OF CLINICAL MICROBIOLOGY, July 1992, p. 1646-1653 0095-1137/92/071646-08$02.00/0 Copyright ©3 1992, American Society for Microbiology

Vol. 30, No. 7

JOURNAL

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

Lyme neuroborreliosis is a frequent and serious manifestation of Lyme borreliosis caused by the tick-borne spirochete Borrelia burgdorferi (7, 22, 29, 49). The best current indicators of active neuroborreliosis are lymphocytic pleocytosis and B. bugdorferi-specific antibody synthesis within the cerebrospinal fluid (CSF) (21, 22, 29, 50). However, a reliable and practical diagnostic assay for the direct demonstration of B. burgdorferi in samples from patients would be preferable. So far the most reliable method for direct detection of B. burgdorferi in clinical samples has been in vitro culture, which is a difficult, time-consuming, and especially with regard to blood (5, 49) but also with regard to CSF (28, 44, 49), a low-yield procedure. Considering the paucity of spirochetes in pathological lesions and clinical specimens, diagnostic amplification of specific DNA sequences by the polymerase chain reaction (PCR) (47) seemed an obvious solution to this problem. Several sensitive PCR assays for detection of either purified B. burgdorferi DNA or in vitrocultivated spirochetes have been reported (34, 39, 46, 51). We recently compared the diagnostic sensitivity of in vitro culture and PCR for the detection of B. burgdorfen in tissues from experimentally infected animals and found that PCR is superior in terms of ease and sensitivity (33). However, only a few reports have described the application of PCR to clinical samples from patients with Lyme borreliosis (11, 14, 16, 32, 38). In those studies, the total number of patients studied was generally low and it was not stated whether they were consecutive patients. As sample sources, some studies used serum (16), whereas others used CSF (11, 32), skin *

biopsy (38), joint fluid (11), or urine (14) specimens. The practical role of PCR in the laboratory diagnosis of Lyme borreliosis is still unclear. It remains unknown in which clinical manifestations PCR might be useful, which sample source is optimal, and what diagnostic sensitivity is achieved. The aim of the present study was (i) to evaluate the diagnostic performance of PCR in a series of consecutive patients with definite Lyme neuroborreliosis by using urine and CSF as sample sources and (ii) to investigate the duration of B. burgdorferi DNA excretion in urine after therapy. MATERIALS AND METHODS Patients with active neuroborreliosis. Urine and CSF samples were obtained from 10 patients with active neuroborreliosis. The patients were successively diagnosed between November 1990 and July 1991. Eight of the patients had early (second-stage) neuroborreliosis (lymphocytic meningoradiculitis), and two patients had late (third-stage) neuroborreliosis (chronic progressive encephalomyelitis) (1, 22, 29). The diagnosis of neuroborreliosis was, in every patient, based on lymphocytic pleocytosis and definite B. burgdorferi-specific intrathecal immunoglobulin M (IgM) and/or IgG antibody synthesis in CSF. All patients received 20 million IU of penicillin G intravenously per day for 10 to 14 days. The clinical and relevant laboratory data for the 10 patients are summarized in Tables 1 and 2. Urine and CSF samples were obtained before antibiotic therapy was begun. Additional urine samples were obtained 3 to 6 days after the start of treatment; 10 to 14 days after the

Corresponding author. 1646

Vol.. 30, 1992

B. BURGDORFERI DETECTION IN URINE AND CSF BY PCR

1647

TABLE 1. Clinical characteristics of patients examined in this study Disease Dureato

Patieno

no.

Sex"/age Sex"r)ge (yr)

duration

1 2

M/53 M/10

4 wk 16 wk

3

F/64

14 wk

4

F/71

24 wk

5 6

F/32 M/20

8 wk 10 wk

7

M/57

3 wk

8

F/29

1 yr

9

F/69

8 mo

10

M/10

1 wk

Patient

Clinical

history

Clinical

Painful radiculitis in lower back, sixth nerve palsy Headache, back pain, general symptoms, nausea, intermittent fever and weight loss Headache and painful radiculitis in neck and shoulder girdle Erythema migrans; pronounced pain and dysesthesia of left leg; severe, flaccid paraparesis inferior, most pronounced on left side Painful dysesthesia in both groin and buttocks Hleadache, low-grade fever, and painful radiculitis in lower back Tick bite, erythema migrans; headache, weight loss, painful radiculitis in the neck and shoulder girdle; bilateral facial palsy and sixth nerve palsy Erythema migrans followed by 1 yr of general symptoms, nausea, vomiting, headache, weight loss, and several episodes compatible with partial complex seizures Transient sixth nerve palsy, fatigue, and weight loss; 4 mo later onset of a progressive ataxic gait; slight paraparesis inferior with bilateral Babinski's sign Tick bite, erythema migrans, headache, and general symptoms

follow-up

Complete recovery Complete recovery Complete recovery Complete recovery Complete recovery Complete recovery Slight residual facial palsy Complete recovery

