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Routine procedures for isolation and identification of enterococci and fecal streptococci.

Over the past 6 years, a revised classification of the streptococci and enterococci, based primarily on molecular techniques such as 16S rRNA sequencing and DNA-DNA hybridization, emerged. However, little attention was placed on routine physiological tests that could be used in food and clinical laboratories to differentiate between species of a new genus, Enterococcus, and fecal Streptococcus spp. The purpose of this study was to devise a convenient and reliable system to identify enterococci and fecal streptococci by using conventional procedures. Fifty-nine strains of 13 Enterococcus spp., including the type strains and many strains used by previous investigators, were characterized by using conventional tube tests, the API Rapid Strep system, and MicroScan Pos ID panels. Results were compared with each other and with previously published results. A comparison of conventional tube tests versus published tube test results yielded 17 discrepancies. Although not all tests were done with each of the three systems, 28 discrepancies between results obtained with the API system and those obtained with conventional tube tests were found. There were 24 discrepancies between results obtained with the MicroScan Pos ID panel and those obtained with conventional tube tests. There were 12 discrepancies between the results with the API Rapid Strep system and those with the MicroScan Pos ID panels. We devised flow charts of key tests that might be used to identify cultures without resorting to nucleic acid analysis and other labor- and equipment-intensive analyses.

Diagnostic Tests, Routine↗

Human microsporidial infections.

Microsporidia are obligate intracellular spore-forming protozoal parasites belonging to the phylum Microspora. Their host range is extensive, including most invertebrates and all classes of vertebrates. More than 100 microsporidial genera and almost 1,000 species have now been identified. Five genera (Enterocytozoon spp., Encephalitozoon spp., Septata spp., Pleistophora sp., and Nosema spp.) and unclassified microsporidia (referred to by the collective term Microsporidium) have been associated with human disease, which appears to manifest primarily in immunocompromised persons. The clinical manifestations of microsporidiosis are diverse and include intestinal, pulmonary, ocular, muscular, and renal disease. Among persons not infected with human immunodeficiency virus, ten cases of microsporidiosis have been documented. In human immunodeficiency virus-infected patients, on the other hand, over 400 cases of microsporidiosis have been identified, the majority attributed to Enterocytozoon bieneusi, an important cause of chronic diarrhea and wasting. Diagnosis of microsporidiosis currently depends on morphological demonstration of the organisms themselves. Initial detection of microsporidia by light microscopic examination of tissue sections and of more readily obtainable specimens such as stool, duodenal aspirates, urine, sputum, nasal discharge, bronchoalveolar lavage fluid, and conjunctival smears is now becoming routine practice. Definitive species identification is made by using the specific fluorescein-tagged antibody (immunofluorescence) technique or electron microscopy. Treatment options are limited, but symptomatic improvement of Enterocytozoon bieneusi infection may be achieved with the anthelmintic-antiprotozoal drug albendazole. Preliminary observations suggest that Septata intestinalis and Encephalitozoon infections may be cured with albendazole. Progress is being made with respect to in vitro propagation of microsporidia, which is crucial for developing antimicrosporidial drugs. Furthermore, molecular techniques are being developed for diagnostic purposes, taxonomic classification, and analysis of phylogenetic relationships of microsporidia.

Animals↗

Electrophysiological classification of somatostatin-positive interneurons in mouse sensorimotor cortex.

