Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction

In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for speciesspecific detection ofEncephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, andEnterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected withEnterocytozoon bieneusi (n=9),Encephalitozoon spp. (n=2), andEncephalitozoon intestinalis (n=1) as well as stool spiked with spores ofEncephalitozoon cuniculi andEncephalitozoon hellem and tissue cultures ofEncephalitozoon cuniculi andEncephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection ofEnterocytozoon bieneusi andEncephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected withEncephalitozoon cuniculi andEncephalitozoon hellem. Moreover, identification ofEncephalitozoon spp. could be specified asEncephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates speciesspecific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.     

Acute and long-term humoral immunity following active immunization of rabbits with inacctivated spores of various Encephalitozoon species

Microsporidia of the genus Encephalitozoon are increasingly being reported as a cause of severe, often disseminated infections, mainly in patients with acquired immunodeficiency syndrome (AIDS). Immunological identification of each of the three recognized species (E. cuniculi, E. hellem, and E. intestinalis) requires the availability of specific immune sera. All sera available thus far have been generated by direct inoculation of rabbits with virulent microsporidian spores. This study demonstrates for the first time that subcutaneous immunization with inactivated spores of E. cuniculi, E. hellem, or E. intestinalis is capable of generating highly active rabbit hyperimmune sera to the homologous antigens, with maximal titers being 1:5,120, 1:1,280, and 1:2,560, respectively, as determined by the indirect immunofluorescence technique (IIF). Broad cross-reactivity of the rabbit antisera with all heterologous Encephalitozoon antigens was determined by IIF and immunogold electron microscopy; however, only the E. hellem immune serum strongly cross-reacted with spores of Enterocytozoon bieneusi. During the 35-month follow-up period the antibody titers to the homologous antigens declined to 1:640, 1:160, and 1:320, respectively. The observed decay curves for antibody titers against E. cuniculi, E. hellem, and E. intestinalis were fitted using mathematical modeling, resulting in a predicted duration for specific immune responses of about 7 years on average. Knowledge of the magnitude and duration of specific immune responses is a prerequisite for further evaluation of the concept of using inactivated microsporidian spores in the quest for vaccines against microsporidian infections. Received: 10 April 2000 / Accepted: 18 July 2000     

Anti-osteopenic effect of alpha-ketoglutarate sodium salt in ovariectomized rats

The purpose of the study was to determine the effect of alpha-ketoglutarate sodium salt (AKG) treatment on the mineralization of the tibia in female rats during the development of osteopenia (Experiment-1) and in the condition of established osteopenia (Experiment-2). Thirty-two female rats were ovariectomized (OVX) to induce osteopenia and osteoporosis and another 32 female rats were sham-operated (SHO) and then randomly divided between the two experiments. In Experiment-1, the treatment with AKG started after a 7-day period of convalescence, whereas in Experiment-2 the rats were subjected to a 60-day period of osteopenia fixation, after which the actual experimental protocol commenced. AKG was administered in the experimental solution for drinking at a concentration of 1.0?mol/l and a placebo (PLC) was used as a control solution. After 60?days of experimental treatment the rats in both experiements were sacrificed, the body weight recorded, and blood serum and isolated tibia were stored for further analysis. The bones were analyzed using tomography and densitometry, and for estimation of mechanical properties the 3-point bending test was used. Serum concentrations of osteocalcin and collagen type I crosslinked C-telopeptide were measured. The anabolic effects of AKG on bone during osteopenia development in Experiment-1 not only stopped the degradation of bone tissue, but also stimulated its mineralization. The usage of AKG in animals with established osteopenia (Experiment-2) was not able to prevent bone atrophy, but markedly reduced its intensity. The stimulation of tibia mineralization after AKG treatment has been also argued in healthy SHO animals. The results obtained prove the effectiveness of AKG usage in the prophylaxis and therapy of osteopenia and osteoporosis, induced by bilateral gonadectomy. Additionally, the results clearly prove that treatment with AKG improves the mineralization of bone tissue in healthy animals.     

Mechanical transport and transmission of Cryptosporidium parvum oocysts by wild filth flies

Over the course of six months wild filth flies were collected from traps left for 7-10 days in a barn with or without a calf shedding Cryptosporidium parvum Genotype 2 oocysts in diarrheic feces. The oocysts of C. parvum transported on the flies' exoskeletons and eluted from their droplets left on visited surfaces were infectious for mice. The mean number of oocysts carried by a fly varied from 4 to 131, and the total oocyst number per collection varied from 56 to approximately 4.56 x 10(3). Fly abundance and intensity of mechanical transmission of infectious C. parvum oocysts were positively correlated, and both increased significantly when an infected calf was in the barn. Molecular data showed that the oocysts shed by infected calves were carried by flies for at least 3 weeks. Filth flies can acquire infectious C. parvum oocysts from unsanitary sites, deposit them on visited surfaces, and therefore may be involved in human or animal cryptosporidiosis.     

Assessment of flow changes in the circle of Willis after stenting for severe internal carotid artery stenosis.

