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INTRODUCTION: An effective fight against cholera requires an in-depth consideration of the knowledge, attitudes and social representations of cholera within a population. Cholera outbreaks persist in the Extreme North of Cameroon because of the inadequate integration of representations of cholera, water and hygiene in the fight against this disease. Through a constructivist intercultural approach not conflicting with the western ethnocentric model, socio-cultural/religious and historical ideologies can be reconciled to provide optimal and sustainable healthcare solutions to the repeated and long lasting cholera epidemics using participative research, intercultural mediation and dialogue in Cameroon. METHODS: Through a cross-sectional, ethnographic and participative study, data was generated using semi-directed in-depth interviews of key informants, collection of videos, pictures and the completion of 2 pre-tested questionnaire types in 3 communities (Maroua I, Maroua II and Mokolo). Quantitative data was entered using Ms Excel and Epi Info 7, and analysed using Epi Info 7. Qualitative data was analysed inductively using the concept of social representations. RESULTS: Results show evidence of the inadequate integration of cultural and socio-cultural factors favouring cholera spread and a respondent population majority unable to identify this (92.82%). Equally identifying environmental and cultural factors, the results bring out the impact of the on-going cholera combating strategy.CONCLUSION: Representations of cholera, cultural and socio-cultural values are not adequately considered in the fight against cholera. We recommend policy-makers and health actors to improve on the integration of these through advocacy, in designing, communicating and implementing effective prevention strategies via participative research, intercultural mediation and dialogue.

Background: Diagnosis of acute ischemia typically relies on evidence of ischemic lesions on magnetic resonance imaging (MRI), a limited diagnostic resource. We aimed to determine associations of clinical variables and acute infarcts on MRI in patients with suspected low-risk transient ischemic attack (TIA) and minor stroke and to assess their predictive ability., Methods: We conducted a post-hoc analysis of the Diagnosis of Uncertain-Origin Benign Transient Neurological Symptoms (DOUBT) study, a prospective, multicenter cohort study investigating the frequency of acute infarcts in patients with low-risk neurological symptoms. Primary outcome parameter was defined as diffusion-weighted imaging (DWI)-positive lesions on MRI. Logistic regression analysis was performed to evaluate associations of clinical characteristics with MRI-DWI-positivity. Model performance was evaluated by Harrel's c-statistic., Results: In 1028 patients, age (Odds Ratio (OR) 1.03, 95% Confidence Interval (CI) 1.01-1.05), motor (OR 2.18, 95%CI 1.27-3.65) or speech symptoms (OR 2.53, 95%CI 1.28-4.80), and no previous identical event (OR 1.75, 95%CI 1.07-2.99) were positively associated with MRI-DWI-positivity. Female sex (OR 0.47, 95%CI 0.32-0.68), dizziness and gait instability (OR 0.34, 95%CI 0.14-0.69), normal exam (OR 0.55, 95%CI 0.35-0.85) and resolved symptoms (OR 0.49, 95%CI 0.30-0.78) were negatively associated. Symptom duration and any additional symptoms/symptom combinations were not associated. Predictive ability of the model was moderate (c-statistic 0.72, 95%CI 0.69-0.77)., Conclusion: Detailed clinical information is helpful in assessing the risk of ischemia in patients with low-risk neurological events, but a predictive model had only moderate discriminative ability. Patients with clinically suspected low-risk TIA or minor stroke require MRI to confirm the diagnosis of cerebral ischemia.

Background: Magnetic resonance imaging infarct topography may assist with determining stroke etiology. The influence of diffusion-weighted imaging (DWI)-positive lesions on etiology determination in patients with transient ischemic attack or minor stroke is not well studied., Methods and Results: We prospectively enrolled patients between 2010 and 2017 in 2 studies; participants with a final diagnosis of probable or definite transient ischemic attack or stroke were pooled for analysis. The primary outcome was the adjudicated ischemic etiology. We compared proportion of each etiology (cardioembolic, large-vessel, small-vessel disease, other) in patients who had DWI positivity compared with DWI negativity. We used logistic regression to determine the adjusted odds ratio (OR) for each etiology compared with undetermined by DWI positivity. The final analysis included 1498 patients: 832 (55.5%) were DWI-positive. DWI-positive patients were more likely to be diagnosed with small-vessel disease (19.1% versus 5.3%) and less likely with undetermined etiology (36.9% versus 53.0%; P <0.001). After adjustment, the presence of any DWI lesion was associated with increased odds of assigning any etiology (OR, 1.8 [95% CI, 1.3-2.5]). A single DWI lesion was associated with increased odds of small-vessel disease diagnosis (OR, 9.5 [95% CI, 6.4-14.0]), and multiple DWI lesions with reduced odds of small-vessel disease (OR, 0.2 [95% CI, 0.1-0.4]) but increased odds of all other etiologies compared with undetermined etiology., Conclusions: Any DWI-positive lesion after suspected transient ischemic attack or minor stroke was associated with increased odds of assigning a etiology. Presence and topography of DWI lesions on magnetic resonance imaging may assist with etiology determination and may impact stroke prevention therapies.