Classification of inhibitory interneurons is critical in determining their role in normal information processing and pathophysiological conditions such as epilepsy. Classification schemes have relied on morphological, physiological, biochemical, and molecular criteria; and clear correlations have been demonstrated between firing patterns and cellular markers such as neuropeptides and calcium-binding proteins. This molecular diversity has allowed generation of transgenic mouse strains in which GFP expression is linked to the expression of one of these markers and presumably a single subtype of neuron. In the GIN mouse (EGFP-expressing Inhibitory Neurons), a subpopulation of somatostatin-containing interneurons in the hippocampus and neocortex is labeled with enhanced green fluorescent protein (EGFP). To optimize the use of the GIN mouse, it is critical to know whether the population of somatostatin-EGFP-expressing interneurons is homogeneous. We performed unsupervised cluster analysis on 46 EGFP-expressing interneurons, based on data obtained from whole cell patch-clamp recordings. Cells were classified according to a number of electrophysiological variables related to spontaneous excitatory postsynaptic currents (sEPSCs), firing behavior, and intrinsic membrane properties. EGFP-expressing interneurons were heterogeneous and at least four subgroups could be distinguished. In addition, multiple discriminant analysis was applied to data collected during whole cell recordings to develop an algorithm for predicting the group membership of newly encountered EGFP-expressing interneurons. Our data are consistent with a heterogeneous population of neurons based on electrophysiological properties and indicate that EGFP expression in the GIN mouse is not restricted to a single class of somatostatin-positive interneuron.

Algorithms↗

The Minnesota Grading System of eye bank eyes for age-related macular degeneration.

PURPOSE: The Minnesota Grading System (MGS) is a method to evaluate human eye bank eyes and determine the level of age-related macular degeneration (AMD), by using criteria and definitions from the Age-Related Eye Disease Study (AREDS). METHODS: Donor eyes (108 pairs) from the Minnesota Lions Eye Bank were cut circumferentially at the pars plana to remove the anterior segment. A 1000 +/- 2.5-microm ruby sphere was placed on the optic nerve as a size reference. A digital, high-resolution, color macular photograph was taken through a dissecting microscope. The neurosensory retina was removed from one globe of the pair. The underlying retinal pigment epithelium was rephotographed, localizing the fovea with a proportional triangle. A grid was superimposed in the macular photographs and images were graded according to AREDS criteria. Twenty pairs were dissected bilaterally and graded for symmetry. RESULTS: Eighty-eight globes were graded into one of four MGS categories. Nineteen (95%) of 20 globes had symmetric grades. CONCLUSIONS: The MGS provides a methodology to grade donor tissue from eye bank eyes to correspond to the AREDS classification system. Donor tissue may be used for subsequent molecular analysis, including genomics and proteomics.

Aged↗

Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop.

Recent neuropathologic autopsy studies found that 15 to 25% of elderly demented patients have Lewy bodies (LB) in their brainstem and cortex, and in hospital series this may constitute the most common pathologic subgroup after pure Alzheimer's disease (AD). The Consortium on Dementia with Lewy bodies met to establish consensus guidelines for the clinical diagnosis of dementia with Lewy bodies (DLB) and to establish a common framework for the assessment and characterization of pathologic lesions at autopsy. The importance of accurate antemortem diagnosis of DLB includes a characteristic and often rapidly progressive clinical syndrome, a need for particular caution with neuroleptic medication, and the possibility that DLB patients may be particularly responsive to cholinesterase inhibitors. We identified progressive disabling mental impairment progressing to dementia as the central feature of DLB. Attentional impairments and disproportionate problem solving and visuospatial difficulties are often early and prominent. Fluctuation in cognitive function, persistent well-formed visual hallucinations, and spontaneous motor features of parkinsonism are core features with diagnostic significance in discriminating DLB from AD and other dementias. Appropriate clinical methods for eliciting these key symptoms are described. Brainstem or cortical LB are the only features considered essential for a pathologic diagnosis of DLB, although Lewy-related neurites, Alzheimer pathology, and spongiform change may also be seen. We identified optimal staining methods for each of these and devised a protocol for the evaluation of cortical LB frequency based on a brain sampling procedure consistent with CERAD. This allows cases to be classified into brainstem predominant, limbic (transitional), and neocortical subtypes, using a simple scoring system based on the relative distribution of semiquantitative LB counts. Alzheimer pathology is also frequently present in DLB, usually as diffuse or neuritic plaques, neocortical neurofibrillary tangles being much less common. The precise nosological relationship between DLB and AD remains uncertain, as does that between DLB and patients with Parkinson's disease who subsequently develop neuropsychiatric features. Finally, we recommend procedures for the selective sampling and storage of frozen tissue for a variety of neurochemical assays, which together with developments in molecular genetics, should assist future refinements of diagnosis and classification.

