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Introduction. Leptospirosis, caused by spirochetes of the genus Leptospira , is present in the Koprivnica-Križevci 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-Križevci 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.

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.

The diversity of 61 Bacillus cereus strains isolated from different clinical specimens, food including raw milk and milk products, and water was evaluated. PFGE analysis could discriminate 61 distinct pulsotypes with similarity levels from 25 to 82%, which were divided into 13 clonal complexes. The similarity between clonal complexes was at least 40%. Clinical strains were divided into 10 clonal complexes, while the strains, isolated from milk, food and water were included in 9, 6 and 6 clonal complexes, respectively. Three clonal complexes were dominated by clinical isolates, while they were absent in two complexes. Bacterial isolates from food, being a probable source of alimentary toxoinfection, showed low similarity to isolates from stool specimens. The isolates from both sources were classified together in only 4 out of 13 clonal complexes. The large circular and linear plasmids with the sizes between 50 and 200 kb were detected in 24 (39.3%) and 14 (23%) B. cereus strains, respectively. Thirteen (21.3%) strains contained only one plasmid, two plasmids were found in 6 (9.8%) of strains, and three or more plasmids were obtained in 5 (8.2%) of tested strains. The plasmids were confirmed in 30.8% and 40% of isolates from clinical specimens and food and milk samples, respectively. No clear correlation between the PFGE profiles, the source as well as plasmid content among all tested strains was observed.

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.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.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.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.Slovenian Research Agency and the University Medical Centre Ljubljana.

Legionella pneumophila is the causative agent of severe Legionnaires’ disease (LD). Although an increasing number of LD cases have been observed, published data from Slovenia are very limited and data on molecular epidemiology are even scarcer. The present retrospective study (2006–2020) reports the results of the microbiological diagnosis of LD, as well as the epidemiology and characterization of the Legionella clinical isolates. We tested urine samples from 15,540 patients with pneumonia symptoms for L. pneumophila infection by urine antigen test, of which 717 (4.6%) tested positive. Isolation of L. pneumophila was successfully performed from 88 clinical specimens, with 82 (93.2%) being identified as L. pneumophila sg 1 and six (6.8%) as L. pneumophila sg 2–14. Sequence-based typing (SBT) identified 33 different sequence types (STs), the most frequent being ST1 and ST23. Sequence type 1 mainly comprised isolates belonging to the Philadelphia subgroup, and ST23 mostly to Allentown/France. The standard SBT scheme, as well as Dresden phenotyping for L. pneumophila, presented a high diversity among isolates.

The diversity of 61 Bacillus cereus strains isolated from different clinical specimens, food including raw milk and milk products, and water was evaluated. PFGE analysis could discriminate 61 distinct pulsotypes with similarity levels from 25 to 82%, which were divided into 13 clonal complexes. The similarity between clonal complexes was at least 40%. Clinical strains were divided into 10 clonal complexes, while the strains, isolated from milk, food and water were included in 9, 6 and 6 clonal complexes, respectively. Three clonal complexes were dominated by clinical isolates, while they were absent in two complexes. Bacterial isolates from foods, being a probable source of alimentary toxoinfection, showed low similarity to isolates from stool specimens. The isolates from both sources were classified together in only 4 out of 13 clonal complexes. The large circular and linear plasmids with the sizes between 50 and 200 kb were detected in 24 (39.3%) and 14 (23%) B. cereus strains, respectively. Thirteen (21.3%) strains contained only one plasmid, two plasmids were found in 6 (9.8%) of strains, and three or more plasmids were obtained in 5 (8.2%) of tested strains. The plasmids were confirmed in 30.8% and 40% of isolates from clinical specimens and food and milk samples, respectively. No clear correlation between the PFGE profiles, the source as well as plasmid content among all tested strains was observed.

Statins have anti-inflammatory and potentially antimicrobial activity, but whether they have a beneficial effect on the course of infectious diseases is controversial. In this study, we assessed the impact of pre-existing statin use on the course and outcome of Lyme neuroborreliosis manifested as meningoradiculitis (Bannwarth&rsquo, s syndrome). One hundred and twenty three consecutive patients with Bannwarth&rsquo, s syndrome, of whom 18 (14.6%) were being treated with statins, were included in the study. To assess the influence of statin use on the course and outcome of the disease, univariate and multivariable analyses were performed. No statistically significant association was found between statin pre-treatment and the clinical manifestations, laboratory test results, and outcome of Bannwarth&rsquo, s syndrome. In conclusion, pre-existing use of statins did not significantly impact either the clinical presentation or the outcome of Bannwarth&rsquo, s syndrome.