Complete recovery from general symptoms but slight residual gait disturbance Complete recovery

M, male; F, female.

start of treatment, when therapy was completed; and 1 to 8.5 months (median, 6 months) after therapy. CSF samples were obtained from five patients immediately after the completion of therapy and from both patients with chronic neuroborreliosis 3 months after therapy. Patients with previous neuroborreliosis. Urine was obtained from 10 patients (median age, 44.5 years; age range, 8 to 75 years) who had been treated for neuroborreliosis 8 months to 5 years (median, 1 year) previously. The patients fulfilled the same diagnostic criteria as those mentioned above. Nine patients had early (second-stage) neuroborreliosis. All had recovered completely. One patient had late (third-stage) chronic progressive encephalomyelitis with a

disease duration of 1 year before treatment. After therapy, no further progression of symptoms was seen and CSF parameters had normalized. Control subjects. To evaluate the specificity of the urine assay, we investigated urine from (i) 25 healthy controls (median age, 41 years; age range, 3 to 66 years) and (ii) 10 patients with urinary tract infections with the following bacteria: Proteus mirabilis (n = 3), Klebsiella pneumonia (n = 3), Eschenichia coli (n = 2), and Streptococcus faecalis (n = 2). In each case a urine culture revealed >i10 bacteria per ml. The specificity of the CSF assay was investigated by using CSF samples from (i) five patients with multiple sclerosis

TABLE 2. Laboratory findings for patients examined in this study

Patient no.

1 2 3 4 5 6 7 8 9 10

Intrathecal antibody production CSF examination 14 days CSF examination therapy (specific capture befoe teray ater nse oftheapybefore after onset of therapy before therapy antibody index)"

No. of leukocytes/pl

No. of leukocytes/,ul

Protein

(0/ mononuclear cells)

Protein concn

(% mononuclear

concn

(g/liter)

cells)

(g/liter)

221 (98) 148 (94) 24 (97) 64 (99) 74 (66) 93 624 (94) 55 (98) 61 (98) 296 (100)

1.38 0.88 1.02 1.06 0.30 0.72 4.0) 4.35 1.55 0.6

88 (96) 6 17 (100) No sample No sample No sample No sample

0.86 0.4 0.68

46

No sample No sample No sample No sample

2.8(l

(95)'

Not examined" No sample

0.72' No

sample

Anti-B.

bwirgdorferi

antibody level in serumh

IgG

IgM

IgG

IgM

28.1 5.5

2.9 0.6

0.220 0.510

0.950 1.280

2.6 2.3 43.3 3.0

6.3 3.5 0 0.32

0.990 0.510 0.032 0.920

0.520 0.342 0.500 0.810

13.2 1.4 2.9 0

3.6 0.43 0 9.7

0.085 0.540 0.860 0.040

1.460 0.230 0.400 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.

1648

LEBECH AND HANSEN

J. CLIN. MIC ROBIOL.

TABLE 3. B. burgdorferi strains Strain

DK1 DK2 DK3 DK4 DK5 DK6 DK7 DK8 DK9 DK21 DK29 P/Ko PIBi P/Tm 297 272 245 B31 SL10 SL14 SL42 ACA-1 B. hermsii

Source'

Skin, Skin, Skin, Skin, Skin, CSF, Skin, Skin, Skin, Skin, Skin, Skin, CSF, Skin, CSF

EM ACA ACA EM ACA LMR ACA ACA ACA EM EM EM LMR ACA

Skin Blood Tick CSF, LMR CSF, LMR CSF, LMR Skin, ACA

Supplier"