Background and Objective: The association between focal vs nonfocal presenting symptom and diffusion-weighted imaging (DWI) positivity in relation to onset-to-imaging time in patients with transient neurologic events remains unclear. We hypothesize that episodes consisting of focal symptoms would have proportionally higher DWI-positive imaging at later onset-to-imaging times., Methods: Patients with transient neurologic symptoms and a normal neurologic examination who had DWI in the combined data set of 3 cohort studies were included. We used logistic regression models to evaluate the association between each type of presenting symptom (motor weakness, speech impairment, sensory symptoms, vision loss, diplopia, gait instability, dizziness, headache, presyncope, and amnesia) and DWI positivity after adjusting for clinical variables (age, sex, history of stroke, dyslipidemia, coronary artery disease, atrial fibrillation, symptoms duration [<10, 10-59, ≥60 minutes, or unclear], and study source). We stratified the results by onset-to-imaging time categories (<6 hours, 6-23 hours, and ≥24 hours)., Results: Of the total 2,411 patients (1,345 male, median age 68 years), DWI-positive lesions were detected in 598 patients (24.8%). The prevalence of DWI positivity was highest in those with motor weakness (34.7%), followed by speech impairment (33.5%). In a multivariable analysis, the presence of motor weakness, speech impairment, and sensory symptoms was associated with DWI positivity, while vision loss and headache were associated with lower odds of DWI positivity, but nevertheless had 13.6% and 15.3% frequency of DWI positive. The odds of being DWI positive varied by onset-to-imaging time categories for motor weakness, with greater odds of being DWI positive at later imaging time (<6 hours: odds ratio [OR] 1.25, 95% confidence interval [CI] 0.84-1.87; 6-23 hours: OR 2.24, 95% CI 1.47-3.42; and ≥24 hours: OR 2.42, 95% CI 1.74-3.36; interaction p = 0.033). Associations of other symptoms with DWI positivity did not vary significantly by time categories., Discussion: We found that onset-to-imaging time influences the relationship between motor weakness and DWI positivity in patients with transient neurologic events. Compared with motor, speech, and sensory symptoms, visual or nonfocal symptoms carry a lower but still a substantive association with DWI positivity.

Background The relationship between duration of transient neurological events and presence of diffusion-weighted lesions by symptom type is unclear. Methods and Results This was a substudy of SpecTRA (Spectrometry for Transient Ischemic Attack Rapid Assessment), a multicenter prospective cohort of patients with minor ischemic cerebrovascular events or stroke mimics at academic emergency departments in Canada. For this study we included patients with resolved symptoms and determined the presence of diffusion-weighted imaging (DWI) lesion on magnetic resonance imaging within 7 days. Using logistic regression, we evaluated the association between symptom duration and DWI lesion, assessing for interaction with symptom type (focal only versus nonfocal/mixed), and adjusting for age, sex, education, comorbidities, and systolic blood pressure. Of 658 patients included, a DWI lesion was present in 232 (35.1%). There was a significant interaction between symptom duration and symptom type. For those with focal-only symptoms, there was a continuous increase in DWI probability up to 24 hours in duration (ranging from ≈40% to 80% probability). In stratified analyses, the increase in probability of DWI lesion with increased duration of focal symptoms was seen in women but not men. For those with nonfocal or mixed symptoms, predicted probability of DWI lesion was ≈35% and was greater in men, but did not increase with longer duration. Conclusions Increased duration of neurological deficits is associated with greater probability of DWI lesion in those with focal symptoms only. For individuals with nonfocal or mixed symptoms, about one-third had DWI lesions, but the probability did not increase with duration. These results may be important to improve risk stratification of transient neurological events.

