The current status and outlook for molecular genetic methods in solving the tasks of medical microbiology

The article deals with modern methods, viz. PCR, molecular display and genotherapy, which permit the new approach to the solution of problems connected with the identification of infective agents, the study of the mechanisms of the pathogenesis of infectious diseases and their treatment. In this article concrete examples, clearly demonstrating how each of the above-mentioned technologies makes it possible to broaden the circle of problems solved in infectious pathology of man, are presented.     

Obtaining antisera to Pseudomonas aeruginosa and Proteus antigens for performing immunoenzyme analyses in clinical practice

The data presented in this work indicate that specific antisera to P. aeruginosa and Proteus antigens can be produced by using extracts from these microorganisms, destroyed by ultrasonic treatment or by multiple freezing and thawing, for the immunization of rabbits. Blood serum samples from patients with purulent septic complications were studied for the presence of P. aeruginosa and Proteus antigens in ELISA with the use of peroxidase-labeled antibodies from antisera to P. aeruginosa and Proteus. This investigation revealed that during the first 3 days from the beginning of the clinical manifestations of the complications P. aeruginosa and Proteus antigens were detected in 86.4% and 83.4% of the patients, respectively. In the subsequent bacteriological study of wound discharge from these patients the corresponding microflora was detected.     

Sorption properties of polystyrene plates used in immunoenzyme analyses

The capacity of polystyrene carriers used in the enzyme immunoassay (EIA) for adsorbing 131I-labeled human serum albumin under different conditions has been studied, and the comparison of the plates manufactured by Dynatech AG (Switzerland) and by the Leningrad Works of Medical Polymers has been made. At the first stages of the reaction the antigen is separated from the carrier and the amount of the desorbed antigen depends on its initial dose and the dilution of the assayed sera. The irregular desorption of the antigen leads to misinterpretation of the results. Comparison of the polystyrene plates has shown that each plate is characterized by individual adsorption capacity, which impedes at present the standardization of EIA-based test systems.     

Usefulness of immunoenzyme diagnostic kits and latex tests for isolation of rotaviruses

The aim of this study was to compare characteristic parameters of 4 diagnostic kits available in Poland-immunoenzymatic Rotazyme II and Enzygnost kits and latex kits Rotalex and Slidex. Studies were performed on 67 samples of feces of children treated because of diarrhea. The sensitivity, specificity, frequency of positive tests, false positives and false negatives and the accuracy of the tests under evaluation were determined. The results obtained were further verified using a reference test electrophoresis of RNA of rotavirus. The highest sensitivity was found for Enzygnost, Rotazyme II and Rotalex, 97%, 92% and 90%, respectively, and the lowest for Slidex- 79%, while the specificity was higher for latex kits than for immunoenzymatic kits. The accuracy of the results was highest for Rotalex kit (92%), next for Enzygnost kit (88%), Rotazyme (87%), and Slidex (84%). The significant correlation between OD value readings in spectrophotometer in Rotazyme II kit and the results of visual reading in latex test was found. All tested kits were found to be useful for diagnostic purposes. Rotalex kit due to the high accuracy of the results obtained, methodological simplicity, short time of testing and relatively low price could be a based test in hospital laboratories.     

Capillary electrophoresis for clinical problem solving: analysis of urinary diagnostic metabolites and serum proteins

Many clinical laboratories employ gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) to detect abnormal compounds occurinng in urine and serum due to disease. The methods, particularly GC-MS, often require laborious sample pre-treatment, and separation times may exceed an hour. We describe the use of capillary electrophoresis (CE) equipped a with a diode-array detector in an attempt to improve the efficiency of an analytical system routinely used for diagnosis of human metabolic disease. It was found that urine samples could be injected directly onto the CE instrument without any pre-treatment, and over 50 metabolites were separated in 15 min. Identification of abnormal metabolites was based on migration times and characteristic diode-array spectra. The method readily diagnosed adenolysuccinase deficiency, 5-oxoprolinuria, propionic acidemia and disorders having orotic acid as diagnostic metabolite (e.g. the HHH-syndrome). The results show that CE may become a useful additional tool for diagnosis of metabolic disease. In a different project CE was used to study sera from the Janus-bank. This large serum bank comprises samples collected at intervals from nearly 300 000 blood donors. As the sera are stored at −25°C and not at a lower temperature, a major concern has been the stability of the specimens. GC-MS, 2D-protein electrophoresis, certain immunological assays and enzyme measurements have previously been used to evaluate the stability of the sera. We can now also show that the protein profile, as determined by CE, is remarkably stable even after 22 years of storage. The results moreover confirmed that the CE-method and traditional gel electrophoresis gave almost identical results, except for small amounts of fibrinogen which did not show up on the CE-pattern.     

