Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis.

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Asking about adherence - from flipping the coin to strong evidence.

In the era of antiretroviral therapy (ART) as prevention for transmission of HIV as well as treatment for HIV-positive individuals irrespective of CD4 cell counts, the importance of adherence has grown. Although adherence is not the only determinant of treatment success, it is one of the only modifiable risk factors. Treatment failure reduces future treatment options and therefore long-term clinical success as well as increases the possibility of developing drug resistant mutations. Drug-resistant strains of HIV can then be transmitted to uninfected or drug-naïve individuals limiting their future treatment options, making adherence an important public-health topic, especially in resource-limited settings. Adherence should be monitored as a part of routine clinical care; however, no gold standard for assessment of adherence exists. For use in daily clinical practice, self-report is the most likely candidate for widespread use due to its many advantages over other measurement methods, such as low cost and ease of administration. Asking individuals about their adherence behaviour has been shown to yield valid and predictive data - well beyond the mere flip of a coin. However, there is still work to be done. This article reviews the literature and evidence on self-reported adherence, identifies gaps in adherence research, and makes recommendations for clinicians on how to best utilise self-reported adherence data to support patients in daily clinical practice.

Paediatric oral peanut challenges: a comparison of practice in London and Western Switzerland.

BACKGROUND: There are guidelines on how to develop a food challenge protocol, but at present there is no gold standard guidance on method, and separate units produce differing protocols. METHODS: We performed a retrospective analysis of 200 patients' data from the paediatric allergy units in Lausanne and Geneva, Western Switzerland, and St Thomas' Hospital (STH), UK. RESULTS: St Thomas' Hospital has a younger cohort with a lower overall mean spIgE (2.36 kU/l vs 8.00 kU/l, P = 0.004). The target peanut protein volumes differed: Switzerland 4.4 g vs STH 8.4 g. Despite this, the dose actually achieved in positive challenges was not significantly different (2.33 g vs 1.49 g, P = 0.16). 26% of challenges reacted at 4 g or more of peanut protein. CONCLUSIONS: The differences in results highlight how the variation in reasoning behind food challenge alters the outcome. Standardization of food challenges would allow easy comparison between hospitals and geographical areas for research purposes.

Vertebral microarchitecture and fragility fracture in men: a TBS study.

INTRODUCTION: Although osteoporosis is considered a disease of women, 25% of the individuals with osteoporosis are men. BMD measurement by DXA is the gold standard used to diagnose osteoporosis and assess fracture risk. Nevertheless, BMD does not take into account alterations of microarchitecture. TBS is an index of bone microarchitecture extracted from the spine DXA. Previous studies have reported the ability of the spine TBS to predict osteoporotic fractures in women. This is the first case-controlled study in men to evaluate the potential diagnostic value of TBS as a complement to bone mineral density (BMD), by comparing men with and without fractures. METHODS: To be eligible for this study, subjects had to be non-Hispanic US white men aged 40 and older. Furthermore, subjects were excluded if they have or have had previously any treatment or illness that may influence bone metabolism. Fractured subjects were included if the presence of at least one fracture was confirmed. Cases were matched for age (±3 years) and BMD (±0.04 g/cm(2)) with three controls. BMD and TBS were first retrospectively evaluated at AP spine (L1-L4) with a Prodigy densitometer (GE-Lunar, Madison, USA) and TBS iNsight® (Med-Imaps, France) in Lausanne University Hospital blinded from clinical outcome. Inter-group comparisons were undertaken using Student's t-tests or Wilcoxon signed rank tests. Odds ratios were calculated per one standard deviation decrease as well as areas under the receiver operating curve (AUC). RESULTS: After applying inclusion/exclusion criteria, a group of 180 male subjects was obtained. This group consists of 45 fractured subjects (age=63.3±12.6 years, BMI=27.1±4.2 kg/m(2)) and 135 control subjects (age=62.9±11.9 years, BMI=26.7±3.9 kg/m(2)) matched for age (p=0.86) and BMD (p=0.20). A weak correlation was obtained between TBS and BMD and between TBS and BMI (r=0.27 and r=-0.28, respectively, p<0.01). Subjects with fracture have a significant lower TBS compared to control subjects (p=0.013), whereas no differences were obtained for BMI, height and weight (p>0.10). TBS OR per standard deviation is 1.55 [1.09-2.20] for all fracture type. When considering vertebral fracture only TBS OR reached 2.07 [1.14-3.74]. CONCLUSION: This study showed the potential use of TBS in men. TBS revealed a significant difference between fractured and age- and spine BMD-matched nonfractured subjects. These results are consistent with those previously reported on for men of other nationalities.

