Persistence of Borrelia garinii and Borrelia afzelii in patients with lyme arthritis

Zent.bl. Bakteriol. 289, 301-318 (1999) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/zbl bakteriol

Zentralblatt fUr

Persistence of Borrelia garinii and Borrelia afzelii in Patients with Lyme Arthritis D. Hulinska, J. Votypka, and M. Valesova 1 National Institute of Public Health, Prague 1 Internal Clinic, 3 rd Faculty of Medicine, Charles University, Prague, Czech Republic

Received July 25, 1998 . Revision received January 22, 1999 . Accepted February 23, 1999

Summary We repeatedly detected DNA of Borrelia garinii or B. afzelii and Borrelia-like struc­ tures in the blood, joint fluid or in the synovium of 10 patients with Lyme arthritis by means of the polymerase chain reaction and immunoelectron microscopy at 2-4month intervals in the course of two years. All samples were analyzed using primers which amplified the 165 rRNA gene sequence of Borrelia burgdorferi sensu lato and nucleotide sequences for the OspA gene. No cross hybridization occurred with DNA from human cells and with DNA from other bacteria. Capture and labelling with monoclonal antibodies of aggregated antigens, membranes and flagellae were evident in the blood of 7 patients, in 4 synovial membranes and 2 synovial fluids. Borreliae were found in blood capillaries, in collagen and in clusters surrounding inflammatory cells in the synovium of patients with recurrent infections who carried IgM and IgG antibodies to OspA and to 83 kDa core protein. After significant improvement for sev­ eral weeks after treatment, arthritis recurred in six patients. 5ynoviocyte hyperplasia, inflammatory infiltration and concentric adventitial fibroplasia were seen in the syn­ ovium of the patients with persisting borreliae. Only two patients were infected with B. afzelii, the others with B. garinii.

Introduction Lyme borreliosis (LB) is an increasingly important public health problem throughout the world. It is endemic in the Czech Republic, where it is caused by the heterogenic strains of Borrelia burgdorferi sensu lato which are trans­ mitted to human and animal hosts by Ixodes ticks (4, 6). Early infection is 21

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D. Huifnska,]. Votypka, and M. Valesova

characterized by the appearance of erythema migrans (EM). In many cases, however, this early symptom is not noticed. If left untreated, more serious symptoms of late Lyme borreliosis (LB) may develop (22). A major problem with managing LB is the difficulty in recognizing periods of active infection. Antibody serology for LB is useful for confirming probable exposure to the Borrelia antigen but does not distinguish between active and inactive infection (13). A positive antibody response may persist long after the borreliae have been eradicated (23) or may result from cross reactivity with other infections (7). Furthermore, serological assays for LB have so far not been standardized (12). In Europe, three species pathogenic for humans (3) and at least eight serotypes of B. burgdorferi sensu lato (26) are known. This heterogeneity complicates the comparison of interpretation criteria for sera from patients in the early and late stages of LB. Different genospecies of Bor­ relia are associated with distinct clinical manifestations of LB (2). It is known that detection or even cultivation of borreliae in the case of late LB, namely from the synovium or synovial fluid, is successful only in excep­ tional cases (21,24). Nevertheless, borreliae have been reported to persist (9), even after treatment (17). The application of the polymerase chain reaction (peR) for the detection of borrelial DNA in blood, cerebrospinal fluid, urine, skin (28), and synovial fluid (21) of patients in the early and late stages of LB can provide significant advantages also prior to the appearance of a specific antibody response (1). Disadvantages of peR detection of borrelial DNA have been reported following treatment (17). In this study, we investigated a group of 10 patients who had been select­ ed from 51 patients with serology positive for LB and who had repeatedly shown clinical symptoms of arthritis in the course of their illness. The group was completed by six patients with late Lyme arthritis documented previ­ ously (27) in the study following treatment with ceftriaxone and who agreed to a repeated taking of samples for investigation by means of peR, immu­ nosorbent electron microscopy (IEM) and Western blot (WB) analysis at reg­ ular intervals for two years to monitor any persistence of Borrelia burgdor­ feri sensu lato and to identify possible recurrent active infection even after treatment.

