Effective Stakeholder Engagement for Collation, Analysis and Expansion of Antimicrobial Resistance (AMR) Data: A CAPTURA Experience.

BACKGROUND: An effective implementation of antimicrobial resistance (AMR) surveillance projects requires sustainable and multidisciplinary engagement with stakeholders from various backgrounds, interests and aims. The "Capturing Data on Antimicrobial resistance Patterns and Trends in Use in Regions of Asia" (CAPTURA) project, funded by the Fleming Fund, initially targeted 12 countries in South Asia (SA) and Southeast Asia (SEA) to "expand the volume of historical and current data on AMR and antimicrobial usage" and support local agencies through capacity building activities. METHODS: In this article, we focus on early stakeholder engagement activities and present overall statistics on AMR data collated from 72 laboratories across seven countries. This included 2.3 million records of antimicrobial susceptibility testing (AST) data, which were curated, analyzed, and shared back to the facilities for informed decision making. RESULTS: Approximately 98% of the data collated by CAPTURA originated from laboratories based in SA countries. Furthermore, country-wide data were analyzed to identify commonly reported pathogens in each country, followed by descriptions of AST practices and multidrug-resistant (MDR) pathogens. Overall, we found meager adherence to standard guidelines to perform and record AST results, and a significant number of MDR pathogens were reported. CONCLUSIONS: We conclude that close collaboration with the existing national mechanisms for identifying AMR data sources was crucial for the project's success. Although we show a vast retrospective dataset on AMR is available for data sharing in Asia, there remain critical gaps in data generation/management practice and analysis capacity for AMR data at most facilities.

CAPTURA Regional Workshop Proceedings (28-30 June 2022, Virtual).

In response to the global threat of antimicrobial resistance (AMR), the Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) project worked with microbiology laboratories, pharmacies, and local governments in South Asia and Southeast Asia to expand the volume of historical and current data available on AMR and antimicrobial use and to identify gaps in data and areas for quality improvement. When the CAPTURA project completed its country-level engagement in the first half of 2022, the consortium brought together local, regional, and global AMR stakeholders for a virtual regional workshop to review data outputs from the project and share strategies to inform national and regional efforts to combat AMR. This paper summarizes the main topics presented in the workshop held from 28 to 30 June 2022. As such, it highlights lessons learned from the project and strategies to fight AMR. Although CAPTURA has been invaluable to countries and information from the project is already being used, barriers concerning data quality and sharing remain. Regional-level initiatives should continue to build on the momentum gained from the CAPTURA project in supporting national-level surveillance and data quality improvements to inform critical decisions around planning, policies, and clinical care. Project findings have highlighted that issues with antimicrobial resistance and use are wide ranging across countries. Going forward, building on the current foundations and tailoring approaches to meet local needs and capacities will be fundamental in combatting AMR.

Methodological Approach to Identify and Expand the Volume of Antimicrobial Resistance (AMR) Data in the Human Health Sector in Low- and Middle-Income Countries in Asia: Implications for Local and Regional AMR Surveillance Systems Strengthening.

Antimicrobial resistance (AMR) is a multifaceted global health problem disproportionately affecting low- and middle-income countries (LMICs). The Capturing data on Antimicrobial resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) project was tasked to expand the volume of AMR and antimicrobial use data in Asia. The CAPTURA project used 2 data-collection streams: facility data and project metadata. Project metadata constituted information collected to map out data sources and assess data quality, while facility data referred to the retrospective data collected from healthcare facilities. A down-selection process, labelled "the funnel approach" by the project, was adopted to use the project metadata in prioritizing and selecting laboratories for retrospective AMR data collection. Moreover, the metadata served as a guide for understanding the AMR data once they were collected. The findings from CAPTURA's metadata add to the current discourse on the limitation of AMR data in LMICs. There is generally a low volume of AMR data generated as there is a lack of microbiology laboratories with sufficient antimicrobial susceptibility testing capacity. Many laboratories in Asia are still capturing data on paper, resulting in scattered or unused data not readily accessible or shareable for analyses. There is also a lack of clinical and epidemiological data captured, impeding interpretation and in-depth understanding of the AMR data. CAPTURA's experience in Asia suggests that there is a wide spectrum of capacity and capability of microbiology laboratories within a country and region. As local AMR surveillance is a crucial instrument to inform context-specific measures to combat AMR, it is important to understand and assess current capacity-building needs while implementing activities to enhance surveillance systems.

