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J Clin Microbiol. 2002 December; 40(12): 4581–4584.
doi: 10.1128/JCM.40.12.4581-4584.2002. PMCID: PMC154646

Copyright © 2002, American Society for Microbiology

Importance of Sample Preparation for Molecular Diagnosis of Lyme Borreliosis from Urine

A. R. Bergmann,1 B. L. Schmidt,2 A.-M. Derler,1† and E. Aberer1*
Department of Dermatology, Karl Franzens University Hospital, A-8036 Graz,1 Department of Dermatology, Lainz Hospital, Vienna, Austria2
*Corresponding author. Mailing address: Auenbrugger Platz 8, A-8036 Graz, Austria. Phone: 0043/316-385-80317. Fax: 0043/316-385-2466. E-mail: elisabeth.aberer@uni-graz.at.
†Present address: County Hospital, Feldbach, Austria.
Received July 2, 2002; Revised August 8, 2002; Accepted September 24, 2002.

Urine PCR has been used for the diagnosis of Borrelia burgdorferi infection in recent years but has been abandoned because of its low sensitivity and the irreproducibility of the results. Our study aimed to analyze technical details related to sample preparation and detection methods. Crucial for a successful urine PCR were (i) avoidance of the first morning urine sample; (ii) centrifugation at 36,000 × g; and (iii) the extraction method, with only DNAzol of the seven different extraction methods used yielding positive results with patient urine specimens. Furthermore, storage of frozen urine samples at −80°C reduced the sensitivity of a positive urine PCR result obtained with samples from 72 untreated erythema migrans (EM) patients from 85% in the first 3 months to <30>3 months. For confirmation of the PCR products, hybridization by GEN-ETI-K-DEIA can be recommended.


We thank G. Gorkiewicz, Institute of Molecular Biology, Biochemistry, and Microbiology, Karl Franzens University, Graz, Austria, for sequencing.

This work was supported by the Austrian Science Foundation (project P13668).


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Clin Diagn Lab Immunol. 2005 Aug;12(8:910-7

Critical evaluation of urine-based PCR assay for diagnosis of lyme borreliosis.

Rauter C, Mueller M, Diterich I, Zeller S, Hassler D, Meergans T, Hartung T.

Biochemical Pharmacology, University of Konstanz, Fach M655, 78457 Konstanz, Germany. Thomas.Hartung@uni-konstanz.de.

Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole Borrelia or Borrelia DNA was established. The development of a nested real-time PCR targeting ospA enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at -20 degrees C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 x g for 30 min resulted in a concentration of both spikes, i.e., whole Borrelia and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin.

The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis.

PMID: 16085907 [PubMed - in process]
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Całość tutaj:

http://cvi.asm.org/cgi/content/full/12/8/910

etection of Borrelia burgdorferi DNA in urine of patients with ocular Lyme borreliosis

Uwe Pleyera, Susanne Priemb, Lars Bergmanna, Gerd Burmesterb, Christian Hartmanna, Andreas Krauseb
a Department of Ophthalmology, Charité, Humboldt University, Campus Virchow Hospital, Augustenburger Platz 1, D-13353 Berlin, Germany, b Department of Medicine, Rheumatology and Clinical Immunology, Charité, Humboldt University, Campus Charité Mitte, Schumannstrasse 20/21, D-10098 Berlin


Correspondence to: Dr Uwe Pleyer uwe.pleyer@charite.de

Accepted for publication 13 December 2000


AIMTo evaluate the diagnostic value of the polymerase chain reaction (PCR) to detect Borrelia burgdorferi DNA in patients with ocular Lyme borreliosis.
METHODSOf 256 consecutive uveitis patients six selected individuals with clinical evidence for Lyme borreliosis and 30 patients with non-Lyme uveitis were enrolled. Lyme serology was performed by ELISA and western blotting. Urine samples were examined by an optimised nested polymerase chain reaction (PCR) protocol.
RESULTSOnly four of six uveitis patients suspected for Lyme borreliosis were ELISA positive, while all six subjects showed a positive western blot. B burgdorferi PCR was positive in all of these six patients. Whereas two of the 30 controls had a positive Lyme serology, B burgdorferi DNA was not detectable by PCR in any sample from these patients.
CONCLUSIONSPCR for the detection of B burgdorferi DNA in urine of uveitis patients is a valuable tool to support the diagnosis of ocular Lyme borreliosis. Moreover, these patients often show a weak humoral immune response which may more sensitively be detected by immunoblotting.