Objective: To describe sex differences in the presentation, diagnosis, and revision of diagnosis after early brain MRI in patients who present with acute transient or minor neurologic events., Methods: We performed a secondary analysis of a prospective multicenter cohort study of patients referred to neurology between 2010 and 2016 with a possible cerebrovascular event and evaluated with brain MRI within 8 days of symptom onset. Investigators documented the characteristics of the event, initial diagnosis, and final diagnosis. We used multivariable logistic regression analyses to evaluate the association between sex and outcomes., Results: Among 1,028 patients (51% women, median age 63 years), more women than men reported headaches and fewer reported chest pain, but there were no sex differences in other accompanying symptoms. Women were more likely than men to be initially diagnosed with stroke mimic (54% of women vs 42% of men, adjusted odds ratio (OR) 1.60, 95% confidence interval [CI] 1.24-2.07), and women were overall less likely to have ischemia on MRI (10% vs 17%, OR 0.52, 95% CI 0.36-0.76). Among 496 patients initially diagnosed with mimic, women were less likely than men to have their diagnosis revised to minor stroke or TIA (13% vs 20%, OR 0.53, 95% CI 0.32-0.88) but were equally likely to have acute ischemia on MRI (5% vs 8%, OR 0.56, 95% CI 0.26-1.21)., Conclusions: Stroke mimic was more frequently diagnosed in women than men, but diagnostic revisions were common in both. Early brain MRI is a useful addition to clinical evaluation in diagnosing transient or minor neurologic events., (© 2020 American Academy of Neurology.)

Introduction: Biospecimens and associated data are invaluable tools in Genomics and Personalized Health (GAPH) research and can aid in the discovery of disease etiology and the development of therapeutics. Objective: To examine the experiences of patients invited to a particular GAPH study, Spectrometry in TIA Rapid Assessment (SpecTRA), and to explore broader biospecimen and data sharing preferences among a larger group of patients who had opted into a Permission to Contact for research program. Methods: An electronic survey was e-mailed to 515 participants. The survey was completed by 38% of participants, an unspecified number of whom were also SpecTRA participants. Results: Of those respondents who recalled participating in SpecTRA, 96% strongly agreed, agreed, or were neutral when asked if they received enough information to make an informed decision. Seventy-two percent agreed and 20% were neutral when asked if their study questions were addressed. Ninety-six percent of all respondents felt that SpecTRA's aim to develop a proteomic test for stroke was a worthwhile investment for health care, 98% said they were willing to provide a sample and/or information to facilitate the project's goals, and 96% to health research in general. Fifty-three percent of all participants suggested they would be comfortable sharing health information collected during SpecTRA with for-profit organizations, 87% with nonprofit organizations, and 38% said it matters to them where in the world their sample/information would be sent. Conclusions: Our results suggest that while there is room for improvement in providing adequate information to enable participants' understanding of the purpose of GAPH studies such as SpecTRA, patients are supportive of GAPH in general. Results also suggest that willingness to participate would likely be impacted by factors such as the study's commercial and national affiliations. This study indicates that further work is required to guide improvements on how the GAPH research community describes studies to potential participants, and to enable participation options that incorporate variable participant preferences.

Background and Purpose- Acute minor neurological deficits are a common complaint in the emergency department and differentiation of transient ischemic attack/minor stroke from a stroke mimic is difficult. We sought to assess the ability of white matter hyperintensity (WMH) volume to aid the diagnosis in such patients. Methods- This is a post hoc analysis of the previously published SpecTRA study (Spectrometry in TIA Rapid Assessment) of adult patients that presented to the emergency department with acute minor neurological deficits between December 2013 and March 2017. WMH volumes were measured if fluid-attenuated inversion recovery imaging was available. Outcomes of interest were final diagnosis, symptoms at presentation, and 90-day stroke recurrence. Results- WMH volume was available for 1485 patients. Median age was 70 years (interquartile range, 59-80), and 46.7% were female. Mean WMH volume was higher in transient ischemic attack/minor strokes compared with stroke mimics (1.71 ln mL [95% CI, 1.63-1.79 ln mL] versus 1.15 ln mL [95% CI, 1.02-1.27 ln mL], P <0.001). In multivariable-adjusted logistic regression analysis, WMH volume was not associated with final diagnosis. However, the combination of both diffusion-weighted imaging positivity and high WMH volume led to lower odds of focal symptoms at presentation ( P =0.035). Conclusions- The combination of diffusion-weighted imaging positivity and high WMH volume was associated with lower odds of focal symptoms at presentation in patients seen with minor neurological deficits in the emergency department. This suggests that WMH volume might be an important consideration and the absence of focal symptoms at presentation should not discourage clinicians from further investigating patients with suspected cerebral ischemia.

