Vol. 30, No. 7JOURNAL OF CLINICAL MICROBIOLOGY, July 1992, p. 1646-16530095-1137/92/071646-08$02.00/0Copyright ©3 1992, American Society for Microbiology

Detection of Borrelia burgdorfieri DNA in Urine Samples andCerebrospinal Fluid Samples from Patients with Early andLate Lyme Neuroborreliosis by Polymerase Chain Reaction

ANNE-METTE LEBECH* AND KLAUS HANSENBorrelia Laboratory, Department of Infection-Immunology, Division of Biotechnology,

Statens Seruminstitut, Copenhagen, Denmark

Received 17 January 1992/Accepted 26 March 1992

A polymerase chain reaction (PCR) was developed for use in the identification of a 248-bp fragment of theBorrelia burgdorferi flagellin gene in urine and cerebrospinal fluid (CSF) from patients with Lyme neurobor-reliosis. The specificities of the PCR products were confirmed by DNA-DNA hybridization with an internalprobe. The assay had a detection limit of 10 in vitro-cultivated B. burgdorferi. The PCR assay seemed to bespecies wide as well as species specific, since DNA from all 21 B. burgdorferi isolates from humans tested butnot from Borrelia hermsii or Treponema pallidum could be amplified. We tested 10 consecutively diagnosedpatients with untreated neuroborreliosis. There was lymphocytic pleocytosis and intrathecal B. burgdorferi-specific 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 ninepatients; five patients, including two patients with chronic neuroborreliosis, were PCR positive prior totreatment, whereas urine samples from the remaining four patients obtained 3 to 6 days after the onset oftherapy became PCR positive. All urine samples obtained >4 weeks after therapy were negative by PCR. PCRof 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 DNAdetection by PCR. Normalization of inflammatory CSF changes and the negative PCR results during follow-upeven in patients with chronic neuroborreliosis do not point to a persistent infection. The future role of PCR asa diagnostic tool for Lyme neuroborreliosis is still uncertain.

Lyme neuroborreliosis is a frequent and serious manifes-tation of Lyme borreliosis caused by the tick-borne spiro-chete Borrelia burgdorferi (7, 22, 29, 49). The best currentindicators of active neuroborreliosis are lymphocytic pleo-cytosis and B. bugdorferi-specific antibody synthesis withinthe cerebrospinal fluid (CSF) (21, 22, 29, 50). However, areliable and practical diagnostic assay for the direct demon-stration of B. burgdorferi in samples from patients would bepreferable. So far the most reliable method for direct detec-tion of B. burgdorferi in clinical samples has been in vitroculture, which is a difficult, time-consuming, and especiallywith regard to blood (5, 49) but also with regard to CSF (28,44, 49), a low-yield procedure. Considering the paucity ofspirochetes in pathological lesions and clinical specimens,diagnostic amplification of specific DNA sequences by thepolymerase chain reaction (PCR) (47) seemed an obvioussolution to this problem. Several sensitive PCR assays fordetection of either purified B. burgdorferi DNA or in vitro-cultivated spirochetes have been reported (34, 39, 46, 51).We recently compared the diagnostic sensitivity of in vitroculture and PCR for the detection of B. burgdorfen in tissuesfrom experimentally infected animals and found that PCR issuperior in terms of ease and sensitivity (33). However, onlya few reports have described the application of PCR toclinical samples from patients with Lyme borreliosis (11, 14,16, 32, 38). In those studies, the total number of patientsstudied was generally low and it was not stated whether theywere consecutive patients. As sample sources, some studiesused serum (16), whereas others used CSF (11, 32), skin

* Corresponding author.

biopsy (38), joint fluid (11), or urine (14) specimens. Thepractical role of PCR in the laboratory diagnosis of Lymeborreliosis is still unclear. It remains unknown in whichclinical manifestations PCR might be useful, which samplesource is optimal, and what diagnostic sensitivity isachieved.The aim of the present study was (i) to evaluate the

diagnostic performance of PCR in a series of consecutivepatients with definite Lyme neuroborreliosis by using urineand CSF as sample sources and (ii) to investigate theduration of B. burgdorferi DNA excretion in urine aftertherapy.

MATERIALS AND METHODS

Patients with active neuroborreliosis. Urine and CSF sam-ples were obtained from 10 patients with active neuroborre-liosis. The patients were successively diagnosed betweenNovember 1990 and July 1991. Eight of the patients hadearly (second-stage) neuroborreliosis (lymphocytic meningo-radiculitis), and two patients had late (third-stage) neurobor-reliosis (chronic progressive encephalomyelitis) (1, 22, 29).The diagnosis of neuroborreliosis was, in every patient,based on lymphocytic pleocytosis and definite B. burgdor-feri-specific intrathecal immunoglobulin M (IgM) and/or IgGantibody synthesis in CSF. All patients received 20 millionIU of penicillin G intravenously per day for 10 to 14 days.The clinical and relevant laboratory data for the 10 patientsare summarized in Tables 1 and 2.

Urine and CSF samples were obtained before antibiotictherapy was begun. Additional urine samples were obtained3 to 6 days after the start of treatment; 10 to 14 days after the

1646

on April 17, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

B. BURGDORFERI DETECTION IN URINE AND CSF BY PCR

TABLE 1. Clinical characteristics of patients examined in this study

Patient Sex"/age DiseasePatieno Sex"r)ge Dureato Clinical history Clinical follow-upno. (yr) duration

1 M/53 4 wk Painful radiculitis in lower back, sixth nerve palsy Complete recovery2 M/10 16 wk Headache, back pain, general symptoms, nausea, Complete recovery

intermittent fever and weight loss3 F/64 14 wk Headache and painful radiculitis in neck and shoul- Complete recovery

der girdle4 F/71 24 wk Erythema migrans; pronounced pain and dysesthesia Complete recovery

of left leg; severe, flaccid paraparesis inferior,most pronounced on left side

5 F/32 8 wk Painful dysesthesia in both groin and buttocks Complete recovery6 M/20 10 wk Hleadache, low-grade fever, and painful radiculitis in Complete recovery

lower back7 M/57 3 wk Tick bite, erythema migrans; headache, weight loss, Slight residual facial palsy

painful radiculitis in the neck and shoulder girdle;bilateral facial palsy and sixth nerve palsy

8 F/29 1 yr Erythema migrans followed by 1 yr of general symp- Complete recoverytoms, nausea, vomiting, headache, weight loss,and several episodes compatible with partial com-plex seizures

