Identification of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in patients with erythema migrans.

Anaplasma phagocytophilum has been first isolated from the blood of two Czech patients simultaneously with a cultivation of Borrelia burgdorferi sensu lato from their erythema migrans lesions. Cultivation of different Borrelia spp. from 12 erythema migrans biopsies, from 2 blood, one liquor and one placenta sample in BSK-H medium was successful. Adapted conventional methods targeting 16S rRNA and OspA genes for real-time polymerase chain reaction (PCR) and partial sequencing of these genes together with microscopical examinations of the blood smears provided a direct detection of the B. afzelii, B. burgdorferi, B. garinii, B. valaisiana and B. bissettii in the skin, B. garinii in the blood, placenta and liquor in 24 (36.3 %) patients, and A. phagocytophilum in 10 (15 %) patients with erythema migrans. Positive indirect IgM immunofluorescence against Anaplasma sp. was obtained in 7 cases, specific IgG antibodies were detected in 12 patients. Three women suffering from erythema migrans in the first trimester had positive PCR for Anaplasma and/or for Borrelia in the blood and two of them, later, in the placenta. Interpretation of laboratory data can bring important contribution to establishing the role of Anaplasma sp. in erythema migrans and forming the principle of precaution with laboratory diagnosis during pregnancy which always should be reflected in the resistance of Anaplasma sp. toward penicillins.

Phenotypic and genotypic analysis of Borrelia spp. isolated from Ixodes ricinus ticks by using electrophoretic chips and real-time polymerase chain reaction.

The genotype of Borrelia burgdorferi sensu lato was detected in 371 out of 1244 ticks. Borrelia determination was based on partial sequencing of the 16S rRNA gene and real-time polymerase chain reactions for identification and quantitation of ospA and recA genes. Different Borrelia spp. were identified; B. garinii in 40% ticks followed by B. afzelii (36.3%), B. burgdorferi sensu stricto (12.9%), B. valaisiana (3.5%), B. lusitaniae (0.8%), B. bissettii (0.5%) and B. miyamotoi-like (0.5%). Cultivation of 30 borrelia strains in BSK-H medium, among them B. valaisiana, B. bissettii-like and B. miyamotoi-like strains was unique in Czechia. Calibrated microfluidic-based quantification showed differences in the concentration of the nucleic acids and molar mass of the outer surface proteins of different Borrelia spp. with standard sensitivity and specificity and was helpful for their identification. The outer surface protein OspA was absent in B. miyamotoi-like and the OspB protein in B. valaisiana, B. lusitaniae and in three subtypes of B. garinii.

PCR in lyme neuroborreliosis: a prospective study.

OBJECTIVES: DNA proof is the only widely available direct diagnostic tool in Lyme borreliosis. Sensitive PCR detecting of spirochetal DNA was prepared and a prospective study in neuroborreliosis was performed. MATERIALS AND METHODS: 57 hospitalised patients with active neuroborreliosis and proved CSF antibodies synthesis were examined. Nested-PCR (utilizing three targets) was used for the detection of specific DNA in plasma, CSF and urine. RESULTS: Before treatment 36 positive patients (63.1%) were found in all tested specimens in parallel, 28 patients (49.1%) were positive in urine, 20 in CSF (35.0%) and 16 in plasma 28.0%). Later only urine was tested and the following results were obtained: 17 positive patients (30.0%) immediately after treatment, 8 (14.0%) after 3 months and one patient persisted positivity after 6 months. CONCLUSIONS: The highest sensitivity of PCR was achieved in the acute period of neuroborreliosis - 63.1% in three body fluids comparing with CSF antibody synthesis.

Molecular and microscopical evidence of Ehrlichia spp. and Borrelia burgdorferi sensu lato in patients, animals and ticks in the Czech Republic.

We report moderately severe cases of human ehrlichiosis and a lethal one caused by human granulocytic Ehrlichia, the HGE agent, closely related to Ehrlichia phagocytophila and Ehrlichia equi. Their vector is the Ixodes ricinus tick, which also transmits Borrelia burgorferi sensu lato in central, west and east regions of the Czech Republic. The diagnosis was established by PCR with sequence analysis of the genes encoding 16S rRNA of Ehrlichia and with reverse hybridization by using enzyme linked immunosorbent assay with different covalently coupled probes to the activated plate. Ten out of 47 patients and 10 huntsmen were PCR positive and 7 of them seroconverted to the HGE. Coinfection of Ehrlichia phagocytophila with Borrelia burgdorferi sensu lato was detected in 3 patients. Ehrlichia spp., the HGE agent, was isolated and propagated only from one patient in the HL-60 promyelocytic cell line. The maintenance of Ehrlichia in culture and in patients was assayed also by immunocytological staining and electron microscopy. Sequence or hybridization analysis of PCR results in different wild mammals and birds showed significant sources of Ehrlichia fagocytophila in nature. Three variants of E. phagocytophila in wild roe deer and boars, as well as for the first time in birds, have been described. Cultures from the blood of horses, and from the spleen and kidney specimens of roes and boars, PCR positive for Ehrlichia spp., displayed a disappearing level of the pathogen or contamination with other bacteria.

[Long-term treatment of combined hyperlipidemia with a combination of fluvastatin and fenofibrate]. / Dlouhodobá lécba kombinované hyperlipidémie kombinací fluvastatinu s fenofibrátem.

The long-term efficacy and safety of fluvastatin monotherapy was compared with that of the combination of fluvastatin and fenofibrate in 104 patients with coronary heart disease and combined hyperlipidemia in an open, randomised, parallel group, clinical study of 78 weeks duration. Combined hyperlipidemia was defined as LDL-cholesterol 4.1 mmol/l and higher and triglycerides between 2.5 and 4.5 mmol/l after 8 weeks of dietary intervention. The patients were treated with either fluvastatin 40 mg daily or with the combination of fluvastatin (20 mg daily) and micronized fenofibrate 200 mg daily. Mean values of total and LDL-cholesterol decreased by 19.3% and 29.7% respectively after fluvastatin treatment and by 21.5% and 29.1% respectively after the combination of fluvastatin and fenofibrate treatment. The differences between the treated groups were not significant. Mean values of HDL-cholesterol increased significantly more after the combination of fluvastatin and fenofibrate than after fluvastatin monotherapy (26% vs. 9.9%). The mean values of triglycerides decreased significantly more after the combination treatment than after fluvastatin monotherapy (-40.2% vs. -19.7%). The treatment in both groups was well tolerated and no signs of myopathy were observed in any patient. The study was discontinued in 1 patient due to the increase of liver enzymes. The most frequently observed side effects were minor gastrointestinal symptoms, which were more frequent in patients treated by the combination of fluvastatin and fenofibrate. Thus our results demonstrate that the combination of fluvastatin and fenofibrate is an effective and safe treatment option for patients with coronary heart disease and mild to moderate combined hyperlipidemia if a more radical lowering of triglycerides and increase of HDL-cholesterol is desired.

[A single-center controlled clinical study with Sandimmune Neoral after kidney transplantation]. / Monocentrická kontrolovaná klinická studie se Sandimmunem Neoral u nemocných po transplantaci ledviny.

The authors compared in a controlled clinical study two groups of patients after a first renal transplantation treated by triple drug immunosuppressive therapy. In a group of 31 patients the triple combination comprised Sandimmune Neoral. In the control group there were 30 patients who received Sandimmune. No differences were found between the two groups as regards the effectiveness of this treatment and the authors did not confirm a lower incidence of rejections described in patients treated with Sandimmune Neoral. They confirmed, however, a lower interindividual variability of Cy-A levels assessed specifically in patients treated with Sandimmune Neoral.

[Treatment of hypertension with a fixed combination of bopindolol and chlorthalidone (Sandoretic)]. / Lécba hypertenze fixní kombinací bopindololu a chlorthalidonu (Sandoretic).

A multicentric, prospective, 16-week open study evaluated the effectivity and tolerance of the fixed combination of the beta-blocking agent bopindolol with the diuretic chlorthalidone--Sandoretic in 81 patients with mild to moderate hypertension. The combination of these two drugs is appropriate, since both drugs have long-term effects. Sandoretic induced a decrease of the mean sitting initial systolic blood pressure of 162.5 +/- 16.5 mmHg to 134.2 +/- 12.8 mmHg at the end of the study, a decrease of 28.3 mmHg. Diastolic blood pressure decreased after 16 weeks of treatment from the initial value of 103.9 +/- 4.9 mmHg to 85.3 +/- 6.0, a decrease of 18.6 mmHg. Changes of the standing systolic and diastolic blood pressures were of similar magnitude. Sandoretic treatment led to a normalization of the diastolic blood pressure (90 mmHg and lower) in 80.3% of patients. In 49.4% of patients treatment with Sandoretic led even to a diastolic blood pressure of 85 mmHg and lower and 29.6% patients had at the end of treatment diastolic blood pressure 80 mmHg and lower. Tolerance of the drug was excellent in 75.3% patients. Sandoretic induced a mild, however, significant decrease of potassium plasma levels. The increase of the uric acid plasma level was also significant. Monitoring of potassium plasma levels is therefore necessary during the treatment with Sandoretic. In patients showing a decrease of the potassium plasma level, potassium sparing diuretic-amiloride should be added or the dosage of the drug should be halved.

[Spirapril in the therapy of mild and moderately severe hypertension. A Czech and Slovak multicenter study]. / Spirapril v lécbe mírné az stredne tezké hypertenze. Jednorocní multicentrická ceská a slovenská studie.

