engleski

Diagnosis of Lyme borreliosis by polymerase chain reaction

Borrelia burgdorferi (B. burgdorferi), a spirochete mostly transmitted by ticks of the Ixodidae family, causes Lyme borreliosis (LB), a multisystemic disease that affects the skin, joints, central nervous system, and heart. Typical and the most common skin manifestation of Lyme borreliosis is erythema migrans (EM), whereas neurologic symptoms, joint involvement, and chronic skin alterations can develop at later stages of the disease. In northwestern Croatia, an endemic area for Lyme borreliosis, four genomic B. burgdorferi sensu lato groups were identified in the Ixodes ricinus ticks: B. afzelii, B. garinii, B. valaisiana (group VS116), and B. burgdorferi sensu stricto. The classification of B. burgdorferi sensu lato into genomic groups has clinical relevance for LB. The association of B. afzelii with skin manifestations, of B. garinii with neurologic symptoms, and of B. burgdorferi sensu stricto with arthritis has been demonstrated. The pathogenetic potential of B. valaisiana has not yet been identified. In Croatia, B. burgdorferi was first isolated in 1991 at the Department of Dermatology and Venereology, Zagreb University Hospital Center, from the skin of an EM patient, and was named P1 Zagreb. Electrophoretic analysis of B. burgdorferi proteins revealed six of the most important proteins of different molecular mass: OspA, OspB, OspC, p41, p60, and p100, classifying the isolate into the B. burgdorferi sensu lato group. Sequence data and phylogenetic analysis confirmed that DNA isolates from the sera of patients with EM coming from northwestern Croatia belonged to the B. afzelii genospecies. The diagnosis of LB is generally based on clinical picture and demonstration of specific antibodies to B. burgdorferi by indirect serologic tests. The immunofluorescence assay (IFA) measuring circulating serum antibody binding to B. burgdorferi antigen is most commonly used. Specific IgM antibodies to B. burgdorferi occur 2 to 4 weeks after infection (in most patients, initial IgM response decreases after 1-2 months), whereas the presence of specific IgG antibodies in serum can be demonstrated only at 6 to 8 weeks of infection, from whence they are detectable for months and years thereafter. The shortcomings of this assay are subjective interpretation of results, false-positive reaction in patients with treponemal infection and some autoimmune disorders, and low sensitivity in stage I disease. Low number of B. burgdorferi in pathologic lesions and tissue fluids, low antigen level, and the ability of B. burgdorferi to escape the host's immune response result in slow, poor, and unreliable formation of specific antibodies. The interpretation of serologic test results in endemic populations with subclinical B. burgdorferi infection may frequently prove quite difficult. The method of target-sequence DNA amplification by repetitive cycles of DNA synthesis, polymerase chain reaction (PCR), has proved useful in the diagnosis of LB. It has proved especially valuable in the diagnosis of early dissemination of B. burgdorferi, spirochetemia, and from primary skin lesions, which is rarely manifested by clinical signs of advanced LB. In these cases, LB poses a diagnostic problem just because of the low B. burgdorferi concentration in blood, which makes isolation of B. burgdorferi unsuccessful, whereas PCR has proved to be fast, reliable, sensitive, and species-specific even in samples containing <10 bacteria per milliliter of tested fluid. PCR has also been found useful when the clinical manifestations of LB are not typical, eg, absence of erythema chronica migrans (ECM), presence of isolated asymmetric joint lesions, or neuroborreliosis in the early stage of disease. Early results provided by PCR allow for and help in timely initiation of LB treatment as well as in the follow-up of therapeutic success. In addition, PCR has been used in typing of as-yet-unclassified B. burgdorferi genotypes as well as in assessing the possible persistence of B. burgdorferi infection activity. Therefore, the method is highly valuable in patients with chronic symptoms that are not specific exclusively for LB, such as arthritis and encephalomyelitis. The aim of the present study was to analyze the sensitivity of IFA and PCR methods in the diagnosis of LB. Sensitivity testing was performed in three target groups of subjects at a high risk of LB infection (two groups of the risk population of forestry workers) or with existent clinical picture of LB. A group of subjects from the risk population of forestry workers from an LB-endemic area in northwestern Croatia, a group of subjects from the risk population of forestry workers from an LB-nonendemic area in Istria, and a group of subjects suffering from clinically manifest early LB in the form of ECM were included in the study. The fact that the inflammatory stage of circumscribed scleroderma (CS) and subsequent sclerotization resemble a typical late manifestation of LB, acrodermatitis chronica atrophicans, has stimulated some researchers to analyze sera of CS patients to identify the possible etiopathogenetic role of B. burgdorferi sensu lato in its development. Urged by this concept, we included CS patient serum testing in this study to demonstrate the presence of antibodies or Borrelia DNA.

Linked immunosorbent-assay; Burgdorferi sensu-lato; Tick-borne spirochetosis; Localized scleroderma; Erytherma migrans; Immunofluorescence asay; Genomic groups; Disease; Prevalence; Morphea

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