9 F/69 8 mo Transient sixth nerve palsy, fatigue, and weight loss; Complete recovery from general4 mo later onset of a progressive ataxic gait; slight symptoms but slight residualparaparesis inferior with bilateral Babinski's sign gait disturbance

10 M/10 1 wk Tick bite, erythema migrans, headache, and general Complete recoverysymptoms

M, male; F, female.

start of treatment, when therapy was completed; and 1 to 8.5 disease duration of 1 year before treatment. After therapy,months (median, 6 months) after therapy. CSF samples were no further progression of symptoms was seen and CSFobtained from five patients immediately after the completion parameters had normalized.of therapy and from both patients with chronic neuroborre- Control subjects. To evaluate the specificity of the urineliosis 3 months after therapy. assay, we investigated urine from (i) 25 healthy controls

Patients with previous neuroborreliosis. Urine was ob- (median age, 41 years; age range, 3 to 66 years) and (ii) 10tained from 10 patients (median age, 44.5 years; age range, 8 patients with urinary tract infections with the followingto 75 years) who had been treated for neuroborreliosis 8 bacteria: Proteus mirabilis (n = 3), Klebsiella pneumonia (nmonths to 5 years (median, 1 year) previously. The patients = 3), Eschenichia coli (n = 2), and Streptococcus faecalis (nfulfilled the same diagnostic criteria as those mentioned = 2). In each case a urine culture revealed >i10 bacteria perabove. Nine patients had early (second-stage) neuroborreli- ml.osis. All had recovered completely. One patient had late The specificity of the CSF assay was investigated by using(third-stage) chronic progressive encephalomyelitis with a CSF samples from (i) five patients with multiple sclerosis

TABLE 2. Laboratory findings for patients examined in this study

CSF examination CSF examination 14 days Intrathecal antibody production Anti-B. bwirgdorferibefoeterayaternseoftheapybefore therapy (specific capture antibody level in

Patient before therapy after onset of therapy antibody index)" serumhno. No. of leukocytes/pl Protein No. of leukocytes/,ul Protein

(0/ mononuclear concn (% mononuclear concn IgG IgM IgG IgMcells) (g/liter) cells) (g/liter)

1 221 (98) 1.38 88 (96) 0.86 28.1 2.9 0.220 0.9502 148 (94) 0.88 6 0.4 5.5 0.6 0.510 1.2803 24 (97) 1.02 17 (100) 0.68 2.6 6.3 0.990 0.5204 64 (99) 1.06 No sample No sample 2.3 3.5 0.510 0.3425 74 (66) 0.30 No sample No sample 43.3 0 0.032 0.5006 93 0.72 No sample No sample 3.0 0.32 0.920 0.8107 624 (94) 4.0) No sample No sample 13.2 3.6 0.085 1.4608 55 (98) 4.35 46 (95)' 2.8(l 1.4 0.43 0.540 0.2309 61 (98) 1.55 Not examined" 0.72' 2.9 0 0.860 0.40010 296 (100) 0.6 No sample No sample 0 9.7 0.040 0.450

"A specific capture antibody index of .t).3 indicates B. bwirgdorferi-specific intrathecal antibody production (21)."98K specific cutoff level in indirect IgG ELISA, OD = 0.160) (20); 98% specific cutoff level in capture IgM ELISA, OD = t).5t)0 (23).The value was 13 leukocytes per ,ul 3 months after therapy.

" The value was 0).97 g/liter 3 months after therapy.' The value was 2 leukocytes per ,l 3 months after therapy.'The value was t).6t) g/liter 3 months after therapy.

Vol.. 30, 1992 1647

on April 17, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

1648 LEBECH AND HANSEN

TABLE 3. B. burgdorferi strains

Strain Source' Supplier"

DK1 Skin, EM SSDK2 Skin, ACA SSDK3 Skin, ACA SSDK4 Skin, EM SSDK5 Skin, ACA SSDK6 CSF, LMR SSDK7 Skin, ACA SSDK8 Skin, ACA SSDK9 Skin, ACA SSDK21 Skin, EM SSDK29 Skin, EM SSP/Ko Skin, EM V. Preac-MursicPIBi CSF, LMR V. Preac-MursicP/Tm Skin, ACA V. Preac-Mursic297 CSF A. C. Steere272 Skin A. C. Steere245 Blood A. C. SteereB31 Tick A. G. BarbourSL10 CSF, LMR M. KarlssonSL14 CSF, LMR M. KarlssonSL42 CSF, LMR M. KarlssonACA-1 Skin, ACA E. AsbrinkB. hermsii K. Hovind-Hougen

a EM, erythema migrans; ACA, acrodermatitis chronica atrophicans; CSF,cerebrospinal fluid; LMR, lymphocytic meningoradiculitis.6 SS, Statens Seruminstitut, Copenhagen, Denmark; V. Preac-Mursic, Maxvon Pettenkofer Institute, Munich, Germany; A. C. Steere, Tufts NewEngland Medical Center, Boston, Mass. (49); A. G. Barbour, Health ScienceCenter, San Antonio, Tex.; M. Karlsson, Danderyd Sjukhus, Stockholm,Sweden (28); E. Asbrink, Sodersjukhuset, Stockholm, Sweden (2); K. Hov-ind-Hougen, Statens Veterinaere Serumlaboratorium, Copenhagen, Den-mark.

and (ii) 10 patients with the following central nervous systeminfections: varicella-zoster meningoencephalitis (n = 1),herpes simplex encephalitis (n = 2), meningococcal menin-gitis (n = 2), fungal encephalitis (n = 1), and other forms ofviral encephalitis (n = 4). The range of pleocytosis in theCSF of these patients was 12 x 106 to 3,797 x 106 cells perliter. All serum and CSF specimens from control individualswere seronegative for B. burgdorferi, and none had B.burgdorferi-specific intrathecal antibody production.

All urine and CSF samples were stored at -20°C until use.Spirochetal strains. The analytical sensitivity of our PCR

assay and the range of different B. burgdorferi strains whichwere detectable were tested by using the panel of 22 differentin vitro-cultivated B. burgdorfen isolates listed in Table 3.Except for the U.S. type strain B31, which is an isolate froma tick, all other strains were isolated from patients withLyme borreliosis; 18 strains were from Europe and 3 strainswere from North America. For the investigation of thespecies specificity of our PCR assay, we used DNA ex-tracted from Borrelia hermsii (a causative agent of relaps-ing fever) and Treponema pallidum (Nichols pathogenicstrains). All Borrelia strains were grown in BSK medium (4),and T. pallidum was grown in rabbit testicles (42).