Utility and Evaluation of Applied Project Management Processes Within a Large Multicountry Health Systems Development Project Conducted During the Coronavirus Disease 2019 (COVID-19) Pandemic.

The increasing trends in antimicrobial resistance (AMR) continue to pose a significant threat to human health, with grave consequences in low- and middle-income countries. In collaboration with local governments and microbiology laboratories in South Asian and Southeast Asian countries, the Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) project worked to identify gaps and expand the volume of existing AMR data to inform decision-makers on how to best strengthen their national AMR surveillance capacity. This article describes overall project management processes and the strategies implemented to address the disruptive impact of the coronavirus disease 2019 (COVID-19) pandemic on the project activities across diverse contexts in different countries. Also, it assesses in-country team's feedback on the conduct of activities and their overall impact on project completion. The strategies employed were tailored to the specific context of each country and included increased communication and collaboration among consortium partners and in-country teams, as well as hiring of additional in-country team members. This paper highlights the importance of local representation and capacities as well as real-time (virtual) engagement with stakeholders, ensuring close monitoring of the local situation and ability to tailor context-specific mitigation strategies to continue project implementation during disruptive external circumstances.

Recording and Reporting of Antimicrobial Resistance (AMR) Priority Variables and Its Implication on Expanding Surveillance Sites in Nepal: A CAPTURA Experience.

Data on antimicrobial resistance (AMR) from sites not participating in the National AMR surveillance network, conducted by National Public Health Laboratory (NPHL), remain largely unknown in Nepal. The "Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia" (CAPTURA) assessed AMR data from previously untapped data sources in Nepal. A retrospective cross-sectional data review was carried out for the AMR data recorded between January 2017 and December 2019 to analyze AMR data from 26 hospital-based laboratories and 2 diagnostic laboratories in Nepal. Of the 56 health facilities initially contacted to participate in this project activity, 50.0% (28/56) signed a data-sharing agreement with CAPTURA. Eleven of the 28 hospitals were AMR surveillance sites, whereas the other 17, although not part of the National AMR surveillance network, recorded AMR-related data. Data for 663 602 isolates obtained from 580 038 patients were analyzed. A complete record of the 11 CAPTURA priority variables was obtained from 45.5% (5/11) of government hospitals, 63.6% (7/11) of private hospitals, and 54.6% (6/11) of public-private hospitals networked with NPHL for AMR surveillance. Similarly, 80% (8/10) of clinics and 54.6% (6/11) of laboratories outside the NPHL network recorded complete data for the 10 Global Antimicrobial Resistance and Use Surveillance System (GLASS) priority variables and 11/14 CAPTURA priority variables. Retrospective review of the data identified areas requiring additional resources and interventions to improve the quality of data on AMR in Nepal. Furthermore, we observed no difference in the priority variables reported by sites within or outside the NPHL network, thus suggesting that policies could be made to expand the surveillance system to include these sites without substantially affecting the government's budget.

Evaluating Quality Management and Diagnostics Microbiology Performance Within an International External Quality Assessment (EQA) Program Serving National One Health Sector Reference Laboratories Across Asia: Experience Amid the Coronavirus Disease 2019 (COVID-19) Pandemic.

