The aim of the present study was to assess concentrations of different cytokines and chemokines in serum and CSF of patients with Lyme neuroborreliosis, and to identify possible marker(s) that would enable distinction between clinically evident and suspected Lyme neuroborreliosis as well as between Lyme neuroborreliosis and tick-borne encephalitis (TBE). Our additional interest was to evaluate relationship between cytokine/chemokine concentrations and B. burgdorferi sensu lato isolation from CSF as well as intrathecal synthesis of specific borrelial antibodies. We found that higher concentrations of CXCL13 and lower concentrations of IL-10 in sera were associated with higher odds for clinically evident Lyme neuroborreliosis in comparison to suspected Lyme neuroborreliosis as well as to TBE. CSF concentrations of IL-2, IL-5, IL-6, IL-10 and CXCL13 in the CSF were higher in patients with evident Lyme neuroborreliosis in comparison to those with suspected Lyme neuroborreliosis. Comparison of CSF cytokines and chemokines levels in patients with and without intrathecal synthesis of specific borrelial antibodies revealed that CXCL13 CSF concentration is significantly associated with intrathecal synthesis of borrelial antibodies. Comparison of cytokine and chemokine CSF concentrations in patients with clinically evident Lyme neuroborreliosis according to CSF culture result revealed that higher concentrations of IFN-gama were associated with lower odds for borrelia isolation.Although several differences in serum and CSF concentrations of various cytokines and chemokines between the groups were found, the distinctive power of the majority of these findings was low. Further research on well-defined groups of patients is needed to appraise their potential diagnostic usefulness.
F.02 Acquisition of new scientific knowledge
COBISS.SI-ID: 30765273Broth microdilution and macrodilution assays were used to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of six antimicrobial agents (ceftriaxone, cefuroxime sodium, azithromycin, amoxicillin, doxycycline and amikacin) for nine European human isolates of Borrelia burgdorferi sensu stricto (s.s.). Strains were obtained from patients diagnosed with Lyme borreliosis in Slovenia. Modified Kelly-Pettenkofer medium with a final inoculum of 10(5)Borrelia cells/mL and incubation periods of 72h and of 3 weeks and 6 weeks were used in the determination of MICs and MBCs, respectively. Observed MICs indicated that all isolates were susceptible to all the tested antimicrobial agents with the exception of amikacin. Cefuroxime sodium (MIC(90)=0.063mg/L), azithromycin (MIC(90)=0.22mg/L) and ceftriaxone (MIC(90)=0.25mg/L) displayed the lowest MICs, followed by amoxicillin (MIC(90)=1mg/L) and doxycycline (MIC(90)=2mg/L); no strain was susceptible to amikacin (MIC(90)=256mg/L). MBCs after incubation for 3 weeks and 6 weeks were determined for amoxicillin (MBC(90)=32mg/L), doxycycline (MBC(90)=32mg/L) and amikacin (MBC(90)=1024mg/L) and were found to be high (but not defined) for azithromycin (MBC(90))0.88mg/L), cefuroxime sodium (MBC(90))4mg/L) and ceftriaxone (MBC(90))4mg/L). In determination of borrelial susceptibility to antimicrobial agents, intrinsic low susceptibility or methodological factors could result in low in vitro susceptibility of individual strains. This study is the first report on the antibiotic susceptibility of a series of European human isolates of B. burgdorferi s.s.
F.02 Acquisition of new scientific knowledge
COBISS.SI-ID: 30408409B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. bavariensis are the principal species which account for Lyme borreliosis (LB) globally. We have developed an internally controlled duplex quantitative real time PCR assay targeting the Borrelia 16S rRNA and the human RNAseP genes. This assay is well-suited for laboratory confirmation of suspected cases of LB and will be used to assess the efficacy of a vaccine against LB in clinical trials. The assay is highly specific, successfully detecting DNA extracted from 83 diverse B. burgdorferi sensu lato strains representing all major species causing LB, while 21 unrelated microbial species and human genomic DNA tested negative. The assay was highly reproducible and sensitive, with a lower limit of detection of 6 copies per PCR reaction. Together with culture, the assay was used to evaluate paired 3 mm skin biopsy samples taken from 121 patients presenting with solitary erythema migrans (EM) lesion. PCR testing identified more positive biopsy samples than culture (77.7% PCR positive versus 55.1% culture positive) and correctly identified all specimens scored as culture positive. OspA-based typing identified the majority of isolates as B. afzelii (96.8%) and the bacterial load was significantly higher in culture positive biopsies than in culture negative biopsies (P = 0.001). The quantitative data also enabled relationships between Borrelia burden and patient symptoms to be evaluated. The bacterial load was significantly higher among patients with systemic symptoms than without (P = 0.02) and was significantly higher for biopsies retrieved from patients with EM lesions with central clearing (P,0.001). 16S copy numbers were moderately lower in samples from patients reporting a history of LB (P = 0.10). This is the first quantitative PCR study of human skin biopsies predominantly infected with B. afzelii and the first study to demonstrate a clear relationship between clinical symptoms in B. afzelii-infected patients and Borrelia burden.
F.21 Development of new health/diagnostic methods/procedures
COBISS.SI-ID: 30608857Clinical 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.
F.02 Acquisition of new scientific knowledge
COBISS.SI-ID: 31060953Tick-borne encephalitis (TBE) developed in 3 persons in Slovenia who drank raw milk; a fourth person, who had been vaccinated against TBE, remained healthy. TBE virus RNA was detected in serum and milk of the source goat. Persons in TBE-endemic areas should be encouraged to drink only boiled/pasteurized milk and to be vaccinated.
F.01 Acquisition of new practical knowledge, information and skills
COBISS.SI-ID: 30563289