Purification of spirochetal DNA and sample preparation forPCR from in vitro-cultivated B. burgdorferi. Total DNA fromall Borrelia strains and T. pallidum was extracted as de-scribed previously (18, 25). DNA concentrations were deter-mined spectrophotometrically by measuring the A260 (35).An amount of 100 ng of the preparations was used astemplate DNA.The analytical sensitivity of the PCR assay was investi-

gated on serially diluted, purified B. burgdorfeni DK1 DNA;

the dilutions ranged from 1 pRg/10 ,u to 0.001 pg/10 [L.Furthermore, the sensitivity was determined by using invitro-cultivated B. burgdorferi without prior DNA extrac-tion. For this purpose, samples with a cell density rangingfrom 106 to 10 B. burgdorferi per 10 RI were used. Thesewere obtained by a 10-fold dilution series with a phosphate-buffered saline (PBS) solution containing 108 B. burgdorferiDK1 per 10 RI, as determined by dark-field microscopy. Thesamples containing 1% (vol/vol) Triton X-100 were heated to100°C for 10 min. An aliquot of 10 RI was used as templateDNA.Sample preparation for PCR from patient specimens. (i)

Urine. Five milliliters of urine was heated to 100°C for 5 min,pelleted (15,000 x g for 30 min), and resuspended in 1 ml ofPBS (pH 7.4). The solution was then centrifuged at 20,000 xg for 20 min, and the pellet was dried under vacuum andresuspended in 20 RI of redistilled water. Twenty microlitersof a 5% Chelex-100-resin solution (catalog no. 142-2832;Bio-Rad, Richmond, Calif.) was added to the sample beforeit was heated to 100°C for 5 min, centrifuged for 1 min at3,000 x g, and subsequently chilled on ice.

Supernatants in amounts of 1, 5, 10, or 20 RI were used assources of template DNA.

(ii) CSF. Two different methods for the preparation ofDNA from CSF were investigated. The first technique (3)was based on DNA precipitation. Three hundred microlitersof CSF was heated to 100°C for 15 min; the DNA was thenprecipitated with 2 volumes of 99% (vol/vol) ethanol for 30min at - 20°C and finally pelleted by centrifugation for 30 minat 20,000 x g. The DNA pellet was washed with cold 70%ethanol, dried under vacuum, and resuspended in 20 ,ul ofredistilled water. The samples were heated to 100°C for 10min in the presence of 1% (vol/vol) Triton X-100 and weresubsequently chilled on ice. An aliquot of 20 RI was thenused as template DNA. In the second approach (15), 200 RIof CSF was added to 800 RI to PBS (pH 7.4), and the solutionwas centrifuged at 15,000 x g for 15 min. The dried pelletwas resuspended in 50 ,ul of redistilled H20, heated to 100°Cfor 10 min in the presence of 1% Triton X-100, and subse-quently chilled on ice. Twenty microliters was then sub-jected to PCR.

For optimization of the PCR conditions for clinical spec-imens, normal urine and CSF were artificially seeded with invitro-cultivated B. burgdorferi. A controlled number of B.burgdorferi DK1, ranging from 106 to 10 spirochetes, wasadded to either 5 ml of urine or 200 pul of CSF. Thesesimulated samples were treated as described above.PCR. In order to obtain PCR primers specific for all B.

burgdorfeni strains, we decided to amplify a segment of theflagellin-encoding gene of B. burgdorferi (13), which hasbeen shown to be highly conserved among strains (10, 13,51). To obtain a B. burgdorferi-specific amplification, theprimers were placed in areas nonhomologous to the se-quences of B. hennsii (45) and T. pallidum (9, 41) flagellingenes. The sequences and positions of the oligonucleotideprimers that amplified a 248-bp fragment are shown in Table4.PCR was performed in a reaction volume of 50 RI contain-

ing 1 U of Taq DNA polymerase (Amplitaq; Perkin-ElmerCetus, Norwalk, Conn.), 10 mM Tris hydrochloride (pH8.3), 50 mM KCI, 0.01% gelatin, 5.5 mM MgCl2, 200 puM(each) deoxynucleotide triphosphates (dATP, dCTP, dTTP,and dGTP), and 40 pmol of each primer. The PCR mixturewas overlaid with 45 p.l of mineral oil (M-3516; Sigma, St.Louis, Mo.). All reactions were performed in a thermalcycler (Techne PH-C-1; Techne Ltd., Cambridge, United

J. CLIN. MIC ROBIOL.

on April 17, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

B. BURGDORFERI DETECTION IN URINE AND CSF BY PCR 1649

TABLE 4. Sequences and positions of oligonucleotide primers for PCR

Gene and primer Sequence Coding strand Locationa

Gene encoding the B. burg-dorferi flagellin

F-7 5'-CTC TGG TGA GGG AGC TCA AAC-3' + 594-614F-3 5'-GTA CTA TTC TTT ATA GAT TC-3' - 823-842F-8b 5'-CAT CAC TTG CTA AAA TTG AAA ATG C-3' + 743-767

Gene encoding the humangastrin gene

12 5'-CCC CCA CAC CTC GTG GCA G-3' + 6481-649915 5'-GGC ACT CAG ATC TTC TCC CT-3' - 6885-6905

' Base positions are numbered according to the published sequence of the 41-kDa B. burgdorferi flagellin gene (13) or the sequence of the human gastrin gene(27).

b 32P-labeled oligonucleotide probe.

Kingdom). After an initial denaturation at 94°C for 4 min, thePCR conditions were denaturation at 94°C for 1 min, anneal-ing at 39°C for 2 min, and extension at 66°C for 3 min, for 40cycles. After the final cycle, the temperature was maintainedat 66°C for 5 min to complete the extension. The PCRs wereanalyzed for amplified products by agarose gel electropho-resis (1.5%) (Sea-Kem FMC, Rockland, Maine).

In order to evaluate a possible PCR inhibition because ofcomponents in urine, we performed an amplification of a444-bp fragment of the human gastrin gene (27) in parallel onsamples from two patients. The amplification was confirmedby agarose gel electrophoresis. The sequences and positionsof the gastrin oligonucleotide primers are shown in Table 4;they were kindly provided by Jens Bundgaard, Departmentof Infection-Immunology, Statens Seruminstitut, Copen-hagen. PCR conditions were as follows: denaturation at 94°Cfor 1 min, annealing at 50°C for 1 min, and extension at 72°Cfor 2 min, for 35 cycles.

