Comparison of different Borrelia burgdorferi sensu lato strains for detection of immune response in patients with erythema migrans

The aim of the present study was to establish which combination of serological method and Borrelia strain used as an antigen would provide the most appropriate demonstration of borrelial infection in patients with eythema migrans residing in Slovenia. Four different strains were chosen as antigens: two strains of B. afzelii and two strains of B. garinii which differed in their expression of the outer proteins OspA, OspB and OspC. Each individual strain was used as antigen in immunofluorescence test (IFT), enzyme-linked immunosorbent assay (EIA) with whole borrelial cells, and EIA with ultrasonicated borrelial cells. With these 12 different tests, 100 samples were examined for the presence of specific IgM and IgG antibodies: 50 sera of blood donors and 50 sera of patients with erythema migrans. The latter were further subdivided into skin culture-positive and -negative subgroups. A commercial Western blot (WB) test was performed for 26 sera of the control group and 25 sera of patients with erythema migrans. The four different methods had distinct specificity and sensitivity. The most specific approaches were IFT (100% for IgM and 90–92% for IgG) and the WB test (100% for IgM and 73% for IgG), followed by EIA with whole borrelial cells (80–98% for IgM and 76–84% for IgG) and EIA with ultrasonicated borrelial cells (76–94% for IgM and 72–80% for IgG). The sensitivity levels of all these tests were low. The most sensitive were EIA tests with whole borrelial cells (28–36% for IgM and 32–42% for IgG) followed by EIA with ultrasonicated borrelial cells (22–32% for IgM and 24–36% for IgG), the WB test (16% for IgM and 32% for IgG) and IFT (0–2% for IgM and 14–20% for IgG). The following methods gave significant differences between patients and negative controls in detecting IgM antibodies: EIA with whole borrelial cells with both B. afzelii antigens and with antigen B. garinii that expressed OspA and OspC, EIA with ultrasonicated borrelial cells with antigen B. afzelii that expressed OspA, OspB and OspC. In detecting IgG antibodies, significant differences were observed between EIA with whole borrelial cells and with antigen B. afzelii that expressed OspA and OspB. Borreliae were isolated from the skin of 34/50 (68%) patients with erythema migrans: two strains failed to grow, while 26/32 (81%) strains were identified as B. afzelii, 5/32 (16%) as B. garinii and 1/32 (3%) as B. burgdorferi sensu stricto. No statistically significant differences in serologic test results between culture-positive and -negative patients with erythema migrans were found.     

Thrombocytopenia — A common finding in the initial phase of tick-borne encephalitis

Summary The aim of this prospective study was to assess the frequency of thrombocytopenia and abnormal liver function tests in the initial phase of tick-borne encephalitis. In 1994, 248 adult patients presented at the Department of Infectious Diseases, University Medical Centre Ljubljana, with acute lymphocytic meningitis or meningoencephalitis and serologically confirmed tick-borne encephalitis virus infection. In 180/248 (72.6%) patients, typical biphasic course of the illness was found and 28/180 (15.6%) patients with biphasic course were examined in both phases of the illness. In 20 out of these 28 (71.4%) patients, thrombocytopenia and leukopenia were found initially, in one (3.6%) patient only leukopenia was recorded and three (10.7%) patients had thrombocytopenia without leukopenia. In four (14.3%) patients leukocyte and thrombocyte values were within the normal range. The lowest leukocyte number was 1.4×10⁹/l and the lowest recorded thrombocyte number was 60×10⁹/l. Abnormal liver function tests were discovered in four out of 18 patients tested (22.2%). In conclusion, in the initial phase of tick-borne encephalitis thrombocytopenia and abnormal liver function tests may be found. Thrombocytopenia is a common finding with a frequency similar to that of well-known leukopenia, while abnormal liver function tests are relatively rare.

---

Thrombozytopenie — ein häufiger Befund in der Initialphase der Frühsommer-Meningoencephalitis