SS SS SS SS SS SS SS SS SS SS SS V. Preac-Mursic V. Preac-Mursic V. Preac-Mursic A. C. Steere A. C. Steere A. C. Steere A. G. Barbour M. Karlsson M. Karlsson M. Karlsson E. Asbrink 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, Max von Pettenkofer Institute, Munich, Germany; A. C. Steere, Tufts New England Medical Center, Boston, Mass. (49); A. G. Barbour, Health Science Center, San Antonio, Tex.; M. Karlsson, Danderyd Sjukhus, Stockholm, Sweden (28); E. Asbrink, Sodersjukhuset, Stockholm, Sweden (2); K. Hovind-Hougen, Statens Veterinaere Serumlaboratorium, Copenhagen, Denmark.

and (ii) 10 patients with the following central nervous system infections: varicella-zoster meningoencephalitis (n = 1), herpes simplex encephalitis (n = 2), meningococcal meningitis (n = 2), fungal encephalitis (n = 1), and other forms of viral encephalitis (n = 4). The range of pleocytosis in the CSF of these patients was 12 x 106 to 3,797 x 106 cells per liter. All serum and CSF specimens from control individuals were 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 which were detectable were tested by using the panel of 22 different in vitro-cultivated B. burgdorfen isolates listed in Table 3. Except for the U.S. type strain B31, which is an isolate from a tick, all other strains were isolated from patients with Lyme borreliosis; 18 strains were from Europe and 3 strains were from North America. For the investigation of the species specificity of our PCR assay, we used DNA extracted from Borrelia hermsii (a causative agent of relapsing fever) and Treponema pallidum (Nichols pathogenic strains). 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 for PCR from in vitro-cultivated B. burgdorferi. Total DNA from all Borrelia strains and T. pallidum was extracted as described previously (18, 25). DNA concentrations were determined spectrophotometrically by measuring the A260 (35). An amount of 100 ng of the preparations was used as template DNA. The analytical sensitivity of the PCR assay was investigated 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 in vitro-cultivated B. burgdorferi without prior DNA extraction. For this purpose, samples with a cell density ranging from 106 to 10 B. burgdorferi per 10 RI were used. These were obtained by a 10-fold dilution series with a phosphatebuffered saline (PBS) solution containing 108 B. burgdorferi DK1 per 10 RI, as determined by dark-field microscopy. The samples containing 1% (vol/vol) Triton X-100 were heated to 100°C for 10 min. An aliquot of 10 RI was used as template DNA. 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 of PBS (pH 7.4). The solution was then centrifuged at 20,000 x g for 20 min, and the pellet was dried under vacuum and resuspended in 20 RI of redistilled water. Twenty microliters of a 5% Chelex-100-resin solution (catalog no. 142-2832; Bio-Rad, Richmond, Calif.) was added to the sample before it was heated to 100°C for 5 min, centrifuged for 1 min at 3,000 x g, and subsequently chilled on ice. Supernatants in amounts of 1, 5, 10, or 20 RI were used as sources of template DNA. (ii) CSF. Two different methods for the preparation of DNA from CSF were investigated. The first technique (3) was based on DNA precipitation. Three hundred microliters of CSF was heated to 100°C for 15 min; the DNA was then precipitated with 2 volumes of 99% (vol/vol) ethanol for 30 min at - 20°C and finally pelleted by centrifugation for 30 min at 20,000 x g. The DNA pellet was washed with cold 70% ethanol, dried under vacuum, and resuspended in 20 ,ul of redistilled water. The samples were heated to 100°C for 10 min in the presence of 1% (vol/vol) Triton X-100 and were subsequently chilled on ice. An aliquot of 20 RI was then used as template DNA. In the second approach (15), 200 RI of CSF was added to 800 RI to PBS (pH 7.4), and the solution was centrifuged at 15,000 x g for 15 min. The dried pellet was resuspended in 50 ,ul of redistilled H20, heated to 100°C for 10 min in the presence of 1% Triton X-100, and subsequently chilled on ice. Twenty microliters was then subjected to PCR. For optimization of the PCR conditions for clinical specimens, normal urine and CSF were artificially seeded with in vitro-cultivated B. burgdorferi. A controlled number of B. burgdorferi DK1, ranging from 106 to 10 spirochetes, was added to either 5 ml of urine or 200 pul of CSF. These simulated 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 the flagellin-encoding gene of B. burgdorferi (13), which has been shown to be highly conserved among strains (10, 13, 51). To obtain a B. burgdorferi-specific amplification, the primers were placed in areas nonhomologous to the sequences of B. hennsii (45) and T. pallidum (9, 41) flagellin genes. The sequences and positions of the oligonucleotide primers that amplified a 248-bp fragment are shown in Table 4. PCR was performed in a reaction volume of 50 RI containing 1 U of Taq DNA polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk, Conn.), 10 mM Tris hydrochloride (pH 8.3), 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 mixture was overlaid with 45 p.l of mineral oil (M-3516; Sigma, St. Louis, Mo.). All reactions were performed in a thermal cycler (Techne PH-C-1; Techne Ltd., Cambridge, United

