Ultrasound-enhanced latex immunoagglutination and PCR as complementary methods for non-culture-based confirmation of meningococcal disease

Preadmission administration of antibiotics to patients with suspected meningococcal infection has decreased the likelihood of obtaining an isolate and has stimulated development of rapid and reliable non-culture-based diagnostic methods. The sensitivity of the conventional test card latex agglutination test (TCLAT) for detection of capsular polysaccharide has been reported to be suboptimal. In the United Kingdom meningococcal DNA detection by PCR has become readily available and is now used as a first-line investigation. Recently, the performance of latex antigen detection has been markedly improved by ultrasound enhancement. Three tests for laboratory confirmation of meningococcal infection, (i) PCR assays, (ii) TCLAT, and (iii) ultrasound-enhanced latex agglutination test (USELAT), were compared in a retrospective study of 125 specimens (serum, plasma, and cerebrospinal fluid specimens) from 90 patients in whom meningococcal disease was suspected on clinical grounds. Samples were from patients with (i) culture-confirmed meningococcal disease, (ii) culture-negative but PCR-confirmed meningococcal disease, and (iii) clinically suspected but non-laboratory-confirmed meningococcal disease. USELAT was found to be nearly five times more sensitive than TCLAT. Serogroup characterization was obtained by both PCR and USELAT for 44 samples; all results were concordant and agreed with the serogroups determined for the isolates when the serogroups were available. For 12 samples negative by USELAT, the serogroup was determined by PCR; however, for 12 other specimens for which PCR had failed to indicate the serogroup, USELAT gave a result. USELAT is a rapid, low-cost method which can confirm a diagnosis, identify serogroups, and guide appropriate management of meningococcal disease contacts. A complementary non-culture-based confirmation strategy of USELAT for local use supported by a centralized PCR assay service for detection of meningococci would give the benefits of timely information and improved epidemiological data

Interfacial instability at cell membranes

SCOPUS: re.jinfo:eu-repo/semantics/publishe

Far-from-equilibrium phenomena in bioadhesion processes

This review is concerned with the physical chemistry of processes occurring during bioadhesion i.e. when living cells approach and contact other cells or when living cells approach and contact nonliving or solid surfaces. A good deal is known at present about biological membrane composition pointing towards a heterogeneous composition of this medium. There is now a clear need to detailed specific and quantitative understanding of interactions between biological membranes. Experiments and theory begin to converge in order to provide a better understanding of the cell functions depending on these interactions: anchorage spreading locomotion differentiation activation of immune response and so on. In this review equilibrium processes are first recalled and interpreted by several models (mechanical or thermodynamical models). Far-from-equilibrium processes are then described for mammalian cells or more precisely for red blood cells. These processes are interpreted in terms of the interfacial instability theory taking into account the role of external contraints leading to dissipative structures. Examples of such far-from-equilibrium processes are the appearance of nonuniform morphologies due to the bridging of surface polymers the clustering of surface receptors following a surface reaction between cell receptors and fixed binding sites on a substrate etc. The approach remains always macroscopic but opens vistas to correlate with molecular approaches. Several important and challenging research directions regarding non-equilibrium processes in bioadhesion are the relation between adhesion and fusion the metabolic regulation controlled or induced by adhesion the process of reversible/irreversible adhesion and separation etc. The importance of integrated studies combining the efforts of different disciplines (biology chemistry physics computer science etc.) is stressed throughout this review and will provide new answers to fundamental problems. © 1996 by John Wiley & Sons Ltd.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Membrane-Membrane contact: Involvement of interfacial instability in the generation of discrete contacts

The classical approach to understanding the closeness of approach of two membranes has developed from consideration of the net effect of an attractive van der Waals force and a repulsive electrostatic force. The repulsive role of hydration forces and stereorepulsion glycocalyx forces have been recently recognized and an analysis of the effect of crosslinking molecules has been developed. Implicit in these approaches is the idea of an intercellular water layer of uniform thickness which narrows but retains a uniform thickness as the cells move towards an equilibrium separation distance. Most recently an attempt has been made to develop a physical chemical approach to contact which accommodates the widespread occurrence of localized spatially separated point contacts between interacting cells and membranes. It is based on ideas drawn from analysis of the conditions required to destabilize thin liquid films so that thickness fluctuations develop spontaneously and grow as interfacial instabilities to give spatially periodic contact. Examples of plasma membrane behaviour which are consistent with the interfacial instability approach are discussed and experiments involving polycation, polyethylene glycol, dextran and lectin adhesion and agglutination of erythrocytes are reviewed. © 1989 Plenum Publishing Corporation.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Influence of polymer concentration and molecular weight and of enzymic glycocalyx modification on erythrocyte interaction in dextran solutions