Materials and Methods Patients We describe a selected group of 16 out of 51 patients who had positive serology and recurrent clinical symptoms of arthritis in early or late LB. Pain and swelling in the knee or other joint, bilateral knee or shoulder pain without reddening or swelling or multiple joints pains and tenderness with negative testing for rheumatoid arthritis were the main signs. The patients were enrolled at the Rheumatologic, Infectious, Neuro­ logic, and Dermatologic Clinics of the Faculty Hospitals in Prague between March 1995 and December 1997. A group of 10 patients was repeatedly tested by direct and

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indirect laboratory methods and clinically observed at regular intervals for two years. The six patients suffering from late stage Lyme borreliosis with involvement of the joints was followed for up to one year after a 14-day course of 2 g ceftriaxone once daily i. v. The peripheral nervous system was affected in the course of the illness in 7 patients. There were polyneuropathy and abnormalities of the radicular motor func­ tion. Only five patients had previously suffered from erythema migrans and one had acrodermatitis chronica atrophicans. Blood specimens were collected at regular inter­ vals over a 2-year period and examined for the presence of Lyme-specific antibody and borrelial DNA or Borrelia cells. Biopsy of the synovial membrane (SM) was performed during arthroscopy in 5 patients. Synovial fluid (SF) was collected using arthrocente­ sis in 7 patients. Decisions for administration of therapy were made largely on the basis of manifestations. Data collected by physicians include manifestations such as erythema migrans (EM), arthritis (A), or neurological symptoms (N) and beginning of therapy (pointed in Tables 2-3). The control group of 4 patients with positive rheu­ matoid arthritis and negative serology for LB, as well as 4 healthy blood donors were used for PCR testing of the specificity of the primer sets applied.

DNA isolation and PCR Whole blood (5 ml) and synovial fluid were collected in tubes with citrate for PCR and IEM investigation. The buffy coat on top of the red cells was collected separately. The DNA was extracted using QiaAmp columms (QIAGEN Tissue DNA isolation kit, QIAGEN Inc., CA, USA) according to the manufactor's instructions. The cells (buffy coat) or synovial biopsy material were prepared with proteinase K added to a DNA lysis buffer and incubated for 1 hour at 55°C. DNA was extracted with phenol/chlo­ roform and the aqueous layer was precipitated with isopropanol. The DNA pellet was rinsed with 70 % ethanol and resuspended in water. The primers used for amplification were selected according to Marcone and Garon (19), who defined phylogenetically significant target sequences, based upon 16S rRNA signature nucleotide analysis, i. e. the LD primers known to amplify all species asso­ ciated with LB to generate an amplification product of 357 bases. The BB primer set, BG and VS461 primer sets generate amplification products of 169, 574 and 519 bp and differentiate between B. garinii and B. afzelii. The other primers used were select­ ed specifically to target a 600 bp or 448 bp region of the OspA gene (Table 1) and a 110 bp region of the OspC gene (kindly donated by B. J. Luft, SUNY). All PCR com­ ponents were obtained from the GeneAmp kit and used as recommended by the sup­ plier (Perkin-Elmer Cetus). PCRs were performed with a PTC-200 DNA Engine (MJResearch, Inc. Massachusetts, USA) thermocycler by denaturing for 1 min at 94°C, followed by 35 three-step cycles recommended. Control samples included DNA exracted from control patients, two blank control samples with 5 !-II of water subsitut­ ed for DNA, and positive control samples with 1.5 and 15 pg of total B. garinii DNA (strain Ml92), of B. afzelii DNA (strain Kc90), also together with negative control samples from patients with rheumatoid arthritis and four healthy donors. All positive samples were tested by at least two different primer sets. Ten percent of the products amplified by PCR were analyzed by horizontal electrophoresis on 1.5 % agarose gels using standard electrophoresis conditions (20) with molecular size markers pUC18 HAEIII, and PCR Low ladders 100 bp (Sigma). An oligonucleotide corresponding to an internal sequence among the OspA prim­ ers was selected as a probe and labelled with digoxigenin-ll-dUTP using the random primed method (Boehringer Mannheim) for Southern blots. PCR products were trans-

304

D. Hulinska, J. Votypka, and M. Valesova

Table 1. Oligonucleotide Primer and Probe Sequences Oligonucleotide (Base No.)