VOL. 30, 1992

1649

B. BURGDORFERI DETECTION IN URINE AND CSF BY PCR

TABLE 4. Sequences and positions of oligonucleotide primers for PCR Gene and primer

Gene encoding the B. burgdorferi flagellin F-7 F-3

F-8b Gene encoding the human gastrin gene 12 15 '

Sequence

Locationa

+

5'-CTC TGG TGA GGG AGC TCA AAC-3' 5'-GTA CTA TTC TTT ATA GAT TC-3' 5'-CAT CAC TTG CTA AAA TTG AAA ATG C-3'

+

594-614 823-842 743-767

5'-CCC CCA CAC CTC GTG GCA G-3' 5'-GGC ACT CAG ATC TTC TCC CT-3'

+ -

6481-6499 6885-6905

Base positions are numbered according to the published sequence of the 41-kDa B.

(27). b 32

Coding strand

burgdorferi flagellin gene (13) or the sequence of the human gastrin gene

P-labeled oligonucleotide probe.

Kingdom). After an initial denaturation at 94°C for 4 min, the PCR conditions were denaturation at 94°C for 1 min, annealing at 39°C for 2 min, and extension at 66°C for 3 min, for 40 cycles. After the final cycle, the temperature was maintained at 66°C for 5 min to complete the extension. The PCRs were analyzed for amplified products by agarose gel electrophoresis (1.5%) (Sea-Kem FMC, Rockland, Maine). In order to evaluate a possible PCR inhibition because of components in urine, we performed an amplification of a 444-bp fragment of the human gastrin gene (27) in parallel on samples from two patients. The amplification was confirmed by agarose gel electrophoresis. The sequences and positions of the gastrin oligonucleotide primers are shown in Table 4; they were kindly provided by Jens Bundgaard, Department of Infection-Immunology, Statens Seruminstitut, Copenhagen. PCR conditions were as follows: denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min, for 35 cycles. Consecutive samples from one patient were always included within the same experiment. While preparing samples as well as PCR mixtures, samples from patients and controls were handled alternately within the same run. PCR mixtures were made in a laminar airflow hood in a remote PCRdedicated area which was never exposed to products of B. burgdorfen or final PCR products. The PCR facility was exposed to UV illumination for at least 15 h between experiments. Each PCR experiment included a negative control in which water replaced template DNA and a positive control which contained 1 ng of purified DNA from B. burgdorferi DK1. Pipetting was carried out with autoclavable pipettes (one-pettes; Costar Corporation, Cambridge, Mass.) and aerosol-resistant tips (Scandinavian Diagnostic Services, Falkenberg, Sweden). Southern blotting and slot blot hybridization. The specificity of the amplified PCR product was confirmed by DNADNA hybridization. Southern blotting was performed as described previously (33). For slot blot analysis, 15 ,ul of the PCR 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 15 min at 4°C. Using a Slot blot minifold II (reference no. 447 800; Schleicher & Schuell, Dassel, Germany), samples were directly 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 15 min. 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 then baked at 80°C for 2 h. The oligonucleotide probe F-8 (Table 4) 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 were subsequently autoradiographed as described previously (33). Lyme borreliosis serology. Anti-B. burgdorferi IgG and IgM in serum were measured by an indirect enzyme-linked immunosorbent assay (ELISA) and a ,u-capture ELISA (20, 23). B. burgdorferi-specific intrathecal IgG and IgM antibody production 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 single amplified fragment of the expected size of 248 bp when purified DNA of B. burgdorfen DK1 was used as the template. The authenticity of the amplification product was confirmed by Southern blotting, in which the PCR-amplified product hybridized to the probe F-8, which is complementary to the central part of the target sequence. A 248-bp DNA fragment could be amplified, from all 22 B. burgdorfen strains listed in Table 3, and all strains were shown to hybridize with the specific probe F-8. The species specificity of the PCR assay for B. burgdorferi Ni

1

2 3 4 M

1

i

A

2 3 4 s

B

4029*22-1

-_

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

1650

J. Cl-IN. MlICROBIO0L.

LEBECH AND HANSEN TREATMENT ::: i, U r i rn e

PATIENT No.