Importance: Early treatment of patients with transient ischemic attack (TIA) reduces the risk of stroke. However, many patients present with symptoms that have an uncertain diagnosis. Patients with motor, speech, or prolonged symptoms are at the highest risk for recurrent stroke and the most likely to undergo comprehensive investigations. Lower-risk patients are much more likely to be cursorily investigated., Objective: To establish the frequency of acute infarct defined by diffusion restriction detected on diffusion-weighted imaging (DWI) magnetic resonance imaging (MRI) scan (DWI positive)., Design, Setting, and Participants: The Diagnosis of Uncertain-Origin Benign Transient Neurological Symptoms (DOUBT) study was a prospective, observational, international, multicenter cohort study of 1028 patients with low-risk transient or minor symptoms referred to neurology within 8 days of symptom onset. Patients were enrolled between June 1, 2010, and October 31, 2016. Included patients were 40 years or older and had experienced nonmotor or nonspeech minor focal neurologic events of any duration or motor or speech symptoms of short duration (≤5 minutes), with no previous stroke., Exposures: Patients underwent a detailed neurologic assessment prior to undergoing a brain MRI within 8 days of symptom onset., Main Outcomes and Measures: The primary outcome was restricted diffusion on a brain MRI scan (acute stroke)., Results: A total of 1028 patients (522 women and 506 men; mean [SD] age, 63.0 [11.6] years) were enrolled. A total of 139 patients (13.5%) had an acute stroke as defined by diffusion restriction detected on MRI scans (DWI positive). The final diagnosis was revised in 308 patients (30.0%) after undergoing brain MRI. There were 7 (0.7%) recurrent strokes at 1 year. A DWI-positive brain MRI scan was associated with an increased risk of recurrent stroke (relative risk, 6.4; 95% CI, 2.4-16.8) at 1 year. Absence of a DWI-positive lesion on a brain MRI scan had a 99.8% negative predictive value for recurrent stroke. Factors associated with MRI evidence of stroke in multivariable modeling were older age (odds ratio [OR], 1.02; 95% CI, 1.00-1.04), male sex (OR, 2.03; 95% CI, 1.39-2.96), motor or speech symptoms (OR, 2.12; 95% CI, 1.37-3.29), ongoing symptoms at assessment (OR, 1.97; 95% CI, 1.29-3.02), no prior identical symptomatic event (OR, 1.87; 95% CI, 1.12-3.11), and abnormal results of initial neurologic examination (OR, 1.71; 95% CI, 1.11-2.65)., Conclusions and Relevance: This study suggested that patients with transient ischemic attack and symptoms traditionally considered low risk carry a substantive risk of acute stroke as defined by diffusion restriction (DWI positive) on a brain MRI scan. Early MRI is required to make a definitive diagnosis.

Background: Elevated blood pressure (BP) at emergency department (ED) presentation and advancing age have been associated with risk of ischemic stroke; however, the relationship between BP, age, and transient ischemic attack/minor stroke (TIA/MS) is not clear., Methods: A multi-site, prospective, observational study of 1084 ED patients screened for suspected TIA/MS (symptom onset < 24 h, NIHSS< 4) between December 2013 and April 2016. Systolic and diastolic BP measurements (SBP, DBP) were taken at ED presentation. Final diagnosis was consensus adjudication by stroke neurologists; patients were diagnosed as either TIA/MS or stroke-mimic (non-cerebrovascular conditions). Conditional inference trees were used to define age cut-points for predicting binary diagnosis (TIA/MS or stroke-mimic). Logistic regression models were used to estimate the effect of BP, age, sex, and the age-BP interaction on predicting TIA/MS diagnosis., Results: Over a 28-month period, 768 (71%) patients were diagnosed with TIA/MS: these patients were older (mean 71.6 years) and more likely to be male (58%) than stroke-mimics (61.4 years, 41%; each p < 0.001). TIA/MS patients had higher SBP than stroke-mimics (p < 0.001). DBP did not differ between the two groups (p = 0.191). SBP was predictive of TIA/MS diagnosis in younger patients, after accounting for age and sex; an increase of 10 mmHg systolic increased the odds of TIA/MS 18% (odds ratio [OR] 1.18, 95% CI 1.00-1.39) in patients < 60 years, and 23% (OR 1.23, 95% CI 11.12-1.35) in those 60-79 years, while not affecting the odds of TIA/MS in patients ≥80 years (OR 0.99, 95% CI 0.89-1.07)., Conclusions: Raised SBP in patients younger than 80 with suspected TIA/MS may be a useful clinical indicator upon initial presentation to help increase clinicians' suspicion of TIA/MS., Trial Registration: ClinicalTrials.gov NCT03050099 (10-Feb-2017) and NCT03070067 (3-Mar-2017). Retrospectively registered.