BACKGROUND: Strengthening external quality assessment (EQA) services across the One Health sector supports implementation of effective antimicrobial resistance (AMR) control strategies. Here we describe and compare 2 different approaches for conducting virtual laboratory follow-up assessments within an EQA program to evaluate quality management system (QMS) and procedures for pathogen identification and antimicrobial susceptibility testing (AST). METHODS: During the coronavirus disease 2019 (COVID-19) pandemic in 2021 and 2022, 2 laboratory assessment approaches were introduced: virtual-based and survey-based methodologies. The evaluation of 2 underperforming Animal Health laboratories through a virtual-based approach occurred between May and August 2021. This evaluation encompassed the utilization of 3 online meetings and document reviews, performed subsequent to the execution of EQA procedures. Within a distinct group of laboratories, the survey-based assessment was implemented from December 2021 to February 2022, also following EQA procedures. This phase encompassed the dissemination of an online survey to 31 participating laboratories, alongside a sole online consultation meeting involving 4 specific underperforming laboratories. RESULTS: The virtual-based assessment post-EQA aimed to identify gaps and areas for improvement in the laboratory's practices for pathogen identification and AST. This approach was, however, time-intensive, and, hence, only 2 laboratories were assessed. In addition, limited interactions in virtual platforms compromised the assessment quality. The survey-based post-EQA assessment enabled evaluation of 31 laboratories. Despite limitations for in-depth analysis of each procedure, gaps in QMS across multiple laboratories were identified and tailored laboratory-specific recommendations were provided. CONCLUSIONS: Reliable internet and plans for efficient time management, post-EQA virtual laboratory follow-up assessments are an effective alternative when conducting onsite evaluation is infeasible as observed during the COVID-19 pandemic, although the successful implementation of remediation plans will likely require in person assessments. We advocate application of hybrid approaches (both onsite and virtual) for targeted capacity building of AMR procedures with the ability to implement and oversee the process.

Antimicrobial Resistance Surveillance Methods in Bangladesh: Present and Way Forward.

The Institute of Epidemiology, Disease Control and Research (IEDCR) conducts active, case-based national antimicrobial resistance (AMR) surveillance in Bangladesh. The Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) project accessed aggregated retrospective data from non-IEDCR study sites and 9 IEDCR sites to understand the pattern and extent of AMR and to use analyzed data to guide ongoing and future national AMR surveillance in both public and private laboratories. Record-keeping practices, data completeness, quality control, and antimicrobial susceptibility test practices were investigated in all laboratories participating in case-based IEDCR surveillance and laboratory-based CAPTURA sites. All 9 IEDCR laboratories recorded detailed case-based data (n = 16 816) in electronic format for a priority subset of processed laboratory samples. In contrast, most CAPTURA sites (n = 18/33 [54.5%]) used handwritten registers to store data. The CAPTURA sites were characterized by fewer recorded variables (such as patient demographics, clinical history, and laboratory findings) with 1 020 197 individual data, less integration of patient records with the laboratory information system, and nonuniform practice of data recording; however, data were collected from all available clinical samples. The analyses conducted on AMR data collected by IEDCR and CAPTURA in Bangladesh provide current data collection status and highlight opportunities to improve ongoing data collection to strengthen current AMR surveillance system initiatives. We recommend a tailored approach to conduct AMR surveillance in high-burden, resource-limited settings.

Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA).

BACKGROUND: In 2015, the UK government established the Fleming Fund with the aim to address critical gaps in surveillance of antimicrobial resistance (AMR) in low- and middle-income countries in Asia and Africa. Among a large portfolio of grants, the Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) project was awarded with the specific objective of expanding the volume of historical data on AMR, consumption (AMC), and use (AMU) in the human healthcare sector across 12 countries in South and Southeast Asia. METHODS: Starting in early 2019, the CAPTURA consortium began working with local governments and >100 relevant data-holding facilities across the region to identify, assess for quality, prioritize, and subsequently retrieve data on AMR, AMC, and AMU. Relevant and shared data were collated and analyzed to provide local overviews for national stakeholders as well as regional context, wherever possible. RESULTS: From the vast information resource generated on current surveillance capacity and data availability, the project has highlighted gaps and areas for quality improvement and supported comprehensive capacity-building activities to optimize local data-collection and -management practices. CONCLUSIONS: The project has paved the way for expansion of surveillance networks to include both the academic and private sector in several countries and has actively engaged in discussions to promote data sharing at the local, national, and regional levels. This paper describes the overarching approach to, and emerging lessons from, the CAPTURA project, and how it contributes to other ongoing efforts to strengthen national AMR surveillance in the region and globally.