Choice of the suitability criteria of polystyrene-based solid-phase carriers for performing immunoenzyme analyses

The authors discuss a tentative approach to the choice of criteria indicating the optimal suitability of different solid-phase carriers made of polystyrene for use in the enzyme immunoassay (EIA), viz. the dependence of specificity, sensitivity, reproducibility and reliability of EIA results on the adsorption properties, transparency expressed in percent and transparency variations of the plates under test. The evaluation of the carriers by four parameters is proposed with the use of assay plates manufactured by Nunc A/S (Denmark) for control. To ensure the objective evaluation of the suitability of polystyrene plates for use in EIA, the choice of uniform criteria is necessary.     

The diagnostic effectiveness of an immunoenzyme test system for determining diphtheria toxin antigens in the blood serum in a clinical laboratory trial

The data on the approbation of the diagnostic value of the enzyme immunoassay (EIA) system for the determination of diphtheria toxin in the blood sera of diphtheria patients and persons suspected for diphtheria are presented. The EIA system was prepared on the basis of F(ab)2 fractions of purified antidiphtheria antibodies. 240 serum samples from diphtheria and tonsillitis patients and from healthy persons were studied. Diphtheria toxin was determined in all patients with the toxic form of diphtheria and in 41.3% of patients with its localized forms. Blood was taken mainly of the first week of the disease. In healthy persons the results of EIA were negative. Thus, the trial of the assay system in a clinical laboratory showed its good diagnostic effectiveness. The use of this EIA system in medical practice is believed to be quite promising.     

Brain mechanisms of imagination in solving creative verbal tasks

The brain’s mechanisms of imagination were studied using electroencephalography (EEG) spectral analysis in student actors and student non-actors under three experimental conditions: when they generated coherent stories on the basis of art reproductions (STORY task); listed the details of art reproductions presented (DETAIL task); and performed simple arithmetic calculations while observing a neutral background (COUNT task). Statistical analysis showed that, in α₁ (7.5–10 Hz) and α₂ (10–12.5 Hz) frequency bands, in both groups, execution of the STORY task, in contrast to the DETAIL task, was accompanied by significantly higher spectral power (synchronization) in most of the cortical areas studied; while, the contrasts STORY-COUNT and DETAILS-COUNT, were associated with a decrease in the EEG’s power (desynchronization) in all of the areas studied. Topographic mapping of the EEG’s power showed that, in both groups, maximal differences between the STORY and DETAILS tasks were related to the central parietal area. Maximal differences between the STORY and COUNT tasks, as well as those between the DETAILS and COUNT tasks, were related mainly to the occipital areas. Based on these findings, we consider parietal areas to be stable elements of integrated brain mechanisms underlying verbal creativity in actors and nonactors. Comparing our data with previous studies, we suggest that the parietal areas are involved in the selective inhibition of visual information processing during the involvement of brain structures in the processes of imagination.     

Sediment quality assessment: status and outlook

This paper is a synthesis of the 44 presentations at the First International Symposium on Sediment Quality Assessment (Sweden, August 1994). The paper includes Initial Premises, Sediments, Tools (with particular emphasis on bioassays), Strategies and Challenges. Major testable hypotheses are proposed as follows (ranging in complexity, recognizing differences apparent at the Symposium in level of expertise and knowledge): (1) there is no single perfect method of sediment assessment, there are only tools in the toolbox; (2) significant sediment pollution (contamination resulting in adverse biological effects) comes from non-anthropogenic sources; (3) artificial sediments will provide future reference comparisons; (4) knowledge of suspended sediments is required to understand bedded sediments; (5) ammonia and/or hydrogen sulfide cannot explain all sediment toxicity, in particular non-acute responses; (6) subcellular (e.g., genetic) responses are a research tool, not yet appropriate for monitoring or assessment; (7) although the effects of sediment storage cannot be predicted, non-toxic and highly toxic sediments are less affected by prolonged storage than are moderately toxic sediments; (8) sediment ingestion is a more important route of exposure than pore water for some organisms; (9) water column organisms and aqueous exposures should not be used for whole-organism sediment tests; (10) validation of sediment bioassays is not always simple or possible. Two major conclusions are: (i) generalizations are not [yet] possible regarding sediment quality; (ii) correctly assessing sediment quality is primarily a function of the correct reference comparison.