Consecutive samples from one patient were always in-cluded within the same experiment. While preparing samplesas well as PCR mixtures, samples from patients and controlswere handled alternately within the same run. PCR mixtureswere made in a laminar airflow hood in a remote PCR-dedicated area which was never exposed to products of B.burgdorfen or final PCR products. The PCR facility wasexposed to UV illumination for at least 15 h betweenexperiments. Each PCR experiment included a negativecontrol in which water replaced template DNA and a posi-tive control which contained 1 ng of purified DNA from B.burgdorferi DK1. Pipetting was carried out with autoclav-able pipettes (one-pettes; Costar Corporation, Cambridge,Mass.) and aerosol-resistant tips (Scandinavian DiagnosticServices, Falkenberg, Sweden).

Southern blotting and slot blot hybridization. The specific-ity of the amplified PCR product was confirmed by DNA-DNA hybridization. Southern blotting was performed asdescribed previously (33). For slot blot analysis, 15 ,ul of thePCR products was heated to 100°C for 2 min, chilled on ice,and denatured in 100 p.1 of 0.4 M NaCl-24 mM EDTA for 15min at 4°C. Using a Slot blot minifold II (reference no. 447800; Schleicher & Schuell, Dassel, Germany), samples weredirectly applied to GeneScreen membranes (catalog no.NEF-976; Dupont, NEN Research Products, Boston, Mass.)which were presoaked in 0.4 M NaOH-0.6 M NaCl for 15min. The slots were washed twice with 200 p.l of 0.5 M Tris(pH 7)-i M NaCl and air dried. The membranes were thenbaked at 80°C for 2 h. The oligonucleotide probe F-8 (Table4) was end-labeled with [ct-32P]dCTP by using a terminus-

labeling kit (Enzo Diagnostics, Inc., New York, N.Y.).Membranes were hybridized and washed at 42°C and weresubsequently autoradiographed as described previously (33).Lyme borreliosis serology. Anti-B. burgdorferi IgG and

IgM in serum were measured by an indirect enzyme-linkedimmunosorbent assay (ELISA) and a ,u-capture ELISA (20,23). B. burgdorferi-specific intrathecal IgG and IgM antibodyproduction was measured as recently described by using a p.-and y-capture ELISA (21). All three assays used purified B.burgdorfen flagellum as the test antigen.

RESULTS

Sensitivity and specificity of the PCR for detection of B.burgdorferi DNA. As shown in Fig. 1, PCR yielded a singleamplified fragment of the expected size of 248 bp whenpurified DNA of B. burgdorfen DK1 was used as thetemplate. The authenticity of the amplification product wasconfirmed by Southern blotting, in which the PCR-amplifiedproduct hybridized to the probe F-8, which is complemen-tary to the central part of the target sequence. A 248-bpDNA fragment could be amplified, from all 22 B. burgdorfenstrains listed in Table 3, and all strains were shown tohybridize with the specific probe F-8.The species specificity of the PCR assay for B. burgdorferi

Ni 1 2 3 4 Mi

A

4029-

*22-1 -_

1 2 3 4s

B

FIG. 1. (A) PCR amplification of B. burgdorferi DK1 (1 ng) (lane1), T. pallidum (1 ng) (lane 2), and B. hermsii (1 ng) (lane 3)demonstrated by agarose gel electrophoresis after ethidium bromidestaining. Lane 4, negative control. pBR322 cleaved with Hindlll-Hinfl was included as a DNA size marker (in base pairs; lanes M).(B) Southern blot hybridization of PCR amplification of B. burgdor-feri DK1 (1 ng) (lane 1), T. pallidum (1 ng) (lane 2), and B. hermsii(1 ng) (lane 3). Lane 4, negative control. The amplification productswere identified by using the 32P-end-labeled probe F-8 (Table 4).

VOL. 30, 1992

on April 17, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

1650 LEBECH AND HANSEN

i,::: U r i rne

PATIENT No.

10

.9

*8I..

6 +

5 +

4 _

3 -

2 +

I

FIG. 2. Slot blot hybridization of PCR products obtained fromartificially seeded urine and CSF samples. As described in the text,volumes of 5 ml of urine (A) and 200 [LI of CSF (B) were seeded with106 spirochetes (lanes 1), 104 spirochetes (lanes 2), 103 spirochetes(lanes 3), 102 spirochetes (lanes 4), 10 spirochetes (lanes 5), or 0spirochetes (lanes 6). Lanes 7, positive control (1 ng of purified B.blurgdorferi DK1 DNA); lanes 8, negative control. The specificitiesof the amplification products were confirmed by using the 32P-end-labeled probe F-8. All PCRs shown here originated from the sameexperiment.

TREATMENT

I ++ ++ + _-

+ + -

+

+

+ +

I I., I I I0 3-6 10-14 1 2 3 4 5 6 7 8 9 10

DAYS MONTHS

FIG. 3. Detection by PCR of B. burgdorferi DNA in urine frompatients with active neuroborreliosis before and after treatment. +,positive by PCR; -, negative by PCR; *, the patients had chronicneuroborreliosis; O, patient CSF was positive for B. burgdorferibefore treatment. For patients 2 and 3, CSF was positive aftertherapy.

by using DNA from T. pallidum or B. hermsii as the templateis illustrated in Fig. 1. No amplification products of 248 bpcould be seen either in the agarose gel or in the Southernblot. Thus, the primers used in our PCR were B. burgdorferispecific and presumably reacted with all B. burgdorferistrains.The sensitivity of the PCR assay was determined on

serially diluted purified B. burgdorferi DNA; dilutionsranged from 1 p.g to 0.001 pg. The analytical sensitivityobtained was 0.01 pg when the amplified fragments weredetected with the radioactively labeled probe F-8. To esti-mate the sensitivity of the PCR assay in terms of theminimum number of spirochetes which could be detected,spirochetal solutions containing 106 to 10 spirochetes per 10pAl were used. By adding 10 .1l of these dilutions as templateDNA, a reproducible amplification was achieved when only10 spirochetes were added to the PCR mixture.With regard to artificially seeded urine and CSF samples,

the specific amplification could still be obtained when 100spirochetes were added to 5 ml of urine or 200 p.l of CSF(Fig. 2). Thus, the assay sensitivity corresponds to 20 to 50B. burgdorferi per ml of urine or CSF.