Purpose. Increasing consumption of colistin as treatment for infections with multidrug-resistant (MDR) Gram-negative bacilli (GNB) has been accompanied by increasingly frequent reports of colistin-resistant (ColR) MDR GNB. Higher selective pressure creates a favourable environment that can facilitate the spread of ColR isolates. Monitoring of asymptomatic ColR GNB carriage can give us a better understanding of this emerging healthcare problem, particularly in wards with higher polymyxin selective pressure and prevalence of carbapenem-resistant GNB. Our aim was to assess the ColR GNB colonization rate in intensive care unit (ICU) patients and evaluate the performance of two surveillance protocols using a selective medium. Methodology. A prospective study included 739 surveillance samples (rectal swabs and tracheal aspirates) from 330 patients that were screened for ColR GNB carriage using SuperPolymyxin medium. Two approaches were used: direct sample plating and overnight pre-enrichment of samples followed by plating. Colistin resistance was confirmed with broth microdilution. ColR isolates were molecularly screened for plasmid-mediated mcr genes. Results. A total of 44/739 samples (45 ColR GNB isolates) were positive for ColR GNB, which included 31/330 (9.4 %) colonized patients; mcr genes were not detected. The direct plating method only identified 17/45 (37.8 %) isolates correctly, whereas the pre-enrichment protocol identified all 45 ColR GNB. Conclusion. The colonization rate among our ICU patients was 9.4 %. Based on our findings, the pre-enrichment step is necessary for the determination of ColR GNB carriage – even though the time to result takes an additional day, fewer than half of ColR GNB carriers were detected using the direct plating protocol.

Acinetobacter baumannii primarily causes colonization, yet it can be an opportunistic pathogen associated with hospital-acquired infections. Many countries report rapid spread of carbapenem-resistant Acinetobacter baumannii (CRAb) which limits treatment options, with colistin frequently being the last line treatment option. The aim of our study was to evaluate a recently developed rapid method, namely the Rapid ResaPolymyxin test, for detection of colistin resistance (ColR) in Acinetobacter baumannii. This test was used for rapid screening of colistin resistance in a clinical setting where there is endemicity of CRAb isolates. A total of 82 A. baumannii clinical isolates were included in the evaluation. The majority of them were resistant to carbapenems (75/82, 91.5%). A total of 37 isolates (45.1%) were resistant to colistin, all being resistant to carbapenems. None of the ColR isolates carried the plasmid- mediated mcr-1 to -5 genes. The Rapid ResaPolymyxin NP test reached a 95.1% categorical agreement with results of reference broth microdilution method, with 93.3% sensitivity and specificity, and positive and negative predictive values being respectively at 92.3% and 97.7%. The Rapid ResaPolymyxin NP test performed well on our collection of clinical and surveillance CRAb isolates from the Central Slovenia region. The test is inexpensive and easy to integrate into laboratory workflow. The main value of the test is rapid categorization of susceptibility and resistance which has important implications with respect to the treatment strategy as well as the infection control measures.

Twenty-five percent to 50% of acute gastroenteritis (AGE) cases remain etiologically undiagnosed. Our main aim was to determine the most appropriate list of enteric pathogens to be included in the daily diagnostics scheme of AGE, ensuring the lowest possible diagnostic gap.Two hundred ninety seven children ≤6 years of age, admitted to hospital in Slovenia, October 2011 to October 2012, with AGE, and 88 ≤6 years old healthy children were included in the study. A broad spectrum of enteric pathogens was targeted with molecular methods, including 8 viruses, 6 bacteria and 2 parasites.At least one enteric pathogen was detected in 91.2% of cases with AGE and 27.3% of controls. Viruses were the most prevalent (82.5% and 15.9%), followed by bacteria (27.3% and 10.2%) and parasites (3.0% and 1.1%) in cases and controls, respectively. A high proportion (41.8%) of mixed infections was observed in the cases. For cases with undetermined etiology (8.8%), stool samples were analyzed with next generation sequencing, and a potential viral pathogen was detected in 17 additional samples (5.8%).Our study suggests that tests for rotaviruses, noroviruses genogroup II, adenoviruses 40/41, astroviruses, Campylobacter spp. and Salmonella sp. should be included in the initial diagnostic algorithm, which revealed the etiology in 83.5% of children tested. The use of molecular methods in diagnostics of gastroenteritis is preferable because of their high sensitivity, specificity, fast performance and the possibility of establishing the concentration of the target. The latter may be valuable for assessing the clinical significance of the detected enteric, particularly viral pathogens.