Dementia↗

Microarray analysis in drug discovery and clinical applications.

DNA microarray analyzes genome-wide gene expression patterns and is used in many areas including drug discovery and clinical applications. This chapter summarizes some of these applications such as identification and validation of anti-infective drug target, study mechanisms of drug action and drug metabolism, classification of different types of tumors, and use of molecular signatures for prediction of disease outcome. A step-by-step protocol is provided for sample preparation, sample labeling and purification, hybridization and washing, feature extraction, and data analysis. Important considerations for a successful experiment are also discussed with emphasis on drug discovery and clinical applications. Finally, a clinical study is presented as an example to illustrate how DNA microarray technology can be used to identify gene signatures, and to demonstrate the promise of DNA microarray as a clinical tool.

Computational Biology↗

ProLysED: an integrated database and meta-server of bacterial protease systems.

UNLABELLED: Bacterial proteases are an important group of enzymes that have very diverse biochemical and cellular functions. Proteases from prokaryotic sources also have a wide range of uses, either in medicine as pathogenic factors or in industry and therapeutics. ProLysED (Prokaryotic Lysis Enzymes Database), our meta-server integrated database of bacterial proteases, is a useful, albeit very niche, resource. The features include protease classification browsing and searching, organism-specific protease browsing, molecular information and visualisation of protease structures from the Protein Data Bank (PDB) as well as predicted protease structures. AVAILABILITY: ProLysED is integrated into the ProLysES (Prokaryotic Lysis Enzymes Site) website at http://genome.ukm.my/prolyses/. Access to the ProLysED database is free for academic users upon registration.

Amino Acid Sequence↗

An update of the epidemiology of non-Hodgkin's lymphoma.

The incidence of non-Hodgkin's lymphoma (NHL) has increased approximately 80% since the 1970s, and is now the fifth most common cancer in the United States. The incidence of NHL is approximately 50% higher among men than women and 35% higher among white people than black people. The incidence rates of all subtypes of NHL have increased, especially diffuse large-cell and immunoblastic subtypes. Extranodal NHL has increased more rapidly than nodal NHL. The overall NHL incidence rates stabilized in the early 1990s and then began to decrease between 1996 and 2000, resulting in part from a decrease in the incidence of AIDS. The incidence of NHL types not associated with AIDS and NHL in groups at low risk of AIDS has continued to increase throughout the 1990s. The increasing incidence of NHL is poorly understood. Improved diagnostic techniques, the effects of the human immunodeficiency virus epidemic, and immunosuppressive therapies accounted for only one third of the increase. Increase in NHL may be attributed to immunodeficiency, various infections, familial aggregation, blood transfusion, genetic susceptibility to NHL, diet, and chemical exposures to pesticides and solvents. Some studies also suggest that associations between risk factors and specific NHL subtypes may be stronger than associations between the same risk factors and NHL in aggregate. Future epidemiologic studies should incorporate the new World Health Organization classification of NHL and new techniques such as cytogenetic molecular analyses to identify subtype-specific etiologic factors. Evaluation of polymorphisms in genes involved in immune function, inflammation, and the activation or detoxification of environmental and occupational chemicals is also warranted.

Female↗

Comparison of genetic changes between interphase and metaphase nuclei in monitoring CML and APL treatment using DC-FISH technique.

