Knowledge of and attitudes towards medical research ethics among first year doctoral students in Slovenia at the Faculty of Medicine, University of Ljubljana.

BACKGROUND: Research ethics and attitudes should be the main concern of those who are conducting and publishing research in medicine. METHODS: A cross-sectional study was conducted using a questionnaire among first year postgraduate doctoral students in Biomedicine at the Faculty of Medicine, University of Ljubljana during the academic year 2022/2023. RESULTS: There were 54 out of 57 doctoral students included in the study, with a mean age (SD) of 29.7 (4.7) years, with predominantly female doctoral students, 66.7%. The number of correct answers out of 39 considered to illustrate students' knowledge of medical research ethics was 31, meaning that they gave correct answers to 80% of all the questions. The mean number (SD) of correct answers was 18.9 (5.8), which significantly differed from 31 (p < 0.001). The previous experience of the doctoral students in research was significantly correlated with their knowledge of medical research ethics, even when controlling for the age, gender and workplace of respondents. CONCLUSION: This study clearly showed that insufficient knowledge and a poor level of attitudes exist about the main questions pertaining to medical research ethics. Overall knowledge is well below the expected positive answers. Further studies are needed to compare the knowledge of doctoral students with that of their tutors and what implications this might have for further teaching of research ethics.

Solitary and multiple erythema migrans in children: comparison of demographic, clinical and laboratory findings.

BACKGROUND: Data on European children with erythema migrans (EM) are limited. PATIENTS AND METHODS: 553 patients, 333 with solitary and 220 with multiple EM, diagnosed between 1996 and 2000, were included in the prospective study. Demographic, clinical and laboratory data including borrelial serum immunofluorescence assay antibody titers and Borrelia burgdorferi sensu lato blood culture results were obtained; findings in solitary and multiple EM were compared. RESULTS: Comparison revealed that children with multiple EM were younger (4.5 vs 6.5 years; p = 0.0000), less often reported a tick bite at the site of later skin lesion (25% vs 46%; p = 0.0000), had a longer incubation period (22 vs 13 days; p = 0.0028), more frequently presented with a ringlike lesion (99% vs 86%; p = 0.0000), less often reported associated local (15% vs 41%; p = 0.0000) but not systemic symptoms (28% vs 26%, p = 0.6913), more frequently had abnormal findings on physical examination (35% vs 26%; p = 0.0264), and a higher frequency of laboratory abnormalities including the presence of borrelial serum antibodies as well as B. burgdorferi sensu lato isolated from blood (12% vs 6%; p = 0.0267); younger age and male sex were identified as risk factors for the isolation of Borrelia. 40/44 isolates were Borrelia afzelii. CONCLUSION: Analysis of a large group of European children with solitary and multiple EM revealed several demographic, clinical and laboratory differences between the two groups.

Isolation of Borrelia burgdorferi sensu lato from blood of children with solitary erythema migrans.

OBJECTIVES: To establish the frequency of isolation of Borrelia burgdorferi sensu lato from blood of children with solitary erythema migrans (EM) in Europe, to determine the strains of the isolated borreliae and to compare the clinical course and the outcome of the disease according to positive and negative blood culture result. METHODS: In the prospective study we included 134 consecutive patients younger than 15 years with solitary EM, referred to our institution in 1996 and 1997. One milliliter of blood was withdrawn before treatment and cultured in modified Kelly-Pettenkofer medium. Isolated borreliae were typed according to LRFP analysis. Patients were treated with either penicillin V or cefuroxime axetil for 14 days. The posttreatment course was surveyed by follow-up visits during 1 year. RESULTS: B. burgdorferi sensu lato was isolated in 12 of 134 (9%) patients. Eleven blood isolates were typed: 10 were found to be B. afzelii and 1 was Borrelia garinii. Comparison of blood culture-positive and -negative patients revealed no differences in pretreatment characteristics or in posttreatment clinical course. However, worsening of local and/or systemic signs and symptoms at the beginning of antibiotic therapy (Jarish-Herxheimer's reaction) was identified more often in the blood culture-positive than in the blood culture-negative group (5 of 12 vs. 17 of 122, respectively; P = 0.0274). CONCLUSIONS: The isolation rate of B. burgdorferi sensu lato from the blood of children with solitary EM was 9%. The majority of the isolates were B. afzelii. Blood culture-positive patients treated with oral antibiotics were not at greater risk for unfavorable course of the disease than patients with negative blood culture result.

