A sensitive homologous radioimmunoassay for human relaxin-2 (h-RLX-2) based on antibodies characterized by epitope mapping studies

We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.     

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.     

Mutational analysis of GJB1, MPZ, PMP22, EGR2, and LITAF/SIMPLE in Serbian Charcot-Marie-Tooth patients

We report the results of mutational analysis in the following genes: GJB1 , MPZ , PMP22 , EGR2 , and LITAF/SIMPLE in 57 Charcot-Marie-Tooth (CMT) patients of Serbian origin without the PMP22 duplication. We found 10 different mutations in 14 CMT patients: 6 mutations in GJB1 , 3 in MPZ , and 1 in PMP22 . Five of six GJB1 mutations are reported for the first time, and the most frequent one appears to be a founder mutation in the Serbian population. No mutations were found in EGR2 or LITAF . Thus, GJB1 mutation analysis should be done in patients without the PMP22 duplication and male-to-male transmission of CMT.     

Epidemiology of myotonic dystrophy type 1 (Steinert disease) in Belgrade (Serbia)

The aim of this study was to estimate the incidence and prevalence of myotonic dystrophy type 1 (DM1) in Belgrade during the period 1983–2002.

The patients who had DM1 were ascertained through hospital records from all neurological departments in Belgrade during 1983–2002. The molecular genetic analysis was performed in all patents included in the study.

We identified 101 DM1 patients (52 males and 49 females). The average annual incidence rate of DM1 in Belgrade for the period observed was 2.0/1,000,000 (95% confidence interval (CI), 0.3–8.3), 2.1/1,000,000 (95% CI, 0.3–8.3) for males and 2.0/1,000,000 (95% CI, 0.3–8.3) for females. The highest age-specific DM1 incidence was registered in the age group 20–49: 3.4/1,000,000 (95% CI, 0.5–7.6), 4.0/1,000,000 (95% CI, 1.1–10.2) in males and 2.5/1,000,000 (95% CI, 0.5–7.6) in females. In the population of Belgrade, a cumulative probability of acquiring DM1 was 1 per 8621 for men and 1 per 9259 for women (1 per 8940 of the population for both sexes). The prevalence of DM1 in Belgrade on 31 December 2002 was 5.3/100,000 (95% CI, 4.2–6.6).     

Doxycycline versus ceftriaxone for the treatment of patients with chronic Lyme borreliosis

BACKGROUND: Therapeutic guidelines for treatment of late manifestations of Lyme borreliosis have not yet become well established. Patients with symptoms suggesting central nervous system involvement are often treated with courses of intravenous ceftriaxone. This is an expensive treatment approach with potentially severe side effects. We compared the efficacy, side effects and costs of doxycycline and ceftriaxone in the treatment of such patients. PATIENTS AND METHODS: Adult patients qualified for the study if they had nonspecific symptoms suggesting central nervous system involvement for more than six months (but without overt clinical signs of the involvement), had positive serum borrelial antibody titers and/or erythema migrans prior to the onset of symptoms, had not been previously treated with antibiotics and did not have pleocytosis in the cerebrospinal fluid. Patients were given either 100 mg of oral doxycycline twice daily for 4 weeks (23 patients) or 2 g of intravenous ceftriaxone daily for 2 weeks followed by 100 mg of doxycycline twice daily for another 2 weeks (23 patients). Clinical outcome was assessed during a 12-month follow-up period. RESULTS: Improvement in the frequency and/or the intensity of symptoms was reported by more than two-thirds of the 46 patients enrolled in the study. The two treatment regimens were found to be correspondingly effective. Photosensitivity reactions and gastrointestinal symptoms were noted more often among patients receiving doxycycline than in those receiving ceftriaxone. Treatment with doxycycline proved to be much cheaper than with ceftriaxone. CONCLUSIONS: In patients with previously untreated chronic Lyme borreliosis with symptoms suggesting central nervous system involvement but without overt clinical signs of it, and without pleocytosis in the cerebrospinal fluid, treatment with doxycycline is as effective as with ceftriaxone. Treatment with doxycycline is cheap and relatively safe, but gastrointestinal symptoms and photosensitivity reactions can be expected more often than with ceftriaxone.     