In leukemias, the monitoring techniques on the response after the treatment have clinical importance for evaluating new therapeutic approaches and identifying the risk of relapse. In this study, genetic changes before and after chemotherapy in interphase and metaphase nuclei of bone morrow of adults with provisional diagnosis of leukemia were compared to understand the molecular characterization and pathogenesis of the leukemia for the classification of diagnosis and prognosis. We examined bone morrow cells of 47 chronic myeloid leukemia (CML) cases (29 of 47 at the time of diagnosis, 31 of 47 after chemotherapy) with the bcr/abl translocation probes and of 10 acute promyelocytic leukemia (APL) cases (7 of 10 at the time of diagnosis, 4 of 10 after chemotherapy) with the PML/RARalpha translocation probes by using dual color-flourescence in situ hybridization (DC-FISH). For each case, 400 interphase nuclei and 11 to 25 metaphases nuclei were analysed. The ratios of translocations before and after chemotherapy were compared between interphase and metaphase nuclei. After chemotherapy, though, translocations were detected in interphase nuclei of 29 of the 31 CML and 4 of the 4 APL cases, these translocations were determined in metaphase nuclei of only 14 of the 31 CML and 1 of the 4 APL cases with very low ratios (p < 0.01). The results showed that the rates of translocation positive interphase nuclei were higher than the rates of translocation positive metaphase nuclei (p < 0.01) after chemotherapy, so there may be some factors effecting proliferative activity of metaphase formation in leukemias.

Bone Marrow Cells↗

Analysis and classification of cerebellar malformations.

BACKGROUND AND PURPOSE: Because of improved visualization of posterior fossa structures with MR imaging, cerebellar malformations are recognized with increasing frequency. Herein we attempt to describe and propose a rational classification of cerebellar malformations. METHODS: MR images obtained in 70 patients with cerebellar malformations were retrospectively reviewed. The cerebellar malformations were initially divided into those with hypoplasia and those with dysplasia. They were then divided into focal and diffuse malformations. Finally, they were separated according to other features, such as brain stem involvement and cerebral involvement. RESULTS: All patients with diffuse cerebellar dysplasia (muscular dystrophy [n = 10], cytomegalovirus [n = 6], lissencephaly [n = 3],) had abnormalities of the cerebrum. Patients with focal cerebellar dysplasia of the Joubert (n = 12) and rhombencephalosynapsis (n = 8) types had variable cerebral dysplasia. Patients with nonsyndromic focal cerebellar dysplasia (isolated focal cerebellar cortical dysplasia [n = 2], cerebellar heterotopia with cerebellar cortical dysplasia [n = 1], idiopathic diffuse cerebellar dysplasia [n = 1], Lhermitte-Duclos syndrome [n = 1]) and those with cerebellar hypoplasia (isolated cerebellar hypoplasia [n = 6], pontocerebellar hypoplasia type 1 [n = 1]) had normal cerebra. Patients with features of Dandy-Walker malformation (n = 19) had both hypoplasia and dysplasia of the cerebellum. No notable difference was found between the cerebella of patients with large fourth ventricle cysts (Dandy-Walker malformations) and those without large fourth ventricle cysts (isolated cerebellar hypoplasia). Therefore, the Dandy-Walker malformation seems to be heterogeneous. CONCLUSION: Use of this classification system helps in the segregation and understanding of the relationship among cerebellar malformations. Although it will undoubtedly require revisions, this classification is a first step in combining imaging with molecular biology to facilitate understanding of cerebellar development and maldevelopment.

Adolescent↗

[Gap junctions in the nervous tissue].

In recent years, prominent progresses have been made in the study of the localization and function of gap junctions in the nervous tissue. The application of molecular approaches has advanced the disclosure of the structure, classification and biophysical properties of gap junctions. Dye-coupling and Ca2+ imaging techniques have provided explicit and effective ways for the study of gap junctional functions. The regulation of gap junctions involves the expression of gap junction genes and changes in the conductivity of gap junction channels. Gap junctional intercellular communication includes the exchange of second-messengers and electrical coupling between coupled cells. GJ plays an important role in the function of neuroglial cells, neurons in some brain regions and probably in the formation of local neuronal circuits.

