[Microbiological point of care tests]

It is well known that the early initiation of a specific antiinfective therapy is crucial to reduce the mortality in severe infection. Procedures culturing pathogens are the diagnostic gold standard in such diseases. However, these methods yield results earliest between 24 to 48 hours. Therefore, severe infections such as sepsis need to be treated with an empirical antimicrobial therapy, which is ineffective in an unknown fraction of these patients. Today's microbiological point of care tests are pathogen specific and therefore not appropriate for an infection with a variety of possible pathogens. Molecular nucleic acid diagnostics such as polymerase chain reaction (PCR) allow the identification of pathogens and resistances. These methods are used routinely to speed up the analysis of positive blood cultures. The newest PCR based system allows the identification of the 25 most frequent sepsis pathogens by PCR in parallel without previous culture in less than 6 hours. Thereby, these systems might shorten the time of possibly insufficient antiinfective therapy. However, these extensive tools are not suitable as point of care diagnostics. Miniaturization and automating of the nucleic acid based method is pending, as well as an increase of detectable pathogens and resistance genes by these methods. It is assumed that molecular PCR techniques will have an increasing impact on microbiological diagnostics in the future.

Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of "non-alcoholic" beer

In abstinence maintenance programs, for reissuing the driving licence and in workplace monitoring programs abstinence from ethanol and its proof are demanded. Various monitoring programs that mainly use ethyl glucuronide (EtG) as alcohol consumption marker have been established. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer has become more and more popular. In Germany, these "alcohol-free" beverages may still have an ethanol content of up to 0.5vol.% without the duty of declaration. Due to severe negative consequences resulting from positive EtG tests, a drinking experiment with 2.5L of non-alcoholic beer per person was performed to address the question of measurable concentrations of the direct metabolites EtG and EtS (ethyl sulphate) in urine and blood. Both alcohol consumption markers - determined by LC-MS/MS - were found in high concentrations: maximum concentrations in urine found in three volunteers were EtG 0.30-0.87mg/L and EtS 0.04-0.07mg/L, i.e., above the often applied cut-off value for the proof of abstinence of 0.1mg EtG/L. In the urine samples of one further volunteer, EtG and EtS concentrations cumulated over-night and reached up to 14.1mg/L EtG and 16.1mg/L EtS in the next morning's urine. Ethanol concentrations in blood and urine samples were negative (determined by HS-GC-FID and by an ADH-based method).

Ethyl sulphate and ethyl glucuronide in vitreous humor as postmortem evidence marker for ethanol consumption prior to death

To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices.

[Current concepts in tobacco cessation and prevention]

In the recent years, for oral care in general, both improving oral hygiene and tobacco use cessation have been identified as necessary measures to gain and maintain long-term periodontal health. This growing evidence has given the dental team a whole new task to tackle when achieving and maintaining oral health with their patients. In order to support dental patients to quit tobacco use, it is helpful for the clinician to have a clear understanding of the genesis of 'tobacco use disease' in general. At present, the evidence-based method for tobacco use cessation consists of professional counselling on behavioural change using the so called "5A Method" (Ask, Advise, Assess, Assist and Arrange") in combination with pharmacotherapy. A suitable model for behavioural support in tobacco use cessation would help patients to move from one stage to the next. People who want to quit the smoking habit do not always participate in carefully controlled nicotine withdrawal programs, e.g. in linear fashion and from start to finish. Nevertheless, simple instructions - like those offered in the "Assist" (to help) and "Arrange" (to organize follow-up visits) - can be valuable tools for dental professionals supporting their patients to quit smoking. On the basis of significant evidence on the recovery of the oral mucosa and the periodontal tissue following tobacco use cessation, a new task has been emerged in dentistry: the role of oral health professionals providing counselling for patients who ought to quit tobacco use.

An integrated nano-scale approach to profile miRNAs in limited clinical samples

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

Range of motion in anterior femoroacetabular impingement

The range of motion of normal hips and hips with femoroacetabular impingement relative to some specific anatomic reference landmarks is unknown. We therefore described: (1) the range of motion pattern relative to landmarks; (2) the location of the impingement zones in normal and impinging hips; and (3) the influence of surgical débridement on the range of motion. We used a previously developed and validated noninvasive 3-D CT-based method for kinematic hip analysis to compare the range of motion pattern, the location of impingement, and the effect of virtual surgical reconstruction in 28 hips with anterior femoroacetabular impingement and a control group of 33 normal hips. Hips with femoroacetabular impingement had decreased flexion, internal rotation, and abduction. Internal rotation decreased with increasing flexion and adduction. The calculated impingement zones were localized in the anterosuperior quadrant of the acetabulum and were similar in the two groups and in impingement subgroups. The average improvement of internal rotation was 5.4 degrees for pincer hips, 8.5 degrees for cam hips, and 15.7 degrees for mixed impingement. This method helps the surgeon quantify the severity of impingement and choose the appropriate treatment option; it provides a basis for future image-guided surgical reconstruction in femoroacetabular impingement with less invasive techniques.

