A paperfluidic platform to detect Neisseria gonorrhoeae in clinical samples

Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be readily treated with antibiotics, but high-risk patients often do not return to the clinic for treatment if results are not provided at the point of care. A rapid, sensitive molecular diagnostic would help increase NG treatment and reduce the prevalence of this sexually transmitted disease. Here, we report on the design and development of a rapid, highly sensitive, paperfluidic device for point-of-care diagnosis of NG. The device integrates patient swab sample lysis, nucleic acid extraction, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and visual lateral flow detection within an 80 min run time. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept study. This paperfluidic diagnostic has a clinically relevant limit of detection of 500 NG cells per device with analytical sensitivity down to 10 NG cells per device. In triplicate testing of 40 total urethral and vaginal swab samples, the device had 95% overall sensitivity and 100% specificity, approaching current laboratory-based molecular NG diagnostics. This diagnostic platform could increase access to accurate NG diagnoses to those most in need.This work was funded by the National Institute of Health National Institute of Allergy and Infectious Diseases award number R01 AI113927 to Boston University and the NIH National Institute of Biomedical and Bioengineering award number U54 EB007958 to Johns Hopkins University. (R01 AI113927 - National Institute of Health National Institute of Allergy and Infectious Diseases; U54 EB007958 - NIH National Institute of Biomedical and Bioengineering)Accepted manuscrip

The impact on domestic prices and government costs of limiting wheat procurement in India

India's government plays a major role in the Indian wheat market, procuring wheat at a minimum support price for distribution to consumers at a subsidized rate through fair price shops. The costs of this program have been growing recently in real terms. Studies suggest different options for addressing these growing costs through means such as: reducing per-unit operating expenses, changing the scope of the program, altering pricing schemes, or limiting procurement operations. This study assesses the impact of limiting government procurements on the domestic market and government expenditures. A partial equilibrium model is developed to help estimate the impact of this policy change. The model represents the Indian wheat market and the relevant government policy variables necessary to estimate the change in government costs from this policy alternative. Results suggest that there are saving available from limiting government procurements of wheat. The domestic market would likely see lower prices because of these reduced government interventions. Sensitivity analysis suggests that limiting import could help to ease the pressure on domestic prices caused by the policy change. The market effects, including both greater use and less production, the cost savings, and potential winners and losers are all relevant facts for decision makers in India who contemplate procurement and PDS system reforms

Pattern of paresis in ALS is consistent with the physiology of the corticomotoneuronal projections to different muscle groups

OBJECTIVE: A recent neuroanatomical staging scheme of amyotrophic lateral sclerosis (ALS) indicates that a cortical lesion may spread, as a network disorder, both at the cortical level and via corticofugal tracts, including corticospinal projections providing direct monosynaptic input to α-motoneurons. These projections are involved preferentially and early in ALS. If these findings are clinically relevant, the pattern of paresis in ALS should primarily involve those muscle groups that receive the strongest direct corticomotoneuronal (CM) innervation. METHODS: In a large cohort (N=436), we analysed retrospectively the pattern of muscle paresis in patients with ALS using the UK Medical Research Council (MRC) scoring system; we subsequently carried out two independent prospective studies in two smaller groups (N=92 and N=54). RESULTS: The results indicated that a characteristic pattern of paresis exists. When pairs of muscle groups were compared within patients, the group known to receive the more pronounced CM connections was significantly weaker. Within patients, there was greater relative weakness (lower MRC score) in thumb abductors versus elbow extensors, for hand extensors versus hand flexors and for elbow flexors versus elbow extensors. In the lower limb, knee flexors were relatively weaker than extensors, and plantar extensors were weaker than plantar flexors. CONCLUSIONS: These findings were mostly significant (p<0.01) for all six pairs of muscles tested and provide indirect support for the concept that ALS may specifically affect muscle groups with strong CM connections. This specific pattern could help to refine clinical and electrophysiological ALS diagnostic criteria and complement prospective clinicopathological correlation studies

Implementation of a population-based epidemiological rare disease registry: study protocol of the amyotrophic lateral sclerosis (ALS) - registry Swabia

