,12

As an internal amplification control, a primer pair specific for thepyruvate dehydrogenase gene (PDH primer) was used (5′ GGT ATG GAT GAG GAG CTG GA; 3′ CAG CCC TCG ACT AAC CTT GT; length of the amplicon 185 bp; EMBL accession numberJ03576).13

The PCR reaction mixture (total volume 25 μl) contained 2.5 μl of the isolated DNA, 2.5 μl 10-fold PCR buffer (Perkin Elmer Cetus, Norwalk, CT; final concentration 10 mM TRIS-HCl, 50 mM KCl, 1.5 mM MgCl₂, 0.001% (w/v) gelatine), 0.3 μM of each primer, 200 μM of each nucleotide (Boehringer Mannheim, Mannheim, Germany), and 0.8 U of Taq polymerase (Ampli Taq, Perkin Elmer Cetus). The outer PCR was carried out with a total of 40 cycles: DNA was denatured at 94°C for one minute, primers annealed at 42°C for one minute, and extended at 72°C for one minute. Nested PCR was performed with 2.5 μl of the outer amplicons as template. Twenty five cycles were carried out with the temperature profile described above. Amplicons were visualised on a 3% agarose gel stained with ethidium bromide and photodocumented using a gel documentation system (Appligene imager, Appligene Oncor, Heidelberg, Germany).