A Phase 3, Multicenter, Randomized, Controlled Trial to Evaluate Immune Equivalence and Safety of Multidose and Single-dose Formulations of Vi-DT Typhoid Conjugate Vaccine in Healthy Filipino Individuals 6 Months to 45 Years of Age.

Trial Design: Phase 3, randomized, controlled, multicenter, equivalence trial. Methods: Recruitment of participants occurred between 04Februray2020 and 15July2020 at four centers in the Philippines: University of the East - Ramon Magsaysay Memorial Medical Center Inc., Quezon City; University of Philippines Manila - National Institute of Health, Ermita Manila; Asian Hospital and Medical Center, Metro Manila, Philippines Study; and Medical Research Unit, Tropical Disease Foundation, Makati City, Metro Manila, Philippines. Participants: 1800 adults and children 6-months to 45-years of age. Interventions: Participants received a single injection of multidose (MD) or single dose (SD) Vi-DT as test vaccines or meningococcal conjugate vaccine as a comparator. Objective: To evaluate immune equivalence of SD and MD formulations of Vi-DT, and to assess the safety of both formulations compared with comparator vaccine. Outcome Measurement: Blood draw for immunogenicity was performed at baseline prior to vaccine receipt and at four weeks after vaccination for a subset of participants to determine anti-Vi IgG geometric mean titers (GMT) and seroconversion rates. The primary outcome was comparison of anti Vi-IgG seroconversion and GMT between the two formulations of Vi-DT at 4 weeks following vaccine administration. Immune equivalence of MD and SD formulations was confirmed when the two-tailed 95% confidence interval (CI) of the GMT ratio is within [0.67, 1.5] at a two-sided significance level of 0.05. All participants were followed for safety events for six months after vaccine administration. Randomization: Participants were randomized to receive SD Vi-DT, MD Vi-DT, or meningococcal conjugate vaccines in 2.5:2.5:1 allocation ratio. Blinding: Study participants and observers were blinded to treatment assignment. Findings: Immune equivalence of SD (n=252) and MD (n=247) formulations was confirmed by anti-Vi IgG GMT ratio of 1.14 (95%CI: 0.91, 1.43) with respective GMTs in the MD and SD groups of 640.62 IU/mL (95%CI: 546.39, 751.11) and 562.57 IU/mL (95%CI: 478.80, 661.00) (p=0.259). Similarly, anti-Vi IgG seroconversion rate difference between the two formulations of ‒0.43% (95%CI: -4.42, 3.56) confirmed immune equivalence with corresponding seroconversion rates of 98.38% (95%CI: 95.91, 99.37) and 98.81% (95%CI: 96.56, 99.59) in MD and SD Vi-DT formulations, respectively (p=0.722). Both formulations of Vi-DT had a satisfactory safety profile - all five serious adverse events reported during the study were unrelated to the investigational product. Interpretation: The MD and SD formulations of Vi-DT elicited robust and equivalent immune responses following one dose vaccination, and both formulations demonstrated a favorable safety profile. Trial Registration: ClinicalTrials.gov: NCT04204096. Funding: This study was funded by the Bill & Melinda Gates Foundation (OPP 1115556).

What is the median volume of intracerebral hemorrhage and is it changing?