Diagnostic value of PCR for detection of Borrelia burgdorferi in skin biopsy and urine samples from patients with skin borreliosis

Brettschneider S, Bruckbauer H, Klugbauer N, Hofmann H

J Clin Microbiol 1998 Sep;36(9):2658-65

Klinik fur Dermatologie und Allergologie am Biederstein, Technische Universitat Munchen, Munich, Germany.

Abstract:
Skin biopsies of 36 patients with erythema migrans and acrodermatitis chronica atrophicans (ACA) before therapy and those of 8 patients after therapy were examined for Borrelia burgdorferi DNA by PCR. Skin biopsies of 27 patients with dermatological diseases other than Lyme borreliosis and those of 10 healthy persons were examined as controls. Two different primer sets targeting 23S rRNA (PCR I) and 66-kDa protein (PCR II) genes were used. PCR was performed with freshly frozen tissue (FFT) and paraffin-embedded tissue (PET). For FFT specimens of erythema migrans, 73% were positive by PCR I, 79% were positive by PCR II, and 88% were positive by combining PCR I and II. For PET specimens, PCR was less sensitive (PCR I, 44%; PCR II, 52%). For FFT specimens of ACA, PCR I was positive for two of five patients and PCR II was positive for four of five patients. B. burgdorferi was cultured from 79% of the erythema migrans specimens but not from any of the ACA lesions. Elevated B. burgdorferi antibodies were detected in sera of 74% of erythema migrans patients and 100% of ACA patients. All urine samples were negative by PCR II, whereas PCR I was positive for 27%. However, hybridization of these amplicons was negative. Sequencing of three amplicons identified nonborrelial DNA. In conclusion, urine PCR is not suitable for the diagnosis of skin borreliosis. A combination of two different primer sets achieves high sensitivity with skin biopsies. In early erythema migrans infection, culture and PCR are more sensitive than serology.

Keywords:
Adult, Aged, Bacterial Proteins, BIOSYNTHESIS, GENETICS, Base Sequence, Biopsy, Borrelia burgdorferi, GENETICS, ISOLATION & PURIF, Chromosome Mapping, Chromosomes, Bacterial, DNA, Bacterial, GENETICS, DNA, Ribosomal, GENETICS, Female, Human, Lyme Disease, DIAGNOSIS, PATHOLOGY, URINE, Male, Middle Age, Molecular Sequence Data, Polymerase Chain Reaction, METHODS, Reference Values, RNA, Ribosomal, 23S, GENETICS, Sensitivity and Specificity, Sequence Alignment, Skin, CYTOLOGY, MICROBIOLOGY, PATHOLOGY, Skin Diseases, Bacterial, DIAGNOSIS, PATHOLOGY, URINE, Urine, MICROBIOLOGY

Molecular and Cellular Probes
Volume 11, Issue 2, April 1997, Pages 89-94

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in urine specimens of patients with erythema migrans lesions

G. Merciera, A. Burckela and G. Lucotteb, f1

a Burckel Laboratory, 27 rue Taine, Paris, France

b Regional Center of Neurogenetics, Service of Neurology, Maison Blanche Hospital, 45 rue Cognacq-Jay, Reims, France


Received 6 November 1996; accepted 7 November 1996. ; Available online 17 April 2002.

Abstract
A polymerase chain reaction (PCR) assay was developed for the detection ofBorrelia burgdorferi-specific DNA in the urine of patients with erythema migrans (EM). The target for the PCR was a specific region of the flagellin gene, and DNA was extracted from urine by Chelex resin. The detection limit was 1–10 genomes ofB. burgdorferi,B. gariniiorB. afzelii. A prospective study was performed with 12 consecutively diagnosed patients with EM, to evaluate the PCR assay on clinical samples.Borrelia burgdorferi-specific DNA could be detected in urine specimens from the 12 patients with EM before antibiotic therapy. Five weeks after therapy all the patients were negative by PCR of urine. Results of the present study confirm that the described PCR assay is sensitive and that this sort of test allows monitoring of the efficacy of therapy in patients with early Lyme borreliosis.

Author Keywords: Borrelia burgdorferi DNA; polymerase chain reaction (PCR); detection in urine; Lyme borreliosis (LB); erythema migrans (EM); flagellin sequences; monitoring; antibiotic therapy

f1 Author to whom all correspondence should be addressed.

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in
the urine and breast milk of patients with Lyme borreliosis

Diagn Microbiol Infect Dis. 1995 Mar;21(3):121-8.
PMID: 7648832 [PubMed - indexed for MEDLINE]

Schmidt BL, Aberer E, Stockenhuber C, Klade H, Breier F, Luger A.

