A disposable chemical heater and dry enzyme preparation for lysis and extraction of DNA and RNA from microorganisms.

Sample preparation, including bacterial lysis, remains a hurdle in the realization of complete point-of-care tests for many pathogens. Here, we developed a sample preparation methodology for enzymatic lysis and sample heating for low-resource, point-of-care applications. We show an instrument-free chemical heater system for rapid lysis of a gram-positive bacterium (Staphylococcus aureus) and an RNA virus (human respiratory syncytial virus) using a dried lysis enzyme mixture (achromopeptidase) for S. aureus. After a lysis step (<1 minute), lysis enzymes are heat deactivated (<5 minutes) using a simple disposable chemical heater. We demonstrated that both DNA and RNA in the heat-treated sample could be directly amplified without purification, even in the presence of a clinically-obtained human nasal sample. This simple approach to dry enzyme storage and sample heating is adaptable to many applications where samples need to be lysed, including use in low-resource laboratories and in single-use or cartridge-based point-of-care diagnostic devices.

Precision chemical heating for diagnostic devices.

Decoupling nucleic acid amplification assays from infrastructure requirements such as grid electricity is critical for providing effective diagnosis and treatment at the point of care in low-resource settings. Here, we outline a complete strategy for the design of electricity-free precision heaters compatible with medical diagnostic applications requiring isothermal conditions, including nucleic acid amplification and lysis. Low-cost, highly energy dense components with better end-of-life disposal options than conventional batteries are proposed as an alternative to conventional heating methods to satisfy the unique needs of point of care use.

[An innovative system for analysis and further development of long-term care for older people - results of the European PROject INTERLINKS]. / Eine innovative Systematik zur Analyse und Weiterentwicklung der Langzeitpflege und -betreuung älterer Menschen - Ergebnisse des EU-Projekts INTERLINKS.

In the light of recent demographic changes, the continued development of structures of long-term care (LTC) for older people is needed across Europe. In Germany, as in many other European countries, existing provisions of LTC are neither adequately coordinated nor user-oriented. The integration of relevant institutions and actors constitutes a central challenge for a high quality of care. The European research project INTERLINKS asked how the interfaces within fragmented structures of LTC are currently managed. Thematic foci included quality assessment and assurance, embedding preventive and rehabilitative aspects into LTC, the involvement and support of informal care-givers, and questions regarding the governance and financing of LTC. Using a framework developed by INTERLINKS, the project included the construction of an instrument for a detailed and comprehensive analysis of LTC systems. As an online platform with more than 100 examples of innovative approaches to coordination and integration in practice, this framework facilitates an exchange among experts - both within countries and at a European level - that will help stimulate the further development of LTC. The website also offers a German translation of the database in order to facilitate its use by German speakers.

New screening technologies for type 2 diabetes mellitus appropriate for use in tuberculosis patients.

Type 2 diabetes mellitus (DM), which is epidemic in low- and middle-income countries (LMICs), may threaten gains made in tuberculosis (TB) control, as DM is both a major risk factor for developing active TB and it can lead to adverse TB treatment outcomes. Despite World Health Organization guidance that all TB patients should be screened for DM, most facilities in LMICs that manage TB patients do not currently perform screening for DM, due in part to the cost and complexity involved. DM screening is further complicated by the presentation of transient hyperglycemia in many TB patients, as well as differences in diabetes risk factors (e.g., body mass index) between TB patients and the general public. In this article, we review existing and new technologies for DM screening that may be more suitable for TB patients in LMICs. Such methods should be rapid, they should not require fasting, and they should allow the provider to differentiate between transient and longer-term hyperglycemia, using inexpensive tools that require little training and no specialized infrastructure. Several methods that are currently under development, such as point-of-care glycated hemoglobin and glycated albumin assays, non-invasive advanced glycation end-product readers, and sudomotor function-based screening devices, offer interesting performance characteristics and warrant evaluation in populations with TB.