OBJECTIVES: Population-level estimates of the median intracerebral hemorrhage (ICH) volume would allow for the evaluation of clinical trial external validity and determination of temporal trends. We previously reported the median ICH volume in 1988. However, differences in risk factor management, neuroimaging, and demographics may have affected ICH volumes. The goal of this study is to determine the median volume of ICH within a population-based cross-sectional study, including whether it has changed over time. METHODS: The Genetic and Environmental Risk Factors for Hemorrhagic Stroke study was a population-based study of ICH among residents of the Greater Cincinnati/Northern Kentucky region from 2008 through 2012. This study utilizes those data and compares with ICH cases from the same region in 1988. Initial computed tomography images of the head were reviewed, and ICH volumes were calculated using consistent methodology. RESULTS: From 2008 through 2012, we identified 1117 cases of ICH. The median volume of ICH was 14.0 mL and was lower in black (11.6) than in white (15.5) patients. Median volumes of lobar and deep ICH were 28.8 mL and 9.8 mL, respectively. Median ICH volume changed significantly from 1988 to 2008-2012, with age-and-race-adjusted volume decreasing from 18.3 mL to 13.76 mL (p = 0.025). CONCLUSIONS: Median volume of ICH was 13.76 mL, and this should be considered in clinical trial design. Median ICH volume has apparently decreased from 1988 to 2008-2012.

Exploring Socio-Demographic Factors Affecting Psychological Symptoms in Humidifier Disinfectant Survivors.

This study aimed to compare the psychological symptoms of humidifier disinfectant survivors to the general population and explore socio-demographic factors influencing survivors' psychological symptoms. A one-way Multivariate Analysis of Covariance (MANCOVA) and a series of two-way MANCOVA were conducted with a sample of 228 humidifier disinfectant survivors and 228 controls. The results demonstrated that the survivor group displayed higher anxious/depressed symptoms, withdrawn symptoms, somatic complaints, thought problems, attention problems, aggressive behavior and rule-breaking behavior than the general group. Moreover, among the socio-demographic factors, the two-way interaction effects of group × family economic status and group × number of friends were found to be statistically significant. The limitations and implications of this study are discussed.

Risk factors for seizures after intracerebral hemorrhage: Ethnic/Racial Variations of Intracerebral Hemorrhage (ERICH) Study.

OBJECTIVE: We aimed to identify risk factors for seizures after intracerebral hemorrhage, and to validate the prognostic value of the previously reported CAVE score (0-4 points: cortical involvement, age <65, volume >10 mL, and early seizures within 7 days of hemorrhage). PATIENTS AND METHODS: Ethnic/Racial Variations of Intracerebral Hemorrhage (ERICH) was a prospective study of spontaneous intracerebral hemorrhage. We included patients who did not have a prior history of seizure and survived to discharge. Univariate analysis and multiple logistic regression modeling were used to identify risk factors for seizure. RESULTS: From 2010-2015, 3000 cases were recruited, and 2507 patients were included in this study. Seizures after hospital discharge developed in 77 patients 3.1 %). Patients with lobar (cortical) hemorrhage (OR 3.0, 95 % CI 1.8-5.0), larger hematoma volume (OR 1.5 per cm3, 95 % CI 1.2-2.0), and surgical evacuation of hematoma (OR 2.6, 95 % CI 1.4-4.8) had a higher risk of late seizure, and older patients had a lower risk (OR 0.88 per 5-year interval increase, 95 % CI 0.81-0.95). The CAVE score was highly associated with seizure development (OR 2.5 per unit score increase, 95 % CI 2.0-3.2, p < 0.0001). The CAVS score, substituting surgical evacuation for early seizure, increased the OR per unit score to 2.8 (95 % CI 2.2-3.5). CONCLUSIONS: Lobar hemorrhage, larger hematoma volume, younger age, and surgical evacuation are strongly associated with the development of seizures. We validated the CAVE score in a multi-ethnic population, and found the CAVS score to have similar predictive value while representing the current practice of AED use.