Noninvasive three-dimensional assessment of femoroacetabular impingement

A CT-based method ("HipMotion") for the noninvasive three-dimensional assessment of femoroacetabular impingement (FAI) was developed, validated, and applied in a clinical pilot study. The method allows for the anatomically based calculation of hip range of motion (ROM), the exact location of the impingement zone, and the simulation of quantified surgical maneuvers for FAI. The accuracy of HipMotion was 0.7 +/- 3.1 degrees in a plastic bone setup and -5.0 +/- 5.6 degrees in a cadaver setup. Reliability and reproducibility were excellent [intraclass correlation coefficient (ICC) > 0.87] for all measures except external rotation (ICC = 0.48). The normal ROM was determined from a cohort of 150 patients and was compared to 31 consecutive hips with FAI. Patients with FAI had a significantly decreased flexion, internal rotation, and abduction in comparison to normal hips (p < 0.001). Normal hip flexion and internal rotation are generally overestimated in a number of orthopedic textbooks. HipMotion is a useful tool for further assessment of impinging hips and for appropriate planning of the necessary amount of surgical intervention, which represents the basis for future computer-assisted treatment of FAI with less invasive surgical approaches, such as hip arthroscopy.

Calibration of a surgical microscope with automated zoom lenses using an active optical tracker

BACKGROUND: In this paper, we present a new method for the calibration of a microscope and its registration using an active optical tracker. METHODS: Practically, both operations are done simultaneously by moving an active optical marker within the field of view of the two devices. The IR LEDs composing the marker are first segmented from the microscope images. By knowing their corresponding three-dimensional (3D) position in the optical tracker reference system, it is possible to find the transformation matrix between the referential of the two devices. Registration and calibration parameters can be extracted directly from that transformation. In addition, since the zoom and focus can be modified by the surgeon during the operation, we propose a spline based method to update the camera model to the new setup. RESULTS: The proposed technique is currently being used in an augmented reality system for image-guided surgery in the fields of ear, nose and throat (ENT) and craniomaxillofacial surgeries. CONCLUSIONS: The results have proved to be accurate and the technique is a fast, dynamic and reliable way to calibrate and register the two devices in an OR environment.

ABO blood group incompatible haematopoietic stem cell transplantation and xenograft rejection

The current organ shortage in transplantation medicine stimulates the exploration of new strategies to expand the donor pool including the utilisation of living donors, ABO-incompatible grafts, and xenotransplantation. Preformed natural antibodies (Ab) such as anti-Gal or anti-A/B Ab mediate hyperacute graft rejection and thus represent a major hurdle to the employment of such strategies. In contrast to solid organ transplantation (SOT), ABO blood group incompatibilities are of minor importance in haematopoietic stem cell transplantation (HSCT). Thus, ABO incompatible HSCT may serve as an in vivo model to study carbohydrate antigen (Ag)-mismatched transplantations such as ABO-incompatible SOT or the effect of preformed Ab against Gal in xenotransplantation. This mini-review summarises our clinical and experimental studies performed with the support of the Swiss National Science Foundation program on Implants and Transplants (NFP-46). Part 1 describes data on the clinical outcome of ABO-incompatible HSCT, in particular the incidence of several immunohaematological complications, acute graft-versus-host-disease (GvHD), and the overall survival. Part 2 summarises the measurements of anti-A/B Ab in healthy blood donors and ABO-incompatible HSCT using a novel flow cytometry based method and the potential mechanisms responsible for the loss of anti-A/B Ab observed following minor ABO-incompatible HSCT, ie the occurrence of humoral tolerance. Part 3 analyses the potential of eliminating Gal expression as well as specific complement inhibitors such as dextran sulfate and synthetic tyrosine analogues to protect porcine endothelial cells from xenoreactive Ab-mediated damage in vitro and in a hamster-to-rat heart transplantation model. In conclusion, due to similarities of the immunological hurdles of ABO incompatible transplantations and xenotransplantation, the knowledge obtained from both fields might lead to new strategies to overcome humoral rejection in transplantation.

