Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Assessment of three criteria to establish borrelial infection in suspected lyme neuroborreliosis.

PURPOSE: Diagnosis of (European) Lyme neuroborreliosis has been based on clinical presentation, cerebrospinal fluid (CSF) pleocytosis and demonstration of intrathecal borrelial antibody synthesis (ITBAS) to document Borrelia burgdorferi s. l. INFECTION: It is not known if other criteria to document Borrelia infection may contribute to the diagnosis. METHODS: We compared the sensitivity of three individual criteria (ITBAS, CSF Borrelia culture, and the presence of erythema migrans [EM]) to confirm the diagnosis of early Lyme neuroborreliosis in 280 patients ≥ 15 years of age evaluated at a Lyme borreliosis outpatient clinic in Slovenia. The patients had either radicular pain of new onset or involvement of a cranial nerve but without radicular pain, each in conjunction with CSF pleocytosis. Evaluation was of patients who had each of the three confirmatory criteria assessed, and for whom at least one criterion was positive. RESULTS: Analysis of 280 patients, 120 women and 160 men, median age 57 (range 15-84) years, revealed that ITBAS was the most frequently observed positive criterion (85.4%), followed by EM (52.9%), and by a positive CSF Borrelia culture (9.6%). Of the 280 patients, 154 (55%) met only one criterion (43.2% ITBAS only, 10.7% EM only, and 1.1% positive CSF culture only), whereas 42.1% met two criteria. Only 2.9% of patients were positive by all three criteria. CONCLUSION: Although ITBAS was the most frequent criterion for confirmation for Borrelia infection, the presence of EM alone confirmed an additional 10.7% of patients and a positive CSF Borrelia culture alone added another 1.1%.

Colocalization of Radicular Pain and Erythema Migrans in Patients With Bannwarth Syndrome Suggests a Direct Spread of Borrelia Into the Central Nervous System.

BACKGROUND: There is a general assumption that after deposition into skin, Lyme borreliae disseminate hematogenously to other organs, resulting in extracutaneous manifestations of Lyme borreliosis, including Lyme neuroborreliosis. However, our experience over the past 40 years, along with several published case reports that observed colocalization of radicular pain and erythema migrans (EM) in patients with borrelial meningoradiculoneuritis (Bannwarth syndrome), argues against hematogenous dissemination in Lyme neuroborreliosis. METHODS: We compared the location of EM in 112 patients with Bannwarth syndrome to 12315 EM patients without neurological involvement. Moreover, we assessed the colocalization of EM and radicular pain in patients with Bannwarth syndrome. RESULTS: Compared to >12000 EM patients without neurological involvement, patients with Bannwarth syndrome had a significantly higher frequency of EM on head/neck (6% vs 1%; P=.0005) and trunk (47% vs 24%; P<.0001), similar frequency on arms (16% vs 16%; P=.91), but lower frequency on legs (30% vs 59%; P<.0001). Moreover, in 79% (89/112) of patients the site of EM matched the dermatomes of radicular pain. The odds for a congruent location of EM and radicular pain were highly significant with the highest odds ratios (OR) observed for head (OR=221), followed by neck (OR=159), legs (OR=69), arms (OR=48), and trunk (OR=33). CONCLUSIONS: The greater frequency of EM on head/neck and trunk and the colocalization of EM with radicular pain in patients with Bannwarth syndrome suggest that central nervous system involvement in Lyme neuroborreliosis is due to a retrograde spread of borrelia from skin to the spinal cord via peripheral nerves.

Clinical manifestations and long-term outcome of early Lyme neuroborreliosis according to the European Federation of Neurological Societies diagnostic criteria (definite versus possible) in central Europe. A retrospective cohort study.