Acute Febrile Illness Among Children in Butajira, South-Central Ethiopia During the Typhoid Fever Surveillance in Africa Program.

BACKGROUND: Clearly differentiating causes of fever is challenging where diagnostic capacities are limited, resulting in poor patient management. We investigated acute febrile illness in children aged ≤15 years enrolled at healthcare facilities in Butajira, Ethiopia, during January 2012 to January 2014 for the Typhoid Fever Surveillance in Africa Program. METHODS: Blood culture, malaria microscopy, and blood analyses followed by microbiological, biochemical, and antimicrobial susceptibility testing of isolates were performed. We applied a retrospectively developed scheme to classify children as malaria or acute respiratory, gastrointestinal or urinary tract infection, or other febrile infections and syndromes. Incidence rates per 100 000 population derived from the classification scheme and multivariate logistic regression to determine fever predictors were performed. RESULTS: We rarely observed stunting (4/513, 0.8%), underweight (1/513, 0.2%), wasting (1/513, 0.2%), and hospitalization (21/513, 4.1%) among 513 children with mild transient fever and a mean disease severity score of 12 (95% confidence interval [CI], 11-13). Blood cultures yielded 1.6% (8/513) growth of pathogenic agents; microscopy detected 13.5% (69/513) malaria with 20 611/µL blood (95% CI, 15 352-25 870) mean parasite density. Incidences were generally higher in children aged ≤5 years than >5 to ≤15 years; annual incidences in young children were 301.3 (95% CI, 269.2-337.2) for malaria and 1860.1 (95% CI, 1778.0-1946.0) for acute respiratory and 379.9 (95% CI, 343.6-420.0) for gastrointestinal tract infections. CONCLUSIONS: We could not detect the etiological agents in all febrile children. Our findings may prompt further investigations and the reconsideration of policies and frameworks for the management of acute febrile illness.

Occurrence of Typhoid Fever Complications and Their Relation to Duration of Illness Preceding Hospitalization: A Systematic Literature Review and Meta-analysis.

BACKGROUND: Complications from typhoid fever disease have been estimated to occur in 10%-15% of hospitalized patients, with evidence of a higher risk in children and when delaying the implementation of effective antimicrobial treatment. We estimated the prevalence of complications in hospitalized patients with culture-confirmed typhoid fever and the effects of delaying the implementation of effective antimicrobial treatment and age on the prevalence and risk of complications. METHODS: A systematic review and meta-analysis were performed using studies in the PubMed database. We rated risk of bias and conducted random-effects meta-analyses. Days of disease at hospitalization (DDA) was used as a surrogate for delaying the implementation of effective antimicrobial treatment. Analyses were stratified by DDA (DDA <10 versus ≥10 mean/median days of disease) and by age (children versus adults). Differences in risk were assessed using odds ratios (ORs) and 95% confidence intervals (CIs). Heterogeneity and publication bias were evaluated with the I2 value and funnel plot analysis, respectively. RESULTS: The pooled prevalence of complications estimated among hospitalized typhoid fever patients was 27% (95% CI, 21%-32%; I2 = 90.9%, P < .0001). Patients with a DDA ≥ 10 days presented higher prevalence (36% [95% CI, 29%-43%]) and three times greater risk of severe disease (OR, 3.00 [95% CI, 2.14-4.17]; P < .0001) than patients arriving earlier (16% [95% CI, 13%- 18%]). Difference in prevalence and risk by age groups were not significant. CONCLUSIONS: This meta-analysis identified a higher overall prevalence of complications than previously reported and a strong association between duration of symptoms prior to hospitalization and risk of serious complications.

Association of Multivitamin and Mineral Supplementation and Risk of Cardiovascular Disease: A Systematic Review and Meta-Analysis.