Animals↗

[Prognosis and prognostic factors in oncology and oncologic surgery ].

At present time, we can see increasing interest in prognosis and prognostic factors. This review refers to the problems of prognostics in oncology and oncosurgery. Authors emphasize importance of prognosis and its integrating role in issues of treatment tactics and strategy selection. This article presents main classification of prognostic factors, e.g. the subject-based one, which consists of three groups-tumor-related, host-related and environment related factors. Factors that belong to the first group are anatomic extent of disease that is classified according to the TNM classification, tumor histopathologic factors and a great number of molecular and genetic factors. The question of clinical relevance of those factors is discussed. Host-related prognostic factors include inherent demographic characteristics, performance status, comorbid conditions and others. Environment-related prognostic factors involve physician, the health care system and society as a whole. This group of prognostic factors is less studied, despite the fact that namely these factors can have fundamental importance in questions of disease outcome.

Biomarkers, Tumor↗

[Breast carcinoma and genomic technology: contribution of DNA microarrays].

Genome wide expression analyses techniques are now applied to breast carcinoma. Several results have been achieved : molecular clustering of breast carcinomas, implementation of powerful prognostic classifications, and in some way predictive classification. Unfortunately, translation to bedside remains limited due to standardization difficulties.

Breast Neoplasms↗

Association of genetic variability within the Borrelia burgdorferi sensu lato with the ecology, epidemiology of Lyme borreliosis in Europe.

Lyme borreliosis (LB) represents the most common vector-borne zoonotic disease in the Northern Hemisphere. The infection is caused by the spirochetes of the Borrelia burgdorferi sensu lato (s.l.) complex which circulate between tick vectors and vertebrate reservoir hosts. The complex of Borrelia burgdorferi s.l. encompasses at least 12 species. Genetic variability within and between each species has a considerable impact on pathogenicity, clinical picture, diagnostic methods, transmission mechanisms and its ecology. The distribution of distinct genospecies varies with the different geographic area and over a time. In recent years, new molecular assays have been developed for direct detection and classification of different Borrelia strains. Profound studies of strain heterogeneity initiated a new approach to vaccine development and routine diagnosis of Lyme borreliosis in Europe. Although great progress has been made in characterization of the organism, the present knowledge of ecology and epidemiology of B. burgdorferi s.l. is still incomplete. Further information on the distribution of different Borrelia species and subspecies in their natural reservoir hosts and vectors is needed.

Animals↗

Patterns of hematological malignancies in Chernobyl clean-up workers (1996-2005).

AIM: The question as to whether the incidence of leukemias and malignant lymphomas among the Chernobyl clean-up workers increased in 20 years after the catastrophe is still a point of much controversy. Precise diagnosis of the main forms of hematopoietic malignancies according to FAB classification and new WHO classification and comparison of these data with that in the general population will be helpful in estimating the relative contribution of the radiation factor to the overall incidence of such pathologies. PATIENTS AND METHODS: The data on 218 consecutive cases of malignant diseases of hematopoietic and lymphoid tissues in Chernobyl clean-up workers diagnosed in 1996-2005 are given in comparison with the data of 2697 consecutive patients of general population of the same age group. The morphology and cytochemistry of bone marrow and peripheral blood cells were studied. Immunocytochemical techniques (APAAP, LSAB-AP) and the broad panel of monoclonal antibodies to lineage specific and differentiation antigens of leukocytes were employed for immunophenotyping leukemic cells. RESULTS: Various types of oncohematological diseases developing 10-20 years after Chernobyl accident were registered in a group of clean-up workers under study including myelodysplastic syndromes (MDS), acute leukemias (ALL and AML), chronic myelogenous leukemia (CML) and other chronic myeloproliferative diseases, chronic lymphocytic leukemia (B-CLL) and other chronic lymphoproliferative diseases of B and T cell origin. MDS percentage among patients of clean-up workers group tended to exceed MDS percentage in the group of patients representing the general population examined at the same period (4.58 vs. 3.70%). Among 34 AML cases, leukemia was preceded by MDS in seven patients. The relative contribution of CML to the total number of clean-up workers with leukemia was higher than the corresponding percentage value in general population examined at the same period (9.17 vs. 6.59%). B-CLL was a predominant form of hematopoietic malignancies in clean-up workers under study (25.68%). Nevertheless, B-CLL percentage in patients of clean-up workers group did not differ significantly from that in the patients of general population. The multiple myeloma percentage (7.79%) in the group of patients belonging to clean-up workers in our study turned out to be twice as much as in the patients of general population (4.0%). CONCLUSION: The verified diagnosis of tumors of hematopoietic and lymphoid tissue according to modern classification (EGIL, WHO) could be the prerequisite for further molecular genetic and analytical epidemiology study of leukemias that may be related to Chernobyl NPP accident consequences.