We validate our previously developed (DOI: 10.1101/089227) clinical prediction rule for diagnosing transient ischemic attack on the basis of presenting clinical symptoms and compare its performance with the ABCD2 score in first-contact patient settings. Two independent and prospectively collected patient validation cohorts were used: (a) referral cohort-prospectively referred emergency department and general practitioner patients ( N = 877); and (b) SpecTRA cohort-participants recruited as part of the SpecTRA biomarker project ( N = 545). Outcome measure consisted of imaging-confirmed clinical diagnosis of mild stroke/transient ischemic attack. Results showed that our clinical prediction rule demonstrated significantly higher accuracy than the ABCD2 score for both the referral cohort (70.5% vs 59.0%; p < 0.001) and SpecTRA cohort (72.8% vs 68.3%; p = 0.028). We discuss the potential of our clinical prediction rule to replace the use of the ABCD2 score in the triage of transient ischemic attack clinic referrals.

Importance: Sex differences have been described in the presentation, care, and outcomes among people with acute ischemic strokes, but these differences are less understood for minor ischemic cerebrovascular events. The present study hypothesized that, compared with men, women are more likely to report nonfocal symptoms and to receive a stroke mimic diagnosis., Objective: To evaluate sex differences in the symptoms, diagnoses, and outcomes of patients with acute transient or minor neurologic events., Design, Setting, and Participants: This prospective cohort study of patients with minor ischemic cerebrovascular events or stroke mimics enrolled at multicenter academic emergency departments in Canada between December 2013 and March 2017 and followed up for 90 days is a substudy of SpecTRA (Spectrometry for Transient Ischemic Attack Rapid Assessment). In total, 1729 consecutive consenting patients with acute transient or minor neurologic symptoms were referred for neurologic evaluation; 66 patients were excluded for protocol violation (n = 46) or diagnosis of transient global amnesia (n = 20)., Exposures: The main exposure was female or male sex., Main Outcomes and Measures: The main outcome was the clinical diagnosis (cerebral ischemia vs stroke mimic). Secondary outcomes were 90-day stroke recurrence and 90-day composite outcome of stroke, myocardial infarction, or death. The association between presenting symptoms (focal vs nonfocal) and clinical diagnosis was also assessed. Research hypotheses were formulated after data collection., Results: Of 1648 patients included, 770 (46.7%) were women, the median (interquartile range) age was 70 (59-80) years, 1509 patients (91.6%) underwent brain magnetic resonance imaging, and 1582 patients (96.0%) completed the 90-day follow-up. Women (522 of 770 [67.8%]) were less likely than men (674 of 878 [76.8%]) to receive a diagnosis of cerebral ischemia (adjusted risk ratio [aRR], 0.88; 95% CI, 0.82-0.95), but the 90-day stroke recurrence outcome (aRR, 0.90; 95% CI, 0.48-1.66) and 90-day composite outcome (aRR, 0.86; 95% CI, 0.54-1.32) were similar for men and women. No significant sex differences were found for presenting symptoms. Compared with patients with no focal neurologic symptoms, those with focal and nonfocal symptoms were more likely to receive a diagnosis of cerebral ischemia (aRR, 1.28; 95% CI, 1.15-1.39), but the risk was highest among patients with focal symptoms only (aRR, 1.45; 95% CI, 1.34-1.53). Sex did not modify these associations., Conclusions and Relevance: The results of the present study suggest that, despite similar presenting symptoms among men and women, women may be more likely to receive a diagnosis of stroke mimic, but they may not have a lower risk than men of subsequent vascular events, indicating potentially missed opportunities for prevention of vascular events among women.

Objectives: The Canadian Stroke Best Practice Recommendations suggests that patients suspected of transient ischemic attack (TIA)/minor stroke receive urgent brain imaging, preferably computed tomography angiography (CTA). Yet, high requisition rates for non-cerebrovascular patients overburden limited radiological resources, putting patients at risk. We hypothesize that our clinical decision support tool (CDST) developed for risk stratification of TIA in the emergency department (ED), and which incorporates Canadian guidelines, could improve CTA utilization., Methods: Retrospective study design with clinical information gathered from ED patient referrals to an outpatient TIA unit in Victoria, BC, from 2015-2016. Actual CTA orders by ED and TIA unit staff were compared to hypothetical CTA ordering if our CDST had been used in the ED upon patient arrival., Results: For 1,679 referrals, clinicians ordered 954 CTAs. Our CDST would have ordered a total of 977 CTAs for these patients. Overall, this would have increased the number of imaged-TIA patients by 89 (10.1%) while imaging 98 (16.1%) fewer non-cerebrovascular patients over the 2-year period. Our CDST would have ordered CTA for 18 (78.3%) of the recurrent stroke patients in the sample., Conclusions: Our CDST could enhance CTA utilization in the ED for suspected TIA patients, and facilitate guideline-based stroke care. Use of our CDST would increase the number of TIA patients receiving CTA before ED discharge (rather than later at TIA units) and reduce the burden of imaging stroke mimics in radiological departments.