Reliable tidal volume estimates at the airway opening with an infant monitor during high-frequency oscillatory ventilation.

OBJECTIVE: To assess the suitability of a hot-wire anemometer infant monitoring system (Florian, Acutronic Medical Systems AG, Hirzel, Switzerland) for measuring flow and tidal volume (Vt) proximal to the endotracheal tube during high-frequency oscillatory ventilation. DESIGN: In vitro model study. SETTING: Respiratory research laboratory. SUBJECT: In vitro lung model simulating moderate to severe respiratory distress. INTERVENTION: The lung model was ventilated with a SensorMedics 3100A ventilator. Vt was recorded from the monitor display (Vt-disp) and compared with the gold standard (Vt-adiab), which was calculated using the adiabatic gas equation from pressure changes inside the model. MEASUREMENTS AND MAIN RESULTS: A range of Vt (1-10 mL), frequencies (5-15 Hz), pressure amplitudes (10-90 cm H2O), inspiratory times (30% to 50%), and Fio2 (0.21-1.0) was used. Accuracy was determined by using modified Bland-Altman plots (95% limits of agreement). An exponential decrease in Vt was observed with increasing oscillatory frequency. Mean DeltaVt-disp was 0.6 mL (limits of agreement, -1.0 to 2.1) with a linear frequency dependence. Mean DeltaVt-disp was -0.2 mL (limits of agreement, -0.5 to 0.1) with increasing pressure amplitude and -0.2 mL (limits of agreement, -0.3 to -0.1) with increasing inspiratory time. Humidity and heating did not affect error, whereas increasing Fio2 from 0.21 to 1.0 increased mean error by 6.3% (+/-2.5%). CONCLUSIONS: The Florian infant hot-wire flowmeter and monitoring system provides reliable measurements of Vt at the airway opening during high-frequency oscillatory ventilation when employed at frequencies of 8-13 Hz. The bedside application could improve monitoring of patients receiving high-frequency oscillatory ventilation, favor a better understanding of the physiologic consequences of different high-frequency oscillatory ventilation strategies, and therefore optimize treatment.

Animal Tissue Response To Mid-Term Electronic Modular Artificial Sphincter Implantation

Introduction and objectives: The AMS 800TM is considered the gold standard for sphincter replacement. However, the one-ring design can erode the urethra and lead to severe complications. A mechanism that could alternatively compress successive segments of the urethra would limit such deleterious outcome. We report 12 weeks animal urethral tissue analysis following implantation of a new modular artificial sphincter. METHODS: The device is composed by three parts: the contractile unit, two rings and an integrated microprocessor. The contractile unit is made of Nitinol fibers. The rings are placed around the urethra to control the flow of urine by squeezing the urethra. They work in a sequential alternative mode and are controlled by a microprocessor connected to an external computer. The computer can reveal specific failure of device components. The device was impkanted in eight male sheep. The rings were positioned around the urethra and the control unit was placed 5cm away. The device was working twenty hours per day; it was open 10min. per hour to allow urination. The animals were sacrificed after 12 weeks. The urethra and the tissues surrounding the control unit were macroscopically and microscopically examined. Two transversal sections crossing the sphincter and two transversal sections crossing the urethra alone were obtained and stained with modified Paragon after resin embedding. Urethra was also embedded in paraffin. The first section was stained with safranin-hematoxylin-eosin, the second section was stained with Masson's Trichrome and the remaining eight sections were available for immunolabelling of the macrophages.Results: The chronic study went uneventful. No clinical infection or pain was observed. The computer registered no specific failure in ring function, Nitinol wires and tube connectors. At explantation, except for a slight grade of lymphocytes in two out of eight specimens, no urethral stricture or atrophy could be observed. Immunohistochemistry confirmed the absence of macrophages. Tissue structure and organization of the urethra with and without artificial sphincter were similar. No migration of the device was observed.Conclusions: The study clearly showed no tissue damage or inflammation of the urethra. Electronic design, preservation of urethral vascularisation and adjustability after implantation are the key ideas to improve the actual AUS. Further studies will be carried out to evaluate this potential.