Demographic features, clinical characteristics and laboratory findings in children with multiple erythema migrans in Slovenia.

OBJECTIVE: To identify demographic, clinical and laboratory characteristics of children with multiple erythema migrans (EM) in Slovenia. METHODS: We prospectively studied patients aged 15 years and less, examined at our department for multiple EM in 1996 and 1997. Demographic and clinical data were collected by means of a questionnaire. In addition, basic haematological and biochemical investigations, serologic testing, and Borrelia cerebrospinal fluid and blood cultures were performed. RESULTS: Ninety-five children (44 girls, 55 boys) aged 1 to 13.5 (median, 4.5) years fulfilled the inclusion criteria. A tick bite was recalled by 23%. The incubation period was 10.5 (range, 1 to 150) days, the duration of skin lesions before the initial examination 4 (range, 1 to 54) days, and the median number of skin lesions, 4.5 (range, 2 to 35). The initial disease was mild in 81% of patients. Local and systemic symptoms were reported by 11% and 30% of children, respectively. Clinical signs accompanying EM lesions were found in 42%. Cerebrospinal pleocytosis (predominantly lymphocytic) was seen in 18% of patients; none of them had frank clinical evidence of central nervous system involvement. Serum IgM and IgG antibodies were detected in 28% and 22% of children, respectively. In 3/79 (4%) patients, Borreliae were isolated from the blood and in 2/83 (2%) from the cerebrospinal fluid. In 2/81 (2%) children, borrelial IgG intrathecal antibody production was demonstrated. CONCLUSIONS: Multiple EM in Slovene children is a mild disease. However, some patients had an associated, usually asymptomatic, infection of the central nervous system.

Analysis of Borrelia burgdorferi sensu lato isolated from cerebrospinal fluid.

Involvement of the nervous system in Lyme borreliosis may occur with or without erythema migrans and it may present with a variety of neurological symptoms. In this study we analysed phenotypic and genotypic characteristics of 40 Borrelia strains isolated from cerebrospinal fluid (CSF) of 38 Slovenian patients with different clinical manifestations of Lyme borreliosis. In seven of the patients, Borreliae were also isolated from skin lesions. Species identification and plasmid profiles were determined by pulsed-field gel electrophoresis and protein profiles by SDS-PAGE. MluI digestion profiles of Borrelia burgdorferi sensu lato DNA showed that 25 (62.5%) isolates were B. garinii, 14 (35%) B. afzelii, and one (2.5%) B. burgdorferi sensu stricto. All strains, except one, possessed a large plasmid and a varying number of smaller plasmids. Three (7.5%) isolates exhibited an unusual plasmid profile, with a large plasmid dimer or three copies of the large plasmid. In protein analyses, all strains expressed OspA protein. OspB was present significantly more often in B. afzelii than B. garinii strains (p=0.0000), while OspC was more often present in B. garinii than B. afzelii strains (p=0.0052). In the seven patients with Borreliae isolated also from the skin, the CSF and skin isolates were identical, either B. garinii (six patients) or B. afzelii (one patient). Species and plasmid heterogeneity as well as antigen diversity could play a role in the pathogenesis of the infection. When combined with our own earlier data, the results suggest species-related organotropism.

Characterisation of Borrelia burgdorferi sensu lato strains isolated from patients with skin manifestations of Lyme borreliosis residing in Slovenia.