A diagnostic blood test for stroke is desirable but will likely require multiple proteins rather than a single "troponin." Validating large protein panels requires large patient numbers. Mass spectrometry (MS) is a cost-effective tool for this task. We compared differences in the abundance of 147 protein markers to distinguish 20 acute cerebrovascular syndrome (ACVS) patients who presented to the Emergency Department of one urban hospital within < 24 h from onset) and from 20 control patients who were enrolled via an outpatient neurology clinic. We targeted proteins from the stroke literature plus cardiovascular markers previously studied in our lab. One hundred forty-one proteins were quantified using MS, 8 were quantified using antibody protein enrichment with MS, and 32 were measured using ELISA, with some proteins measured by multiple techniques. Thirty proteins (4 by ELISA and 26 by the MS techniques) were differentially abundant between mimic and stroke after adjusting for age in robust regression analyses (FDR < 0.20). A logistic regression model using the first two principal components of the proteins significantly improved discrimination between strokes and controls compared to a model based on age alone (p < 0.001, cross-validated AUC 0.93 vs. 0.78). Significant proteins included markers of inflammation (47%), coagulation (40%), atrial fibrillation (7%), neurovascular unit injury (3%), and other (3%). These results suggest the potential value of plasma proteins as biomarkers for ACVS diagnosis and the role of plasma-based MS in this area.

Objective: To validate our previously developed 16 plasma-protein biomarker panel to differentiate between transient ischaemic attack (TIA) and non-cerebrovascular emergency department (ED) patients., Method: Two consecutive cohorts of ED patients prospectively enrolled at two urban medical centers into the second phase of SpecTRA study (training, cohort 2A, n = 575; test, cohort 2B, n = 528). Plasma samples were analyzed using liquid chromatography/multiple reaction monitoring-mass spectrometry. Logistic regression models which fit cohort 2A were validated on cohort 2B., Results: Three of the panel proteins failed quality control and were removed from the panel. During validation, panel models did not outperform a simple motor/speech (M/S) deficit variable. Post-hoc analyses suggested the measured behaviour of L-selectin and coagulation factor V contributed to poor model performance. Removal of these proteins increased the external performance of a model containing the panel and the M/S variable., Conclusions: Univariate analyses suggest insulin-like growth factor-binding protein 3 and serum paraoxonase/lactonase 3 are reliable and reproducible biomarkers for TIA status. Logistic regression models indicated L-selectin, apolipoprotein B-100, coagulation factor IX, and thrombospondin-1 to be significant multivariate predictors of TIA. We discuss multivariate feature subset analyses as an exploratory technique to better understand a panel's full predictive potential.

Objective: To derive a plasma biomarker protein panel from a list of 141 candidate proteins which can differentiate transient ischaemic attack (TIA)/minor stroke from non-cerebrovascular (mimic) conditions in emergency department (ED) settings., Design: Prospective clinical study (#NCT03050099) with up to three timed blood draws no more than 36 h following symptom onset. Plasma samples analysed by multiple reaction monitoring-mass spectrometry (MRM-MS)., Participants: Totally 545 participants suspected of TIA enrolled in the EDs of two urban medical centres., Outcomes: 90-day, neurologist-adjudicated diagnosis of TIA informed by clinical and radiological investigations., Results: The final protein panel consists of 16 proteins whose patterns show differential abundance between TIA and mimic patients. Nine of the proteins were significant univariate predictors of TIA [odds ratio (95% confidence interval)]: L-selectin [0.726 (0.596-0.883)]; Insulin-like growth factor-binding protein 3 [0.727 (0.594-0.889)]; Coagulation factor X [0.740 (0.603-0.908)]; Serum paraoxonase/lactonase 3 [0.763 (0.630-0.924)]; Thrombospondin-1 [1.313 (1.081-1.595)]; Hyaluronan-binding protein 2 [0.776 (0.637-0.945)]; Heparin cofactor 2 [0.775 (0.634-0.947)]; Apolipoprotein B-100 [1.249 (1.037-1.503)]; and von Willebrand factor [1.256 (1.034-1.527)]. The scientific plausibility of the panel proteins is discussed., Conclusions: Our panel has the potential to assist ED physicians in distinguishing TIA from mimic patients.