IgG subclass distribution of anti-ADAMTS13 antibodies in patients with acquired thrombotic thrombocytopenic purpura

BACKGROUND: ADAMTS13-neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To analyze the IgG subclass distribution of anti-ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis. METHODOLOGY: An enzyme-linked immunosorbent assay-based method was used to quantify the relative amounts of IgG subclasses of anti-ADAMTS13 antibodies in acquired TTP plasma. RESULTS: IgG(4) (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG(1) (52%), IgG(2) (50%), and IgG(3) (33%). IgG(4) was found either alone (17/52) or with other IgG subclasses (35/52). IgG(4) was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG(4) and IgG(1) antibodies (P < 0.01). Patients with high IgG(4) levels and undetectable IgG(1) are more prone to relapse than patients with low IgG(4) levels and detectable IgG(1). CONCLUSIONS: All IgG subclasses of anti-ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG(4), followed by IgG(1), antibodies dominating the anti-ADAMTS13 immune response. Levels of IgG(4) could be useful for the identification of patients at risk of disease recurrence.

Human intervertebral disc stiffness correlates better with the Otsu threshold computed from axial T2 map of its posterior annulus fibrosus than with clinical classifications

Degeneration of the intervertebral disc, sometimes associated with low back pain and abnormal spinal motions, represents a major health issue with high costs. A non-invasive degeneration assessment via qualitative or quantitative MRI (magnetic resonance imaging) is possible, yet, no relation between mechanical properties and T2 maps of the intervertebral disc (IVD) has been considered, albeit T2 relaxation time values quantify the degree of degeneration. Therefore, MRI scans and mechanical tests were performed on 14 human lumbar intervertebral segments freed from posterior elements and all soft tissues excluding the IVD. Degeneration was evaluated in each specimen using morphological criteria, qualitative T2 weighted images and quantitative axial T2 map data and stiffness was calculated from the load-deflection curves of in vitro compression, torsion, lateral bending and flexion/extension tests. In addition to mean T2, the OTSU threshold of T2 (TOTSU), a robust and automatic histogram-based method that computes the optimal threshold maximizing the distinction of two classes of values, was calculated for anterior, posterior, left and right regions of each annulus fibrosus (AF). While mean T2 and degeneration schemes were not related to the IVDs' mechanical properties, TOTSU computed in the posterior AF correlated significantly with those classifications as well as with all stiffness values. TOTSU should therefore be included in future degeneration grading schemes.

A case of distinct difference between the measured blood Ethanol concentration and the concentration estimated by Widmark's equation

In the last century, several mathematical models have been developed to calculate blood ethanol concentrations (BAC) from the amount of ingested ethanol and vice versa. The most common one in the field of forensic sciences is Widmark's equation. A drinking experiment with 10 voluntary test persons was performed with a target BAC of 1.2 g/kg estimated using Widmark's equation as well as Watson's factor. The ethanol concentrations in the blood were measured using headspace gas chromatography/flame ionization and additionally with an alcohol Dehydrogenase (ADH)-based method. In a healthy 75-year-old man a distinct discrepancy between the intended and the determined blood ethanol concentration was observed. A blood ethanol concentration of 1.83 g/kg was measured and the man showed signs of intoxication. A possible explanation for the discrepancy is a reduction of the total body water content in older people. The incident showed that caution is advised when using the different mathematical models in aged people. When estimating ethanol concentrations, caution is recommended with calculated results due to potential discrepancies between mathematical models and biological systems

Comparative Evaluation of Pelvic Allograft Selection Methods

This paper presents a firsthand comparative evaluation of three different existing methods for selecting a suitable allograft from a bone storage bank. The three examined methods are manual selection, automatic volume-based registration, and automatic surface-based registration. Although the methods were originally published for different bones, they were adapted to be systematically applied on the same data set of hemi-pelvises. A thorough experiment was designed and applied in order to highlight the advantages and disadvantages of each method. The methods were applied on the whole pelvis and on smaller fragments, thus producing a realistic set of clinical scenarios. Clinically relevant criteria are used for the assessment such as surface distances and the quality of the junctions between the donor and the receptor. The obtained results showed that both automatic methods outperform the manual counterpart. Additional advantages of the surface-based method are in the lower computational time requirements and the greater contact surfaces where the donor meets the recipient.

In situ fiber-optical monitoring of cytosolic calcium in tissue explant cultures

We present a fluorescence-lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber-optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green-5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s.

Application of real-time fluorescent PCR for quantitative assessment of Neospora caninum infections in organotypic slice cultures of rat central nervous system tissue

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection.