Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu

Predicting Hemolytic Uremic Syndrome and Renal Replacement Therapy in Shiga Toxin-producing Escherichia coli-infected Children.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) infections are leading causes of pediatric acute renal failure. Identifying hemolytic uremic syndrome (HUS) risk factors is needed to guide care. METHODS: We conducted a multicenter, historical cohort study to identify features associated with development of HUS (primary outcome) and need for renal replacement therapy (RRT) (secondary outcome) in STEC-infected children without HUS at initial presentation. Children agedeligible. RESULTS: Of 927 STEC-infected children, 41 (4.4%) had HUS at presentation; of the remaining 886, 126 (14.2%) developed HUS. Predictors (all shown as odds ratio [OR] with 95% confidence interval [CI]) of HUS included younger age (0.77 [.69-.85] per year), leukocyte count ≥13.0 × 103/μL (2.54 [1.42-4.54]), higher hematocrit (1.83 [1.21-2.77] per 5% increase) and serum creatinine (10.82 [1.49-78.69] per 1 mg/dL increase), platelet count \u3c250 \u3e× 103/μL (1.92 [1.02-3.60]), lower serum sodium (1.12 [1.02-1.23 per 1 mmol/L decrease), and intravenous fluid administration initiated ≥4 days following diarrhea onset (2.50 [1.14-5.46]). A longer interval from diarrhea onset to index visit was associated with reduced HUS risk (OR, 0.70 [95% CI, .54-.90]). RRT predictors (all shown as OR [95% CI]) included female sex (2.27 [1.14-4.50]), younger age (0.83 [.74-.92] per year), lower serum sodium (1.15 [1.04-1.27] per mmol/L decrease), higher leukocyte count ≥13.0 × 103/μL (2.35 [1.17-4.72]) and creatinine (7.75 [1.20-50.16] per 1 mg/dL increase) concentrations, and initial intravenous fluid administration ≥4 days following diarrhea onset (2.71 [1.18-6.21]). CONCLUSIONS: The complex nature of STEC infection renders predicting its course a challenge. Risk factors we identified highlight the importance of avoiding dehydration and performing close clinical and laboratory monitoring

Assessing the Validity Evidence of an Objective Structured Assessment Tool of Technical Skills for Neonatal Lumbar Punctures

Background The lumbar puncture ( LP ) is a procedural competency deemed necessary by the Accreditation Council for Graduate Medical Education and the Emergency Medicine and Pediatric Residency Review Committees. The emergency department ( ED ) is a primary site for residents to be evaluated performing neonatal LP s. Current evaluation methods lack validity evidence as assessment tools. Objectives This was a pilot study to develop an objective structured assessment of technical skills for neonatal LP ( OSATS ‐ LP ) and to document validity evidence for the instrument in regard to five sources of test validity: content, response process, relation to other variables, inter‐rater reliability, and consequences of testing. Methods Pediatric residents were videotaped in the fall of 2011 for comparison of faculty evaluation of resident performance during a neonatal LP using a video‐delayed format. Residents completed a demographic experience survey evaluating relations to other variables. Content and response process validity was obtained through expert panel meetings and resulted in the following seven domains of performance for the OSATS ‐ LP : preparation, positioning, analgesia, needle insertion, cerebrospinal fluid ( CSF ) collection, management of laboratory studies, and sterility. t‐tests assessed significance between level of training, previous intensive care unit experience, and residents' self‐assessed confidence in comparison with their total performance score. The inter‐rater agreement of the OSATS ‐ LP was obtained using the Fleiss' kappa for each domain. Results Sixteen pediatric residents completed the simulation with six raters evaluating each resident (96 ratings). The domains of sterility and CSF collection had moderate statistical reliability (κ = 0.41 and 0.51, respectively). The domains of preparation, analgesia, and management of laboratories had substantial reliability (κ = 0.60, 0.62, and 0.62, respectively). The domains of positioning and needle insertion were less reliable (κ = 0.16 and 0.16, respectively). Individuals who had completed one or more rotations in the neonatal intensive care unit ( NICU ) had a higher total score (12.5 vs. 16.9; p < 0.01). The residents' own perception of ability to perform an LP unsupervised did not result in a higher total score. Conclusions The OSATS ‐ LP has reasonable evidence in four of the five sources for test validity. This study serves as a launching point for using this tool in clinical environments such as the ED and, therefore, has the potential to provide real‐time formative and summative feedback to improve resident skills and ultimately lead to improvements in patient care. Resumen Comprobación de la Validez de una Herramienta de Evaluación Estructurada Objetiva de Habilidades Técnicas para las Punciones Lumbares Neonatales Introducción La punción lumbar ( PL ) es un procedimiento cuya competencia se considera necesaria por el Accreditation Council for Graduate Medical Education y los Emergency Medicine and Pediatric Residency Review Committees . El servicio de urgencias ( SU ) es un lugar primario para evaluar a los residentes en la realización de la PL neonatal. Los métodos de evaluación actuales carecen de evidencia válida como herramientas de evaluación. Objetivos Éste fue un estudio piloto para desarrollar una evaluación estructurada objetiva de las habilidades técnicas para la PL neonatal ( OSATS ‐ LP ) y para documentar la validez para el instrumento respecto a cinco fuentes de la validez de un test: el contenido, el proceso respuesta, la relación a otras variables, la fiabilidad interobservador y las consecuencias del test. Metodología Los residentes de pediatría fueron grabados en otoño de 2011 para la comparación de la evaluación docente del rendimiento del residente durante una PL neonatal usando un formato de video diferido. Los residentes completaron una encuesta de experiencia demográfica mediante la evaluación de las relaciones con otras variables. La validez del contenido y del proceso de respuesta se obtuvo a través de las reuniones de un panel de expertos y resultó en los siguientes siete dominios de la realización para el OSATS ‐ LP : la preparación, la posición, la analgesia, la inserción de la aguja, la recogida del líquido cefalorraquídeo ( LCR ), el manejo de los estudios de laboratorio y la esterilidad. La significación entre el nivel de entrenamiento, la experiencia previa en una unidad de cuidados intensivos y la confianza autoevaluada del residente en comparación con su puntuación de rendimiento total se analizó con el test de la t de Student. La concordancia interobservador del OSATS ‐ LP se obtuvo con el índice kappa de Fleiss para cada dominio. Resultados Dieciséis residentes de pediatría completaron la simulación con seis evaluadores que examinaron a cada residente (96 clasificaciones). Los dominios de esterilidad y recogida de LCR tuvieron una fiabilidad moderada (k = 0,41 y 0,51, respectivamente). Los dominios de preparación, analgesia y manejo de laboratorio tuvieron una fiabilidad sustancial (k = 0,60, 0,62, y 0,62, respectivamente). Los dominios de la posición e inserción de la aguja fueron menos fiables (k = 0,16 y 0,16, respectivamente). Los residentes que habían realizado más PL se correlacionaron con una puntuación total mayor (coeficiente de correlación de Pearson = 0,5, p < 0,05). Los sujetos que habían completado una o más rotaciones en la unidad de cuidados intensivos neonatal tuvieron una puntuación total más alta (12,5 vs. 16,9; p < 0,01). La autopercepción de los residentes de la capacidad para realizar una PL no supervisada no resultó en una puntuación total más alta. Conclusiones La OSATS ‐ LP tiene una evidencia razonable en cuatro de las cinco fuentes para la validez del test. Este estudio sirve como un punto de partida para usar esta herramienta en los ambientes clínicos como el SU , y por ello, tiene la potencialidad para proporcionar una formación a tiempo real y una retroalimentación sumativa para mejorar las habilidades de los residentes, y conducir finalmente a mejoras en la atención del paciente.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97233/1/acem12093.pd

Assessing the Validity Evidence of an Objective Structured Assessment Tool of Technical Skills for Neonatal Lumbar Punctures