I ++

10

.9

+

+

_-

*8

+

+

-

6

+

+

I..

,"

......

5

+

+

4

_

+

3

-

2

+

+

+

I

FIG. 2. Slot blot hybridization of PCR products obtained from artificially 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 with 106 spirochetes (lanes 1), 104 spirochetes (lanes 2), 103 spirochetes (lanes 3), 102 spirochetes (lanes 4), 10 spirochetes (lanes 5), or 0 spirochetes (lanes 6). Lanes 7, positive control (1 ng of purified B. blurgdorferi DK1 DNA); lanes 8, negative control. The specificities of the amplification products were confirmed by using the 32P-endlabeled probe F-8. All PCRs shown here originated from the same experiment.

by using DNA from T. pallidum or B. hermsii as the template is illustrated in Fig. 1. No amplification products of 248 bp could be seen either in the agarose gel or in the Southern blot. Thus, the primers used in our PCR were B. burgdorferi specific and presumably reacted with all B. burgdorferi strains. The sensitivity of the PCR assay was determined on serially diluted purified B. burgdorferi DNA; dilutions ranged from 1 p.g to 0.001 pg. The analytical sensitivity obtained was 0.01 pg when the amplified fragments were detected with the radioactively labeled probe F-8. To estimate the sensitivity of the PCR assay in terms of the minimum number of spirochetes which could be detected, spirochetal solutions containing 106 to 10 spirochetes per 10 pAl were used. By adding 10 .1l of these dilutions as template DNA, a reproducible amplification was achieved when only 10 spirochetes were added to the PCR mixture. With regard to artificially seeded urine and CSF samples, the specific amplification could still be obtained when 100 spirochetes were added to 5 ml of urine or 200 p.l of CSF (Fig. 2). Thus, the assay sensitivity corresponds to 20 to 50 B. burgdorferi per ml of urine or CSF. Detection of B. burgdorferi DNA in clinical specimens. (i) Urine specimens. B. burgdorferi DNA was detectable in urine samples from 9 of 10 patients with active neuroborreliosis tested. A 248-bp fragment was seen on agarose gel electrophoresis, and the amplification products were probed on a slot blot with the probe F-8 (Fig. 3 and 4). In five patients B. burgdorferi DNA was detected prior to treatment, whereas in the remaining patients PCR was positive for B. burgdorferi DNA only for the second urine sample, which was obtained within 3 to 6 days after the onset of therapy. A urine sample was obtained from seven patients immediately after therapy was completed. Urine samples from two of the seven patients still contained detectable amounts of B. burgdorferi DNA. B. burgdorferi DNA was found in the urine of only one patient 4 weeks after therapy. All follow-up urine samples obtained 2 to 8.5 months (medi-

0

I

I I., 3-6 10-14 1

2

3

4

5

I 6

I 7

8

DAYS

9

10 MONTHS

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

an, 6 months) after therapy were negative for B. burgdorferi DNA by PCR. Similarly, no B. burgdorferi DNA could be detected by PCR in urine samples obtained from 10 individuals with a previously diagnosed and treated neuroborreliosis 8 months to 5 years before urine sampling. When the frequencies of positive PCR results before treatment (5 of 10 patients) and later than 4 weeks after therapy (0 of 20 patients) were compared, the difference is statistically significant (P < 0.05; Fisher's test). This result does not favor the hypothesis of a persistent infection. None of the urine samples obtained from 25 healthy controls or from the 10 patients with various bacterial urinary tract infections were PCR positive. By using DNA prepared from in urine as template DNA, several samples led to additional amplification of one or several DNA fragments which did not hybridize with the specific F-8 probe. The PCR results were dependent on the amount of template DNA that was added to the PCR mixture. Various amounts (1, 5, 10, or 20 [lI) of the DNA

A

B

C

D

89-

NC PC

---

_..