BACKGROUND AND PURPOSE: The characteristics and long-term outcome of Lyme neuroborreliosis (LNB) according to diagnostic certainty (definite vs. possible) are incompletely understood. METHODS: In this retrospective cohort study of adults with definite or possible LNB, clinical and microbiological characteristics and long-term outcome over 12 months were evaluated at a single medical center. Severity of acute disease and long-term outcome were assessed using a composite clinical score encompassing clinical findings and symptoms and by the probability of incomplete recovery. RESULTS: Amongst 311 adult patients enrolled from 2008 to 2017, 139 (44.7%) had definite LNB and 172 (55.3%) had possible LNB. The most frequent LNB manifestation was cranial neuropathy with or without meningitis (53.4%). Patients with definite LNB more often had Bannwarth syndrome (53.2% vs. 18.6%), more severe disease (6 points vs. 4 points), longer pre-treatment duration (median 21 days vs. 13.5 days), higher cerebrospinal fluid pleocytosis (median 139 × 106 /L vs. 11 × 106 /L) and higher rate of Borrelia seropositivity (84.2% vs. 68.6%) than those with possible LNB. Ceftriaxone was prescribed more often than oral doxycycline in definite LNB than in possible LNB (96.4% vs. 65.7%). Unfavorable outcomes decreased during follow-up, being higher in patients with more severe disease at enrollment and in those with possible LNB, but were not associated with antibiotic therapy. CONCLUSIONS: Early LNB, most often presenting as cranial neuropathy, was definitively diagnosed in less than half of cases. A better diagnostic approach is needed to confirm borrelial etiology. Ceftriaxone was not superior to doxycycline in the treatment of early LNB, regardless of diagnostic certainty. In this retrospective cohort study of 311 adults with Lyme neuroborreliosis (LNB), allocated according to diagnostic certainty, early LNB was definitively diagnosed in less than half of cases and the most frequent LNB manifestation was cranial neuropathy with or without meningitis. Patients with definite LNB more often had Bannwarth syndrome, more severe disease, longer pre-treatment duration, higher cerebrospinal fluid pleocytosis and higher rate of Borrelia seropositivity than those with possible LNB. A better diagnostic approach is needed to confirm borrelial etiology. Ceftriaxone was not superior to doxycycline in the treatment of early LNB, regardless of diagnostic certainty.

In vitro efficacy of antibiotics against different Borrelia isolates.

In the present study, the effectiveness of six antimicrobial agents have been tested against 24 borrelia strains isolated from Ixodes ricinus ticks (11 Borrelia lusitaniae, eight Borrelia afzelii, three Borrelia garinii and two Borrelia valaisiana) and one B. lusitaniae strain isolated from human skin. The minimum inhibitory concentration range of antimicrobial agents was as follows: amoxicillin, 0.125-2 mg/L; doxycycline, 0.125-1 mg/L, ceftriaxone, 0.016-0.063 mg/L; cefuroxime, 0.063-1 mg/L; azithromycin, 0.0017-0.11 mg/L; amikacin 32-512 mg/L. Potentially pathogenic B. lusitaniae and B. valaisiana species were more susceptible to amoxicillin and azithromycin than pathogenic B. afzelii and B. garinii (P < 0.05); B. garinii, B. lusitaniae and B. valaisiana were more susceptible to doxycycline than B. afzelii (P < 0.05) while all species showed same susceptibility to ceftriaxone and cefuroxime (P > 0.05). This study is the first report on in vitro susceptibility of isolates from Serbia to antimicrobial agents and the first report on susceptibility of larger number of isolates of potentially pathogenic species B. lusitaniae. We showed that antimicrobial agents in vitro inhibit growth of borrelia strains very effectively, indicating the potential of their equally beneficial use in the treatment of Lyme borreliosis.

Evaluation of real-time PCR targeting the lipL32 gene for diagnosis of Leptospira infection.

BACKGROUND: Different diagnostic methods have been used for the laboratory confirmation of leptospirosis. Molecular diagnostic techniques are not only faster and more sensitive than culture analysis, but can also detect a Leptospira infection before the appearance of antibodies. The aim of the present study was to analyze and compare two different PCR approaches applied to blood and urine specimens obtained from patients with clinical manifestations that were suggestive of leptospirosis. Furthermore, the results of these different PCR approaches were compared with the results of culture and serology analyses. RESULTS: A total of 400 samples (234 blood or 58.5% and 166 urine of 41.5%) from 310 Slovenian patients with clinical manifestations suggestive of leptospirosis were tested using conventional PCR assays targeting the rrs gene and RT-PCR targeting the lipL32 gene. Additionally, culture, serology and sequence analysis were performed for the majority of these samples. The PCR and RT-PCR results were concordant in 376 out of 400 of these samples (94.0%). Conventional PCR was positive for 27 out of 400 samples (6.8%) and RT-PCR was positive for 47 out of 400 samples (11.8%). Culture and microscopic agglutination tests supported these diagnoses. CONCLUSIONS: A comparison of the two PCR methods indicated that the RT-PCR targeting of the lipL32 gene was faster, more sensitive and more specific for the determination of Leptospira DNA in these clinical samples.