BACKGROUND: Multiple studies have attempted to identify the association between multivitamin/mineral (MVM) supplementation and cardiovascular disease (CVD) outcomes, but the benefits remain controversial. We performed a systematic review and meta-analysis of the associations between MVM supplementation and various CVD outcomes, including coronary heart disease (CHD) and stroke. METHODS AND RESULTS: We conducted a comprehensive search of Medline, Embase, and the Cochrane Library for studies published between January 1970 and August 2016. We included clinical trials and prospective cohort studies in the general population evaluating associations between MVM supplementation and CVD outcomes. Data extraction and quality assessment were independently conducted by 2 authors, and a third author resolved discrepancies. Eighteen studies with 2 019 862 participants and 18 363 326 person-years of follow-up were included in the analysis. Five studies specified the dose/type of MVM supplement and the rest did not. Overall, there was no association between MVM supplementation and CVD mortality (relative risk [RR], 1.00; 95% confidence interval [CI], 0.97-1.04), CHD mortality (RR, 1.02; 95% CI, 0.92-1.13), stroke mortality (RR, 0.95; 95% CI, 0.82-1.09), or stroke incidence (RR, 0.98; 95% CI, 0.91-1.05). There was no association between MVM supplements and CVD or CHD mortality in prespecified subgroups categorized by mean follow-up period, mean age, period of MVM use, sex, type of population, exclusion of patients with history of CHD, and adjustment for diet, adjustment for smoking, adjustment for physical activity, and study site. In contrast, MVM use did seem to be associated with a lower risk of CHD incidence (RR, 0.88; 95% CI, 0.79-0.97). However, this association did not remain significant in the pooled subgroup analysis of randomized controlled trials (RR, 0.97; 95% CI, 0.80-1.19). CONCLUSIONS: Our meta-analysis of clinical trials and prospective cohort studies demonstrates that MVM supplementation does not improve cardiovascular outcomes in the general population.

Post-IV thrombolytic headache and hemorrhagic transformation risk in acute ischemic stroke.

Background: Headache during or soon after administration of IV tissue plasminogen activator (tPA) in patients with acute ischemic stroke (AIS) is a concern for hemorrhagic transformation (HT). However, no data are available regarding the incidence of HT in these patients or the prognostic indication of these headaches. We examine the importance of tPA-associated headaches among AIS patients in terms of HT rates and clinical outcomes. Methods: AIS patients treated with IV tPA at a comprehensive stroke center between January 2007 and November 2012 were retrospectively reviewed for documented tPA-associated headache in the first 24 hours post-tPA. We compared the headache and nonheadache groups for differences in various clinical and radiologic outcomes. Results: Of the 193 patients, 63 (32.6%) had tPA-associated headache. Headache patients (HP) were younger than nonheadache patients (NHP) (mean ± SD, 59.5 ± 17.4 years vs 69.9 ± 15.5 years, p < 0.0001), and 53% of HP were men, compared to 49.2% of NHP (p = 0.537). Comorbid conditions did not differ between the 2 groups. There were no statistical differences between HP and NHP in admission NIH Stroke Scale (NIHSS) score (11.2 ± 5.7 vs 11.5 ± 5.5, p = 0.646), NIHSS score at 24 hours (6.5 ± 5.7 vs 7.4 ± 6.9, p = 0.466), NIHSS score at discharge (6.7 ± 10.1 vs 8.1 ± 11.6, p = 0.448), HT (12.7% vs 18.4%, p = 0.3), cervical artery dissection (4.7% vs 5.38%, p = 0.764), length of hospitalization (6.29 ± 5 days vs 6.35 ± 4.7 days, p = 0.935), and disposition. Conclusion: tPA-associated headache does not predict increased risk of HT and has no other prognostic importance in patients with AIS. Prospective studies with a larger cohort may be needed to further explore this relationship.

Effects of ß-sitosterol derived from Artemisia capillaris on the activated human hepatic stellate cells and dimethylnitrosamine-induced mouse liver fibrosis.