Sequence

Set 1 (prof. B. Luft, SUNY) 3'pC (BamHl) Primers for OspC 5'pC (Nde1)

GTC GGC GGA TCC-ITA AGG TIT TIT TGG ACT ITC TGC CTG CGC CAC CAT-ATG AAA AGA ATA CAT TAA GTG CG

Set 2 (prof. B. Luft, SUNY) Primers 5'pA (+) for OspA 5'pA (-)

AAA AAA TAT TTA TTG GGA ATA GG-3' GT TTT TIT GCT CIT TAC ACT AAT TGT TAA-3'

Set 3 (M. M. Manak et al.) Primers (+) Strand 5'pA for OspA (-) Strand 5'pA Probe 5'pA

TGG ATC TGG AGT ACT TGA AGG CGT-3' AGT GCC TGA AIT CCA AGC TGC AGT-3' -NH2 - TAA-CAA GAG CAG ACG GAA CCA GAC-3'

Set 4 (Rosa and Caron) Universal BB primers (+) Strand 5' (-) Strand 5'

GGG ATG TAG CAA TAC ATC T-3' ATA TAG TIT CCA ACA TAG G-3'

Primers for BG B.garinii (+) Strand 5' (-) Strand 5'

GGG ATG TAG CAA TAC ATC T-3' ATA TAG TIT CCA CA TAG T-3'

Primers for VS451 B. afzelii (+) Strand 5' (-) Strand 5'

GCA TGC AAG TCA AAC GGA-3' ATA TAG TIT CCA ACA TAG T-3'

ferred from the agarose gels to a nylon membrane by using standard blotting con­ ditions (Bio-Rad, Trans-blot SD cell for semi-dry electrophoretic DNA transfer). Blotting was performed on a nylon membrane (Hybond-N, Amersham). Following transfer, the membranes were washed, DNA-fixed, denatured and neutralized as rec­ ommended. Southern blots were prehybridized for 2 h and then hybridized for 12 h at 42°C in 50 % formamide-5X SSC (5 X 0.15 M NaCI plus 0.015 M sodium citrate) 0.1 % N laurylsarcosine -5 % blocking agent. The bound probe was visualized with digoxigenin antisera conjugated to alkaline phosphatase. Colour development was produced with 4-Nitro Blue Tetrazolium in substrate buffer (Bio-Rad). In order to simplify procedures for testing, large numbers of samples were measu~ed using the solid phase sandwich hybridization system with a specific oligonucleotide covalently linked to microtiter wells and with a specific biotinylated revelation probe (Biocode, Prob-Lyme, Belgium). The presence of borrelial DNA in a sample was deter­ mined by relating the optical density (OD = 450 nm) of the unknown sample to that of the cut-off value, which had been defined as 3.5 X standard deviation of 4 negative controls + average of 4 negative controls. For the inhibitor test, each specimen was

Persistence of Borrelia

305

spiked with genomic DNA of E. coli and related primers recommended by Perkin­ Elmer Cetus. Electron microscopy Synovium biopsy specimens, blood and synovial fluid, and cultures were studied by electron microscopy using negative staining techniques, immunosorbent electron mi­ croscopy (8) and sectioning techniques. Tissue samples were fixed in 4 % paraformaldhyde with 1.5 % glutaraldehyde in 0.1 M cacodylate buffer, washed, dehydrated and embedded into Epon 812 and for immunode into Lowicryl K4M resin (Polyscience, Warrington, PAl. Semithick and semithin sections were cut using ultracut Reichert and LKB equipment and stained by the modified Bosma technique with amylase pretreatment or processed sections (Low­ icryl K4M) with monoclonal antibodies (Mab) and secondary goat anti-mouse anti­ bodies conjugated with peroxidase (Sigma) for detection of borrelial structures in sem­ ithick sections under the light microscope or with uranyl acetate and lead citrate for ultrastructural examination under the Jeol PC XII electron microscope. The samples of blood plasma and synovial fluid were assayed by the immunocap­ ture of immunosorbent method (IEM) as described previously (8, 26). The blood sam­ ples of healthy donors spiked with different amounts of Borrelia and reference blood samples from mice prepared for IEM served as positive controls, respectively. Serology IgG and IgM titres to the Lyme spirochete were determined by ELISA using standard conditions as recommended by the supplier (Test-Line Ltd, CR).The 98 th percentile of absorbance values for the controls was used as the cut-off level. In ELISA with soni­ cated antigens, a positive value was defined as -0.730 for IgG and -0.850 for IgM. In the IgM capture ELISA (Dako Diagnostika GmbH), a positive value was -0.380. Western blot (WB) with commercial Borrelia garinii Western blot IgG and IgM kits (Biowestern Ltd, CR) was performed, or WB IgM was prepared "in-house" and stan­ dardized as recommended (12). Sonicated antigens (strains M192 and Kc90) were sep­ arated in 10.5 % polyacrylamide gel (acrylamide-bisacrylamide 37.5: 1, Bio-Rad) and transferred to a nitrocellulose sheet (4.5 !-1m, Sigma) by a semi-dry blotter (Trans blot SD Semi-dry cell transfer, Bio-Rad). The strips were incubated with 1 : 200 dilutions of sera or fluids after a blocking step, washed and incubated with swine anti-human IgM conjugated to peroxidase under standard conditions. Monoclonal antibodies (Mab) against OspA, OspB, P83 and flagellin of B. garinii and B. afzelii (Mab 184, 336.36, 148) were kindly donated by Professor B. Luft, SUNY, USA. Cultivation Cultures were established from 10 blood samples, from 4 joint fluid samples and 2 samples of synovium in modified BSK-H (Barbour-Stonner-Kelly) medium (Sigma). A 0.2 f1m membrane filter (Sartorius No.SM) was used for the filtration of the medium and horse serum supplement. All culture tubes were incubated at 33°C and examined by darkfield microscopy once in 6 weeks. After two or three passages, the isolates were stored at -70°C.

