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Aim of the Study: The main goal of this study was to find the best method for Aspergillus DNA isolation from peripheral blood samples. The method should be very effective but not expensive or time consuming to be suitable for routine diagnostics use., Material and Methods: We compared four different methods for Aspergillus DNA isolation - one method with enzymatic lysis of the fungal cell wall and three methods that combine chemical and mechanical lysis (using high speed cell disruption with glass beads). Peripheral blood samples were inoculated with defined amount of Aspergillus fumigatus suspension and used for DNA isolation. Isolated DNA was then quantitatively analyzed with in-house real-time PCR method using specific probe., Results: Enzymatic method seems not to be useful in a routine diagnostics mainly because of its low efficiency and too long processing time. Better could be the methods using both chemical and mechanical cell disruption that can isolate fungal DNA with high efficiency in a relatively short time., Conclusions: The method using ZR Fungal/Bacterial DNA Kit (Zymo Research, USA) was chosen for routine use in our laboratory because it is cheap, fast and very efficient.

Since many people all around the world are suffering from genetic disorders, modern therapeutic approaches are focused on the search of new pharmaceutical products. These products will be able to act on the gene level, more accurately on the nucleotide sequences themselves. RNA interference (RNAi) is an evolutionary conserved process that is caused by double stranded RNA (dsRNA). MicroRNA (miRNA) and small interfering RNA (siRNA) are the most important dsRNAs, which have been identified so far. Short (19-25 bp) non-coding dsRNAs are responsible for regulation of cellular development, heterochromatin formation and genomic stability in eukaryotes. Most importantly they are able to silence cognate genes. Therefore, they can provide new insights into the gene function and pathway analysis. Furthermore, they are believed to be new potential targets for diagnosis and therapeutics, especially for the treatment of genetic disorders, which can be caused by nucleotides insertions, deletions and translocations.

PCR detection of fungal pathogens in clinical samples has been discussed in journals for more than two decades. However, its use for diagnosing invasive aspergillosis is still controversial, despite the fact that molecular methods are routinely used in various fields of modern microbiology. These are e. g. genotyping of bacterial strains resistant to antibiotics, molecular epidemiology or routine detection of viral infections in clinical material. PCR methods have made the diagnostic applications faster, simpler and more accurate. This review deals with issues related to molecular methods for diagnosing invasive fungal infections and the main factors limiting their use in everyday clinical practice.

Aim of the Study: The main goal was to explain the discrepancies between two PCR methods used for detection of cytomegalovirus (CMV) in peripheral blood samples of patients of Department of Internal Medicine-Hematooncology, University Hospital, Brno., Materials and Methods: In past we used exon 4 of major immediate-early (MIE) gene as the target for quantitative detection of the CMV in clinical samples, but sometimes this method failed to detect the viral load in samples that were positively tested using less sensitive qualitative method targeting another region (exon 2-4) of the same gene. From January 2004 to January 2005 we totally tested samples from 363 patients. 64 patients were at least once CMV positive using quantitative method, but 20 patients were repeatedly false negative.To find the cause of this discrepancy we performed partial sequence analysis of this region (nt positions 2719-2919, GenBank M21295) and glycoprotein B (gB) genotyping. We sequenced samples from 35 patients-15 giving true positive (both in qualitative and quantitative method) and 20 giving false negative (negative in quantitative but positive in qualitative method) results in several consecutive blood samples., Results: The 15 true positive samples were 100% homological, whereas all 20 false negative samples showed high degree of variation from the laboratory strain AD169. These changes are not random and indicate that the two groups of patients were infected by different CMV genotypes. Moreover, sequence alignment showed similarity to laboratory strains Toledo and Towne. No preferential concordance was observed between clinical context, MIE exon 4 sequence and gB groups., Conclusions: Because of high sequence variability exon 4 of MIE gene can not be used for routine diagnostics. Genetic varibility among the pathogenic strains may seriously affect its proper diagnostics.

Introduction: Acute upper gastrointestinal tract bleeding is a cause of significant morbidity and mortality and is a reason for urgent endoscopy. Besides an age and associated diseases, prognosis of patients influence also localisation and type of bleeding. The aim of our retrospective analysis was to discover causes of bleeding into upper GI tract and its characteristics over a 4 year period., Methodology: A survey of urgent upper GI tract endoscopies in the Clinic of Internal Medicine in Motol in Prague because of an acute GI tract bleeding (hematemesis or melena) was done. Found ulcers were assessed using Forrest classification. Moreover, number and causes of recurrences of bleeding were also assessed., Results: Within years 1998-2001 an urgent upper GI endoscopy because of bleeding (hematemesis or melena) was done in the Clinic of Internal Medicine in Prague in Motol in 1639 patients of an average age 62.2. 56% were men (average age 59) and 44% were women (average age 65.3). An endoscopy finding without pathology was present in 21.4%. The most frequent sources of bleeding were ulcers in duodenal bulb (20%), stomach ulcers (18.2%), and hemorrhagic gastropathy (16.5%) and varices (10.3%). Results of the Forrest classification in the ulcerative disease of stomach and duodenum were as follows: Forrest Ia 9.5%, Ib 24%, IIa 14.6%, IIb 18.7%, IIc 22.9%, III 10.3%. Recurrent bleeding was identified in 8.4% of patients, thereof bleeding from esophageal varices experienced 2.9% of patients (average age 45.8), bleeding from ulcers in bulbus 2.7% (Forrest Ib, IIa a IIb) of patients of an average age 62.6, and bleeding from ulcers in stomach 2.1% (Forrest Ia, IIa a Ib) of patients of an average age 62.5. Causes of recurrent bleeding were in one case bleeding from Barrett's oesophageal ulcer and in one case bleeding from ulcer in diaphragmatic hernia. Within 48 hours recurrent bleeding appeared in 65% of patients., Conclusion: Urgent endoscopy in gastrointestinal tract bleeding is an essential part of a complex medical care. It is highly reliable in identifying cause of bleeding, it enables to start treatment immediately and to consider prognosis of a patient.

The authors administered to two groups (56 and 44) patients before gastroscopy analgosedation-5 mg diazepam and local anaesthesia of the throat. To the first group diazepam was administered by the i.v., to the second group by the sublingual route. The objective was to find out whether analgosedation is desirable from the patients aspect and whether the sublingual form of premedication is accepted by the patients. The authors found that 90% of the patients wish to have analgosedation and anaesthesia before the procedure. The sublingual form of administration was accepted by 89% of the patients.