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Journal of Clinical Microbiology, April 2001, p. 1294-1298, Vol. 39, No. 4
Department of Infectious Diseases and Medical
Microbiology, Lund University, Lund,1 and
Department of Microbiology, Umeå University,
Umeå,2 Sweden
Received 16 August 2000/Returned for modification 11 November
2000/Accepted 11 January 2001
Southern Sweden is an area of Lyme borreliosis (LB) endemicity,
with an incidence of 69 cases per 100,000 inhabitants. The most
frequent clinical manifestations are erythema migrans (77%) and
neuroborreliosis (16%). There was no record of human
Borrelia strains being isolated from patients in this
region before the prospective study reported here. Borrelia
spirochetes were isolated from skin and cerebrospinal fluid (CSF) from
LB patients living in the region. A total of 39 strains were
characterized by OspA serotype analysis, species-specific PCR, and
signature nucleotide analysis of the 16S rRNA gene. Of 33 skin
isolates, 31 (93.9%) were Borrelia afzelii strains and 2 (6.1%) were Borrelia garinii strains. Of six CSF isolates,
five (83.3%) were B. garinii and one (16.7%) was
B. afzelii. Neither Borrelia burgdorferi
sensu stricto strains nor multiple infections were observed. The
B. afzelii isolates were of OspA serotype 2. Three
B. garinii strains were of OspA serotype 5, and the remaining four strains were of OspA serotype 6. All of the
B. garinii strains belonged to the same 16S
ribosomal DNA ribotype class. Our findings agree with earlier findings
from other geographic regions in Europe where B. afzelii and B. garinii have been recovered
predominately from skin and CSF cultures, respectively. To further
study the possible presence in Sweden of the genotype B. burgdorferi sensu stricto, which is known to be present in Europe
and to occur predominately in patients with Lyme arthritis, molecular
detection of Borrelia-specific DNA in synovial samples from
Lyme arthritis patients should be performed.
Lyme borreliosis (LB) or Lyme
disease (15) is probably the most common tick-borne human
disease in Europe and the United States. The disease is a multisystemic
infection caused by the tick-borne spirochete Borrelia
burgdorferi sensu lato. Cases of LB and isolates of spirochetes
have been identified in most countries in the Northern Hemisphere
(1, 6, 12, 17, 20). The distribution of the disease is
associated with the distribution of the Ixodes tick complex
and, in Europe, Ixodes ricinus (26). This tick
is known to be abundant especially in the southern coastal areas and
central regions of Sweden, with a 5 to 26% prevalence of
borrelia-infected ticks (11, 12, 23). Typical borrelial infections primarily involve the skin, nervous system, and joints. Involvement of the nervous system neuroborreliosis is the predominant result of morbidity caused by the Borrelia bacteria in
Europe, and Borrelia bacteria are also the most common
bacterial pathogen of the nervous system in Sweden (43).
Borrelia isolates that cause LB are divided into three
different genospecies of the B. burgdorferi sensu lato
group, B. burgdorferi sensu stricto, Borrelia
afzelii, and Borellia garinii, on the basis of DNA
sequence identity (6, 18, 46). However, human pathogenic
Borrelia species that cannot be classified into any of these
groups have recently been reported (45). The three B. burgdorferi sensu lato genospecies have been
isolated from human skin, cerebrospinal fluid (CSF), and synovial fluid
as well as from ticks in Europe (16, 17, 22, 25), whereas
only the B. burgdorferi sensu stricto genotype has been
found in humans in North America. The agents of LB are divided into
eight serotypes based on their reactivity to OspA monoclonal antibodies
(MAbs) (47, 48). Furthermore, clinical data suggest an
association between infecting Borrelia species and clinical
manifestations in Europe (4, 5, 16, 17, 21, 22, 27, 39, 40, 44). B. burgdorferi sensu stricto is the most
common spirochete species associated with Lyme arthritis, and
B. afzelii predominates in the skin, especially in
cases of acrodermatitis chronica atrophicans (ACA) (39).
B. garinii is most often associated with
neuroborreliosis (5, 44). Most European skin isolates
belong to OspA serotype 2 of B. afzelii, whereas a
similar but less-stringent association has emerged between CSF isolates
and B. garinii (47).
Southern Sweden has been identified as an area where LB is highly
endemic, with an incidence of 69 cases per 100,000 inhabitants (10). The most frequent clinical manifestations are
erythema migrans (EM) (77%), neuroborreliosis (16%), and arthritis
(7%). No LB isolates had been recovered in this area of endemicity
before our present study.