306

D. Hulinska, J. VotYpka, and M. Valesova

Results Initially, 51 patients who had shown evidence of Lyme disease (LD) were entered into the study. Inclusion criteria were a confirmed diagnosis of late Lyme borreliosis based on the presence of at least one joint manifestation and positive immune response confirmed by WB. Only 10 patients in the group continued to have recurrent bouts of arthritis during the follow-up period. Six patients had long-term chronic arthritis (27). The ten patients' histories and results of laboratory tests are outlined in Tables 2-3. Five patients had pre­ ceding dermatological symptoms presenting as erythema migrans (EM), one patient had acrodermatitis chronic a atrophicans (ACA) after untreated EM, and 7 patients had at least once a nervous system manifestation in the course of the illness. . Borrelial DNA was detected with at least one primer-probe set in samples of the blood of ten patients (Fig. 1), in the synovial fluid of six patients, and in the synovial membrane of four patients (Tables 2-3). The other six chron­ ic patients previously tested had borreliae that could be assigned to European strains (BB and LD primers). They were investigated at irregular intervals and therefore have not been included in the tables. Borrelia garinii DNA (BG primers) was detected in 8 patients, B. afzelii DNA (VS461 primers) in 2 patients (Nos.4 and 7). Cross-amplifications between species with the species-specific primers BG and VS461 did not occur. All control samples taken from blood donors and from 4 patients with rheumatoid arthritis were PCR-negative. All blank control samples included with each sample tested were negative. The sensitivity of the chromosomal primer-probe set 16S rRNA was lower than that of the OspA primer-probe set. Titration of the input amount of template DNA used in the PCR reactions and subsequent agarose gel electophoresis of 10 % of the reaction volume that a template input of 15 fg (OspA primers) and of 35 fg (BG primers) was sufficient to visualize the amplification products (Fig. 2). The lower limits of detection of template B. garinii DNA in gel electrophoresis were made better visible by the use of hybridization. The amount of 3.5 fg of DNA from pa­ tients Nos. 2 and 8 were readily detected by spot blot (Fig.3A) as well as by Southern blot (Fig.3B). The presence of a large excess of nonspecific DNA (E. coli) or human lymphocyte DNA had no effect on the sensitivity of detec­ tion and successfully served as inhibitor test. The solid phase hybridization system using a biotinylated probe was posi­ tive for DNA of Borrelia burgdorferi sensu lato in all confirmed samples from PCR-positive patients. The optical density of these samples was close to that of the positive control. Borrelial antigens were detected by IEM in the blood of 7 patients who were also PCR-positive but at different times. Positive PCR and IEM results were seen during the same period of investigation in 4 patients only. A comparison of these methods showed that detection of borreliae in the blood by IEM was positive also after or at the time of treatment while the PCR was negative.