Emerging borreliae - Expanding beyond Lyme borreliosis.

Lyme borreliosis (or Lyme disease) has become a virtual household term to the exclusion of other forgotten, emerging or re-emerging borreliae. We review current knowledge regarding these other borreliae, exploring their ecology, epidemiology and pathological potential, for example, for the newly described B. mayonii. These bacteria range from tick-borne, relapsing fever-inducing strains detected in some soft ticks, such as B. mvumii, to those from bat ticks resembling B. turicatae. Some of these emerging pathogens remain unnamed, such as the borrelial strains found in South African penguins and some African cattle ticks. Others, such as B. microti and unnamed Iranian strains, have not been recognised through a lack of discriminatory diagnostic methods. Technical improvements in phylogenetic methods have allowed the differentiation of B. merionesi from other borrelial species that co-circulate in the same region. Furthermore, we discuss members that challenge the existing dogma that Lyme disease-inducing strains are transmitted by hard ticks, whilst the relapsing fever-inducing spirochaetes are transmitted by soft ticks. Controversially, the genus has now been split with Lyme disease-associated members being transferred to Borreliella, whilst the relapsing fever species retain the Borrelia genus name. It took some 60 years for the correlation with clinical presentations now known as Lyme borreliosis to be attributed to their spirochaetal cause. Many of the borreliae discussed here are currently considered exotic curiosities, whilst others, such as B. miyamotoi, are emerging as significant causes of morbidity. To elucidate their role as potential pathogenic agents, we first need to recognise their presence through suitable diagnostic approaches.

Borrelial Lymphocytoma in Adult Patients.

BACKGROUND: Information on the course and outcome of borrelial lymphocytoma (BL) is limited. METHODS: The study comprised 144 adult patients (75 female, 69 male; median age, 49 years) who had BL diagnosed at a single center between 1986 and 2014 and were followed up for 1 year. RESULTS: BL was located on the breast in 106 patients (73.6%), on the ear lobe in 27 (18.8%), and elsewhere in 11 (7.6%). The median duration of BL before diagnosis was 27 days (interquartile range [IQR], 9-68 days). Concomitant erythema migrans was registered in 104 of 144 patients (72.2%); other objective manifestations of Lyme borreliosis (LB) were present in 11 (7.6%). Immunoglobulin M and/or G borrelial serum antibodies were present in 72 patients (50%). Borreliae were isolated from BL lesions in 14 of 42 patients (33.3%) who had not received antibiotics before skin biopsy. Of 13 typed Borrelia strains, 11 were B. afzelii, 1 was B. garinii, and 1 was B. bissettii The median duration of BL after starting antibiotic treatment was 21 days ([IQR], 10-30 days); the average duration was longer in patients who were older, had longer BL duration before treatment, or had signs of disseminated LB. Treatment failure occurred in 14 of 144 patients (9.7%). Patients with signs or symptoms of disseminated LB before treatment had nearly 4 times higher odds of treatment failure (95% confidence interval, 1.22-13.07) than those without such symptoms. All patients with treatment failure had uneventful outcome after retreatment. CONCLUSIONS: BL is a rare manifestation of early localized LB. Fourteen-day antibiotic treatment, as used for erythema migrans, is effective.

Course and Outcome of Early European Lyme Neuroborreliosis (Bannwarth Syndrome): Clinical and Laboratory Findings.