Detection of B. burgdorferi DNA in clinical specimens. (i)Urine specimens. B. burgdorferi DNA was detectable inurine samples from 9 of 10 patients with active neuroborre-liosis tested. A 248-bp fragment was seen on agarose gelelectrophoresis, and the amplification products were probedon a slot blot with the probe F-8 (Fig. 3 and 4). In fivepatients B. burgdorferi DNA was detected prior to treat-ment, whereas in the remaining patients PCR was positivefor B. burgdorferi DNA only for the second urine sample,which was obtained within 3 to 6 days after the onset oftherapy. A urine sample was obtained from seven patientsimmediately after therapy was completed. Urine samplesfrom two of the seven patients still contained detectableamounts of B. burgdorferi DNA. B. burgdorferi DNA wasfound in the urine of only one patient 4 weeks after therapy.All follow-up urine samples obtained 2 to 8.5 months (medi-

an, 6 months) after therapy were negative for B. burgdorferiDNA by PCR. Similarly, no B. burgdorferi DNA could bedetected by PCR in urine samples obtained from 10 individ-uals with a previously diagnosed and treated neuroborrelio-sis 8 months to 5 years before urine sampling. When thefrequencies of positive PCR results before treatment (5 of 10patients) and later than 4 weeks after therapy (0 of 20patients) were compared, the difference is statistically sig-nificant (P < 0.05; Fisher's test). This result does not favorthe hypothesis of a persistent infection.None of the urine samples obtained from 25 healthy

controls or from the 10 patients with various bacterialurinary tract infections were PCR positive.By using DNA prepared from in urine as template DNA,

several samples led to additional amplification of one orseveral DNA fragments which did not hybridize with thespecific F-8 probe. The PCR results were dependent on theamount of template DNA that was added to the PCRmixture. Various amounts (1, 5, 10, or 20 [lI) of the DNA

A B C D

8-

9-

NC -PC --- _..

FIG. 4. Slot blot hybridization showing representative PCR re-sults for urine samples from two patients with active neuroborreli-osis. Patient numbers are indicated. Lane A, pretreatment urinesample; lane B, urine samples on days 3 to 6; lane C, urine sampleobtained after antibiotic treatment was completed (days 10 to 14);lane D, urine sample obtained from 1 to 8.5 months after thecompletion of therapy. PC, positive control (1 ng of purified B.burgdoifiri DK1 DNA); NC, negative control. The amplificationproducts were identified with the 32P-end-labeled probe F-8.

J. Cl-IN. MlICROBIO0L.

," ......

on April 17, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

B. BURGDORFERI DETECTION IN URINE AND CSF BY PCR 1651

M 3 3 8 8 PCNCMB IA

PC-

NC

3-

3-Ilul 5u1 20 ui

1 20 1 2OulFIG. 5. (A) Slot blot hybridization of urine samples processed

for PCR. Numbers to the left are patient numbers as described inTables 1 and 2. PC, positive control (1 ng of purified B. burgdorferiDK1 DNA); NC, negative control. The volumes indicated below thepanel reflect the amount of samples from patients that was subjectedto PCR. The amplification products were identified with the 32P-end-labeled probe F-8. (B) PCR amplification of a 444-bp fragment of thehuman gastrin gene. Numbers on top are patient numbers as

described in Tables 1 and 2. PC, positive control (plasmid pG8Eencoding the human gastrin gene); NC, negative control. Thevolumes indicated below the panel reflect the amount of samplesfrom patients that was subjected to PCR. M, DNA size marker. AllPCRs shown here originated from the same experiment.

preparation from urine specimens from all patients were

used as template DNA. Of the 17 PCR-positive samples, 6were found to be PCR positive only when 1 ,u was used as

template DNA, 5 were found to be PCR positive only when20 ,ul was used as template DNA, and 6 were found to bePCR positive when 1 to 20 ,u was used as template DNA. Inorder to investigate the inhibition of Taq polymerase byurine components, PCR amplification of the human gastringene was performed by using 1 or 20 pl of the prepared urinesample from patients 3 and 8 as template DNA. Results ofthis experiment are shown in Fig. 5. The presence of PCRinhibitors in urine was demonstrated in patient 3, for whomthe use of 20 RI of template preparation yielded neither a B.burgdorfen nor a gastrin amplification product, whereas theuse of 1 ,ul resulted in the amplification of both genes. Thesituation in patient 8 illustrated that the specific amplificationof B. burgdorferi needs at least 5 pul of DNA as a template.CSF specimens. Only the second protocol for preparing

DNA from CSF samples yielded positive results; however,B. burgdorfen DNA was detected in only two pretreatmentCSF samples (those from patients 1 and 9) and in two of fiveCSF samples obtained immediately after therapy (those frompatients 2 and 3). The remaining CSF samples from patientswith neuroborreliosis as well as those from patients withdifferent central nervous system infections and multiplesclerosis were PCR negative, regardless of the PCR protocolused.Assay reproducibility. In order to assess the reproducibil-

ity of the PCR assay, aliquots of 5 ml of pretreatment urinefrom four patients (patients 2 to 5) were subjected to thecomplete procedure described above on six independentoccasions. In patients 3 and 4 the urine was found to benegative for B. burgdorferi DNA by PCR of all six prepara-tions. In patients 2 and 5, B. burgdorferi DNA was detectedin five of six preparations.

DISCUSSION

Results of this study indicate that PCR can be applied as a

diagnostic test for Lyme neuroborreliosis. Of 10 consecutive

patients, the urine of 9 patients was positive for B. burgdor-feri DNA by PCR the CSF of 4 patients was positive for B.burgdorfeii DNA by PCR. The sparsity of B. burgdorferi inthe tissues and body fluids of patients with Lyme borreliosishas been the fundamental problem in all attempts to directlydetect the organism. Therefore, when PCR became avail-able, it seemed to be a promising solution to the problem.However, so far there have been only a few reports that havedescribed the use of PCR on clinical samples from patientswith Lyme neuroborreliosis (11, 14, 32). Since the studiesdescribed in those reports were based on only a few and notconsecutively studied patients, the diagnostic performanceof PCR for Lyme neuroborreliosis remains to be clarified.From a diagnostic viewpoint, the optimal target DNA se-quence should be B. burgdorfen specific, but it should alsobe conserved in all B. burgdorferi strains. The OspA gene,which encodes a species-specific outer surface protein (6),has been used as a template in PCR (11, 34, 39, 43).However, not all strains could be amplified (11, 39, 43). Thisis in accordance with recently published sequence datawhich showed that there is only 80% homology betweendifferent B. burgdorfen strains (52). Differences in OspAsequences are most pronounced for European isolates (52).The flagellin-encoding gene, on the other hand, is highlyconserved among different B. burgdorferi strains, with therebeing only a few nucleotide differences even between U.S.and European strains (13, 51). Being aware of the extensiveDNA homology between flagellin genes of B. burgdorfenand even taxonomically remotely related bacteria such asSalmonella typhi and E. coli (10, 41, 51), we placed ouroligonucleotide primers in areas that were nonhomologouswhen we compared them with the sequence data for the twoclosely related spirochetes B. hermsii and T. pallidum (9, 41,45). Our PCR assay proved to be species wide and B.burgdorfen specific (Table 3; Fig. 1).The sensitivity of our PCR depended on the solvent of the