Lyme borreliosis is the most prevalent tick-borne infection in Slovenia. Skin disorders are the most frequent clinical manifestations. The aim of the present study was to assess the phenotypic and genotypic diversity of a large number of human Borrelia burgdorferi sensu lato isolates and to evaluate any association between the isolates and different clinical manifestations. All 103 strains tested were from patients suffering from the skin disorders of Lyme borreliosis. Skin biopsies, cerebrospinal fluid and blood samples from patients were inoculated into modified Kelly Pettenkofer medium. Protein profiles were determined by SDS-PAGE and species identification and plasmid profiles by pulsed-field gel electrophoresis. MluI digestion profiles showed that 87 (84.5%) isolates belonged to B. afzelii, 15 (14.5%) to B. garinii and 1 (1%) to B. burgdorferi sensu stricto. The number of plasmids in each strain varied from three to seven, and the plasmid size ranged from 15 to 65 kb. Four isolates of B. garinii possessed multiple large plasmids and four isolates had a large plasmid dimer (three B. afzelii and one B. garinii). Isolates showed qualitative and quantitative differences in protein expression. The study found differences in the expression of OspB and OspC proteins between B. afzelii and B. garinii strains. OspB was expressed significantly more often by B. afzelii (78 of 87, 89.6%) than by B. garinii (4 of 15, 26.6%) isolates, while OspC protein was expressed significantly more often by B. garinii (14 of 15, 93.3%) than by B. afzelii (51 of 87, 58.6%) isolates. In Slovenia, B. afzelii causes the majority of skin lesions. The isolates investigated showed plasmid and protein diversity. Heterogeneity of the spirochaetes may be important for virulence, and may have implications for pathogenesis and therapy of the infection. Differences in immunodominant proteins also have an important impact on serological testing and vaccine development.

Comparison of cefuroxime axetil and phenoxymethyl penicillin for the treatment of children with solitary erythema migrans.

OBJECTIVE: To compare the clinical efficacy and drug-related adverse effects of 14 days of treatment with cefuroxime axetil 30 mg/kg/day or phenoxymethyl penicillin 100,000 IU/kg/day in the treatment of children with erythema migrans. METHODS: Consecutive patients younger than 15 years, referred to our institution in 1996 with solitary erythema migrans and without prior antibiotic therapy, were included in this prospective study. Basic demographic features and clinical data were collected by questionnaire. The efficacy of the treatment of acute disease, development of major and/or minor manifestations of Lyme borreliosis and drug-related adverse effects were surveyed at follow-up visits during the first year after the initiation of antibiotic treatment. RESULTS: Forty-six patients received cefuroxime axetil (group C) and 44, phenoxymethyl penicillin (group P). The two groups differed in terms of age (patients in group C were younger), but no other differences in demographic and clinical pre-treatment characteristics were present. The clinical course during the post-treatment period revealed no significant differences between the two groups: the duration of erythema migrans (7.1 +/- 7.5 days in group C, 10.6 +/- 19.3 days in group P) and the appearance of minor manifestations of Lyme borreliosis (8.8% in group C, 9.1% in group P) were comparable; no major manifestations were recorded. Twelve months after antibiotic treatment all patients were free of symptoms. The patients treated with cefuroxime axetil had more drug-related adverse effects than did those treated with phenoxymethyl penicillin (26.1% versus 6.8%, p = 0.0301). "Herxheimer's reaction" at the beginning of treatment was identified more often in group C than in group P, but the difference was not statistically significant. CONCLUSIONS: Cefuroxime axetil and phenoxymethyl penicillin are equally effective in the treatment of children with solitary erythema migrans; however drug-related adverse effects were more frequently observed with cefuroxime axetil.

Erythema migrans in pregnancy.