Zusammenfassung Diese prospektive Studie wurde durchgeführt, um die Häufigkeit einer Thrombozytopenie und pathologischer Leberfunktionstests in der Initialphase der Zeckenencephalitis zu bestimmen. 1994 suchten 248 erwachsene Patienten die Abteilung für Infektionskrankheiten am medizinischen Zentrum der Universität Ljubljana auf, die an einer akuten lymphozytären Meningitis oder Meningoencephalitis und einer serologisch bestätigten Infektion durch das Frühsommer-Meningoencephalitis-Virus litten. Bei 180/248 Patienten (72,6%) fand sich ein typischer biphasischer Verlauf. 28/180 Patienten (15,6%) mit biphasischem Verlauf konnten in beiden Phasen untersucht werden. Bei 20 dieser Patienten (71,4%) fand sich initial eine Thrombozytopenie und Leukopenie, bei einem Patienten (3,6%) nur eine Leukopenie und bei drei (10,7%) eine Thrombopenie ohne Leukopenie. Bei vier Patienten (14,3%) waren die Leukozyten- und Thrombozytenwerte im Normalbereich. Die niedrigste Leukozytenzahl war 1,4×10⁹/l und die niedrigste Thrombozytenzahl 60×10⁹/l. Bei vier der 18 untersuchten Patienten (22,2%) fanden sich pathologische Leberwerte. Man findet folglich in der Frühphase der Zeckenencephalitis eine Thrombozytopenie und pathologische Leberwerte. Die Thrombozytopenie ist ein häufiger Befund und ähnlich häufig wie die bekannte Leukopenie. Pathologische Leberfunktionstests werden hingegen relativ selten beobachtet.

     

CTG repeat polymorphism in DMPK gene in healthy Yugoslav population

OBJECTIVES: Myotonic dystrophy type 1 (DM1) is caused by large expansions of cytosine-thymine-guanine (CTG)-repeats in myotonic dystrophy protein kinase (DMPK)-gene. This gene is highly polymorphic in healthy individuals. It has been proposed that expanded alleles originated from the group of large sized normal alleles. If this is correct, one should expect a positive correlation between the frequency of large sized normal alleles and a prevalence of this disorder in a population. In this paper we determined the distribution of alleles of DMPK gene in healthy Yugoslav population. MATERIAL AND METHODS: A sample of 235 healthy individuals of Yugoslav origin have been genotyped for the alleles of DMPK locus. RESULTS: We found 22 different alleles, ranging in size from 5 to 29 repeats. Among 470 chromosomes studied, 41 chromosomes had more than 18 repeats (8.72%). CONCLUSIONS: Relatively high frequency of large sized normal alleles found in our population, suggest that prevalence of DM1 in Yugoslavia should not be different from the prevalence in other European populations.     

Clinical and epidemiological findings for patients with erythema migrans. Comparison of cohorts from the years 1993 and 2000

OBJECTIVES: i) To present clinical and epidemiological findings on adult patients diagnosed with typical erythema migrans (EM) at our institution during the year 2000 and ii) To compare the findings with data obtained by the same approach for an identical group of patients from 1993, with the aim of testing the hypothesis that, because of increased knowledge of Lyme borreliosis in 2000 compared with 1993, patients will visit us earlier and consequently present with smaller skin lesions. PATIENTS AND METHODS: Patients who qualified for inclusion in the study were adults diagnosed with typical EM in 2000 and 1993 at the Lyme borreliosis Outpatients' Clinic, Ljubljana, Slovenia. Epidemiological and clinical data were obtained from a questionnaire. RESULTS: In 2000, 535 patients had typical EM: 309 (57.8%) females and 226 (42.2%) males, aged 15-80 (median 47) years. Tick bite at the site of later EM was recalled by 311 (58.1%) patients a median of 14 days before the onset of the skin lesion, which was localised on the legs in 55.7% of patients. The median duration of EM prior to examination was 8.5 days, the largest diameter of EM was 12 (4-87) cm, and the average area of involved skin was 75 cm2. Thirty-six of 535 (6.7%) patients had more than one skin lesion (maximum number 21), 281/535 (52.5%) patients reported local symptoms (itching 45.2%, burning 12.9%, and pain 7.1%), and 191/535 (35.7%) reported systemic symptoms (fatigue 17.4%, headache 17.2%, myalgia 12.6%, arthralgia 11.2%, vertigo 3.6%, fever 2.5%, and chills 1.5%). In 1993, 892 patients with typical EM were diagnosed. They had similar characteristics to those in 2000 but were younger (44 versus 47 years; p = 0.025), more often remembered a tick bite (654/892 versus 311/535; p = 0.009), had a smaller diameter (10 versus 12 cm; p < 0.001) and surface area of EM (50.2 versus 75.0 cm2), presented more often with homogeneous skin lesions (378/892 versus 191/535; p = 0.010), and more frequently had some of the associated symptoms including nausea (43/892 versus 12/535; p = 0.021) and malaise (124/892 versus 51/535; p = 0.019). CONCLUSIONS: The majority of findings for the cohorts of EM patients seen in 1993 and 2000 were comparable but--contrary to expectations--in 2000 the duration of EM prior to attending our clinic was not shorter, and the skin lesions were larger and less often homogeneous. These findings indicate that knowledge of Lyme borreliosis did not increase during the period from 1993 to 2000.     