PURPOSE: To assess flow velocities in the cerebral arteries after carotid artery stenting (CAS) in patients with unilateral versus bilateral lesions and analyze velocities in patients with neurological complications after CAS. METHODS: Ninety-two patients (68 men; mean age 63.2 +/- 8.4 years, range 44-82) with internal carotid artery (ICA) stenoses were divided according to unilateral (group I, n = 72) or bilateral (group II, n = 20) disease. Fifty age- and gender-matched patients without lesions in the extra- or intracranial arteries served as a control group. Transcranial color-coded Doppler ultrasound was performed prior to and within 24 hours after CAS in the test groups; systolic velocities were assessed ipsilateral (i) and contralateral (c) to the CAS site in the middle cerebral artery (MCA) and anterior cerebral artery (ACA). RESULTS: Collateral flow via the anterior communicating artery (ACoA) was found in all group-II patients and 90% of group-I patients. After CAS, collateral flow through the ACoA ceased, and the velocity increased by 26% in the iMCA in group I compared to controls (p < 0.001). In group II, iMCA flow increased by 30% (p < 0.001) and flow via the ACoA (p < 0.001) increased, resulting in normalization of cMCA velocities (p = 0.928). In 89 (96.7%) subjects, CAS was uncomplicated. Hyperperfusion syndrome occurred in 2 (2.2%) patients, both with bilateral ICA stenoses; 1 (1.1%) transient ischemic attack was seen in a patient with unilateral disease. In the patients with hyperperfusion syndrome, the MCA velocities were 2.7- and 7.4-fold higher, respectively, versus before CAS and 2-fold higher than in controls. CONCLUSION: Uncomplicated CAS results in an iMCA velocity increase >25% compared to controls. MCA velocities in hyperperfusion syndrome were greatly increased versus before CAS and in controls.     

Reversed blood flow during internal carotid artery stenting using Parodi Anti-Emboli System--progress in protection against cerebral stroke

Carotid artery stenosis is one of the main causes of stroke. Nowadays two techniques for treating carotid stenosis are available - surgical endarterectomy and percutaneous angioplasty combined with stent implantation at the site of stenosis. Cerebral protection devices during internal carotid stenting significantly decrease the incidence of periprocedural complications, however, during the introduction of protective devices the cerebral blood flow remains unprotected. Therefore, the quest for a system specifically protecting cerebral flow during the whole procedure is still underway. Temporary reversal of carotid flow during the procedure using the Parodi Anti-Emboli System seems a viable solution. The present study describes the first two patients who underwent internal carotid artery stenting using this technique in our institution.     

Long-term outcome of coronary balloon angioplasty in diabetic patients

Diabetes is recognised to increase morbidity and mortality after coronary revascularization. We compared clinical outcomes in mean 5-year-long follow-up of coronary balloon angioplasty in diabetic and non-diabetic patients. We studied 621 patients undergoing elective angioplasty from 1987 to 1996. There were 60 (9.7%) patients with diabetes who were compared with 561 non-diabetic patients. Diabetics were older, more often obese, less frequently were current smokers, and less frequently had hypercholesterolaemia. Diabetic patients in comparison with non-diabetics had lower ejection fraction and more frequently had angioplasty of complex (B2 or C) lesions, but there were no differences between both groups in the other clinical and angiographic risk factors. Clinical success of angioplasty, as well as complications rate were similar in both groups. In follow-up restenosis occurred more frequently in diabetics (46.3 vs. 32.2%, P=0.03), resulting in significantly higher re-intervention rate (50.0 vs. 35.4%, P=0.03). Especially diabetic patients were more frequently referred to CABG (20.4 vs. 9. 9%, P=0.02). There were no significant differences in deaths (1.9 vs. 2.8%) and myocardial infarction (3.7 vs. 4.4%). Diabetics presented worse CCS status at the end of observation (Class 0 and I - 61.1 vs. 74.4%, P=0.037). Angioplasty proved to be a safe procedure in diabetic patients. Despite higher restenosis and re-intervention rate in diabetics, mortality as well as myocardial infarction rate was the same in both groups during mean 5-year follow-up.     

Comparison of the effect of perindopril and acebutolol on cerebral hemodynamics in hypertensive patients

Purpose: The aim of the study was to compare effect of perindopril (4 mg once a day) versus acebutolol (400 mg once a day) on cerebral hemodynamics in hypertensive patients. Methods: Thirty untreated patients with mild-to-mode-rate hypertension were studied. Drug influences on Doppler-derived parameters from the cerebral blood flow (CBF) velocity wave form were evaluated in a randomized, double blind, cross-over, placebo controlled study. The mean CBF velocity, pulsatility index (PI), cerebrovascular resistance and index of CBF were calculated from concomitant transcranial Doppler measurements and systemic blood pressure. Results: Acebutolol and perindopril significantly decreased systolic, diastolic and mean blood pressure in relation to placebo. The mean value of CBF velocity increased to a comparable level after both drugs (54.9 ± 9.1 cm/s on placebo vs 62.8 ± 14.5 cm/s on perindopril p < 0.01 and 61.4 ± 9.2 cm/s on acebutolol, p < 0.01). Also, the cerebrovascular resistance index decreased similarly after both drugs (2.26 ± 0.35 on placebo vs 1.68 ± 0.42 on perindopril p < 0.01 and 1.7 ± 0.36 on acebutolol p < 0.01). The calculated CBF index increased significantly after each drug (25.23 ± 7.99 on placebo vs 33.98 ± 11.23 p < 0.01 on perindopril and 30.90 ± 8.04 on acebutolol p < 0.01). However, perindopril augmented the CBF index more effectively than acebutolol (p < 0.05). Conclusions: Among patients with mild-to-moderate hypertension both acebutolol and perindopril beneficially decreased cerebrovascular resistance and increased the CBF index in comparison with placebo. The increase of CBF index was greater after perindopril than acebutolol, which suggests a more significant improvement in cerebral perfusion by perindopril. The non-invasive transcranial Doppler ultrasonography method of CBF velocity measurement may contribute to choosing optimal antihypertensive therapies and to monitor their effect.