Erythrocytes adhere to each other when suspended in supra-threshold concentrations of dextran of molecular mass of 40 kD or greater. The plasma membranes are parallel to each other over the entire length of the contact seam at the lower effective polymer concentrations. When cells are pretreated with the proteolytic enzyme pronase or the sialidase neuraminidase the membranes are not parallel but make contact at spatially periodic locations along the membrane surface. Pronase induced reduction of cell electrophoretic mobility rapidly reaches a limiting value. Nevertheless, prolonged pre-exposure to enzyme leads to a continuing reduction in contact separations. This result taken with the observation that, for equal loss of electrophoretic mobility, a shorter contact separation results from pronase rather than neuraminidase pre-treatment implies that a non-electrostatic consequence of pronase pre-treatment dominates membrane interaction in the experimental regimes examined here. The average lateral contact separation for different enzyme regimes lay in the range 3.3 pm to a limiting lower value of about 0.7 pm. There was a good correlation between the logarithm of a contact separation index (the approach of separation distance to its limiting value) against the logarithm of a derived index related to net attractive interaction for a wide range of experimental conditions. Treatments which increased attraction or decreased repulsion (e.g. increased dextrans concentration or enzyme pre-treatment) lead to shorter lateral contact separation. This result is qualitatively consistent with the predicted behaviour for the dominant wavelength arising from interfacial instability of a thin aqueous film between adjacent membranes. © 1993 Springer-Verlag.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

An interfacial instability approach to erythrocyte adhesion by macromolecules

Models of cell plasma membrane interactions in the presence of adhesive macromolecules often follow the general principles initially formalised by Derjaguin, Landau, Verwey and Overbeek for colloidal particles in suspension. In this model the parallel membranes of mutually adhering cells are considered to lie in an energy minimum at an equilibrium separation distance which is determined by the relative size and range of attractive and repulsive interactions. In contrast, the interfacial instability model considered here directs attention to the dynamic processes which can result in deformation of a thin aqueous intermembrane film to give localised spatially periodic contact points. The contact seam of erythrocytes suspended in polycation, polysaccharide or lectin solutions has been examined in this work by light and electron microscopies. The seams formed by erythrocytes in solutions of the lectins concanavalin A and wheat germ agglutinin show spatial periodicity of contacts (range 0.8-0.5 μm). In normal cells the polycation-induced seam is always spatially periodic while it is usually continuous and parallel in dextran solutions. In the latter case, pretreatment of the cells by the protease pronase results in spatially periodic contacts on subsequent exposure of the cells to dextran. For both polycation- and polysaccharide-induced contact the experimental changes which increase or decrease membrane attraction consistently lead to a decrease or increase respectively in the lateral contact separation distance (range 3.4-0.8 μm). These changes in lateral contact separation distances are qualitatively consistent with the predictions of interfacial instability theory. The changes are discussed as particular examples of a general consequence of the form of membrane interaction profiles. © 1994.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Multiple three-dimensional mammalian cell aggregates formed away from solid substrata in ultrasound standing waves

Single and multiple three-dimensional cell aggregates of human red blood cells (RBCs) and HepG2 cells were formed rapidly in low mega-Hertz ultrasound standing wave fields of different geometries. A single discoid aggregate was formed in a half-wavelength pathlength resonator at a cell concentration sufficient to produce a 3D structure. Multiple cell aggregates were formed on the axis of a cylindrical resonator with a plane transducer (discoid aggregates); in a resonator with a tubular transducer and in the cross-fields of plane and tubular transducers and two plane orthogonal transducers (all cylindrical aggregates). Mechanically strong RBC aggregates were obtained by crosslinking with wheat germ agglutinin (WGA, a lectin). Scanning electron microscopy showed aggregate surface porous structures when RBCs were mixed with WGA before sonication and tighter packing when ultrasonically preformed aggregates were subsequently exposed to a flow containing WGA. HepG2 cell aggregates showed strong accumulation of F-actin at sites of cell-cell contact consistent with increased mechanical stability. The aggregates had a porous surface, and yet confocal microscopy revealed a tight packing of cells in the aggregate's inner core. © 2009 American Institute of Chemical Engineers Biotechnol

Meningitis antigen detection: interpretation of agglutination by ultrasound-enhanced latex immunoassay

Detailed instructions for performance and interpretation of ultrasound-enhanced latex agglutination tests for the rapid identification of bacteria causing meningitis are described. This recently developed technique, which enhances the sensitivity of most latex immunoagglutination assays, has been studied mainly in the context of detection of antigens of meningitis-causing bacteria. The test concentrates on the Wellcogen bacterial antigen kit (Murex Diagnostics Ltd) that contains five latex suspensions specific for Haemophilus influenzae type b, Neisseria meningitidis ACYW135, N. meningitidis B/Escherichia coli K1, Streptococcus group B and Streptococcus pneumoniae. Light photomicrographs of positive agglutination are shown. Particular attention is paid to the appearance of the latex in negative control samples following exposure to ultrasound. Guidance is given on interpretation and assessment in clinical samples

Meningitis antigen detection: interpretation of agglutination by ultrasound-enhanced latex immunoassay

Detailed instructions for performance and interpretation of ultrasound-enhanced latex agglutination tests for the rapid identification of bacteria causing meningitis are described. This recently developed technique, which enhances the sensitivity of most latex immunoagglutination assays, has been studied mainly in the context of detection of antigens of meningitis-causing bacteria. The test concentrates on the Wellcogen bacterial antigen kit (Murex Diagnostics Ltd) that contains five latex suspensions specific for Haemophilus influenzae type b, Neisseria meningitidis ACYW135, N. meningitidis B/Escherichia coli K1, Streptococcus group B and Streptococcus pneumoniae. Light photomicrographs of positive agglutination are shown. Particular attention is paid to the appearance of the latex in negative control samples following exposure to ultrasound. Guidance is given on interpretation and assessment in clinical samples