L'épidémie de diabète sucré (DM) de type 2 dans les pays à revenus faibles et moyens (LMIC) peut constituer une menace pour les progrès de la lutte contre la tuberculose (TB), car le DM est à la fois un facteur majeur de risque et de développement d'une TB active et peut aussi entrainer des résultats défavorables du traitement de la TB. En dépit de la directive de l'Organisation mondiale de la Santé selon laquelle tous les patients TB devraient faire l'objet d'un dépistage pour le DM, la plupart des services des LMIC qui traitent les patients TB ne réalisent pas actuellement le dépistage du DM, en partie en raison du coût et de la complexité qu'il implique. Le dépistage du DM est par ailleurs compliqué par l'existence d'une hyperglycémie transitoire chez beaucoup de patients TB ainsi que par les différences de facteurs de risque de DM (par exemple l'indice de masse corporelle) entre les patients TB et la population générale. Dans cet article, nous révisons les technologies existantes et nouvelles pour le dépistage du DM qui pourraient être les plus applicables aux patients TB dans les LMIC. De telles méthodes devraient être rapides, elles devraient ne pas exiger le jeûne et elles devraient permettre aux pourvoyeurs de soins de distinguer entre des hyperglycémies transitoires et de plus longue durée au moyen d'outils peu coûteux, n'exigeant que peu de formation et aucune infrastructure spécialisée. Différentes méthodes sont actuellement en cours de développement, tels que les tests sur l'hémoglobine glycosylée aux lieux de soins et sur l'albumine glycosylée, les lecteurs des produits finaux d'une glycation avancée non-invasive et les outils de dépistage basés sur la fonction sudomotrice ; elles offrent des caractéristiques intéressantes de performance et méritent une évaluation dans les populations TB.

La diabetes (DM) de tipo 2 presenta características epidémicas en los países con ingresos bajos e intermedios (LMIC) y puede poner en peligro los avances alcanzados en materia de control de la tuberculosis (TB); la DM constituye un factor de riesgo importante de padecer la enfermedad TB activa y también puede tener consecuencias desfavorables en el desenlace del tratamiento antituberculoso. Pese a la recomendación de la Organización Mundial de la Salud de realizar la detección sistemática de la DM en todos los pacientes TB, la mayoría de los establecimientos que atienden a estos pacientes en los LMIC no cumplen con esta práctica, en parte debido a los costos y a la complejidad de la misma. La detección de la DM se complica además por la hiperglucemia transitoria que suele observarse en muchos pacientes TB y por las diferencias en los factores de riesgo de DM, presentes en los pacientes con TB y la población general, por ejemplo el índice de masa corporal. En el presente artículo se analizan las técnicas existentes y los nuevos métodos de detección sistemática de la DM que pueden ser más adaptados a los pacientes con TB de los LMIC. Estos métodos deben ser rápidos, no deben precisar el estado de ayuno y deben permitir al profesional de salud diferenciar entre la hiperglucemia transitoria y la hiperglucemia de largo plazo, mediante la utilización instrumentos de bajo costo, que exijan poco entrenamiento y no necesiten infraestructuras especializadas. En la actualidad, se encuentran en curso de desarrollo varios métodos como las pruebas de hemoglobina glucosilada y albúmina glucosilada realizadas en el punto de atención, los lectores no invasivos de productos finales de la glucosilación avanzada y los dispositivos de detección basados en la función sudomotora; estos métodos ofrecen características de rendimiento interesantes y merecen una evaluación en las poblaciones de pacientes con diagnóstico de TB.

A highly sensitive rapid diagnostic test for Chagas disease that utilizes a recombinant Trypanosoma cruzi antigen.

Improved diagnostic tests for Chagas disease are urgently needed. A new lateral flow rapid test for Chagas disease is under development at PATH, in collaboration with Laboratorio Lemos of Argentina, which utilizes a recombinant antigen for detection of antibodies to Trypanosoma cruzi. To evaluate the performance of this test, 375 earlier characterized serum specimens from a region where Chagas is endemic were tested using a reference test (the Ortho T. cruzi ELISA, Johnson & Johnson), a commercially available rapid test (Chagas STAT-PAK, Chembio), and the PATH-Lemos rapid test. Compared to the composite reference tests, the PATH-Lemos rapid test demonstrated an optimal sensitivity of 99.5% and specificity of 96.8%, while the Chagas STAT-PAK demonstrated a sensitivity of 95.3% and specificity of 99.5%. These results indicate that the PATH-Lemos rapid test shows promise as an improved and reliable tool for screening and diagnosis of Chagas disease.

Initial study of using a laminar fluid diffusion interface for sample preparation in high-performance liquid chromatography.

This report describes a new microfluidic device called the H Filter for sample preparation prior to HPLC. The H Filters make possible a diffusional transfer of an analyte from a sample stream into a stream of a "receiver" fluid. Existing mathematical models can be used for optimizing experimental conditions. The authors have selected the extraction of the antibiotic cephradine from blood to demonstrate the utility of the new device. The extracts of blood samples spiked with cephradine levels between 0.2 and 100 microg/ml were analyzed using a C8 reversed-phase column and UV detection at 260 nm. The HPLC results were in good agreement with theory. The recovery of 32.2+/-2.8% was uniform over the entire range of cephradine concentrations. The new method completely avoids the use of centrifuges, that is otherwise typical for most current methodologies for the preparation of blood samples prior to HPLC analysis.