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

B. BURGDORFERI DETECTION IN URINE AND CSF BY PCR

VOL. 30, 1992 M

A

3

3

8

8

PCNCM

I

B

PC NC 3-

aC 3-I

lul

5u1

20 ui 1 20 1

2Oul

FIG. 5. (A) Slot blot hybridization of urine samples processed for PCR. Numbers to the left are patient numbers as described in Tables 1 and 2. PC, positive control (1 ng of purified B. burgdorferi DK1 DNA); NC, negative control. The volumes indicated below the panel reflect the amount of samples from patients that was subjected to PCR. The amplification products were identified with the 32P-endlabeled probe F-8. (B) PCR amplification of a 444-bp fragment of the human gastrin gene. Numbers on top are patient numbers as described in Tables 1 and 2. PC, positive control (plasmid pG8E encoding the human gastrin gene); NC, negative control. The volumes indicated below the panel reflect the amount of samples from patients that was subjected to PCR. M, DNA size marker. All PCRs 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, 6 were found to be PCR positive only when 1 ,u was used as template DNA, 5 were found to be PCR positive only when 20 ,ul was used as template DNA, and 6 were found to be PCR positive when 1 to 20 ,u was used as template DNA. In order to investigate the inhibition of Taq polymerase by urine components, PCR amplification of the human gastrin gene was performed by using 1 or 20 pl of the prepared urine sample from patients 3 and 8 as template DNA. Results of this experiment are shown in Fig. 5. The presence of PCR inhibitors in urine was demonstrated in patient 3, for whom the use of 20 RI of template preparation yielded neither a B. burgdorfen nor a gastrin amplification product, whereas the use of 1 ,ul resulted in the amplification of both genes. The situation in patient 8 illustrated that the specific amplification of 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 pretreatment CSF samples (those from patients 1 and 9) and in two of five CSF samples obtained immediately after therapy (those from patients 2 and 3). The remaining CSF samples from patients with neuroborreliosis as well as those from patients with different central nervous system infections and multiple sclerosis were PCR negative, regardless of the PCR protocol used. Assay reproducibility. In order to assess the reproducibility of the PCR assay, aliquots of 5 ml of pretreatment urine from four patients (patients 2 to 5) were subjected to the complete procedure described above on six independent occasions. In patients 3 and 4 the urine was found to be negative for B. burgdorferi DNA by PCR of all six preparations. In patients 2 and 5, B. burgdorferi DNA was detected in 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

1651

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 in

the tissues and body fluids of patients with Lyme borreliosis has been the fundamental problem in all attempts to directly detect the organism. Therefore, when PCR became available, it seemed to be a promising solution to the problem. However, so far there have been only a few reports that have described the use of PCR on clinical samples from patients with Lyme neuroborreliosis (11, 14, 32). Since the studies described in those reports were based on only a few and not consecutively studied patients, the diagnostic performance of PCR for Lyme neuroborreliosis remains to be clarified. From a diagnostic viewpoint, the optimal target DNA sequence should be B. burgdorfen specific, but it should also be 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). This is in accordance with recently published sequence data which showed that there is only 80% homology between different B. burgdorfen strains (52). Differences in OspA sequences are most pronounced for European isolates (52). The flagellin-encoding gene, on the other hand, is highly conserved among different B. burgdorferi strains, with there being only a few nucleotide differences even between U.S. and European strains (13, 51). Being aware of the extensive DNA homology between flagellin genes of B. burgdorfen and even taxonomically remotely related bacteria such as Salmonella typhi and E. coli (10, 41, 51), we placed our oligonucleotide primers in areas that were nonhomologous when we compared them with the sequence data for the two closely 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 when artificially seeded CSF or urine samples were used compared with the sensitivity when a spirochetal solution in PBS was used. This phenomenon was described previously (8, 40) and is most likely due to Taq polymerase inhibitors in body fluids. Results of our experiment with different amounts of target DNA preparation and the double amplification of B. burgdorferi DNA and the human gastrin gene in parallel illustrate this problem (Fig. 5). Inhibition could not be circumvented by reducing the sample volume, i.e., from 5 to 1 ml of urine, without the loss of sensitivity, since samples from several patients were PCR positive only when large amounts of target DNA preparation were used. The sensitivity of our PCR with CSF from patients with neuroborreliosis was disappointing and lower than the results presented in a previous report (11), in which CSF samples from 10 of 13 patients were positive for B. burgdorferi DNA by PCR. However, that report (11) specified neither the volume of CSF used nor whether the series of 13 patients was consecutive, and urine was not examined in that study. Because of the low number of B. burgdorferi in CSF, it may be beneficial to use, for example, 3 ml of CSF, like Kruger and Pulz (31) did in a series of two patients. To explore this, we tried to prepare DNA from 1 ml of CSF from two patients with neuroborreliosis whose CSF was otherwise PCR negative for B. burgdorferi DNA. This did not improve the results. We believe that the different sensitivities obtained by using urine and CSF was due to the lower number of B. burgdorferi genome copies in CSF, since the sensitivity of our assay should have been equally high for