M

M

M

1

2

3

56

62

58

42

Age

1 2 4 6 8 16 1 2 4 6 8 12 14 18 1 2 4 8 1 4 5 6 8 12 16

Lab outcome Month

++ +/+/-

+/-

IgM

+ +

+ +

+ + + + +/+/-

+++ + + +/+ +

IgG

WB

+/+/+/+/+ +++++ + +/++++ ++++ +/+++ + imc +++ imc +++ +/+/-

lmc

IgG

+/+++ ++++ + + + + + + lmc +/+ imc +

IgM

ELISA

0

+++

0 0

+

0 00 00

00 0 0 00 00 00

+

0

00 00 00

0 0

Blood SF

peR

0 0 0

0 0

0 0

0 0 0 0000

0

0 0

SM

+

+

+

+

0 0

0 00 00

0 0 0 00 00 0

0

0 00 00 00

0 0

Blood SF

!EM

0 0 0

+ +

+

++

0 0 0 0 0 0

+

+

++

0 0 0

++

0 0 0 0

SM

Therapy

+ +/-

+/-

EM

+ + +

+

+ +/-

+

+ + + +/+ +

AN

+

+

Manifestation

0= not done, + = positive, -= negative, imc = immunocomplexes, EM = erythema migrans, A = arthritis, N = neurological symptoms, WB = Western blot.

4F

Sex

No

Table 2. Long-term results of laboratory methods in patients with marked improvement of Lyme arthritis

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0

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t:C

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13 1 3 6 8 13 2 5 7 13

11

15 1 2 4 5 7 13 17 1 5 8 13 1 3 5

II

1 2 5 9

Lab outcome Month

Imc

+ +

Imc

Imc

Imc

+

Imc

Imc

++++

Imc Imc

++

Imc Imc Imc 0

IgM

Imc

IgG

++ +++ ++

+ + + + + +

0

+ +

++

+/-

+ +/-

+

0

+ + +++ ++

+ + + + ++ +

+/+/+/+/+/++ ++++ ++

IgM

WB

Imc

+++

Imc

+

+ + + +/+ + +/+++ +

Imc

+

IgG

ELISA

+

+

+ +

+

+

0

+

0

+

++

00 00 00 0 0 0 00 00 00 0

0

++

00 0 0

0

00 00 00 0

0

+

0

+

00

0

Blood SF

peR

0

0 0 0

0 0

0

+

0 0 0 0

+

0 0 0 0

0 0

SM

+

+

+

+

+

+

+

00 0 00 0 0 0 0 00 00 00

0 0 00 0 0

0

0 00 00 00

0

00

00

0

Blood SF

!EM

0 0 0

0

0 0

+

0 0 0 0

0

+

0 0

0

0 0

SM

+

+

+

++

+

+

+

++

+

+ +

+

+

Therapy EM

+ +/+

+/++ + +

+ + + + + +

+

+

+ + + + + + + +/-

A

+

+ +

+/-

+ +/-

+ +

N

Manifestation

0= not done, + = positive, -= negative, imc = immunocomplexes, EM = erythema migrans, A = arthritis, N = neurological symptoms, therapy + = beginning of the treatment, WB = Western blot.

F

50

9F

10

56

8F

43

M

6

39

57

F

5

7M

Age

Sex

No

Table 3. Long-term results of laboratory methods in patients with persistent LD w 0

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Persistence of Borrelia 123456789

309

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Fig. 1. Agarose gel showing peR products after 35 cycles of amplification with 8 blood, 3 SF and 1 SM templates. Lane 1: DNA molecular weight marker pBR322 DNA-Hae III. Lanes 2-3: 574 bp bands of 35 fg and 1.5 pg template DNA of B. gari­ nii strain M192. Lane 4: negative control without template. Lanes 5-8: samples are from the blood of patients (Nos. 2, 4, 3, 1) at 2 months of infection. Results corre­ spond to results indicated in Table 2. Lanes 9, 11: amplification products from SM and SF of patient No.2. Lane 10, 12: negative results in SF of patients No.1 and 7 at 6 month of infection. Lanes 13, 14: negative results in blood samples of patient No.9. Lanes 15 and17, lanes 19 and 21: 574 bp bands of DNA template from the blood and from SF of patient No.8. Lanes 23, 25, 27: bands of template DNA isolated from SM of patients Nos. 6, 7, 8.