The aims of the study were to determine the frequency of borrelial infection in patients with peripheral facial palsy (PFP) and to compare clinical and laboratory characteristics of patients with borrelial PFP and patients with PFP of unknown etiology. Adult patients with PFP who presented at our department between January 2006 and December 2013 qualified for the study if they had undergone lumbar puncture and also been tested for the presence of borrelial IgM and IgG antibodies in serum and cerebrospinal fluid (CSF) in indirect chemiluminescence immunoassay. Patients with PFP who had obvious signs/symptoms indicating a disease other than Lyme borreliosis (LB) were excluded. Patients who qualified for the study were classified into three groups according to the clinical and microbiological criteria: those having confirmed LB, those with possible LB, and those with PFP of unknown etiology. Of 589 patients diagnosed with PFP during the eight-year period, 436 patients (240 males, 196 females) with median age 42.5 years (15-87 years) qualified for the study. Among these patients, 64 (14.7%) fulfilled criteria for confirmed LB, 120 (27.5%) had a diagnosis of possible LB, and in 252 (57.8%) the cause of their PFP remained unknown. When compared with patients with unknown cause of PFP, the patients with confirmed LB were older, more often presented in summer, more often reported tick bites, more frequently had LB in the past, more often complained of constitutional symptoms and radicular pain, and more often had bilateral palsy and CSF pleocytosis. Among the patients with possible LB and patients with unknown cause of PFP there were no differences in frequency of constitutional symptoms, radicular pain, bilateral palsy or CSF pleocytosis. Presentation in summer, tick bites, constitutional symptoms and radicular pain, bilateral palsy, and CSF pleocytosis strongly suggest borrelial etiology of PFP.

In this retrospective cohort study of patients with tick-borne encephalitis (TBE), the clinical outcome in relation to co-infection with B. burgdorferi sensu lato (s.l.) and, specifically, the effect of antibiotic treatment on clinical outcome in patients with TBE who were seropositive for borreliae but who did not fulfil clinical or microbiologic criteria for proven co-infection, were assessed at a single university medical center in Slovenia, a country where TBE and Lyme borreliosis are endemic with high incidence. Among 684 patients enrolled during a seven-year period from 2007 through 2013, 382 (55.8%) had TBE alone, 62 (9.1%) had proven co-infection with borreliae and 240 (35.1%) had possible co-infection. The severity of acute illness was similar in all the groups. The odds for incomplete recovery decreased during a 12-month follow-up but were higher in women, older patients, and in those with more severe acute illness. Incomplete recovery was not associated with either proven (odds ratio (OR) 1.21, 95% confidence interval (CI) 0.49&ndash, 2.95, p = 0.670) or possible co-infection (OR 0.95, 95% CI 0.55&ndash, 1.65, p = 0.853). Among patients with possible co-infection, older patients were more likely to be prescribed antibiotics, but the odds for incomplete recovery were similar in those who received antibiotics and those who did not (OR 0.82, 95% CI 0.36&ndash, 1.87, p = 0.630), suggesting that routine antibiotic treatment in patients with TBE and possible co-infection may not be warranted.

Background: Isolation and identification of bacterial pathogens enables accurate diagnosis of bacterial infection, allowing rational use of appropriate narrow-spectrum antibiotics. In some cases, the routine bacterial culture can give negative results. In those cases additional use of molecular techniques such as eubacterial (broad-range 16S rRNA) PCR may detect and identify bacterial genetic material.Methods: Between February 2012 and April 2013 42 specimens from 35 patients, already treated with antimicrobials, were taken and tested by eubacterial PCR in addition to routine microbiological culture. Results: Eubacterial PCR yielded positive result in 21/42 specimens in 18 patients (in three mixed sequences). Therefore, in 15 patients the diagnosis of bacterial infection was obtained with DNA identification and the results were interpreted in accordance to patients’ history, laboratory and image diagnostics. Only 4 specimens were culture-positive.Conclusions: Although eubacterial PCR enables the identification of any bacterial DNA in clinical specimens, there are some limitations: no information concerning antimicrobial susceptibility of the causative agents, problem of differentiating living from dead bacteria and problem to differentiate contaminants from pathogenic bacteria. The method is also expensive. In the following article recommendations for appropriate and rational use of eubacterial PCR are presented.