From 1990 through to 1997, 105 pregnant women with typical EM were investigated at the Lyme Borreliosis Outpatients' Clinic of the Department of Infectious Diseases at the University Medical Centre in Ljubljana, Slovenia. Twenty-five (23.8%) patients acquired borrelial infection during the first trimester of pregnancy; eight (7.6%) of them had noticed the skin lesion before they became pregnant. In 43 (40.6%) patients the EM appeared in the second trimester, and in 37 (35%) patients in the third trimester of pregnancy. Two (1.9%) patients received phenoxymethyl penicillin (1 million IU t.i.d.), three (2.9%) benzyl penicillin (10 million IU b.i.d.), and 100 (95.2%) ceftriaxone (2 g daily). All patients were treated for 14 days except three (2.9%) in whom the treatment with ceftriaxone was discontinued because of mild side effects. The outcome of disease was good in all 105 patients. Ninety-three (88.6%) out of 105 patients had normal pregnancies; the infants were delivered at term, were clinically healthy, and subsequently had a normal psychomotor development. In the remaining 12 (11.4%) patients an adverse outcome was observed. Two (1.9%) pregnancies ended with an abortion (one missed abortion at 9 weeks, one spontaneous abortion at 10 weeks), and six (5.7%) with preterm birth. One of the preterm babies had cardiac abnormalities and two died shortly after birth. Four (3.8%) babies born at term were found to have congenital anomalies; one had syndactyly at birth and three had urologic abnormalities which were registered at the age of 5, 7, and 10 months, respectively. A causal association with borrelial infection was not proven in any infant. For at least some unfavourable outcomes a plausible explanation not associated with Lyme borreliosis was found.

Treatment of erythema migrans in pregnancy.

The course of the illness and the outcome of pregnancy were investigated in a prospective study of 58 consecutively enrolled pregnant women with typical erythema migrans at the University Medical Centre of Ljubljana, Slovenia. Thirteen patients (22.4%) acquired borrelial infection in the first trimester of pregnancy, 27 (46.6%) in the second trimester, and 18 (31%) in the third trimester. All patients were treated for 14 days: 2 (3.4%) with phenoxymethylpenicillin (1 million IU thrice per day), 3 (5.2%) with benzylpenicillin (10 million units twice per day), and 53 (91.4%) with ceftriaxone (2 g daily). For 51 of the 58 women (87.9%), the pregnancies were normal and the infants were born at term, were clinically healthy, and had normal psychomotor development. Of the remaining 7 pregnancies, 1 ended with a missed abortion and 5 with preterm birth; 1 of the preterm babies had heart abnormalities. One child born at term was found to have urologic abnormalities at 7 months of age. A causal relationship between borrelial infection and fetal adverse effects has not been proven, and for at least some such cases a reasonable explanation (not associated with Lyme borreliosis) was found.

Kill kinetics of Borrelia burgdorferi and bacterial findings in relation to the treatment of Lyme borreliosis.

For a better understanding of the persistence of Borrelia burgdorferi sensu lato (s.l.) after antibiotic therapy the kinetics of killing B. burgdorferi s.l. under amoxicillin, doxycycline, cefotaxime, ceftriaxone, azithromycin and penicillin G were determined. The killing effect was investigated in MKP medium and human serum during a 72 h exposure to antibiotics. Twenty clinical isolates were used, including ten strains of Borrelia afzelii and ten strains of Borrelia garinii. The results show that the kinetics of killing borreliae differ from antibiotic to antibiotic. The killing rate of a given antibiotic is less dependent on the concentration of the antibiotic than on the reaction time. Furthermore, the data show that the strains of B. afzelii and B. garinii have a different reaction to antibiotics used in the treatment of Lyme borreliosis and that different reactions to given antibiotics also exist within one species. The B. garinii strains appear to be more sensitive to antibiotics used in therapy. Furthermore, the persistence of B. burgdorferi s.l. and clinical recurrences in patients despite seemingly adequate antibiotic treatment is described. The patients had clinical disease with or without diagnostic antibody titers to B. burgdorferi.

Treatment of borrelial lymphocytoma.