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both body fluids (Fig. 2). The assumption of a higher concentration of spirochetal constituents in urine is supported by the previous successful detection of B. burgdorferi antigens in urine from experimentally infected animals (26) and even some patients (12, 26). B. burgdorferi antigen has never been detected in the CSF of patients with neuroborreliosis. Although the diagnostic sensitivity in pretreatment urine samples was only 50%, it is an interesting finding that the sensitivity increased to 90% when urine obtained 3 to 6 days after the onset of therapy was used. This probably reflects spirochetal lysis and increased excretion of spirochetal breakdown products, which also likely explains the apparently increased amounts of the amplification products regularly found in the second urine sample (Fig. 4). The consequently negative PCR results in posttherapy samples from 20 patients with neuroborreliosis, including 3 patients with chronic neuroborreliosis, suggests eradication of the spirochete and does not favor the hypothesis of a persistent, latent infection, which has been considered as a possible explanation for the frequently persistent specific intrathecal antibody responses in patients with neuroborreliosis (17, 19, 21, 31, 36). However, clinical experience has never revealed the occurrence of relapses in patients with neuroborreliosis who were appropriately treated (22, 29). Recently, T. pallidum DNA has successfully been detected in CSF from patients with neurosyphilis (8, 15, 24, 40). The diagnostic sensitivity achieved in patients with this condition seems higher than that achieved in patients with neuroborreliosis. This and the sporadically positive PCR results obtained months to years after therapy in a few patients with neurosyphilis (40) may be attributable to the significantly higher number of spirochetes in patients with syphilis compared with the number in those with Lyme borreliosis. 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 patients with third-stage (late) neuroborreliosis. Our results are thus the first confirmation that even patients with chronic neuroborreliosis have spirochetal DNA in their CSF and urine, indicating an active infection. Thus, the pathogenesis in patients with this type of neuroborreliosis is not only a result of autoimmune mechanisms (37, 48). Our selection of samples from patients with neuroborreliosis with confirmed specific intrathecal antibody production was intended to obtain relevant documentation and a measure of the sensitivity of the PCR. This is necessary as long as strict diagnostic criteria for neuroborreliosis are not generally used (22, 30). We believe that before PCR is regarded as a reliable diagnostic test, its performance should at least be comparable to that of detection of intrathecalspecific antibody synthesis. Only on this condition, and when the deleterious problem of contamination becomes controllable, will PCR be a useful supplement, especially in patients with a short disease duration, and, possibly, will also 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-specific DNA was found in urine as well as CSF even in patients with chronic neuroborreliosis. The lack of positive PCR results during posttreatment follow-up does not support the hypothesis of a persistent infection. The presence of Taq polymerase 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 a diagnostic tool for Lyme neuroborreliosis remains undetermined. ACKNOWLEDGMENTS We 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 Clinical Immunology, Statens Seruminstitut, Copenhagen, for the synthetic oligonucleotide primers and Frank Espersen, Department of Clinical Microbiology, Rigshospitalet, Copenhagen, for supplying us with urine 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 for Medical Biotechnology under the Danish Biotechnological Research and Development program. REFERENCES 1. Ackermann, R., E. Golimer, and B. Rehse-Kupper. 1985. Progressive Borrelien-Enzephalomyelitis. Chronische Manifestation der Erythema-Chronicum-Migrans-Krankheit am Nervensystem. Dtsch. Med. Wochenschr. 110:1039-1042. 2. Asbrink, E., A. Hovmark, and B. Hederstedt. 1984. The spirochetal etiology of acrodermatitis chronica atrophicans Herxhe-

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