Background: To evaluate the performance of a novel triage system for Transient Ischemic Attack (TIA) units built upon an existent clinical prediction rule (CPR) to reduce time to unit arrival, relative to the time of symptom onset, for true TIA and minor stroke patients. Differentiating between true and false TIA/minor stroke cases (mimics) is necessary for effective triage as medical intervention for true TIA/minor stroke is time-sensitive and TIA unit spots are a finite resource., Methods: Prospective cohort study design utilizing patient referral data and TIA unit arrival times from a regional fast-track TIA unit on Vancouver Island, Canada, accepting referrals from emergency departments (ED) and general practice (GP). Historical referral cohort (N = 2942) from May 2013-Oct 2014 was triaged using the ABCD2 score; prospective referral cohort (N = 2929) from Nov 2014-Apr 2016 was triaged using the novel system. A retrospective survival curve analysis, censored at 28 days to unit arrival, was used to compare days to unit arrival from event date between cohort patients matched by low (0-3), moderate (4-5) and high (6-7) ABCD2 scores., Results: Survival curve analysis indicated that using the novel triage system, prospectively referred TIA/minor stroke patients with low and moderate ABCD2 scores arrived at the unit 2 and 1 day earlier than matched historical patients, respectively., Conclusions: The novel triage process is associated with a reduction in time to unit arrival from symptom onset for referred true TIA/minor stroke patients with low and moderate ABCD2 scores.

Multiple reaction monitoring mass spectrometry (MRM-MS) is an emerging technology for blood biomarker verification and validation; however, the results may be influenced by pre-analytical factors. This exploratory study was designed to determine if differences in phlebotomy techniques would significantly affect the abundance of plasma proteins in an upcoming biomarker development study. Blood was drawn from 10 healthy participants using four techniques: (1) a 20-gauge IV with vacutainer, (2) a 21-gauge direct vacutainer, (3) an 18-gauge butterfly with vacutainer, and (4) an 18-gauge butterfly with syringe draw. The abundances of a panel of 122 proteins (117 proteins, plus 5 matrix metalloproteinase (MMP) proteins) were targeted by LC/MRM-MS. In addition, complete blood count (CBC) data were also compared across the four techniques. Phlebotomy technique significantly affected 2 of the 11 CBC parameters (red blood cell count, p = 0.010; hemoglobin concentration, p = 0.035) and only 12 of the targeted 117 proteins (p < 0.05). Of the five MMP proteins, only MMP7 was detectable and its concentration was not significantly affected by different techniques. Overall, most proteins in this exploratory study were not significantly influenced by phlebotomy technique; however, a larger study with additional patients will be required for confirmation.

Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIII-deficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A.

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

Adenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.

Kearns-Sayre syndrome is the most commonly diagnosed mitochondrial cytopathy and produces severe neuromuscular symptoms. The most frequent cause is a mitochondrial DNA deletion that removes a 4977-base pair segment of DNA that includes several genes encoding for respiratory chain subunits. Treatment of AIDS patients with nucleoside analogs has been reported to cause mtDNA depletion and myopathies. Here, we report that azidothymidine, dideoxyguanosine, and dideoxycytidine cause a depletion of wild-type mtDNA while increasing the levels of deleted mitochondria DNA in Kearns-Sayre syndrome fibroblasts. The result of these effects is a large increase in the relative amounts of delta mtDNA in comparison to wild type mtDNA. We found that Kearns-Sayre syndrome fibroblasts are a mixed population of cells with deleted mtDNA comprising from 0 to over 20% of the total mtDNA in individual cells. Treatment of cloned cell lines with dideoxycytidine did not result in increased levels of delta mtDNA. The results suggest that nucleoside analogs may act to increase the average delta mtDNA levels in a mixed population of cells by preferentially inhibiting the proliferation of cells with little or no delta mtDNA. This raises the possibility that modulation of deleted mtDNA levels may occur by similar mechanisms in vivo, in response to the influence of exogenous agents.

We have identified a 15-bp microdeletion in a highly conserved region of the mitochondrially encoded gene for cytochrome c oxidase (COX) subunit III in a patient with severe isolated COX deficiency and recurrent myoglobinuria. The mutant mitochondrial DNA (mtDNA) comprised 92% of the mtDNA in muscle and 0.7% in leukocytes. Immunoblots and immunocytochemistry suggested a lack of assembly or instability of the complex. Microdissected muscle fibres revealed significantly higher portions of mutant mtDNA in COX-negative than in COX-positive fibres. This represents the first case of isolated COX deficiency to be defined at the molecular level.