Treatment results in 65 patients with borrelial lymphocytoma (22 on the ear lobe and 43 on the breast), registered at the Department of Infectious Diseases, University Medical Centre Ljubljana, from January 1986 to March 1995, are presented. When lymphocytoma was the sole manifestation of Lyme borreliosis or associated with erythema migrans only patients were treated orally with doxycycline, phenoxymethylpenicillin or amoxicillin for 14 days, or azithromycin for 5 days (15, 19, six and 12 patients, respectively). When signs and symptoms of disseminated borrelial infection were present (seven patients) or clinically suspected (six patients) patients received ceftriaxone or penicillin G i.v. for 14 days. Lymphocytoma disappeared within a few weeks after the institution of treatment. The speed of regression depended on the duration of lymphocytoma before the institution of therapy. The number of patients was too low and pretreatment characteristics were too heterogeneous to enable a reliable comparison of the efficacy of different antibiotics. It appears that the effectiveness of doxycycline and azithromycin is comparable and that amoxicillin performs well, but some findings may indicate that phenoxymethylpenicillin is less effective than some newer antibiotics. The optimal agent, dosage and duration of therapy for borrelial lymphocytoma have not been determined.

Influence of ceftriaxone on emergence of Clostridium difficile.

The influence of long-term ceftriaxone administration on the emergence of Clostridium difficile was studied with 80 patients receiving ceftriaxone for 14 days. In five patients (6.3%) C. difficile was cultured. The appearance of gastrointestinal disturbances during treatment with ceftriaxone was common, but the rate of emergence of C. difficile in immunocompetent patients was not high.

Solitary borrelial lymphocytoma: report of 36 cases.

Thirty-six cases of borrelial lymphocytoma were detected during the period 1986 to 1990 in Slovenia. Borrelial lymphocytoma was located on the ear lobe in 17 persons, ten female and seven male, with a median age of 12 years (range 2-56). Fourteen of these 17 were children under 14 years of age. A tick bite was remembered by 15 patients a median of 30 days before borrelial lymphocytoma developed. The most frequent month of onset was September. Erythema migrans preceded or accompanied borrelial lymphocytoma in eight cases. In 15 cases, eight female and seven male, borrelial lymphocytoma was localized on the mamilla. Median age of these patients was 42 years (range 15-72). Twelve had a tick bite about 45 days (median value) before the onset of borrelial lymphocytoma, which occurred most frequently in August. Erythema migrans was reported in 13 patients and preceded borrelial lymphocytoma in ten cases. In another four patients borrelial lymphocytoma was localized on the nose, scrotum, upper arm and shoulder. Antibiotic treatment with phenoxymethyl-penicillin (n = 16), ceftriaxone (n = 8), doxycycline (n = 9), azithromycin (n = 2) and penicillin G (1) led to complete recovery within an average of three weeks in all cases.

Aseptic meningitis after vaccination against measles and mumps.

This retrospective study (1979 to 1986) investigated the possible etiologic relationship between vaccination and aseptic meningitis in 115 hospitalized children who became ill within 30 days of vaccination with the Leningrad 3 strain of mumps virus and the Edmonston-Zagreb strain of measles virus. The etiologic viral diagnosis was based on serologic tests and the isolation of virus from cell cultures which distinguished between attenuated and "virulent" mumps virus. The incidence of mumps vaccine-associated meningitis was 1/1000 vaccine recipients. In 92% of children the incubation period was 11 to 25 days and 28% had associated swelling of the salivary glands. Sixteen cases (13.9%) had a positive cerebrospinal fluid culture (attenuated mumps virus, 6 cases; "virulent" mumps virus, 7 cases; echoviruses, 3 cases). Clustering of cases, seasonal occurrence and age of the patients suggested causal relationship with the vaccination in the majority of children. In 4 patients with attenuated virus isolation from cerebrospinal fluid the incubation period ranged from 17 to 20 days. Clinical findings did not differ from natural mumps meningitis. The course was uncomplicated and at discharge the patients had no sequelae. Measles virus was never found as a cause of the meningitis. The mumps vaccine virus should be recognized as one of the causative agents of aseptic meningitis in countries where less attenuated mumps vaccine is used.