The aim of this study was to isolate and characterize the
Borrelia species that cause LB in southern Sweden. LB
isolates were recovered from skin and CSF. The isolated strains were
characterized by species-specific PCR, signature nucleotide analysis by
partial 16S rRNA gene sequencing (14, 34), and tests of
immune reactivity to a panel of MAbs directed against OspA (47,
48).
Study population.
This prospective multicenter study was
carried out at eight different locations in southern Sweden, an area
where LB is endemic, from 1994 to 1997. Patients suspected of having EM
and neuroborreliosis were studied. Only adults were included in the
group that had skin biopsies. The ethics committee of Lund University,
Lund, Sweden, approved the study. The departments involved in this
study were the departments of infectious diseases, pediatrics, and
dermatology at local hospitals in southern Sweden in the provinces of
Skåne, Blekinge, Halland, and Kalmar, and an outpatient primary care center in the province of Blekinge.
Culture of Borrelia spp.
Borrelia
spirochetes were cultured from 4-mm skin biopsy specimens and 1-ml CSF
samples in Barbour-Stoenner-Kelly medium II (7), with 10%
rabbit sera (Sigma Chemical Co., St. Louis, Mo.) and 1.3% Bacto
gelatin (Difco Laboratories, Detroit, Mich.). The medium used for skin
biopsy specimens contained 100 µg of fosfomycin (Sigma) per ml and 50 µg of rifampicin (Sigma) per ml to inhibit the growth of
contaminating microorganisms. Cultures were incubated at 32°C, and
growth was recorded weekly using dark-field microscopy. The reference
strains used were B. burgdorferi sensu stricto B31 (ATCC 35210) (15), a North American tick isolate; ACA1
B. afzelii, isolated from a Swedish ACA patient
(2); and an IP90 B. garinii tick isolate
from Russia (29).
OspA serotyping.
All of the local isolates used for protein
analysis were low-passage strains (<10 passages), and the cells were
grown in Barbour-Stoenner-Kelly medium II until middle to late log
phase. Sonicated whole-cell lysates prepared from Borrelia
cells were separated on a sodium dodecyl sulfate-polyacrylamide gel
electrophoresis 4 to 15% gradient gel (Bio-Rad, Richmond, Calif.), and
the proteins were transferred to polyvinylidene difluoride membranes
(0.45-µm pore size; MSI Inc., Westborough, Mass.), as previously
described (37). These membranes were probed with a panel
of eight OspA MAbs (47, 48), including L32-1F11, L32-1C8,
L32-14G7, L32-1G3, L32-1F7, and L32-1D11, which were provided by
B. Wilske, and H5332 and H3TS (8), which were obtained from
A. G. Barbour. The H3TS is type-specific for B. burgdorferi sensu stricto serotype 1, and L32-14G7 is
type-specific for B. afzelii serotype 2. B. garinii shows a more divergent pattern that is covered by
serotypes 3 to 8.
Genotyping: 16S rRNA gene (16S rDNA) analysis.
DNA was
isolated according to the method of Bunikis et al. (14).
Standard PCR routines were used to prevent contamination (30). Briefly, filter tips (ART, San Diego, Calif.) were
used, and amplified Borrelia DNA was handled in a different
location. Contamination controls included extraction controls and
negative buffer controls in parallel with strain samples.
Genospecies-specific identification of LB isolates was determined by
16S rRNA gene analysis using PCR and primers described by Marconi and
Garon (34): a (LD) primer set specific for B. burgdorferi sensu lato, a (BB) primer set genospecific for
B. burgdorferi sensu stricto, a (BA) primer set
genospecific for B. afzelii, and a (BG) primer set
genospecific for B. garinii. Primers were synthesized
by Interactiva Biotechnologie GmBh, Ulm, Germany. PCR mixtures
contained a 0.2 µM concentration of each primer, and the PCR buffer
solution contained 10 mM Tris-HCl, 1.5 mM MgCl, 50 mM KCl (pH 8.3), 0.2 mM concentrations of each deoxynucleoside triphosphate, and 1 U of
Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany).
PCRs were performed in an MJ PTC-100 thermocycler (MJ Research Inc.,
Waltham, Mass.), as previously described (34). The
amplified DNA was separated by agarose gel electrophoresis, stained by
ethidium bromide, and visualized using a standard UV transilluminator.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1294-1298.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of Lyme Borreliosis Isolates from
Patients with Erythema Migrans and Neuroborreliosis in Southern
Sweden
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of local human pathogen spirochetes. Seventy-nine skin biopsy specimens and 207 CSF cultures from suspected LB cases were included in the study. Seventy-five patients with EM fulfilled the Centers for Disease Control and Prevention (CDC) criteria for Lyme disease (19), with the exception that a skin lesion less than 5 cm in size was accepted. Four skin biopsy specimens were taken from patients who were considered not to have EM. Thirty-three strains were collected from the 75 skin biopsy specimens, which was a success rate of 42%. The skin strains were recovered from biopsy specimens from individuals whose ages ranged from 19 to 69 years. The female/male distribution was 16:17. Twelve patients with EM had general symptoms of disseminated LB with low-grade fever, headache, arthralgia, and myalgia, and one patient had concomitant meningitis (LU253).