BACKGROUND: Information on the course and outcome of early European Lyme neuroborreliosis is limited. METHODS: The study comprised 77 patients (38 males, 39 females; median age, 58 years) diagnosed with painful meningoradiculitis (Bannwarth syndrome) who were followed up for 1 year at a single center. RESULTS: Duration of neurological symptoms before diagnosis was 30 (interquartile range, 14-50) days. The most frequent symptoms/signs were radicular pain (100%), sleep disturbances (75.3%), erythema migrans (59.7%), headache (46.8%), fatigue (44.2%), malaise (39%), paresthesias (32.5%), peripheral facial palsy (PFP) (36.4%), meningeal signs (19.5%), and pareses (7.8%). Cerebrospinal fluid (CSF) analysis revealed lymphocytic/monocytic pleocytosis, elevated protein concentration, and intrathecal synthesis of borrelial immunoglobulin M and immunoglobulin G antibody in 100%, 81.1%, 63%, and 88.7% of patients, respectively. Borreliae (predominantly Borrelia garinii) were isolated from CSF, skin, and blood in 15.6%, 40.6%, and 2.7% of patients, respectively. The outcome after 14-day treatment with ceftriaxone was favorable in 87.8% of patients. Control CSF examination at 3 months showed decreased leukocyte counts in all patients; however, 23.3% still had pleocytosis (>10 × 10(6) cells/L). A model based on pretreatment data and the findings at the end of 14-day antibiotic treatment accurately predicted which patients would have an unfavorable outcome 6 or 12 months after treatment. CONCLUSIONS: Our patients had fewer pretreatment neurological complications (PFP, pareses) than reported for Bannwarth syndrome decades ago, probably as the result of earlier recognition and prompt antibiotic treatment. Unfavorable outcome was rare and was predicted by the continued presence of symptoms 14 days after commencement of treatment.

Differences in Genotype, Clinical Features, and Inflammatory Potential of Borrelia burgdorferi sensu stricto Strains from Europe and the United States.

Borrelia burgdorferi sensu stricto isolates from patients with erythema migrans in Europe and the United States were compared by genotype, clinical features of infection, and inflammatory potential. Analysis of outer surface protein C and multilocus sequence typing showed that strains from these 2 regions represent distinct genotypes. Clinical features of infection with B. burgdorferi in Slovenia were similar to infection with B. afzelii or B. garinii, the other 2 Borrelia spp. that cause disease in Europe, whereas B. burgdorferi strains from the United States were associated with more severe disease. Moreover, B. burgdorferi strains from the United States induced peripheral blood mononuclear cells to secrete higher levels of cytokines and chemokines associated with innate and Th1-adaptive immune responses, whereas strains from Europe induced greater Th17-associated responses. Thus, strains of the same B. burgdorferi species from Europe and the United States represent distinct clonal lineages that vary in virulence and inflammatory potential.

Broad-Range 16S rDNA PCR on Heart Valves in Infective Endocarditis.

BACKGROUND AND AIM OF THE STUDY: Infective endocarditis (IE) is diagnosed by blood and/or resected valve cultivation and echocardiographic findings, as defined by the Duke criteria. Unfortunately, cultures may be negative due to prior antibiotic therapy or fastidious or slow-growing microorganisms. The study aim was to investigate the value of the broad-range polymerase chain reaction (PCR) in addition to blood and valve culture for the detection of causative microorganisms. METHODS: Between February 2012 and March 2015, valve samples from 36 patients undergoing cardiac surgery were analyzed; of these patients, 26 had a preoperative diagnosis of IE and 10 served as controls. Multiple blood cultures were obtained from 34 patients before antibiotic therapy was commenced. Valve samples were inoculated on bacteriological media and underwent analysis using broad-range PCR (16S rDNA). RESULTS: IE was confirmed microbiologically in 21 of the 26 patients (80.7%); in 20 cases (76.9%) this was by positive blood cultures and in 16 (61.5%) by positive valves. Valves were positive in 15 blood culturepositive patients, and in one blood-culture negative patient. Broad-range PCR detected a microorganism in valves significantly more frequently (n = 14; 53.8%) compared to valve culture (n = 8; 30.7%) (chisquare 11.5, p <0.001). The predominant microorganisms were Staphylococcus aureus, Streptococcus of the viridans group, coagulasenegative staphylococci and Enterococcus faecalis. Blood, valve cultures and broad-range PCR were negative in five patients (19.3%) with IE, and in all 10 subjects of the control group. CONCLUSIONS: Broad-range PCR on valves was more sensitive than valve culture. However, blood culture, if taken before the start of antibiotic therapy, was the best method for detecting IE.

Course and outcome of Early Lyme borreliosis in patients with hematological malignancies.

Patients with erythema migrans and underlying hematological malignancy more often had signs of disseminated Lyme borreliosis and more frequently needed antibiotic retreatment than sex-, age-, and antibiotic treatment-matched immunocompetent persons with erythema migrans. However, the outcome was excellent in both groups.

Detection, identification and genotyping of Borrellia spp. in rodents in Slovenia by PCR and culture.