Capture and labelling after reaction with Mab of aggregated flocculent anti­ gens, membranous structures and flagellae were evident in the blood samples of patient No.1, 3, 4, 10. Little gold was observed on control grids incubat­ ed with uninfected control blood from blood donors or from uninfected mice. Borrelia-like cells were detected in the blood samples (Fig.4A) of patients Nos.8 and 9 during the period without treatment. Borrelia afzelii strain (H118) was cultured from the blood of patient No.9. The structure of B. af­ zelii culture in BSK-H medium (Fig.4B) was slightly different from the struc­ ture of directly detected borreliae in the blood of the patient by IEM. The outer surface membrane of cultured spirochetes was always thick, swollen and forming a pleomorphous envelope. Borreliae in the blood had a thin, fragile outer membrane. This fragility of the outer membrane was the reason for some spirochetes to be detected in the blood without flagellae and outer membrane. No evidence of OspA antibody specifically labelled with gold was detected in the blood in the early phase of infection (yd month), contrary to the BSK-H culture of B. afzelii. The embedded synovial tissue from five arthroscopies that we examined was IEM-positive in three patients (No.2, 6, 8). Synovial biopsy revealed hypertrophy of synovial villi. Histology disclosed synoviocyte hyperplasia and

310

D. Hulinska, J. Votypka, and M. Valesova

123456789

10 11 12 13

14 15 16

17 18 19

20 21 22 23

24 25 26

Fig. 2. Test of sensitivity of BG primer set. Thirty-five cycles were performed and dif­ ferent amounts of template DNA were analysed in a 1 % agarose gel. Lane 1 and 26: wide-range DNA marker (Sigma). Lane 3: positive control. Lanes 4-5: negative con­ trols. Lanes 6-13: template DNA of patient No.8 at reaction amounts of 0.35 fg, 3.50 fg (lane 7), 35 fg (lane 8), 350 fg (lane 9), 3.5 pg, 35 pg and 3.5 ng, 35 ng. Lane 16: 35 fg DNA of patient No.2. Lanes 17-24: B. garinii strain Ml92 at the same re­ action amount as for patient No.8. Lane 25: negative control. For description of me­ thods see text.

marked inflammatory infiltration, sometimes with focal fibrosis. Vasculitis was seen in the synovial inner layer. Borrelia-like cells were found in the lumen of the blood capillary (Fig. 5) in patient No.2 suffering from arthritis. The electron-dense spiral structures with a diameter of 0.25-0.30 [tm and a thick surface membrane lying in parallel with collagen fibrils or in clusters around inflammatory cells (Fig. 6) were seen in the sections through the fibrous tissue of patient No.6. Unusual cyst-like structures were seen in the synovium of patient No.8. Six of the 10 patients had a persistent infection which caused persistent joint manifestations and arthralgias despite repeated treatment. A marked improvement of LB was found in 4 patients. In the following, short comments are made concerning some of these patients. Patient No.2 (Table 2) had pos­ itive PCR in the blood, in SF and SM two months after initial clinical and ser­ ological (positive IgG, IgM ELISA, WB) evaluation of the arthritis as LB. Two months after antibiotic treatment with doxycycline (2 X 200 mg daily for 1 week and then 2 X 100 mg daily for 1 week), the patient was PCR negative and had positive IgG and IgM serology in the ELISA. IgM WB showed the re­ action against the OspC and OspA surface antigens. Four months later (8 th month) he presented again with synovitis and positive PCR for B. garinii in SF and therefore he was treated with 2 g of ceftriaxone parenterally once daily for 2 weeks. During the period of four months after treatment, the patient was

Persistence of Borrelia 311

Fig. 3 A. Hybridization of BG gene probe to detect B. garinii DNA. The first row of spots are three negative and two positive controls (3,4). The positive controls are 5 ng and 3 ng of the biotinylated 20-mer probe. The negative control spots are Nonidet P40. The second row of spots are 574 bp amplified products generated in the PCR with 3.50 fg template DNA from two positive patents, Nos. 2 and 8 (spots 6 and 10) and three negative patients, Nos. 3, 4, 7. Fig. 3 B. Hybridization of the OspA gene probe to B. burgdorferi sensu lato in South­ ern blot shows two positive bands of 375 bp amplified products generated in the PCR with 35 fg template DNA from two positive controls (bands 3, 4) and from patients Nos. 2 and 8 (bands 6, 10).