Eighty of the 207 patients included in the study fulfilled the CDC criteria for neuroborreliosis (19). Of these patients, 10 did not have meningitis. Six CSF isolates were recovered from the 70 patients with meningitis who also fulfilled the CDC criteria for neuroborreliosis, which results in an 8.6% culture success rate. The CSF strains were all obtained from children with meningitis who ranged from 4 to 7 years of age. In addition, two of six children had meningitis and facial palsy. Three of the six children presented an EM or had a history of EM in the head region. When contamination and the number of patients that were treated with an antibiotic before sampling were considered, the success rates for culturing EM skin biopsy specimens and CSF were 50% and 10.2%, respectively. The 33 isolates originating from skin biopsy specimens from patients with EM were identified as LU30, LU34, LU35, LU54, LU58, LU65, LU68, LU71, LU72, LU81, LU82, LU85, LU116, LU118, LU156, LU157, LU165, LU169, LU171, LU182, LU188, LU192, LU203, LU207, LU209, LU212, LU215, LU218, LU235, LU253, LU254, LU256, and LU9038. The six CSF isolates are identified as LU59, LU170, LU185, LU190, LU216, and LU222.OspA serotyping.
The results of the OspA serotyping are shown
in Table 1. Three isolates were
contaminated (LU34, LU35, and LU182) and therefore could not be used
for specific protein analysis. Two strains could not be subcultured
(LU30 and LU65), and only enough cells for DNA analysis were obtained.
None of the strains showed any reactivity to the MAb H3TS that is type
specific for B. burgdorferi sensu stricto and
represents the OspA serotype 1. Twenty-six of the 28 strains (92.9%)
isolated from skin and 1 of the 6 strains (16.7%) isolated from CSF
reacted with the MAb L32-14G7 that is type specific for B. afzelii and represents OspA serotype 2. One of the skin strains
(LU116) and two of the six strains isolated from CSF (LU59 and LU170)
were classified as B. garinii OspA serotype 5. One of
the skin strains (LU118) and three of the six strains isolated from CSF
(LU185, LU190, and LU222) reacted as B. garinii OspA serotype 6.
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16S rDNA PCR and 16S rDNA signature nucleotide analysis.
The
results of the species-specific PCRs are summarized in Table
2. All of the locally isolated strains
could be amplified using the B. burgdorferi sensu
lato-specific primer set LD, whereas none of the local strains could be
amplified by the B. burgdorferi sensu stricto primer
set BB. The DNA from 31 of 33 strains isolated from skin (93.9%)
and 1 of 6 strains from CSF (16.7%) was amplified by the
species-specific primer pair for B. afzelii (BA). Two
of 33 strains originating from skin (6.1%) and 5 of 6 strains isolated from CSF (83.3%) were amplified with the species-specific primer pair
for B. garinii (BG). None of the local isolates were
amplified by both BA and BG primer sets.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this prospective study we have for the first time recovered and characterized Borrelia strains from humans with LB in southern Sweden, an area where the high endemicity of LB is well defined (10).
The genospecies that we found to predominate in early skin borreliosis was B. afzelii (93.9%), while B. garinii predominated in neuroborreliosis (16.7%). Few B. garinii strains were recovered from skin. Occasionally, the B. afzelii strain was recovered from CSF, and one patient with EM and concomitant meningitis had a B. afzelii-positive skin culture. The three different methods used for genospecies determination, 16S rDNA PCR, signal nucleotide analysis, and OspA serotyping, all showed the same results. Our findings are in concordance with earlier clinical studies in Europe (4, 5, 16, 21, 22, 39, 44). B. garinii and B. afzelii have also previously been recovered from skin and CSF in other geographic regions in Sweden (2, 3, 6, 16, 28, 31).
B. garinii has previously been shown to form three patterns of variable signal nucleotide positions in the partial 16S rDNA sequence (14). All of the B. garinii isolates in this study fell into the 16S rDNA signal nucleotide pattern seen in the Russian tick isolate B. garinii IP90 ribotype 1. The B. garinii isolates diverged into the two OspA serotypes 5 and 6. OspA serotype 6 has been shown to predominate in CSF isolates from children in Germany (47). The diversity of B. garinii OspA serotypes depends on the geographical distribution of LB strains (16, 17, 47, 48). Comparison of B. garinii OspA serotypes from tick isolates in Japan with B. garinii serotypes isolated in Europe shows that different OspA serotypes exist on these two continents as well (35).