BACKGROUND: Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, is mainly maintained in natural foci through the transmission cycles of competent tick vectors (Ixodes sp.) and a vertebrate reservoir. Specific rodents have been identified as the principal reservoir of Borrelia burgdorferi sensu lato in Europe. Borrelia miyamotoi is the only relapsing fever spirochete transmitted by the same tick. The aim of the present study was to perform an epidemiological survey to determine the presence of B. burgdorferi sensu lato in rodents occurring in Slovenia and to explore the presence of Borrelia miyamotoi. The study was performed in two parts, retrospective and prospective; a total of 297 rodents was analyzed. Detection and identification of borrelia was performed by molecular methods and additionally in the prospective study by isolation and genotyping (MluI-LRFP and MLST). RESULTS: During the prospective part of the study, borrelia was isolated from 2/46 (4.3 %) lung specimens and from 10/46 (21.7 %) heart specimens of rodents. All isolated strains were identified as B. afzelii subtype Mla1, and MLST analysis revealed 5 distinct sequence types. Borrelia DNA was successfully detected by one or other of the PCR methods in 18/46 (39.1 %) and 75/251 (29.9 %) samples in the prospective and retrospective studies, respectively. LightMix® was found to be more sensitive than the ''in-house" nested PCR (91/297 (30.6 %) vs 48/297 (16.1 %)). Borrelia miyamotoi DNA was detected in 1/251 (0.4 %) and in 1/46 (2.2 %) heart specimens, in the retrospective and prospective parts of the study, respectively. CONCLUSION: We determined the prevalence of B. afzelii in rodents and report for the first time the presence of B. miyamotoi in Slovenia.

Erythema Migrans in Patients with Post-Traumatic Splenectomy.

Information on asplenic Lyme borreliosis (LB) patients with erythema migrans (EM) is lacking. We compared the course and outcome of 26 EM episodes in 24 post-trauma splenectomized patients (median age 51 years) diagnosed at a single clinical center in Slovenia during 1994-2023 with those of 52 age- and sex-matched patients with EM but with no history of splenectomy. All patients were followed for one year. A comparison of pre-treatment characteristics revealed that EM in splenectomized patients was of shorter duration before diagnosis (4 vs. 8 days, p = 0.034) with a smaller EM diameter (10.5 vs. 14 cm, p = 0.046), and more frequently fulfilled criteria for disseminated LB (3/26, 11.5% vs. 0%, p = 0.034). Treatment failure occurred in 5/26 (19.2%) EM episodes in splenectomized patients versus 0/52 in non-splenectomized patients (p = 0.003). The five treatment failure cases were retreated with antibiotic regimens used to treat EM and had complete resolution of all symptoms/signs. In conclusion, our study showed that splenectomized adult patients with EM differ somewhat in presentation and more often have treatment failure compared with non-splenectomized patients with EM.

Microbiologic Findings in a Cohort of Patients with Erythema Migrans.

Erythema migrans (EM) is the initial and the most frequent clinical manifestation of Lyme borreliosis (LB). Herein, we report on the capacity of culture and serology for the demonstration of Borrelia infection in a cohort of 292 patients diagnosed with typical EM at a single medical center. The median duration of EM at diagnosis was 12 days, and the largest diameter was 16 cm; 252 (86.3%) patients presented with solitary EM, whereas 40 (13.7%) had multiple EM. A total of 95/292 (32.5%) patients had positive IgM, and 169 (57.9%) had positive IgG serum antibodies; the Borrelia isolation rate was 182/292 (62.3%). The most frequent species by far was B. afzelii (142/148, 95.9%) while B. garinii (2.7%) and B. burgdorferi s.s. (1.4%) were rare. IgM seropositivity was associated with a younger age, multiple EM and the absence of underlying chronic illness; IgG seropositivity was associated with the duration of EM at diagnosis, the diameter of the EM, having had a previous episode of LB and the absence of symptoms at the site of the EM. Furthermore, the Borrelia isolation rate was statistically significantly lower in patients with positive Borrelia IgM antibodies. Although microbiologic analyses are not needed for the diagnosis of typical EM, they enable insights into the etiology and dynamics of the immune response in the course of early LB.

Suspected early Lyme neuroborreliosis in patients with erythema migrans.