Objective: To explore mitochondrial dysfunction in Parkinson's disease (PD) using 31P magnetic resonance spectroscopy of resting muscle., Design: Case-control study (28 PD patients and 28 normal controls) determining resting forearm inorganic phosphate/phosphocreatine (Pi/PCr) ratio., Results: Significant difference (p = 0.004, one-tailed test) in Pi/PCr ratio between PD patients (0.122) and controls (0.104). No correlation of Pi/PCr ratio with duration, severity, or speed of onset of disease. Positive correlation of Pi/PCr ratio with age in control group; reversed in PD group., Conclusions: Suggests small generalized mitochondrial defect in PD. The possibility that earlier onset of disease is associated with more severe mitochondrial dysfunction needs further study.

We report a patient with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes treated with riboflavin and nicotinamide for 18 months, during which time previously frequent encephalopathic spells ceased. To confirm clinical benefit, we withdrew treatment and monitored response with muscle 31P magnetic resonance spectroscopy (MRS) and sural nerve conduction studies. Of three prospectively chosen MRS variables, two changed coincidentally with clinical end points; phosphocreatine (PCr)/adenosine triphosphate recovery rates fell in parallel with sural nerve sensory amplitudes, and a drop in muscle bioenergetic efficiency (relationship of inorganic phosphate/PCr to the accelerating force of contracting muscle) coincided with development of encephalopathy. Investigations revealed a deficiency of respiratory complex I and mutation of the mitochondrial tRNA(Leu)(UUR). We suggest that a defective cellular energy state in mitochondrial disease may be partially treatable and that changes seen in appropriate muscle spectroscopy studies may parallel improvement in brain and peripheral nerve function.

To determine the relative metabolic efficiency (metabolic energy used per unit of mechanical energy output) of negative to positive muscular power, we used 31P magnetic resonance spectroscopy to monitor the cellular energy metabolism of limb muscles in eight healthy subjects during a nonfatiguing, mixed concentric-eccentric activity and during its concentric and eccentric components. We also studied isometric contractions. We found that in terms of the flow of metabolic energy through the muscle cells, the cost of concentric exercise at this intensity was proportional to the mechanical power generated, but the cost of eccentric and isometric exercise did not increase significantly as the apparent intensity of the exercise increased over the range studied. Although the pattern was similar in all subjects, the quantitative relationship between metabolic cost and mechanical output was different in subjects with different muscular strength. The qualitative results can be explained in the context of the known biochemistry and biophysics of the cellular contractile apparatus (sliding filament theory, with independent force generators).

Five patients from two unrelated pedigrees are affected by an inherited form or forms of mitochondrial encephalomyopathy in which the exact site of the block in the respiratory chain has yet to be identified. All five patients regularly exhibit an unusual aminoacidopathy evident both in fasting plasma and in CSF. Alanine concentrations are elevated, reflecting high tissue pyruvate and lactate levels. Concentrations of the four essential amino acids threonine, methionine, tryptophan and lysine are substantially reduced, as are those of citrulline, ornithine and arginine. This pattern of amino-acid deficiency is apparently not due to failure to absorb the dibasic amino acids, to any abnormality of the urea cycle, to excessive synthesis and turnover of creatine, or to protein malnutrition. The aminoacidopathy presumably is a metabolic consequence of one or more impairments in the electron transport chain in mitochondria. A detailed explanation of its aetiology needs to be sought.

We report two brothers with a previously undescribed type of mitochondrial encephalomyopathy and associated aminoacidopathy. Both have growth failure, progressive intellectual decline, deafness, neurologic dysfunction, exercise intolerance, lactic acidosis, and abnormal plasma and cerebrospinal fluid amino acid levels (elevated levels of alanine and low levels of threonine, methionine, citrulline, tryptophan, ornithine, arginine, and lysine). A muscle biopsy specimen taken from the younger, more severely affected brother showed abnormal mitochondrial morphology. Activities of the following enzymes in cultured fibroblasts from both boys were normal: pyruvate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, cytochrome oxidase, reduced nicotinamide-adenine dinucleotide-cytochrome c reductase, and succinate cytochrome c reductase. Fibroblast mitochondria from the younger boy showed undetectable (less than 1% of control values) adenosine triphosphate synthesis with pyruvate and malate, whereas adenosine triphosphate synthesis with succinate was 70% of control values. These data indicate probably deficient activity of complex I of the electron transport chain. The boys' mother has progressive neurosensory hearing loss; their sister is clinically normal. Both mother and sister have many of the biochemical abnormalities found in the boys. It is possible, but not proved, that this disorder is inherited through maternal mitochondria.