negative and without serious complaints, only with positive IgG serology. Six months after treatment the patient had negative peR in the blood, negative IgM immunocomplexes and positive IgG antibody response but lacked a reaction to OspA antigen. The patient had clinically recurrent symptoms but after a second treatment with another antibiotic, he showed response to the change in therapy and thereafter remained PeR-negative. Patient No.4 (Table 2) had EM after a tick bite and later, an unspecific skin lesion. She was treated with doxycyline (2 X 100 mg daily for 3 weeks) dur­ ing the period of high IgM and IgG positivity. peR was positive in the blood with the chromosomal primer set before treatment and later also with the OspA primer set. Aggregated flocculant antigens were evident in the blood in IEM. She was treated with doxycycline again and two months afterwards syn­ ovial fluid was taken. peR was positive in the SF with all primer sets and with

312

D. HuHnska, J. Votypka, and M. Vales ova

Persistence of Borrelia

313

the set complementary to the DNA of B. afzelii. The optical density of the sample in the sandwich hybridization microwell plate assay was greater than 2.S units, similar to that of the positive control. The patient was treated with 2 g of ceftriaxone i. v. daily for 14 days, became PCR-negative and remained PCR-negative at all subsequent visits. IgM positivity appeared shortly follow­ ing EM and persisted throughout the period of PCR positivity; it became neg­ ative after successful treatment. Positive IgG serology persisted for two years. Patient No.8 (Table 3) who had late LB presented with late stage symptoms such as peripheral neuropathy and paresthesia instead of arthritis that could not be cured with oral doxycycline 100 mg b. i. d. over 21 days. Initial obser­ vation of the blood was PCR-positive with OspA primers and with species­ specific primers for B. garinii. Three months later, borreliae were found in the synovial membrane with IEM, PCR was positive in SF and SM. She had neg­ ative IgM antibody in the ELISA but immunocomplexes appeared shortly fol­ lowing onset of the second therapeutic run with doxycycline for 21 days and persisted throughout the period of observation. PCR was positive in the blood despite repeated treatment. Cultivation in BSK-H medium was positive only in one blood sample of patient No.9 who had positive immunocomplexes and positive PCR three months after initial EM. Borrelia afzelii (strain H118) was isolated. The pati­ ent was treated with doxycycline but after 6 months had recurrent complaints and positive PCR in the blood. Sera of all patients affected by late arthritis exhibited strong IgG reactivity to OspA protein, which was detected only with WB using the borrelial strain that had caused the patient's infection. This OspA-induced IgG and rarely, IgM, response was not expressed in the early stages of infection and was also eliminated in patients with resolved symptoms, contrary to patients with recurrent symptoms. On the other hand, IgM and IgG antibody response to OspC (24-21 kDa) was found in allS recurrent infections during late arthritis despite different antigens in WB. The late IgM immune response to OspA and 83-93 kDa extracellular protein, which was usually recognized by IgG anti­ body, behaved like a marker for late recurring arthritis.

Fig.4 A. Borrelia directly detected by IEM in the blood of patient No.9. The outer surface membrane (OM) is thin, without swelling and 6-7 flagellae (F) are beneath it. Basal knobs (B) are seen on the insertion parts of the flagellae. Reaction with Mab against OspA was negative (x 68 ODD, Mab 181, protein A-gold, negative staining with phosphotungstic acid [PTA]). Insert: 7 flagellae across the cell beneath the thin outer membrane (x 120000, PTA). Bar 0.1 ftm. Fig. 4 B. B. afzelii was cultured from the blood of patient No.9. The outer surface membrane (OM) is thick, swollen and positive with Mab 36 against OspA of B. afze­ Iii (arrow) (x 64000, Mab 36 protein A-gold). Insert: Some of the insertion points for the flagella in the cytoplasmic membrane and thick swollen outer membrane are shown (x 100000, PTA). Bar 0.1 ftm.

314

D. Hulinska,

J. VotYpka, and M. Valesova

Fig. 5. Electron micrograph of ultrathin section from Epon-embedded synovial tissue. Borrelial cells which showed indications of being close to division were in a blood cap­ illary surrounded with erythrocytes, fibrin and microvesicles (M). (x 3800, Uranyl acetate, lead citrate). Insert: cross sections of microvesicles around the surface of some parts of borreliae (x 9600, Ua, Lc).