BACKGROUND: Our objective was to obtain data on patients with erythema migrans (EM) who have symptoms/signs suggesting nervous system involvement and to compare epidemiologic, clinical, and microbiologic findings in patients with and without cerebrospinal fluid (CSF) pleocytosis. METHODS: Adult patients with EM and suspected early Lyme neuroborreliosis were included in this study. RESULTS: Of 161 patients, 31 (19%) had elevated and 130 (81%) had normal CSF cell counts. In contrast to patients with normal CSF cell counts, those with pleocytosis (1) more often reported radicular pain and more often presented with meningeal signs but less frequently complained of malaise; (2) had larger EM skin lesions despite similar duration; (3) more commonly had Borrelia garinii isolated from EM skin lesions (odds ratio for pleocytosis was 31 times higher in patients with established B. garinii skin infection compared to patients with other Borrelia species isolated from their EM skin lesion) and from CSF; and (4) more frequently fulfilled microbiologic criteria for established borrelial infection of the central nervous system. The positive predictive value of pleocytosis for microbiologically proven borrelial infection of the central nervous system (defined by isolation of Borrelia from CSF and/or demonstration of intrathecal synthesis of borrelial antibodies) was 67.9%, whereas normal CSF white cell counts ruled out Lyme neuroborreliosis with a predictive value of 91.9%. CONCLUSIONS: Comparison of European patients with EM who had symptoms/signs suggesting early Lyme neuroborreliosis revealed several differences in the clinical presentation and in microbiologic test results according to CSF findings.

Leptospirosis and characterization of Leptospira isolates from patients in Koprivnica-Krizevci County, Croatia from 2000-2004.

Introduction: Leptospirosis, caused by spirochetes of the genus Leptospira , is present in the Koprivnica-Krizevci County area, Croatia. Clinical manifestation can range from asymptomatic, short-term mild, non-specific febrile disease, to severe forms with high mortality rates. Aim: The aim of the study was to valuate culture in front of microscopic agglutination test (MAT) for diagnosis of infection, and to evaluate clinical and laboratory features of the disease. Moreover, we want to characterize Leptospira strains involved in infection in Koprivnica-Krizevci County, Croatia. Methods: We included 68 patients with clinical presentation consistent with leptospirosis collected in a 5-year period (2000-2004). Clinical samples (blood, urine and cerebrospinal fluid, CSF) were inoculated in Kolthoff's medium; species of isolated Leptospira strains was determined with Tm of real-time PCR, serogroup/serovar with MAT and NotI-RFLP analysis. Demonstration of specific antibodies in patients' sera was done using microscopic agglutination test. Results: Leptospira was isolated from the blood of 14/51(27.5 %) patients and the most often identified serogroup/serovar was Icterohaemorrhagiae (8/10, 80%) followed by Grippotyphosa (10%). Regarding to species level, 8/10 isolated belonged to L. interrogans sensu stricto and one to L. kirschneri species. MAT was carried out on 51 patients with suspected leptospirosis, and was positive in 11/51(21.5 %) patients. Most of our patients presented with moderate severe symptoms, were hospitalized from August to October, and were infected mainly during the work or recreation in our county. The frequency of particular clinical features and pathological laboratory findings correlated with the severity of the clinical condition. Conclusions: Leptospirosis can be confirmed microbiologically, culture and MAT contributed almost equally to the diagnosis of infection. Serovar Icterohaemorrhagiae was found as the dominant one, and L. interrogans sensu stricto as dominant species in our county. Epidemiological data shown that leptospirosis occurs seasonally, affects the rural population, and most commonly is presented with moderate severe clinical course.

Detection, identification and genotyping of Borrelia spp. in ticks of Coastal-Karst and Littoral-Inner Carniola regions in Slovenia.

The density and spread of tick vector species have increased throughout Europe in the last 30 years, leading to an increase of Lyme borreliosis cases, including in Slovenia. The aim of this study was to isolate Borrelia strains and determine the prevalence of B. burgdorferi sensu lato and B. miyamotoi in adults of Ixodes ricinus (Linnaeus) collected in 2019 in the two regions of the country (Coastal-Karst and Littoral-Inner Carniola) by cultivation and PCR. We isolated B. burgdorferi s.l. by culture method in 28/559 (5%) ticks from both regions. Culture-negative samples (531/559, i.e., 95%) were additionally tested by real-time PCR. In 155/531 (29.2%) PCR-positive samples, a fragment of flaB or glpQ was amplified and further sequenced to identify species of the Borrelia. Using both methods, cultivation and PCR, Borrelia spp. prevalence was 32.7% in the Coastal-Karst region and 33.0% in the Littoral-Inner Carniola region. Genotyping of the Borrelia spp. isolates revealed that 17/28 (60%) were B. garinii subtype Mlg2. Of all tick samples tested for B. miyamotoi 8/398 (2%) were PCR positive. Based on previous studies in these regions, we had expected more ticks to be infected with B. afzelii, but genotyping revealed that B. garinii was the most abundant.