A skeletal muscle troponin T specific ELISA based on the use of an antibody against the soluble troponin T (16-31) fragment

Proteolytic degradation of muscle, which occurs post-mortem as part of the meat-ageing process, results in the production of protein fragments. In beef, degradation of skeletal muscle troponin T (TnT) results in the generation of a 16-residue long peptide (TnT (16-31)), identified in trichloroacetic acid (TCA) soluble extracts. We report the development of a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of TnT (16-31), using polyclonal antibodies raised against synthetic TnT (16-31). The ELISA procedure is based on inhibition of binding of the antibodies to immobilised TnT (16-31) by TnT (16-31) present in solution. Its useful range is 30 pmol to 2 nmol TnT (16-31)/ml. Quantification of TnT (16-31) in TCA muscle extracts showed that its concentration was enhanced with ageing. Moreover, a correlation between TnT (16-31) levels and meat tenderness was observed. The ELISA developed herein may prove advantageous for future use at the research and industrial level.     

Comparison of patients fulfilling criteria for confirmed and probable human granulocytic ehrlichiosis

In order to assess the validity of definitions for human granulocytic ehrlichiosis (HGE), epidemiological, clinical and laboratory findings for 13 patients with confirmed HGE and 21 patients fulfilling criteria for probable HGE were compared. The patients were identified during a prospective study on the aetiology of febrile illness occurring after a tick bite, performed in Slovenia from 1995 to 2002. Significant differences between the 2 groups were found for age of patients, duration of fever before the first examination, and several clinical and laboratory parameters including the occurrence of chills, myalgia, arthralgia, leukopenia, thrombocytopenia, abnormal liver function tests results, and elevated concentration of C-reactive protein. All the abnormalities were established more often in patients with confirmed HGE than in those with probable HGE. Patients with confirmed HGE exhibited higher geometric mean titre values and higher peak antibody titres to Anaplasma phagocytophilum that developed later in the course of their illness. The differences indicate that in some patients fulfilling criteria for probable HGE the signs and symptoms most probably are not the result of a recent infection with A. phagocytophilum.     

Causes of febrile illnesses after a tick bite in Slovenian children

BACKGROUND: To establish the etiology in Slovenian children with febrile illnesses occurring after a tick bite. METHODS: Eighty-six febrile patients younger than 15 years referred to our institution in 2001 with a history of a tick bite within 6 weeks before onset of the illness were included in this prospective study. Acute and convalescent serum samples were tested for the presence of antibodies to tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Rickettsia conorii, Babesia microti, Bartonella henselae, Bartonella quintana and Francisella tularensis. Cerebrospinal fluid was investigated in patients in whom meningeal involvement was clinically suspected. Blood and/or cerebrospinal fluid from the patients were cultured in modified Kelly-Pettenkofer medium. PCR was performed to detect ribosomal DNA of A. phagocytophilum and E. chaffeensis. RESULTS: Of 86 patients 33 (38%) were excluded because a well-defined febrile illness not associated with tick bite was established. Tick-borne illness was diagnosed in 28 (53%) of the 53 remaining patients. The most common diagnosis was tick-borne encephalitis (64%), followed by Lyme borreliosis (46%), human monocytic ehrlichiosis and human granulocytic ehrlichiosis (serologic evidence of infection in 9 and 4%, respectively). In 6 (21%) patients there was evidence for infection with more than 1 tick-borne agent. CONCLUSIONS: Tick-borne illness was established in 53% of the patients younger than 15 years presenting with febrile illness occurring within 6 weeks after a tick bite. The most common identified illnesses were tick-borne encephalitis and Lyme borreliosis.     

Survival of Huntington’s disease patients in Serbia: longer survival in female patients

The objective of this study was to estimate probability of survival of Huntington’s disease (HD) patients in Serbia as a function of CAG repeat length and selected demographic variables. This follow-up study was carried out at the Institute of Neurology, Clinical Centre of Serbia, Belgrade, 1982–2004. The study group consisted of 112 HD patients. The significant inverse correlation was found between CAG repeat length and age at onset of HD (r = −0.732, P = 0.001) and age at death (r = −0.760, P = 0.001). The cumulative probabilities of survival in a five, ten, fifteen, and twenty-years’ period were 90.9, 63.2, 10.3 and 4.5%, respectively. Higher survival probabilities were registered in female patients, as well as in those with older age at onset and lower number of CAG repeat length (≤46). The Cox regression analysis showed that significantly poorer outcome of HD in our population was related to younger age at onset (HR-hazard ratio = 1.9; P = 0.047), and larger CAG numbers (HR = 2.4; P = 0.071). The female sex was statistically significantly associated with longer survival (HR = 0.4; P = 0.007). These data might be of some importance for further exploration of natural history and prognosis of HD.