Lab-on-a-chip sample preparation using laminar fluid diffusion interfaces--computational fluid dynamics model results and fluidic verification experiments.

Microfluidic structures for the generation of laminar fluid diffusion interfaces (LFDIs) for sample preparation and analysis are discussed. Experimental data and the results of fluid modeling are shown. LFDIs are generated when two or more streams flow in parallel in a single microfluidic structure without any mixing of the fluids other than by diffusion of particles across the diffusion interface. It has been shown that such structures can be used for diffusion-based separation and detection applications. The method has been applied to DNA desalting, the extraction of small proteins from whole blood samples, and the detection of various constituents in whole blood, among other examples. In this paper the design and manufacture of self-contained microfluidic cartridges for the extraction of small molecules from a mixture of small and large molecules by diffusion is demonstrated. The cards are operated without any external instrumentation, and use hydrostatic pressure as the driving force. The performance of the cartridges is illustrated by separating fluorescein from a mixture of fluorescein and dextran of molecular weight 2 x 10(6). In a single pass, 98.6% of dextran was retained in the product whereas 43.1% of fluorescein was removed. The method is adjustable for different separation requirements, and computational fluid dynamics (CFD) models are shown that demonstrate the tuning of various microfluidic parameters to optimize separation performance. Other applications of LFDIs for establishment of stable concentration gradients, and the exposure of chemical constituents or biological particles to these concentration gradients are shown qualitatively. Microfluidic chips have been designed for high-throughput screening applications that enable the uniform and controlled exposure of cells to lysing agents, thus enabling the differentiation of cells by their sensitivity to specific agents in an on-chip cytometer coupled directly to the lysing structure.

A rapid diffusion immunoassay in a T-sensor.

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 microl of reagents and sample, and is applicable to a wide range of analytes.

The significance of stress hormones (glucocorticoids, catecholamines) for eruptions and spontaneous remission phases in psoriasis.

BACKGROUND: In an earlier paper, it was described how acute eruptions of psoriasis may be produced in phases of immune deficiency and in the presence of bacterial antigen-releasing inflammatory foci, whereas clinical spontaneous remissions are produced in phases of immunologic activity. Therefore, it was of interest to investigate whether the stress hormones cortisol/epinephrine are involved in triggering such deficiency and activity phases. METHODS: During a series of investigations lasting up to 3 years in 95 patients, the following were determined: cortisol/epinephrine levels, polyclonal serum immunoglobulins IgM, IgG, and IgA, total serum IgE, complement C3 and C4 proteins, T cells and subpopulations, as well as streptococcal titers ASO/ADNase B, severity index (PASI) RESULTS: Phases of clinical inactivity are associated with the mechanism, "immunologic regulation," where antibacterial titers are elevated, but all other parameters are unremarkable. Eruption phases (in 32 of 95 patients) showed absolute increases in serum cortisol levels and antibacterial titers, and decreases in serum epinephrine (adrenaline) levels. Phases of spontaneous remission (in 25 of 32 patients) showed, in contrast to the eruption phases, absolute increases in serum epinephrine levels, and significant falls in serum cortisol levels and bacterial titers. CONCLUSIONS: On the basis of these results, the participation of the immune system is confirmed in the pathogenesis of psoriasis, which is subject to control by higher neurohormonal systems. Cortisol may be involved in the clinical eruption phase, and epinephrine in the remission phase. Both hormones are true antagonists and have important effects on the human immune system if produced in excess via the pituitary-adrenal axis. Infection with Streptococcus pyogenes is an additional trigger for the dermatosis.

Quantitative analysis of molecular interaction in a microfluidic channel: the T-sensor.

The T-sensor is a recently developed microfluidic chemical measurement device that exploits the low Reynolds number flow conditions in microfabricated channels. The interdiffusion and resulting chemical interaction of components from two or more input fluid streams can be monitored optically, allowing measurement of analyte concentrations on a continuous basis. In a simple form of T-sensor, the concentration of a target analyte is determined by measuring fluorescence intensity in a region where the analyte and a fluorescent indicator have interdiffused. An analytical model has been developed that predicts device behavior from the diffusion coefficients of the analyte, indicator, and analyte--indicator complex and from the kinetics of the complex formation. Diffusion coefficients depend on the local viscosity which, in turn, depends on local concentrations of all analytes. These relationships, as well as reaction equilibria, are often unknown. A rapid method for determining these unknown parameters by interpreting T-sensor experiments through the model is presented.