The B. burgdorferi sensu stricto genotype has yet to be isolated in Sweden from humans, nor have B. burgdorferi sensu stricto genotypes been found among isolates from our patients. This genotype was isolated from a lesion from a patient with ACA (31) in neighboring Denmark and was also identified by DNA analysis in I. ricinus ticks that were collected from migrating birds in southern Sweden (38). B. burgdorferi sensu stricto is known to be present in European LB, and it has been recovered from both skin and CSF but has been found more often than B. afzelii and B. garinii in synovial samples when molecular analytical methods are used (22, 25, 27). Detection of Borrelia-specific DNA in synovial samples from Lyme arthritis patients should be performed in Sweden to examine the possible presence of the genotype B. burgdorferi sensu stricto in LB.
We did not identify any multiple infections of the various genospecies in our human LB isolates. Mixed infections and genetic diversity of B. burgdorferi sensu lato, as determined by culture and/or PCR, have been observed both in Europe and the United States (20, 32, 33). The possibility of identifying multiple infections probably depends on both variation in growth requirements and factors such as the origin of the spirochetes and the technique and method being used.
Isolation of B. burgdorferi by culture is the best diagnostic evidence of LB. The bacteria are not easily recovered from clinical specimens other than biopsy samples from EM lesions. A success rate of more than 80% with skin biopsy specimens from EM patients has been reported (9). Our success rate was lower, partly due to specimen contamination and antibiotic treatment of patients prior to sampling. All of the CSF isolates were recovered from children who contracted neuroborreliosis less than 3 weeks after subclinical symptoms of meningitis or an acute facial palsy were diagnosed. Adults seem to have a tendency to neglect their symptoms, as many delayed seeing a doctor for more than 3 weeks after onset of symptoms, which may have decreased the possibility of recovering Borrelia spp. from CSF. The Borrelia medium used to culture skin biopsy specimens was supplemented with antibiotics, while the medium for CSF was antibiotic-free, which may have influenced the growth of B. garinii in CSF.
Culture is generally not useful for detection of the Borrelia bacteria or confirmation of the LB diagnosis. For laboratory diagnosis of LB, the enzyme-linked immunosorbent assay and Western blot usually are used. The importance of different B. burgdorferi species used in the antigenic preparations for these tests has been studied in LB patients in Germany (24). Results from the present study indicate a variation in test results that favors a locally recovered Borrelia strain due to the different levels of expression of immunodominant proteins. Use of local LB strains, together with information on the seroreactivity in the population to be tested, should be taken into account when developing diagnostic serological assays for clinical diagnosis (13, 36).
Two B. burgdorferi sensu stricto OspA-based Borrelia vaccines have been developed (41, 42). The geographic diversity and the wide range of clinical manifestations found in European B. burgdorferi sensu lato strains present a great challenge for vaccine development. A prerequisite for developing an LB vaccine is access to a variety of microbiologically and clinically well-characterized Borrelia strains.
Our results confirm the presence of B. afzelii and B. garinii in LB in Sweden. Additional studies on synovial samples from Lyme arthritis with molecular methods would be interesting in order to explore the possible presence of the genotype B. burgdorferi sensu stricto in Sweden. An effective Borrelia vaccine for Sweden at the minimum should protect against infection with the two genospecies B. afzelii and B. garinii.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Swedish Medical Research Council (grants 07922 and 04723), the University Hospital of Lund, the medical faculty of Lund University Kungliga Fysiografiska Sällskapet i Lund, Thelma Zoegas and Johanissons Stiftelse.
We thank Bettina Wilske for MAbs L32 1F11, L32 1C8, L32 14G7, L32 1G3, L32 1F7, and L32 1D11 and Alan Barbour for MAbs H5332 and H3TS and for reviewing the manuscript. We express our appreciation to the clinics taking part in this study: Infectious Diseases, Pediatrics, and Dermatology at the University Hospital in Lund and the hospital in Helsingborg; and Infectious Diseases and Pediatrics at the University Hospital in Malmö, the hospital in Kristianstad, the hospital in Halmstad, the hospital in Karlskrona, and the Kalmar County Hospital and the Ronneby Primary Care Clinic in Blekinge Province.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Umeå University, S-901 87 Umeå, Sweden. Phone: 46-(0)90-7856726. Fax: 46-(0)90-772630. E-mail: sven.bergstrom{at}micro.umu.se.
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Abstract
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Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, April 2001, p. 1294-1298, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1294-1298.2001
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