First Report on Leptospira Species Isolated from Patients in Slovenia.

Leptospirosis is an important worldwide zoonosis, and it has also been reported in Slovenia. The cultivation of Leptospira from human material is difficult. Despite that, we successfully isolated 12 human Leptospira strains isolated from patients between 2002 and 2020 and used various methods for the phenotypic and genotypic characterization of the strains, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) using our own MALDI-TOF data library, melting temperature analysis of the amplified lfb1 gene, determination of Leptospira serogroups using rabbit immune sera, NotI-RFLP of the whole Leptospira genome, multilocus sequence typing (MLST) of seven housekeeping genes, and whole-genome sequencing (WGS)-based typing. We confirmed the presence of four pathogenic Leptospira species (L. kirschneri, L. interrogans, L. borgpetersenii, and L. santarosai) and three serogroups: Grippotyphosa, Icterohaemorrhagiae, and Sejroe. MALDI-TOF identified three of seven isolates at the species level and four isolates at the genus level. Serovars of 8 of the 10 strains were determined using NotI-RFLP. MLST showed that the clinical isolates belonged to sequence types ST17, ST110, and ST155. WGS confirmed the analysis of Leptospira strains using conventional methods. In addition, WGS provided better taxonomic resolution for isolate DDA 10944/10.

Treatment of erythema migrans with doxycycline for 7 days versus 14 days in Slovenia: a randomised open-label non-inferiority trial.

BACKGROUND: Lyme borreliosis is the most prevalent vector-borne disease in Europe and the USA. Doxycycline for 10 days is the primary treatment recommendation for erythema migrans. To reduce potentially harmful antibiotic overuse by identifying shorter effective treatments, we aimed to assess whether oral doxycycline for 7 days is non-inferior to 14 days in adults with solitary erythema migrans. METHODS: In this randomised open-label non-inferiority trial, we enrolled patients with a solitary erythema at the University Medical Centre in Ljubljana, Slovenia. Patients were excluded if they were pregnant or lactating, immunosuppressed, allergic to doxycycline, or had received antibiotics with anti-borrelial activity within 10 days preceding enrolment or had additional manifestations of Lyme borreliosis Adults were randomly allocated 1:1 to receive oral doxycycline 100 mg twice a day for 7 days or 14 days. The primary efficacy endpoint was the difference in proportion of patients with treatment failure, defined as persistent erythema, new objective signs of Lyme borreliosis, or borrelial isolation on skin re-biopsy at 2 months, in a per-protocol analysis (the population that completed the assigned doxycycline regimen according to the study protocol and did not receive any other antibiotics with anti-borrelial activity until the 2-month visit). The non-inferiority margin was 6 percentage points. Safety was assessed in all randomly assigned patients who followed the study protocol and were evaluable at the 14-day visit. This study is registered with ClinicalTrials.gov, NCT03153267. FINDINGS: Between July 3, 2017, and Oct 2, 2018, we enrolled 300 patients (150 per group: median age 56 years [IQR 47-65]; 126 [45%] of 300 male; skin culture positive 72 [30%] of 239 assessed). 295 patients completed antibiotic therapy as per protocol and 294 (98%) patients were evaluable 2 months post-enrolment. Five (3%) of 147 patients from the 7-day group versus 3 (2%) of 147 patients from the 14-day group (one patient did not attend the 2-month visit and was unreachable by telephone) had treatment failure manifesting as persistence of erythema (difference 1·4 percentage points; upper limit of one-sided 95% CI 5·2 percentage points; p=0·64). No patients developed new objective manifestations of Lyme borreliosis during follow-up or had positive repeat skin biopsies. Two (1%) of 150 patients in the 7-day and one (1%) of 150 patients in the 14-day group discontinued therapy due to adverse events. INTERPRETATION: Our data support 7 days of oral doxycycline for adult European patients with solitary erythema migrans, permitting less antibiotic exposure than current guideline-driven therapy. FUNDING: Slovenian Research Agency and the University Medical Centre Ljubljana.