Marqueurs de la filtration glomérulaire en pédiatrie [Glomerular filtration markers in pediatrics]

The assessment of glomerular filtration rate (GFR) is critical for the diagnosis and management of renal diseases in pediatric nephrology. Ideally, it requires the measurement of the renal clearance of a filtration marker. Inulin, an exogenous marker, is the only compound the excretion of which occurs exclusively by glomerular filtration, with no tubular handling. Therefore, inulin clearance provides the most accurate method to measure GFR and is considered as the "gold standard", at all ages including very premature neonates. However, inulin dearance is cumbersome and alternative methods are used in clinical practice. If urine is available, endogenous creatinine clearance is the most reliable method. When urine collection is difficult to obtain, GFR can be estimated by the plasma concentration of endogenous markers mainly eliminated by glomerular filtration, such as creatinine, or the more recently described cystatin C and beta 2-microglobulin. When the endogenous production of these markers is constant, their plasma concentration reflects glomerular filtration; it increases with decreasing renal function. However, in pediatric patients creatinine production depends on muscle mass, which significantly increases with linear growth, as well as age and gender. Mathematical formulas taking these parameters into account have thus been developed. Among these, the so-called "Schwartz formula" is often used and is a reliable estimate of GFR in children. Finally, radionuclide renal scans can be used to evaluate the separate glomerular function of each kidney.

Chest pulse-wave velocity: a novel approach to assess arterial stiffness.

Pulse-wave velocity (PWV) is considered as the gold-standard method to assess arterial stiffness, an independent predictor of cardiovascular morbidity and mortality. Current available devices that measure PWV need to be operated by skilled medical staff, thus, reducing the potential use of PWV in the ambulatory setting. In this paper, we present a new technique allowing continuous, unsupervised measurements of pulse transit times (PTT) in central arteries by means of a chest sensor. This technique relies on measuring the propagation time of pressure pulses from their genesis in the left ventricle to their later arrival at the cutaneous vasculature on the sternum. Combined thoracic impedance cardiography and phonocardiography are used to detect the opening of the aortic valve, from which a pre-ejection period (PEP) value is estimated. Multichannel reflective photoplethysmography at the sternum is used to detect the distal pulse-arrival time (PAT). A PTT value is then calculated as PTT = PAT - PEP. After optimizing the parameters of the chest PTT calculation algorithm on a nine-subject cohort, a prospective validation study involving 31 normo- and hypertensive subjects was performed. 1/chest PTT correlated very well with the COMPLIOR carotid to femoral PWV (r = 0.88, p < 10 (-9)). Finally, an empirical method to map chest PTT values onto chest PWV values is explored.

Glomerular filtration rate: measure creatinine and height rather than cystatin C!

AIM: Inulin clearance (Cin) is the gold standard for assessing glomerular filtration rate (GFR). Other methods are based on the plasma creatinine concentration (Pcreat), creatinine clearance (Ccreat), the Haycock-Schwartz formula and the plasma concentration of cystatin C (PcysC), a 13 kDa basic protein produced at a constant rate by all nucleated cells. The present prospective study was thus designed to evaluate the reliability of PcysC as a marker of GFR in comparison with that of Pcreat, Ccreat and the Haycock-Schwartz formula, using Cin as the gold standard. METHODS: Ninety-nine children (51 m/48 f), with a median age of 8.3 y (1.0-17.9) were studied. Using a cut-off for Cin of 100 ml/min per 1.73 m2, 54 children (54.5%) had impaired GFR. Those with normal GFR were comparable for age, height, weight and body mass index. RESULTS: Logistic regression, ROC analysis and linear regression all showed that Ccreat was the best parameter to discriminate between impaired and normal GFR, followed by the Haycock-Schwartz formula, PcysC, and finally Pcreat, each one being significantly more predictive than the next. CONCLUSION: GFR is better assessed by the Haycock-Schwartz formula than by PcysC or Pcreat alone. It is therefore concluded that when urine collection is not possible, simply measuring the child's Pcreat and height is the best, easiest and cheapest way to assess GFR.

Clinical role of coronary magnetic resonance angiography in the diagnosis of anomalous coronary arteries.

Though rare, anomalous coronary artery disease is a well-known cause of myocardial ischemia and sudden death among children and young adults. The projectional nature of conventional x-ray angiography often leads to difficulty in the definition of anomalous vessels. Studies have now documented the high accuracy of coronary magnetic resonance angiography (MRA) for the noninvasive detection and definition of anomalous coronary arteries among patients with suspected anomalous coronary arteries of congenital conditions associated with anomalous coronary arteries. With increasing clinical experience, coronary MRA will likely emerge as the gold standard for the diagnosis of this condition.

When "enough" is not enough: new perspectives on optimal methadone maintenance dose.