target DNA, since the sensitivity decreased 10 times whenartificially seeded CSF or urine samples were used comparedwith the sensitivity when a spirochetal solution in PBS wasused. This phenomenon was described previously (8, 40) andis most likely due to Taq polymerase inhibitors in bodyfluids. Results of our experiment with different amounts oftarget DNA preparation and the double amplification of B.burgdorferi DNA and the human gastrin gene in parallelillustrate this problem (Fig. 5). Inhibition could not becircumvented by reducing the sample volume, i.e., from 5 to1 ml of urine, without the loss of sensitivity, since samplesfrom several patients were PCR positive only when largeamounts of target DNA preparation were used.The sensitivity of our PCR with CSF from patients with

neuroborreliosis was disappointing and lower than the re-sults presented in a previous report (11), in which CSFsamples from 10 of 13 patients were positive for B. burgdor-feri DNA by PCR. However, that report (11) specifiedneither the volume of CSF used nor whether the series of 13patients was consecutive, and urine was not examined inthat study. Because of the low number of B. burgdorferi inCSF, it may be beneficial to use, for example, 3 ml of CSF,like Kruger and Pulz (31) did in a series of two patients. Toexplore this, we tried to prepare DNA from 1 ml of CSF fromtwo patients with neuroborreliosis whose CSF was other-wise PCR negative for B. burgdorferi DNA. This did notimprove the results. We believe that the different sensitivi-ties obtained by using urine and CSF was due to the lowernumber of B. burgdorferi genome copies in CSF, since thesensitivity of our assay should have been equally high for

VOL. 30, 1992

aC -

on April 17, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

1652 LEBECH AND HANSEN

both body fluids (Fig. 2). The assumption of a higherconcentration of spirochetal constituents in urine is sup-ported by the previous successful detection of B. burgdorferiantigens in urine from experimentally infected animals (26)and even some patients (12, 26). B. burgdorferi antigen hasnever been detected in the CSF of patients with neurobor-reliosis.Although the diagnostic sensitivity in pretreatment urine

samples was only 50%, it is an interesting finding that thesensitivity increased to 90% when urine obtained 3 to 6 daysafter the onset of therapy was used. This probably reflectsspirochetal lysis and increased excretion of spirochetalbreakdown products, which also likely explains the appar-ently increased amounts of the amplification products regu-larly found in the second urine sample (Fig. 4). The conse-quently negative PCR results in posttherapy samples from 20patients with neuroborreliosis, including 3 patients withchronic neuroborreliosis, suggests eradication of the spiro-chete and does not favor the hypothesis of a persistent,latent infection, which has been considered as a possibleexplanation for the frequently persistent specific intrathecalantibody responses in patients with neuroborreliosis (17, 19,21, 31, 36). However, clinical experience has never revealedthe occurrence of relapses in patients with neuroborreliosiswho were appropriately treated (22, 29).

Recently, T. pallidum DNA has successfully been de-tected in CSF from patients with neurosyphilis (8, 15, 24,40). The diagnostic sensitivity achieved in patients with thiscondition seems higher than that achieved in patients withneuroborreliosis. This and the sporadically positive PCRresults obtained months to years after therapy in a fewpatients with neurosyphilis (40) may be attributable to thesignificantly higher number of spirochetes in patients withsyphilis compared with the number in those with Lymeborreliosis.Whereas B. burgdorfien DNA has been cultured from CSF

from patients with second-stage (early) neuroborreliosis (28,44, 49), this has never been done with success in patientswith third-stage (late) neuroborreliosis. Our results are thusthe first confirmation that even patients with chronic neu-roborreliosis have spirochetal DNA in their CSF and urine,indicating an active infection. Thus, the pathogenesis inpatients with this type of neuroborreliosis is not only a resultof autoimmune mechanisms (37, 48).Our selection of samples from patients with neuroborreli-

osis with confirmed specific intrathecal antibody productionwas intended to obtain relevant documentation and a mea-sure of the sensitivity of the PCR. This is necessary as longas strict diagnostic criteria for neuroborreliosis are notgenerally used (22, 30). We believe that before PCR isregarded as a reliable diagnostic test, its performance shouldat least be comparable to that of detection of intrathecal-specific antibody synthesis. Only on this condition, andwhen the deleterious problem of contamination becomescontrollable, will PCR be a useful supplement, especially inpatients with a short disease duration, and, possibly, willalso serve as a measure of therapeutic efficacy.We conclude that in patients with neuroborreliosis, urine

is a more suitable source of DNA for the diagnosis of B.burgdorferi infection than is CSF. B. burgdorfen-specificDNA was found in urine as well as CSF even in patients withchronic neuroborreliosis. The lack of positive PCR resultsduring posttreatment follow-up does not support the hypoth-esis of a persistent infection. The presence of Taq polymer-ase inhibitors in clinical specimens seems, for the moment,to be a significant problem to the application of PCR as a

routine diagnostic method. The future role of PCR as adiagnostic tool for Lyme neuroborreliosis remains undeter-mined.

ACKNOWLEDGMENTSWe thank Marianne T0nder for perfect technical assistance, Karin

Larsen for typing the manuscript, and Jens Vuust for helpful advice.Furthermore, we thank David Hougaard, Department of ClinicalImmunology, Statens Seruminstitut, Copenhagen, for the syntheticoligonucleotide primers and Frank Espersen, Department of ClinicalMicrobiology, Rigshospitalet, Copenhagen, for supplying us withurine samples from patients with bacterial urinary tract infections.Klaus Hansen was supported by Thorvald Madsens legat. The

study was supported by a grant from the Research Center forMedical Biotechnology under the Danish Biotechnological Researchand Development program.

REFERENCES1. Ackermann, R., E. Golimer, and B. Rehse-Kupper. 1985. Pro-

gressive Borrelien-Enzephalomyelitis. Chronische Manifesta-tion der Erythema-Chronicum-Migrans-Krankheit am Nerven-system. Dtsch. Med. Wochenschr. 110:1039-1042.

2. Asbrink, E., A. Hovmark, and B. Hederstedt. 1984. The spiro-chetal etiology of acrodermatitis chronica atrophicans Herxhe-imer. Acta Dermatovenereol. 64:506-512.

3. Aurelius, E., B. Johansson, B. Skoldenberg, A. Staland, and M.Forsgren. 1991. Rapid diagnosis of herpes simplex encephalitisby nested polymerase chain reaction assay of cerebrospinalfluid. Lancet 337:189-192.

4. Barbour, A. G. 1984. Isolation and cultivation of Lyme diseasespirochetes. Yale J. Biol. Med. 57:521-525.

5. Benach, J. L., E. M. Boster, J. P. Hanrahan, J. L. Coleman,G. S. Habicht, T. F. Bast, D. J. Cameron, J. L. Ziegler, A. G.Barbour, W. Burgdorfer, R. Edelman, and R. A. Kaslow. 1983.Spirochetes isolated from the blood of two patients with Lymedisease. N. Engl. J. Med. 308:740-742.

6. Bergstrom, S., V. G. Bundoc, and A. G. Barbour. 1989. Molec-ular analysis of linear plasmid-encoded major surface proteins,OspA and OspB, of the Lyme disease spirochaete Borreliaburgdorferi. Mol. Microbiol. 3:479-486.

7. Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E.Grunwaldt, and J. P. Davis. 1982. Lyme disease-a tickbornespirochetosis? Science 216:1317-1319.

8. Burstain, J. M., E. Grimprel, S. A. Lukehart, M. V. Norgard,and J. D. Radolf. 1991. Sensitive detection of Treponemapallidum by using the polymerase chain reaction. J. Clin.Microbiol. 29:62-69.

9. Champion, C. I., J. N. Miller, M. A. Lovett, and D. R. Blanco.1990. Cloning, sequencing, and expression of two class Bendo-flagellar genes of Treponema pallidum subsp. pallidumencoding the 34.5- and 31.0-kilodalton proteins. Infect. Immun.58:1697-1704.

10. Collins, C., and G. Peltz. 1991. Immunoreactive epitopes on anexpressed recombinant flagellar protein of Borrelia burgdorferi.Infect. Immun. 59:514-520.

11. Debue, M., P. Gautier, C. Hackel, A. Van Elsen, A. Herzog, G.Bigaignon, and A. Bollep. 1991. Detection of Borrelia burgdor-feri in biological samples using the polymerase chain reactionassay. Res. Microbiol. 142:565-572.

12. Dorward, D. W., T. G. Schwan, and C. F. Garon. 1991. Immunecapture and detection of Borrelia burgdorferi antigens in urine,blood, or tissues from infected ticks, mice, dogs, and humans. J.Clin. Microbiol. 29:1162-1170.

13. Gassmann, G. S., M. Kramer, U. B. Gobel, and R. Wallich.1989. Nucleotide sequence of a gene encoding the Borreliaburgdorferi flagellin. Nucleic Acids Res. 17:3590.

14. Goodman, J. L., P. Jurkovich, J. M. Kramber, and R. C.Johnson. 1991. Molecular detection of persistent Borrelia burg-dorferi in the urine of patients with active Lyme disease. Infect.Immun. 59:269-278.

15. Grimprel, E., P. J. Sanchez, G. D. Wendel, J. M. Burstain, G. H.McCracken, J. D. Radolf, and M. V. Norgard. 1991. Use of

J. CLIN. MICROBIOIL.

on April 17, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

B. BURGDORFERI DETECTION IN URINE AND CSF BY PCR 1653

polymerase chain reaction and rabbit infectivity testing to detectTreponema pallidum in amniotic fluid, fetal and neonatal sera,and cerebrospinal fluid. J. Clin. Microbiol. 29:1711-1718.

16. Guy, E. C., and G. Stanek. 1991. Detection of Borrelia burg-dorferi in patients with Lyme disease by the polymerase chainreaction. J. Clin. Pathol. 44:610-611.

17. Hammers-Berggren, S., G. Stiernstedt, M. Karisson, K. Hansen,and A. M. Lebech. 1991. Program Abstr. 5th Eur. Congr. Clin.Microbiol. Infect. Dis., abstr. 1084.

18. Hansen, K., J. M. Bangsborg, H. Fjordvang, N. S. Pedersen, andP. Hindersson. 1988. Immunochemical characterization of andisolation of a Borrelia burgdorferi immunodominant 60-kilodal-ton antigen common to a wide range of bacteria. Infect. Immun.56:2047-2053.

19. Hansen, K., M. Cruz, and H. Linlk 1990. Oligoclonal Borreliaburgdorferi-specific IgG antibodies in cerebrospinal fluid inLyme neuroborreliosis. J. Infect. Dis. 161:1194-1202.

20. Hansen, K., P. Hindersson, and N. Strandberg Pedersen. 1988.Measurement of antibodies to the Borrelia burgdorferi flagellumimproves serodiagnosis in Lyme disease. J. Clin. Microbiol.26:338-346.

21. Hansen, K., and A. M. Lebech. 1991. Lyme neuroborreliosis: anew sensitive diagnostic assay for intrathecal synthesis ofBorrelia burgdorferi-specific immunoglobulin G, A, and M.Ann. Neurol. 30:197-205.

22. Hansen, K., and A. M. Lebech. 1992. The clinical and epidemi-ological profile of Lyme neuroborreliosis in Denmark 1985-90: aprospective study of 187 patients with Borrelia burgdorferi-specific intrathecal antibody production. Brain 115:399-423.

23. Hansen, K., K. Pii, and A. M. Lebech. 1991. Improved immu-noglobulin M serodiagnosis in Lyme borreliosis using a ,u-cap-ture enzyme-linked immunosorbent assay with biotinylated Bor-relia burgdorferi flagella. J. Clin. Microbiol. 29:166-173.

24. Hay, P. E., J. R. Clarke, R. A. Strugnell, D. Taylor-Robinson,and D. Goldmeier. 1990. Use of the polymerase chain reaction todetect DNA sequences specific to pathogenic treponemes incerebrospinal fluid. FEMS Microbiol. Lett. 68:233-238.

25. Hindersson, P., J. D. Knudsen, and N. H. Axelsen. 1987. Cloningand expression of Treponema pallidum common antigen (Tp-4)in Escherichia coli K12. J. Gen. Microbiol. 133:587-596.

26. Hyde, F. W., R. C. Johnson, T. J. White, and C. E. Shelburne.1989. Detection of antigens in urine of mice and humans infectedwith Borrelia burgdorferi, etiologic agent of Lyme disease. J.Clin. Microbiol. 27:58-61.

27. Kariya, Y., K. Kato, Y. Hayashizaki, S. Himeno, S. Tarui, andK. Matsubara. 1986. Expression of human gastrin gene innormal and gastrinoma tissues. Gene 50:345-352.

28. Karlsson, M., K. Hovind-Hougen, B. Svenungsson, and G.Stiernstedt. 1990. Cultivation and characterization of spiro-chetes from cerebrospinal fluid of patients with Lyme borrelio-sis. J. Clin. Microbiol. 28:473-479.

29. Kristoferitsch, W. 1989. Neuropathien bei Lyme-Borreliose.Springer-Verlag, Vienna.

30. Kristoferitsch, W. 1991. Neurological manifestations of Lymeborreliosis: clinical definitions and differential diagnosis. Scand.J. Infect. Dis. Suppl. 77:64-73.

31. Kruger, H., R. Reuss, M. Pulz, E. Rohrbach, K.-W. Pflughaupt,R. Martin, and H. G. Mertens. 1989. Meningoradiculitis andencephalomyelitis due to Borrelia burgdorferi: a follow-upstudy of 72 patients over 27 years. J. Neurol. 236:322-328.

32. Kruger, W. H., and M. Pulz. 1991. Detection of Borreliaburgdorferi in cerebrospinal fluid by the polymerase chainreaction. J. Med. Microbiol. 35:98-102.

33. Lebech, A. M., P. Hindersson, J. Vuust, and K. Hansen. 1991.Comparison of in vitro culture and polymerase chain reactionfor detection of Borrelia burgdorferi in tissue from experimen-tally infected animals. J. Clin. Microbiol. 29:731-737.

34. Malloy, D. C., R. K. Nauman, and H. Paxton. 1990. Detection ofBorrelia burgdorferi using the polymerase chain reaction. J.Clin. Microbiol. 28:1089-1093.

35. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular

cloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

36. Martin, R., U. Martens, V. Sticht-Groh, R. Dorries, and H.Kriiger. 1988. Persistent intrathecal secretion of oligoclonal,Borrelia burgdorfen-specific IgG in chronic meningoradiculo-myelitis. J. Neurol. 7:77-89.

37. Martin, R., J. Ortlauf, V. Sticht-Groh, U. Bogdahn, S. F.Goldmann, and H. G. Mertens. 1988. Borrelia burgdorfen-specific and autoreactive T-cell lines from cerebrospinal fluid inLyme radiculomyelitis. Ann. Neurol. 24:509-516.

38. Melchers, W., J. Meis, P. Rosa, E. Claas, L. Nohlmans, R.Koopman, A. Horrevorts, and J. Galama. 1991. Amplification ofBorrelia burgdorfen DNA in skin biopsies from patients withLyme disease. J. Clin. Microbiol. 29:2401-2406.

39. Nielsen, S. L., K. K. Y. Young, and A. G. Barbour. 1990.Detection of Borrelia burgdorferi DNA by the polymerase chainreaction. Mol. Cell. Probes 4:73-79.

40. Noordhoek, G. T., E. C. Wolters, M. E. J. de Jonge, and J. D. A.van Embden. 1991. Detection by polymerase chain reaction ofTreponema pallidum DNA in cerebrospinal fluid from neuro-syphilis patients before and after antibiotic treatment. J. Clin.Microbiol. 29:1976-1984.

41. Pallesen, L., and P. Hindersson. 1989. Cloning and sequencing ofa Treponema pallidum gene encoding a 31.3-kilodalton end-oflagellar subunit (FlaB2). Infect. Immun. 57:2166-2172.

42. Pedersen, N. S., N. H. Axelsen, and C. S. Petersen. 1981.Antigenic analysis of Treponema pallidum: cross-reactions be-tween individual antigens of T. pallidum and T. reiter. Scand. J.Immunol. 13:143-150.

43. Persing, D. H., S. R. Telford III, A. Spielman, and S. W.Barthold. 1990. Detection of Borrelia burgdorfeni infection inLxodes dammini ticks with the polymerase chain reaction. J.Clin. Microbiol. 28:566-572.

44. Pfister, H. W., K. Einhaupl, V. Preac-Mursic, B. Wilske, and G.Schierz. 1984. The spirochetal etiology of lymphocytic meningo-radiculitis of Bannwarth (Bannwarth's syndrome). J. Neurol.231:141-144.

45. Picken, R. N. 1991. Polymerase chain reaction primers andprobes derived from flagellin gene sequences for specific detec-tion of the agent of Lyme disease and North America relapsingfever. J. Clin. Microbiol. 30:99-114.

46. Rosa, P. A., and T. G. Schwan. 1989. A specific and sensitiveassay for the Lyme disease spirochete Borrelia burgdorferiusing the polymerase chain reaction. J. Infect. Dis. 160:1018-1029.

47. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi,G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primerdirected enzymatic amplification of DNA with a thermostableDNA polymerase. Science 239:487-491.

48. Schluesner, H. J., R. Martin, and V. Sticht Groh. 1989. Autoim-munity in Lyme disease: molecular cloning of antigens recog-nized by antibodies in the cerebrospinal fluid. Autoimmunity2:323-330.

49. Steere, A. C., R. L. Grodzicki, A. N. Kornblatt, J. E. Craft,A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, andS. E. Malawista. 1983. The spirochetal etiology of Lyme dis-ease. N. Engl. J. Med. 308:773-740.

50. Stiernstedt, G. T., M. Granstrom, B. Hederstedt, and B. Skold-enberg. 1985. Diagnosis of spirochetal meningitis by enzyme-linked immunosorbent assay and indirect immunofluorescenceassay in serum and cerebrospinal fluid. J. Clin. Microbiol.2:819-825.

51. Wallich, R., S. E. Moter, M. M. Simon, K. Ebnet, A. Heiberger,and M. D. Kramer. 1990. The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning,expression, and amplification of the gene. Infect. Immun. 58:1711-1719.

52. Wilske, B., J. F. Anderson, G. Baranton, A. G. Barbour, K.Hovind-Hougen, R. C. Johnson, and V. Preac-Mursic. 1991.Workshop summary. Taxonomy of Borrelia spp. Scand. J.Infect. Dis. Suppl. 77:108-129.

VOL. 30, 1992

on April 17, 2021 by guest

http://jcm.asm

.org/D

ownloaded from