Polymerase chain reaction in diagnosis of Borrelia burgdorferi infections an

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Polymerase chain reaction in diagnosis of Borrelia burgdorferi

infections and studies on taxonomic classification.

Lebech AM.

Department of Clinical Biochemistry, Statens Seruminstitut, Copenhagen,

Denmark.

Lyme borreliosis caused by the spirochete Borrelia burgdorferi is now

the most common vectorborne disease in North America, Europe and Asia. It is

a multisystemic infection which may cause skin, neurological, cardiac or

rheumatologic disorders. The aims of the present thesis were: (i) to develop

a PCR assay for direct detection of B. burgdorferi DNA and to evaluate the

diagnostic utility of PCR in clinical specimens from patients with Lyme

borreliosis and (ii) to study the taxonomic classification of B. burgdorferi

isolates and its implications for epidemiology and clinical presentation.

Laboratory diagnosis of Lyme borreliosis by direct demonstration of B.

burgdorferi in clinical specimens would compared to current serology allow

(i) optimal specificity, (ii) increased sensitivity during the first weeks

of infection, when the antibody response is not yet detectable and (iii)

discrimination between ongoing and past infection. Due to the extreme

paucity of spirochetes in clinical specimens neither in vitro culture nor

antigen detection had yielded a sufficient diagnostic sensitivity. Thus the

recently introduced highly sensitive PCR methodology could be a solution and

was thus studied. Assays for PCR amplification and subsequent identification

of B. burgdorferi specific sequences were established and used. For all

assays the analytical sensitivity was a few genome copies using purified DNA

as template. The efficacy of PCR was initially evaluated using tissue

samples from experimentally infected gerbils in order to start with

biological samples a priori known to contain B. burgdorferi. B. burgdorferi

DNA was detectable in 88% of the specimens. Thus the diagnostic sensitivity

of PCR was comparable to and even higher than in vitro culture. PCR was

significantly more sensitive than a histological B. burgdorferi specific

immunophosphatase-staining method. The utility of the PCR was then tested

for identification of B. burgdorferi DNA in skin biopsies from 31 patients

with erythema migrans. The sensitivity of PCR was 71%, which was superior to

culture and serology. Based on own and otherwise published results there is

clear evidence for PCR being the most sensitive and specific test for

detection of B. burgdorferi in skin biopsies from patients with both early

and late dermatoborreliosis. However, since the clinical diagnosis of

dermatoborreliosis in most instances is easy, an invasive procedure as a

skin biopsy, will only be justified in patients with an atypical clinical

presentation. The most frequent and serious manifestation of disseminated

Lyme borreliosis is neuroborreliosis. PCR was applied to 190 patients with

untreated and confirmed neuroborreliosis. B. burgdorferi DNA was detectable

in 17-21% of CSF samples from patients with neuroborreliosis. In patients

with very early neuroborreliosis (< 2 weeks), still being negative for

specific intrathecal antibody synthesis, a positive PCR was more frequent

than in patients with longer disease duration. PCR can be used as a

diagnostic aid in these patients. However, in general the measurement of

specific intrathecal antibody production in patients with neuroborreliosis

was superior to PCR. In urine samples from patients with Lyme borreliosis

the diagnostic sensitivity varied, generally showing a low reproducibility.

Urine is thus not regarded as a suitable sample source for B. burgdorferi

PCR. The reason may be the variable presence of Taq polymerase inhibitors.

Based on a semi-quantitative detection system for amplicons, reflecting the

input amount of specific DNA and thus the density of spirochetes in the

clinical samples high amounts of DNA were found in skin biopsies whereas

especially in urine the amount of DNA was low. When the present study was

initiated there was no accepted classification of B. burgdorferi. A

heterogeneity among B. burgdorferi strains might have important implications

for understanding the epidemiology and different clinical presentations

(dermatoborreliosis versus neuroborreliosis) and courses (self-limiting

versus chronic disease). Furthermore, strain differences were of importance

for selection of suitable antigens for diagnostic assays and for vaccine

development. Since then, B. burgdorferi isolates have been studied by

phenotypic and genotypic traits and have been shown to be highly

heterogeneous. Our first approach was to genotype a panel of human B.

burgdorferi isolates by restriction fragment length polymorphism (RFLP) of

three genes. Thereafter, sequencing and dideoxy fingerprinting of ospA was

applied. By RFLP the strains could be differentiated into two to five

groups. The RFLP classification was compared with four different phenotypic

and genotypic methods including the rRNA typing. Results obtained with the

different methods correlated highly and confirmed the meanwhile accepted

taxonomic classification by Baranton et al., According to this the term B.

burgdorferi sensu lato comprises three different human pathogenic

genospecies B. burgdorferi sensu stricto, B. garinii and B. afzelii. All

three genospecies have been isolated among Danish patients with Lyme

borreliosis and are thus prevalent in Denmark. Since isolation of B.

burgdorferi from patients with Lyme borreliosis is laborious and often

unsuccessful molecular typing methods based on PCR are recommended obviating

the need for isolation by prior culture. Of special interest was to study a

possible association of neuroborreliosis to certain B. burgdorferi

genospecies, indicating species depended organotropism. By RFLP all six CSF

isolates tested belonged to B. garinii and that 6 out of 7 isolates from

patients with acrodermatitis chronica atrophicans belonged to B. afzelii.

Due to the low culture yield of B. burgdorferi from CSF, the association of

B. garinii and neuroborreliosis was further studied by sequence analysis and

dideoxyfingerprinting analysis of ospA PCR amplicons obtained from CSF

samples from patients with neuroborreliosis. Phylogenetic analysis showed

that in 11 out of 13 patients B. garinii DNA was found in CSF. These data

strongly supports the hypothesis that B. garinii is the principal agent of

Lyme neuroborreliosis in Europe. Similarly it was shown that B. afzelii is

associated with acrodermatitis chronica atrophicans and thus

dermatoborreliosis. Due to a strain dependent different selection pressure

in culture only PCR based methods can be used to answer whether mixed

infection in patients specimens occur. Our data indicate that mixed

infections in humans if ever are rare.

Publication Types:

* Evaluation Studies

PMID: 11985118 [PubMed - indexed for MEDLINE]