Discussion Direct proof of borreliae is difficult in SF, in contrast to findings in SM, which limits understanding of the pathogenesis of Lyme arthritis (20) especially when arthritis persists despite antibiotic therapy. The cultivation of borreliae from body fluids is more difficult than from tissues. Borreliae have been cul­ tured from the SF of only two patients (23). We quite failed to culture borre­ liae from SF in spite of repeatedly positive PCR. Spirochetal forms were often seen in synovial tissue with the aid of immunohistological techniques (11,14,25) and with electron microscopy (27) but this method has been rat­ ed as not very conclusive. There is also limited clinical material from humans available for comparison. Dorward et al. (1991)developed and evaluated the immunocapture method for demonstrating Borrelia. We estimated that the combination of the immunocapture method or our immunoelectron micro­ scopic (IEM) method together with PCR can effectively reveal borreliae and when it is done in the same sample could confirm the diagnosis. Persisting Borreliae were reported even after treatment (5, 9, 17,23) suggesting that the

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Fig. 6. Borreliae were found in a cluster around a lymphocyte. Some parts of their thin surface membrane tightly adhere to the unchanged host cell cytoplasmic membrane. (x 8400, Ua, Lc). Insert: cross section through cyst-like (C) structures covered with a thick outer membrane, some of them are electron-transparent and contain electron­ dense bodies (x 12000, Ua, Lc).

spirochetes had not been eradicated (27). We found such persistent infection with B. garinii or B. afzelii in 6 of the 10 patients with recurrent clinical symp­ toms who were followed up during a two-year period. peR, WB and some­ times IEM methods were positive for months or years during subsequent episodes of arthritis. In the remaining patients, borrelial DNA or borrelia-like structures initially present were not detected during the last periods of follow­ up in the blood or synovial fluid samples, and also their arthritis receded with­ in a few months after treatment. We have shown that peR is a useful meth­ od for detecting DNA of B. burgdorferi sensu lato in synovial fluid, synovium and blood from patients with late Lyme arthritis and with recurrent episodes of disease. It seems that the detection of DNA in synovial fluid and in blood indicates the presence of viable spirochetes (25, 26, 27), namely when the IEM method has been positive and peR detection systems with primers for 16S rRNA (chromosomal target sequences) were used. Spirochetes shed OspA segments in the form of membrane vesicles containing extrachromosomal DNA (8) into body fluids and thus complicate peR detection of viable spiro-

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chetes. Therefore, we used the OspA primers and probes for hybridization only to confirm positive findings with 16S rRNA target segments (19). Histo­ logical findings (25) and ultrastructural detection of B. garinii in the syno­ vium (26) imply that spirochetes may survive for years in the affected syno­ vium. We do not agree with other authors (5, 8, 9, 11, 14) in that the number of organisms in affected tissues is small. It is true that it has been necessary to observe many microscopic fields to find borreliae because they are arranged around individual lymphocytes or fibrocytes in the form of focal clusters. They are most often extracellularly located and thus more influenced by the host environment in SF and the blood than in their intracellular location. It may be one reason for unsuccessful cultivation from SF and blood in contrast to succesful cultivation from the skin samples where borreliae were sometimes found intracellularly in fibroblasts and Langerhans cells and influenced a short time by the host environment and its immune reaction. Borreliae in the blood have a thin and fragile outer membrane, contrary to the thick outer envelope that covers borreliae inside cells. Collagen can influence successful cultivation in BSK-H medium from tissue. Successful treatment may corre­ spond with the characteristic IgM and IgG response in WB. IgM antibodies against OspC were found early or in the early stage of arthritis which is in agreement with other authors (12, 15,29). IgM reactions to OspA were seen only in recurrent infection in PCR- and IEM-positive patients. All chronic pa­ tients had IgG antibodies against OspA. Most patients were infected with B. garinii, only two patients with B. afze­ Iii, one of them was a patient with ACA lesion and one with EM. In contrast to the findings of other authors B. garinii was detected in all other patients with EM (2, 28). Acknowledgements. We are indebted to Martin Bojar, Departement of Neurology, 2 nd Faculty of Medicine, Charles University for assistance with neurological information concerning our patients. We thank Monika Ranna, Reference laboratory on Borrelio­ sis for serological technical assistance. This work was supported in parts by funds from Ministry of Health, CR grant IGA3491-3.

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