Immunoregulatory mechanisms and stress hormones in psoriasis (part 1)

BACKGROUND: In an earlier paper, the author noted that psoriatic eruptions may be produced in phases of humoral and cellular immunodeficiency and in the presence of streptococcal antigen-releasing inflammatory foci. In this study it was investigated as to whether stress hormones glucocorticoids, catecholamines) are substantially involved in the activity phases (eruptions) of psoriasis. METHODS: During a series of investigations over two years, the following were determined for 70 chronic psoriasis patients and 50 controls: cortisol-adrenaline serum levels, polyclonal serum immunoglobulins IgM, IgG, IgA, total serum IgE, complements C3, C4, T-cells and subpopulations, streptococcal antibody titres ASO/ADNase B. RESULTS: Phases of clinical inactivity are associated with a mechanism called immunologic regulation: elevated antibacterial titres and unremarkable findings for all other parameters. Phases of clinical activity (in 25/70 patient) showed absolutely elevated serum cortisol levels, absolutely decreased serum epinephrine levels, deficient serum IgM or IgG and IgE levels, increased C3, decreased C4 and T4:T8 ratio, and significantly elevated streptococcal titres. CONCLUSIONS: The greatly elevated serum cortisol levels indicate that glucocorticoids are produced in excess via the pituitary adrenal axis and are significantly involved in the triggering of immunosuppressive phases (eruptions) in psoriasis.

Capillary waveguide optrodes: an approach to optical sensing in medical diagnostics.

Glass capillaries with a chemically sensitive coating on the inner surface are used as optical sensors for medical diagnostics. A capillary simultaneously serves as a sample compartment, a sensor element, and an inhomogeneous optical waveguide. Various detection schemes based on absorption, fluorescence intensity, or fluorescence lifetime are described. In absorption-based capillary waveguide optrodes the absorption in the sensor layer is analyte dependent; hence light transmission along the inhomogeneous waveguiding structure formed by the capillary wall and the sensing layer is a function of the analyte concentration. Similarly, in fluorescence-based capillary optrodes the fluorescence intensity or the fluorescence lifetime of an indicator dye fixed in the sensing layer is analyte dependent; thus the specific property of fluorescent light excited in the sensing layer and thereafter guided along the inhomogeneous waveguiding structure is a function of the analyte concentration. Both schemes are experimentally demonstrated, one with carbon dioxide as the analyte and the other one with oxygen. The device combines optical sensors with the standard glass capillaries usually applied to gather blood drops from fingertips, to yield a versatile diagnostic instrument, integrating the sample compartment, the optical sensor, and the light-collecting optics into a single piece. This ensures enhanced sensor performance as well as improved handling compared with other sensors.

Optical triple sensor for measuring pH, oxygen and carbon dioxide.

A triple sensor unit consisting of opto-chemical sensors for measurement of pH, oxygen and carbon dioxide in bioreactors is presented. The pH and the CO2 sensor are based on the color change of a pH-sensitive dye immobilized on a polymeric support. The resulting changes in absorption are monitored through optical fibers. The oxygen sensor is based on the quenching of the fluorescence of a metal-organic dye. All three sensors are fully LED compatible. The sensitive membranes consist of plastic films and can be stored and replaced conveniently. The sensors are sterilizable with hydrogen peroxide and ethanol. In addition, the pH sensor is steam sterilizable. Accuracy, resolution and reproducibility fulfill the requirements for use in biotechnological applications. Calibration procedures for each sensor are presented. The working principle and the performance of all three sensors are described, with particular emphasis given to their application in bioreactors.

[Comparative testing of Kamillosan cream and steroidal (0.25% hydrocortisone, 0.75% fluocortin butyl ester) and non-steroidal (5% bufexamac) dermatologic agents in maintenance therapy of eczematous diseases]. / Vergleichende Prüfung von Kamillosan Creme gegenüber steroidalen (0,25% Hydrocortison, 0,75% Fluocortinbutylester) und nichtsteroidalen (5% Bufexamac) Externa in der Erhaltungstherapie von Ekzemerkrankungen.

We report on 161 patients suffering from inflammatory dermatoses on hands, forearms, and lower legs who had been initially treated with 0.1% difluocortolone valerate. During the maintenance therapy carried out over a period of 3 to 4 weeks, we tested the efficacy of Kamillosan cream vs. 0.25% hydrocortisone, 0.75% fluocortin butyl ester, and 5% bufexamac in a bilateral comparative study. For the indications tested Kamillosan cream showed more or less equieffective therapeutic results as compared to 0.25% hydrocortisone. It is superior, however, to the non-steroidal anti-inflammatory agent 5% bufexamac as well as to 0.75% fluocortin butyl ester, a further glucocorticoid. With regard to neurodermitis, Kamillosan cream not only shows the same therapeutic effect as 0.25% hydrocortisone but is even of marked superiority towards other reference products.