A one-year open study was focused on the therapeutic effect and tolerance of spirapril in 171 patients with mild to moderate hypertension in 11 centres in the Czech and Slovak Republic. The antihypertensive effectiveness was investigated after four weeks, after 12 weeks and after 52 weeks. Only the results recorded after one year are reported. The study was completed after one year by 139 patients, incl. 120 (86.3%) with a normal diastolic pressure of 90 mmHg or less. The study was not completed by 32 patients (18.8%): because treatment was not effective--9.4%, because of undesirable effects--4.7%, 3.5% were eliminated by the investigators for various reasons (change of domicile, poor collaboration) and 1.2% because the protocol was not respected. According to the analysis "intention to treat" the diastolic pressure was normalized by monotherapy with spirapril in 25.1% of the baseline group; combination of spirapril with bopindolol led to normalization of the pressure in another 38.0% patients and in 7.0% patients normal diastolic pressure was achieved by a combination of spirapril with a hydrochlorothiazide; i.e. a total of 70.1% of the baseline group had a normal diastolic pressure after one year of treatment. In another 9.4% of the baseline group the diastolic pressure declined by 10 mmHg or more but not to normal levels. Thus normalization or effective control of pressure was achieved after one year in 79.6% patients according to the analysis "intention to treat". The combination of spirapril and bopindolol proved very effective. Patients where it was necessary to combine spirapril with bopindolol or hydrochlorothiazide had significantly higher baseline readings of blood pressure. During treatment the authors did not record in any of the groups a change of lipids, potassium, uric acid, the haemogram, liver tests or creatinine. Treatment was well tolerated and undesirable effects were rare (most frequent side effects: cough 3.5%, vertigo also 3.5%). The results of the study indicate that spirapril is an effective and well tolerated ACE inhibitor in the treatment of hypertension and its combination with bopindolol is equally suitable as the combination of spirapril with hydrochlorothiazide.

Cadmium influence on the rat liver.

Influence of cadmium on the intestinal and hepatic tissue of rat was studied in acute injection and oral as well as subchronic and chronic oral experiments with the purpose of better understanding of the penetration mechanisms of cadmium into these tissues. Acute single oral dose of CdCl2 32.5 mg.kg-1 inhibited in vitro the respiration of intestinal scrapings (44%) and in liver slices (25.7%). In combined oral doses of 32.5 mg.kg-1 + 162.5 mg.kg-1 inhibition in the intestine increased with exposure time, while in the liver it decreased. Amount of presumable metallothionein in the liver was approximately ten times higher than that in the intestine. Respiration was measured by a Clark electrode. Binding of cadmium to metallothionein was determined by column chromatography and spectrophotometry. Physiological findings are in agreement with morphological ones. In cases when, following an acute single oral dose, a 50% change was detected in the absorptive jejunal zone, a 10% change was observed in the liver parenchyma. In the case of combined oral dose of 60-70% of absorptive villi zone is damaged by cadmium, which penetrates by passive diffusion into lamina propria and by blood to the liver, where it acts toxically in 30%. Active transport of cadmium after chronic application is preserved. Correlation of physiological and morphological findings was evident.

An X-ray, microanalytical and ultrastructural study of cadmium absorption and transport in rat liver.

Accumulation of cadmium in the liver was demonstrated by X-ray microanalysis in every type of experiment, i.e. after injecting Cd into the ligated intestine and after the peroral acute single and combined, subchronic and chronic administration of Cd. Half an hour after its injection, Cd was localized diffusely in the liver; one hour after injection its increased accumulation in the cells caused generalized changes in the endoplasmic reticulum, mitochondria and nuclei. In acute and chronic peroral tests, the hepatocytes of the intermedial and peripheral zone of the lobes were the main storage region. After an acute dose of Cd, the cells in the centrolobular zone were hydropic, or single-cell necrosis developed; after the longer effect of combined doses the latter was manifested as centrolobular focal necrosis. Cd was not demonstrated in the lesions. Chronic administration did not lead to manifest severe degenerative changes in the liver. Accretions in the mitochondria and on the membranes of the endoplasmic reticulum were identified by means of X-ray analysis with cadmium peaks. Cadmium showed up clearly as L alpha- and L beta-lines at 3.135-3.320 keV. We presume that cadmium is bound in the ribosomes of the endoplasmic reticulum, as well as the mitochondria, and is released by the invagination of swelling mitochondria of the peripheral hepatocytes into Disse's spaces.

Effect of cadmium on the rat intestine.

The effect of cadmium on rat jejunal mucosa was observed after application of CdCl2 doses: acute single (32.5 mg.kg-1) or combined (32.5 + after 24 h 162.5 mg.kg-1) and chronic (250 mg.l-1, daily consumption 10 ml for 3 months). Inhibition of mucosal respiration, measured by a Clark electrode was 44, 50.4 and 38.4% respectively, as related to 100% of controls. Metallothionein was determined in the mucosa for combined and chronic doses. Electron X-ray microanalysis has shown Cd2+ distribution in dependence on pathological changes: greater regressive changes caused smaller Cd2+ retention. In acute experiments Cd2+ penetrate by passive diffusion through foci of damaged villi into the lamina propria. In crypt absorptive cells with preserved alcalic phosphatase and succinate dehydrogenase activity and in Paneth cells Cd2+ retention was determined. Combined dose of Cd2+ damaged homeostasis and prevented Cd2+ retention. In chronic experiments occurred a diffuse distribution of Cd2+ in the mucosa.