BACKGROUND: ß-sitosterol is a cholesterol-like phytosterol, which widely distributed in the plant kingdom. Here, anti-fibrotic effect of the ß-sitosterol was studied using the activated human hepatic stellate cell (HSC) model and dimethylnitrosamine (DMN)-induced mouse hepatic fibrosis model. METHOD: HSCs were activated by transforming growth factor-ß (TGF-ß) and the collagen-1 and α-smooth muscle actin (α-SMA) expressions were measured at the mRNA and protein level. We also studied the effect ß-sitosterol using DMN-induced mouse hepatic fibrosis model. We then measured the collagen-1 and α-SMA expression levels in vivo to investigate anti-hepatofibrotic effect of ß-sitosterol, at both of the mRNA and protein level. RESULTS: ß-sitosterol down regulated the mRNA and protein expression levels of collagen-1 and α-SMA in activated HSC. Oral administration of the ß-sitosterol successfully alleviated the DMN-induced mouse liver damage and prevented collagen accumulation. The mRNA and protein expression levels of collagen-1 and α-SMA were also down regulated in ß-sitosterol treated mouse group. CONCLUSIONS: This study shows the effect of ß-sitosterol on the TGF-ß -or DMN-induced hepatofibrosis. Hence, we demonstrate the ß-sitosterol as a potential therapeutic agent for the hepatofibrosis.

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of ScpB (Rv1710) from Mycobacterium tuberculosis.

Structural maintenance of chromosome (SMC) proteins play diverse roles in cellular DNA reassembly by directly interacting with DNA. They require non-SMC proteins for their proper function; these include the conserved segregation and condensation proteins (Scps) in prokaryotes. ScpB from Mycobacterium tuberculosis was crystallized using the sitting-drop vapour-diffusion method in the presence of 2 M NaCl and 10% PEG 6000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 A at a synchrotron beamline. The crystal belongs to the hexagonal space group R32, with unit-cell parameters a = b = 136.69, c = 78.55 A, gamma = 120 degrees . With one molecule per asymmetric unit, the crystal volume per unit protein weight (V(M)) is 2.95 A(3) Da(-1). The structure was solved by the single anomalous dispersion method and structure refinement is in progress.

Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum.

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH(2)PO(4) and K(2)HPO(4) as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 A on a synchrotron beamline. The crystal belongs to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 94.72, c = 359.68 A, gamma = 120 degrees . The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 A(3) Da(-1). The structure was solved by molecular replacement. Structure refinement is in progress.

Highly efficient protein expression and purification using bacterial hemoglobin fusion vector.

Recently developed bacterial hemoglobin (VHb) fusion expression vector has been widely used for the production of many target proteins due to its distinctive properties of expressing fusion protein with red color which facilitates visualization of the steps in purification, and increasing solubility of the target proteins. However, after intensive use of the vector, several defects have been found. In this report, we present a modified VHb fusion vector (pPosKJ) with higher efficiency, in which most of the defects were eliminated. First, it was found that thrombin protease often digests target protein as well as inserted thrombin cleavage site, so it was replaced by a TEV cleavage site for more specific cleavage of VHb from target protein. Second, a glycine-rich linker sequence was inserted between 6x his-tag and VHb to improve the affinity of 6x his-tag to Ni-NTA resin, resulting in higher purity of eluted fusion protein. Third, EcoRI and XhoI restriction sites located elsewhere in the vector were removed to make these restriction sites available for the cloning of target protein coding genes. A pPosKJ vector was fully examined with an anti-apoptotic BCL-2 family member of Caenorhabditis elegans, CED-9. A C-terminal VHb fusion expression vector (pPosKJC) was also constructed for stable expression of target proteins that may be difficult to express with an N-terminal fusion. Vaccinia-related kinase 1 (VRK1) was also successfully expressed and purified using the vector with high yield. Taken together, we suggest that the VHb fusion vector may be well suited for high-throughput protein expression and purification.