Validation of Cultivation and PCR Methods for Diagnosis of Lyme Neuroborreliosis▿

Borrelial infection may manifest with a wide range of clinical signs, and in many cases, microbiological findings are essential for a proper diagnosis. This study included 48 patients with a working clinical diagnosis of Lyme neuroborreliosis, 45 patients with a working clinical diagnosis of suspected Lyme neuroborreliosis, and a control group comprising 42 patients with tick-borne encephalitis and 21 neurosurgical patients. The aim of the study was to analyze and compare findings of two PCR methods and Borrelia burgdorferi sensu lato culture results by examination of prospectively collected cerebrospinal fluid (CSF) and blood specimens from patients with clinical features of Lyme neuroborreliosis. Borrelial DNA was detected with at least one of the PCR approaches in 16/135 (11.9%) blood samples and 24/156 (15.4%) CSF samples. Using MseI restriction of PCR products of the amplified rrf-rrl region, we identified the majority of strains as Borrelia afzelii. Borreliae were isolated from 1/135 (0.7%) blood samples and from 5/156 (3.2%) CSF specimens. Using MluI restriction for characterization of isolated strains, Borrelia garinii was identified in all CSF isolates. Our study revealed that different approaches for direct demonstration of borrelial infection give distinct results, that there is an urgent need for standardization of the methods for direct detection of borrelial infection, and that the design of studies evaluating the validation of such methods should include appropriate control group(s) to enable assessment of both sensitivity and specificity

Comparison of Borrelia burgdorferi Sensu Lato Strains Isolated from Specimens Obtained Simultaneously from Two Different Sites of Infection in Individual Patients

The aim of the present study was to analyze and compare Borrelia strains isolated from two different specimens obtained simultaneously from individual patients with Lyme borreliosis. Fifty such patients and 50 corresponding pairs of Borrelia isolates (100 low-propagated strains) were subjected to genotypic and phenotypic analysis, including pulsed-field gel electrophoresis for species identification and plasmid profile determination and protein profile electrophoresis for the assessment of the presence and molecular masses of separated proteins. The strains were isolated from two distinct skin lesions (12 patients), skin and blood (28 patients), skin and cerebrospinal fluid (8 patients), and blood and cerebrospinal fluid (2 patients). Out of 100 isolates, 63 were typed as B. afzelii and 37 as B. garinii. From each individual specimen only a single Borrelia species was cultured. Comparison of 50 Borrelia strain pairs isolated from two different specimens of an individual patient revealed that 12/50 (24%) patients were simultaneously infected with two different Borrelia strains; in 3/50 (6%) patients strains differed at the species level, in 4 out of the remaining 47 (9%) patients a strain difference in plasmid profile was established, while 5 out of the remaining 43 (11%) patient strain pairs differed in regard to the protein profiles of the two concurrently isolated strains. The results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor different B. burgdorferi sensu lato strains

Clinical characteristics associated with Borrelia burgdorferi sensu lato skin culture results in patients with erythema migrans.

Clinical characteristics associated with isolation of Borrelia burgdorferi sensu lato from skin have not been fully evaluated. To gain insight into predictors for a positive EM skin culture, we compared basic demographic, epidemiologic, and clinical data in 608 culture-proven and 501 culture-negative adult patients with solitary EM. A positive Borrelia spp. skin culture was associated with older age, a time interval of >2 days between tick bite and onset of the skin lesion, EM ≥ 5 cm in diameter, and location of the lesion on the extremities, whereas several other characteristics used as clinical case definition criteria for the diagnosis of EM (such as tick bite at the site of later EM, information on expansion of the skin lesion, central clearing) were not. A patient with a 15-cm EM lesion had almost 3-fold greater odds for a positive skin culture than patients with a 5-cm lesion. Patients with a free time interval between the tick bite and onset of EM had the same probability of a positive skin culture as those who did not recall a tick bite (OR=1.02); however, the two groups had >3-fold greater odds for EM positivity than patients who reported a tick bite with no interval between the bite and onset of the lesion. In conclusion, several yet not all clinical characteristics used in EM case definitions were associated with positive Borrelia spp. skin culture. The findings are limited to European patients with solitary EM caused predominantly by B. afzelii but may not be valid for other situations

Are patients with erythema migrans who have leukopenia and/or thrombocytopenia coinfected with Anaplasma phagocytophilum or tick-borne encephalitis virus?

Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents

Gender disparity between cutaneous and non-cutaneous manifestations of Lyme borreliosis.

Cutaneous manifestations of Lyme borreliosis in Europe include erythema migrans (EM) and acrodermatitis chronica atrophicans (ACA); the most common non-cutaneous manifestations are Lyme neuroborreliosis (LNB) and Lyme arthritis. The purpose of this study was to evaluate the gender distribution of patients with these clinical manifestations of Lyme borreliosis. Data on gender were obtained from the clinical records of patients with Lyme borreliosis aged ≥15 years who had been evaluated at the University Medical Center Ljubljana, Ljubljana, Slovenia. Among 10,539 patients diagnosed with EM, 6,245 (59.3%) were female and among 506 ACA patients 347 (68.6%) were female. In contrast, among the 60 patients with Lyme arthritis only 15 (25%) were female (p<0.0001 for the comparison of gender with EM or ACA) and among the 130 patients with LNB only 51 (39.2%) were females (p<0.0001for the comparison of gender with EM or ACA). Although the proportion that was female in the LNB group was greater than that of patients with Lyme arthritis, this difference did not reach statistical significance (p = 0.10). Although older individuals are more likely to be female in the general Slovenian population, the age of patients with cutaneous versus non-cutaneous manifestations was not the explanation for the observed differences in gender. In conclusion, patients with cutaneous manifestations of Lyme borreliosis were predominantly female, whereas those with non-cutaneous manifestations were predominantly male. This provocative finding is unexplained but may have direct relevance to the pathogenesis of Lyme borreliosis

Characteristics of patients with erythema migrans (EM) with associated leukopenia and/or thrombocytopenia, and patients with EM with normal blood cell count (controls).

<p>EM, erythema migrans; ESR, erythrocyte sedimentation rate; AST, aspartate aminotransferase enzyme; ALT, alanine aminotransferase enzyme; TBE, tick-borne encephalitis.</p>a<p>Calculated for patients with one tick bite.</p>b<p>Duration of erythema migrans (as noticed by patient) prior to diagnosis.</p>c<p>Of 37 patients with leukopenia at the initial visit, 6 (16.2%) also had decreased blood lukocyte counts at the time of convalescet serum sampling.</p>d<p>Of 32 patients with thrombocytopenia at the initial visit, 8 (25%) also had decreased blood platelet counts at the time of convalescet serum sampling.</p

Correlation between the Species of Lyme Borrelia Isolated from the Skin of Patients with Erythema Migrans and the Gender of Infected Slovenian Patients.

<p>Correlation between the Species of Lyme Borrelia Isolated from the Skin of Patients with Erythema Migrans and the Gender of Infected Slovenian Patients.</p

Gender and Age of Slovenian Patients with Lyme Borreliosis According to Clinical Manifestation.

<p>Gender and Age of Slovenian Patients with Lyme Borreliosis According to Clinical Manifestation.</p