Tick-borne encephalitis: Possibly a fatal disease in its acute stage. PCR amplification of TBE RNA from postmortem brain tissue

Summary Tick-borne encephalitis has occurred regularly in Europe since it was first diagnosed in 1931 bySchneider. The mortality rate of patients with this disease is 1–2%. Death usually occurs in the acute stage of illness. A case report of a 28-year-old patient from Slovenia, who died shortly after the onset of tick-borne encephalitis, is described. The clinical course of disease, results of serological tests, neuropathological findings and polymerase chain reaction amplification of parts of viral genome from postmortem brain tissues are presented.     

Evaluation of immunofluorescence test (IFT) and immuno (western) blot (WB) test in patients with erythema migrans

The diagnosis of Lyme borreliosis is based on the recognition of typical clinical signs and is assisted by laboratory confirmation of borrelial infection. The aim of the present study was to assess the value of an immunofluorescence test (IFT) and an immuno (western) blot (WB) test for the detection of Borrelia burgdorferi sensu lato antibodies in patients with erythema migrans residing in Slovenia. We determined specific IgM and IgG antibodies in 117 patients with erythema migrans and 96 healthy persons using an IFT (in-house test) and a commercial WB test. Skin biopsies of erythema migrans lesions were cultured, and isolated strains were identified with PFGE. There were 66/117 (56.4%) culture-positive and 51/117 (43.6%) culture-negative patients. B. afzelii was found in 52/62 (84%) and B. garinii in 10/62 (16%) biopsies. IFT-IgM antibodies were established in 2/117 (1.7%) erythema migrans patients and in none of the control group, while WB-IgM antibodies were present in 56/117 (48%) patients with erythema migrans and 21/96 (22%) members of the control group (p = 0.002). IFT-IgG antibodies were demonstrated in 3/117 (2.2%) erythema migrans patients and 2/96 (4%) persons of the control group, while corresponding values for WB-IgG were 36/117 (31%) and 26/96 (27%), respectively (non-significant differences). IgM antibodies directed against p41 and OspC, and IgG antibodies directed against p41, p18 and OspC were frequently found in both erythema migrans patients and the control group. The only significant difference between erythema migrans patients and the control group in the WB test was in the reaction of IgM antibodies with OspC antigen, which was found in 54/117 (46%) erythema migrans patients and 18/96 (18.8%) healthy persons (p < 0.0001). The immune response in patients with erythema migrans was very similar to that of the control group determined with either the IFT or WB test.     

Development of erythema migrans in spite of treatment with antibiotics after a tick bite

OBJECTIVES: The recent information on the appearance of erythema migrans despite prophylaxis with 200 mg of doxycycline was the stimulus for a search among our patients for those who developed the skin lesion regardless of receiving antibiotics after a tick bite. METHODS: Data were reviewed for adult patients with erythema migrans diagnosed at our institution from 1994 to July 2001, targeting those who received antibiotics after a tick bite. RESULTS: Seven of 5056 (0.14%) patients, diagnosed with typical erythema migrans, developed the skin lesion despite receiving antibiotics after a tick bite. Antibiotics were prescribed by general physicians: in four cases as prophylaxis of Lyme borreliosis within one day after tick detachment and in three cases because of development of acute respiratory tract infection two, five, and eight days after the bite, respectively. The dosages were as follows: azithromycin in a total dose of 3 g in three patients and 1.5 g in the fourth patient, amoxicillin-clavulanic acid 625 mg t.i.d. for ten days in the fifth patient, amoxycillin 500 mg t.i.d. for seven days followed by azithromycin 250 mg o.d. for eight days in the sixth, and amoxycillin 500 mg t.i.d. for eight days in the seventh. The patients (five females and two males, aged 18-61 years) were referred to our Department on average six (1-19) days after the appearance of skin lesions. They had typical solitary (five patients) or multiple (two patients) erythema migrans with the characteristics usually seen in European patients, except for a rather long incubation period (median value 28 days, range 10-40 days). All laboratory tests, including the examination of cerebrospinal fluid in three patients with the disseminated form of the illness, were within normal range. Borrelial antibodies were demonstrated in only one patient. A skin biopsy specimen obtained from the site of the erythema migrans was culture positive for Borrelia in 2/4 patients. CONCLUSIONS: Our study did not enable us to assess the frequency of antimicrobial prophylaxis failure or the efficacy of individual antibiotics for the prevention of Lyme borreliosis. However, the seven patients presented demonstrate that antibiotic prophylaxis for Lyme borreliosis after a tick bite, at least in Europe, is not entirely effective.