Ludwig Boltzmann Institute for Dermato-Venerological Serodiagnosis,
University of Vienna, Austria.

Current laboratory diagnosis of Lyme borreliosis relies on tests for the
detection of antibodies to Borrelia burgdorferi with known limitations.
By using a simple extraction procedure for urine samples, B. burgdorferi
DNA was amplified by a nested PCR with primers that target the specific
part of the flagellin gene. To control possible inhibition of the enzyme
(polymerase), a special assay using the same primers was developed. We
examined 403 urine samples from 185 patients with skin manifestations of
Lyme borreliosis. Before treatment, B. burgdorferi DNA was detected in
88 of 97 patients with Lyme borreliosis. After treatment, all but seven
patients became nonreactive. Six of these seven persons suffered from
intermittent migratory arthralgias or myalgias, and one from
acrodermatitis chronica atrophicans. Two of 49 control patients with
various dermatologic disorders and none out of 22 presumably healthy
persons were reactive in the PCR. In addition to urine, breast milk from
two lactating women with erythema migrans was tested and also found
reactive. Borrelia burgdorferi DNA can be detected with high sensitivity
(91%) by a nested PCR in urine of patients with Lyme borreliosis. In
addition, this test can be a reliable marker for the efficacy of treatment.

89048796
AU - Burgess EC
IN - School of Veterinary Medicine, University of Wisconsin, Madison 53706.

Borrelia burgdorferi infection in Wisconsin horses and cows

Annals of the New York Academy of Sciences 1988;539:235-43

Blood samples from Wisconsin horses and cows suspected of having clinical
disease due to Borrelia burgdorferi infection were submitted by
veterinary practitioners. All serum, milk, colostrum, and synovial
samples were tested for B. burgdorferi antibodies by immunofluorescence.
Whole blood, milk, colostrum, and synovial fluid samples were cultured
for B. burgdorferi. Records were kept on the clinical signs of
antibody-positive animals, date of sample, and location of the animal by
county. Of the samples tested for antibodies 282/430 cow sera, 118/190
horse sera, 5/10 cow synovial fluids, 3/6 horse synovial fluids, 2/3 cow
colostrums, 0/44 cow milk samples and 1 aborted fetus serum were antibody
positive at a titer of 1:128 or greater. Of samples cultured 7/156 cow
bloods, 2/35 horse bloods, 1/14 cow synovial fluids, 0/4 synovial fluids,
1/3 cow colostrums, 0/44 cow milk, and 2/10 cow urine samples were B.
burgdorferi culture positive. For both cows and horses October and May
were the two peak months for the number of antibody-positive samples. The
most frequent clinical signs in antibody-positive horses and cows were
lameness and swollen joints, but many also had stiffness, laminitis,
abortions, and fevers. Not all antibody-positive animals showed clinical
signs. These findings show that B. burgdorferi infection occurs in horses
and cows and can cause clinical illness in some but not all animals.
Infection in cows and horses occurs most frequently 1 month after the
emergence of adult I. dammini. Because spirochetes could be isolated from
blood, synovial fluid, colostrum, and urine, these animals could be
important in providing an infected blood meal for ticks and bringing B.
burgdorferi in direct contact with humans.
IS - 0077-8923

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Journal of Spirochetal and Tick-Borne Diseases 1998; 5(4):54-62

Evidence for in utero Transmission of Borrelia burgdorferi from Naturally
Infected Cows

Leibstein MM, Khan MI, Bushmich SL


Five of 15 adult cows were spirochetemic at parturition; 4 of the calves from
these cows were also spirochetemic at birth (PCR). Spirochetes were cultured
from the placentas in 2 of 10 cows and from the uterine fluid in 1 of 8 cows.
Borrelia burgdorferi DNA was detected in the colostrum in 4 of 12 cows. Three
of 15 calves were stillborn; Borrelia burgdorferi DNA was detected by PCR in 3
of 3 and spirochetes cultured from 2 of 3 stillborn calves. Fetal tissues from
which Borrelia burgdorferi DNA was detected include blood, spleen, bladder,
kidney, synovial fluid and tissue, heart, cerebrum, and aqueous humor. Borrelia
burgdorferi was cultured from the spleen of one stillborn calf and the kidney
of another. Detection of Borrelia burgdorferi DNA from the tissues of stillborn
calves, as well as spirochetemia in neonatal liveborn and stillborn calves,
gives evidence for in utero transmission of Borrelia burgdorferi in naturally
infected dairy cattle.)