BACKGROUND: The social and medical impact of rare diseases is increasingly recognized. Amyotrophic lateral sclerosis (ALS) is the most prevalent of the motor neuron diseases. It is characterized by rapidly progressive damage to the motor neurons with a survival of 2–5 years for the majority of patients. The objective of this work is to describe the study protocol and the implementation steps of the amyotrophic lateral sclerosis (ALS) registry Swabia, located in the South of Germany. METHODS/DESIGN: The ALS registry Swabia started in October 2010 with both, the retrospective (01.10.2008-30.09.2010) and prospective (from 01.10.2010) collection of ALS cases, in a target population of 8.6 million persons in Southern Germany. In addition, a population based case–control study was implemented based on the registry that also included the collection of various biological materials. Retrospectively, 420 patients (222 men and 198 women) were identified. Prospectively data of ALS patients were collected, of which about 70% agreed to participate in the population-based case–control study. All participants in the case–control study provided also a blood sample. The prospective part of the study is ongoing. DISCUSSION: The ALS registry Swabia has been implemented successfully. In rare diseases such as ALS, the collaboration of registries, the comparison with external samples and biorepositories will facilitate to identify risk factors and to further explore the potential underlying pathophysiological mechanisms

Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras

Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the α-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy

A Locked Nucleic Acid Antisense Oligonucleotide (LNA) Silences PCSK9 and Enhances LDLR Expression In Vitro and In Vivo

The proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in the etiology of familial hypercholesterolemia (FH) and is also an attractive therapeutic target to reduce low density lipoprotein (LDL) cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR) and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol.The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA) antisense oligonucleotide (LNA ASO) that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT) levels revealed that long term LNA ASO treatment (7 weeks) does not cause hepatotoxicity.LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome

Prognosis for patients with amyotrophic lateral sclerosis: development and validation of a personalised prediction model

Summary Background Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive, fatal motor neuron disease with a variable natural history. There are no accurate models that predict the disease course and outcomes, which complicates risk assessment and counselling for individual patients, stratification of patients for trials, and timing of interventions. We therefore aimed to develop and validate a model for predicting a composite survival endpoint for individual patients with ALS. Methods We obtained data for patients from 14 specialised ALS centres (each one designated as a cohort) in Belgium, France, the Netherlands, Germany, Ireland, Italy, Portugal, Switzerland, and the UK. All patients were diagnosed in the centres after excluding other diagnoses and classified according to revised El Escorial criteria. We assessed 16 patient characteristics as potential predictors of a composite survival outcome (time between onset of symptoms and non-invasive ventilation for more than 23 h per day, tracheostomy, or death) and applied backward elimination with bootstrapping in the largest population-based dataset for predictor selection. Data were gathered on the day of diagnosis or as soon as possible thereafter. Predictors that were selected in more than 70% of the bootstrap resamples were used to develop a multivariable Royston-Parmar model for predicting the composite survival outcome in individual patients. We assessed the generalisability of the model by estimating heterogeneity of predictive accuracy across external populations (ie, populations not used to develop the model) using internal–external cross-validation, and quantified the discrimination using the concordance (c) statistic (area under the receiver operator characteristic curve) and calibration using a calibration slope. Findings Data were collected between Jan 1, 1992, and Sept 22, 2016 (the largest data-set included data from 1936 patients). The median follow-up time was 97·5 months (IQR 52·9–168·5). Eight candidate predictors entered the prediction model: bulbar versus non-bulbar onset (univariable hazard ratio [HR] 1·71, 95% CI 1·63–1·79), age at onset (1·03, 1·03–1·03), definite versus probable or possible ALS (1·47, 1·39–1·55), diagnostic delay (0·52, 0·51–0·53), forced vital capacity (HR 0·99, 0·99–0·99), progression rate (6·33, 5·92–6·76), frontotemporal dementia (1·34, 1·20–1·50), and presence of a C9orf72 repeat expansion (1·45, 1·31–1·61), all p<0·0001. The c statistic for external predictive accuracy of the model was 0·78 (95% CI 0·77–0·80; 95% prediction interval [PI] 0·74–0·82) and the calibration slope was 1·01 (95% CI 0·95–1·07; 95% PI 0·83–1·18). The model was used to define five groups with distinct median predicted (SE) and observed (SE) times in months from symptom onset to the composite survival outcome: very short 17·7 (0·20), 16·5 (0·23); short 25·3 (0·06), 25·2 (0·35); intermediate 32·2 (0·09), 32·8 (0·46); long 43·7 (0·21), 44·6 (0·74); and very long 91·0 (1·84), 85·6 (1·96). Interpretation We have developed an externally validated model to predict survival without tracheostomy and non-invasive ventilation for more than 23 h per day in European patients with ALS. This model could be applied to individualised patient management, counselling, and future trial design, but to maximise the benefit and prevent harm it is intended to be used by medical doctors only. Funding Netherlands ALS Foundation