Some methadone maintenance treatment (MMT) programs prescribe inadequate daily methadone doses. Patients complain of withdrawal symptoms and continue illicit opioid use, yet practitioners are reluctant to increase doses above certain arbitrary thresholds. Serum methadone levels (SMLs) may guide practitioners dosing decisions, especially for those patients who have low SMLs despite higher methadone doses. Such variation is due in part to the complexities of methadone metabolism. The medication itself is a racemic (50:50) mixture of 2 enantiomers: an active "R" form and an essentially inactive "S" form. Methadone is metabolized primarily in the liver, by up to five cytochrome P450 isoforms, and individual differences in enzyme activity help explain wide ranges of active R-enantiomer concentrations in patients given identical doses of racemic methadone. Most clinical research studies have used methadone doses of less than 100 mg/day [d] and have not reported corresponding SMLs. New research suggests that doses ranging from 120 mg/d to more than 700 mg/d, with correspondingly higher SMLs, may be optimal for many patients. Each patient presents a unique clinical challenge, and there is no way of prescribing a single best methadone dose to achieve a specific blood level as a "gold standard" for all patients. Clinical signs and patient-reported symptoms of abstinence syndrome, and continuing illicit opioid use, are effective indicators of dose inadequacy. There does not appear to be a maximum daily dose limit when determining what is adequately "enough" methadone in MMT.

A rapid and effective vignetting correction for quantitative microscopy

Images acquired using optical microscopes are inherently subject to vignetting effects due to imperfect illumination and image acquisition. However, such vignetting effects hamper accurate extraction of quantitative information from biological images, leading to less effective image segmentation and increased noise in the measurements. Here, we describe a rapid and effective method for vignetting correction, which generates an estimate for a correction function from the background fluorescence without the need to acquire additional calibration images. We validate the usefulness of this algorithm using artificially distorted images as a gold standard for assessing the accuracy of the applied correction and then demonstrate that this correction method enables the reliable detection of biologically relevant variation in cell populations. A simple user interface called FlattifY was developed and integrated into the image analysis platform YeastQuant to facilitate easy application of vignetting correction to a wide range of images.

Performance of an automated multiplex immunofluorescence assay for detection of Chlamydia trachomatis immunoglobulin G.

Chlamydia serology is indicated to investigate etiology of miscarriage, infertility, pelvic inflammatory disease, and ectopic pregnancy. Here, we assessed the reliability of a new automated-multiplex immunofluorescence assay (InoDiag test) to detect specific anti-C. trachomatis immunoglobulin G. Considering immunofluorescence assay (IF) as gold standard, InoDiag tests exhibited similar sensitivities (65.5%) but better specificities (95.1%-98%) than enzyme-linked immunosorbent assays (ELISAs). InoDiag tests demonstrated similar or lower cross-reactivity rates when compared to ELISA or IF.

On making causal claims: A review and recommendations

Social scientists often estimate models from correlational data, where the independent variable has not been exogenously manipulated; they also make implicit or explicit causal claims based on these models. When can these claims be made? We answer this question by first discussing design and estimation conditions under which model estimates can be interpreted, using the randomized experiment as the gold standard. We show how endogeneity--which includes omitted variables, omitted selection, simultaneity, common methods bias, and measurement error--renders estimates causally uninterpretable. Second, we present methods that allow researchers to test causal claims in situations where randomization is not possible or when causal interpretation is confounded, including fixed-effects panel, sample selection, instrumental variable, regression discontinuity, and difference-in-differences models. Third, we take stock of the methodological rigor with which causal claims are being made in a social sciences discipline by reviewing a representative sample of 110 articles on leadership published in the previous 10 years in top-tier journals. Our key finding is that researchers fail to address at least 66 % and up to 90 % of design and estimation conditions that make causal claims invalid. We conclude by offering 10 suggestions on how to improve non-experimental research.

Pharmacological treatment of heterotopic ossification following hip and acetabular surgery.

Heterotopic ossification is a common complication following total hip arthroplasty and surgery following acetabular trauma. It is associated with pain and a decreased range of movement. Prophylaxis is achieved by either non-steroidal anti-inflammatory drug treatment or localised irradiation therapy. The objective of this study was to evaluate the evidence for pharmacological agents used for the prophylaxis of heterotopic ossification following hip and acetabular surgery. The study used a comprehensive literature search to identify all major clinical studies investigating the pharmacological agents used in the prophylaxis of heterotopic ossification following hip and acetabular surgery. It was concluded that indometacin remains the 'gold standard' for heterotopic ossification prophylaxis following total hip arthroplasty and is the only drug proven to be effective against heterotopic ossification following acetabular surgery. Following total hip arthroplasty, other non-steroidal anti-inflammatory drugs, including naproxen and diclofenac, are equally as effective as indometacin and can be considered as alternative first-line treatments. Celecoxib is also of equal efficacy to indometacin and is associated with significantly fewer gastrointestinal side effects. However, serious concerns were raised over the safety of selective cyclooxygenase-2 inhibitors for the cardiovascular system and these should be used cautiously.