Antigens, CD↗

[Molecular analysis in 23 patients with T-lymphomas].

Gene rearrangement analysis has been performed in 23 patients with T-cell lymphoproliferative diseases: 4 cases with T-gamma lymphocytosis, one case of a Sezary's syndrome, one case of T-cell angioimmunoblastic lymphoma, two cases of T-cell lymphoepitheloid lymphoma, 11 patients with T-cell pleomorphic lymphoma, 3 cases of large anaplastic T-cell lymphoma and one case of T-cell lymphoblastic lymphoma. Rearranged banding patterns have been observed for at least one of the T-cell receptors (TCR) in 19 of the cases, and germ line configuration of the TCR and Ig genes in the other four. Likewise, both Ig and TCR rearrangements have been observed in three cases (one case of T-cell pleomorphic lymphoma, one case of large anaplastic T-cell lymphoma and one case of T-cell lymphoblastic lymphoma). Molecular genetic techniques have been used in order to direct monoclonal proliferations of T cell in the tumoral tissues, to determine the T- or B-cell lineage of the neoplasia and also to outline the molecular characteristics of each group that constitutes the complex classification of T-cell malignancies.

Gene Rearrangement, B-Lymphocyte↗

Mixed-lineage leukemia revisited: acute lymphocytic leukemia with myeloperoxidase-positive blasts by electron microscopy.

Seven adult patients with untreated acute lymphocytic leukemia (ALL) who manifested 5% to 40% myeloperoxidase (MPO)-positive blasts by electron microscopy (EM) are reported. Six patients had an L2 morphology, and one had an L1 morphology by the French-American-British (FAB) classification. The immunophenotype was T cell in four patients. Molecular analysis showed rearrangement of the immunoglobulin JH in four patients, three of them also having rearrangement of the T-cell receptor beta or gamma. Induction chemotherapy with vincristine-doxorubicin-dexamethasone (VAD) produced a complete remission in five of six patients (83%). Our findings suggest the existence of a previously undescribed subtype of mixed-lineage leukemia, which by morphology and immunophenotype often appears as T-cell ALL but exhibits MPO-positive blasts by EM.

Adolescent↗

[Study of morphology and genome structure of Pseudomonas putida bacteriophages for their classification].

A group of 27 bacteriophages specific for Pseudomonas putida strains PpG1 and PpN has been isolated. The phages were characterized and compared with the previously described virulent (pf 16, af, tf and PMW) and temperate (PP56 and PP71) phages. The new phages belong to B1 and C1 morphotypes, according to Ackerman's classification. Phage DNAs were digested with several endonucleases; the molecular weights and homology of the DNAs were determined. All phages of P. putida isolated up to now were distributed into 10 species (groups), on the basis of particle morphology, genome size and the results of homology studies. Recombination processes are believed to participate in formation of phages belonging to certain species.

Bacteriophages↗