Background The lumbar puncture ( LP ) is a procedural competency deemed necessary by the Accreditation Council for Graduate Medical Education and the Emergency Medicine and Pediatric Residency Review Committees. The emergency department ( ED ) is a primary site for residents to be evaluated performing neonatal LP s. Current evaluation methods lack validity evidence as assessment tools. Objectives This was a pilot study to develop an objective structured assessment of technical skills for neonatal LP ( OSATS ‐ LP ) and to document validity evidence for the instrument in regard to five sources of test validity: content, response process, relation to other variables, inter‐rater reliability, and consequences of testing. Methods Pediatric residents were videotaped in the fall of 2011 for comparison of faculty evaluation of resident performance during a neonatal LP using a video‐delayed format. Residents completed a demographic experience survey evaluating relations to other variables. Content and response process validity was obtained through expert panel meetings and resulted in the following seven domains of performance for the OSATS ‐ LP : preparation, positioning, analgesia, needle insertion, cerebrospinal fluid ( CSF ) collection, management of laboratory studies, and sterility. t‐tests assessed significance between level of training, previous intensive care unit experience, and residents' self‐assessed confidence in comparison with their total performance score. The inter‐rater agreement of the OSATS ‐ LP was obtained using the Fleiss' kappa for each domain. Results Sixteen pediatric residents completed the simulation with six raters evaluating each resident (96 ratings). The domains of sterility and CSF collection had moderate statistical reliability (κ = 0.41 and 0.51, respectively). The domains of preparation, analgesia, and management of laboratories had substantial reliability (κ = 0.60, 0.62, and 0.62, respectively). The domains of positioning and needle insertion were less reliable (κ = 0.16 and 0.16, respectively). Individuals who had completed one or more rotations in the neonatal intensive care unit ( NICU ) had a higher total score (12.5 vs. 16.9; p < 0.01). The residents' own perception of ability to perform an LP unsupervised did not result in a higher total score. Conclusions The OSATS ‐ LP has reasonable evidence in four of the five sources for test validity. This study serves as a launching point for using this tool in clinical environments such as the ED and, therefore, has the potential to provide real‐time formative and summative feedback to improve resident skills and ultimately lead to improvements in patient care. Resumen Comprobación de la Validez de una Herramienta de Evaluación Estructurada Objetiva de Habilidades Técnicas para las Punciones Lumbares Neonatales Introducción La punción lumbar ( PL ) es un procedimiento cuya competencia se considera necesaria por el Accreditation Council for Graduate Medical Education y los Emergency Medicine and Pediatric Residency Review Committees . El servicio de urgencias ( SU ) es un lugar primario para evaluar a los residentes en la realización de la PL neonatal. Los métodos de evaluación actuales carecen de evidencia válida como herramientas de evaluación. Objetivos Éste fue un estudio piloto para desarrollar una evaluación estructurada objetiva de las habilidades técnicas para la PL neonatal ( OSATS ‐ LP ) y para documentar la validez para el instrumento respecto a cinco fuentes de la validez de un test: el contenido, el proceso respuesta, la relación a otras variables, la fiabilidad interobservador y las consecuencias del test. Metodología Los residentes de pediatría fueron grabados en otoño de 2011 para la comparación de la evaluación docente del rendimiento del residente durante una PL neonatal usando un formato de video diferido. Los residentes completaron una encuesta de experiencia demográfica mediante la evaluación de las relaciones con otras variables. La validez del contenido y del proceso de respuesta se obtuvo a través de las reuniones de un panel de expertos y resultó en los siguientes siete dominios de la realización para el OSATS ‐ LP : la preparación, la posición, la analgesia, la inserción de la aguja, la recogida del líquido cefalorraquídeo ( LCR ), el manejo de los estudios de laboratorio y la esterilidad. La significación entre el nivel de entrenamiento, la experiencia previa en una unidad de cuidados intensivos y la confianza autoevaluada del residente en comparación con su puntuación de rendimiento total se analizó con el test de la t de Student. La concordancia interobservador del OSATS ‐ LP se obtuvo con el índice kappa de Fleiss para cada dominio. Resultados Dieciséis residentes de pediatría completaron la simulación con seis evaluadores que examinaron a cada residente (96 clasificaciones). Los dominios de esterilidad y recogida de LCR tuvieron una fiabilidad moderada (k = 0,41 y 0,51, respectivamente). Los dominios de preparación, analgesia y manejo de laboratorio tuvieron una fiabilidad sustancial (k = 0,60, 0,62, y 0,62, respectivamente). Los dominios de la posición e inserción de la aguja fueron menos fiables (k = 0,16 y 0,16, respectivamente). Los residentes que habían realizado más PL se correlacionaron con una puntuación total mayor (coeficiente de correlación de Pearson = 0,5, p < 0,05). Los sujetos que habían completado una o más rotaciones en la unidad de cuidados intensivos neonatal tuvieron una puntuación total más alta (12,5 vs. 16,9; p < 0,01). La autopercepción de los residentes de la capacidad para realizar una PL no supervisada no resultó en una puntuación total más alta. Conclusiones La OSATS ‐ LP tiene una evidencia razonable en cuatro de las cinco fuentes para la validez del test. Este estudio sirve como un punto de partida para usar esta herramienta en los ambientes clínicos como el SU , y por ello, tiene la potencialidad para proporcionar una formación a tiempo real y una retroalimentación sumativa para mejorar las habilidades de los residentes, y conducir finalmente a mejoras en la atención del paciente.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97233/1/acem12093.pd

Pandemic Influenza and Acute Care Centers: Taking Care of Sick Patients in a Nonhospital Setting

The ongoing spread of H5N1 avian influenza in Southeast Asia has raised concern about a worldwide influenza pandemic and has made clear the need to plan in advance for such an event. The federal government has stressed the importance of planning and, in particular, has asked hospitals and public health agencies to develop plans to care for patients outside of traditional healthcare settings. These alternative or acute care centers (ACCs) would be opened when hospitals, emergency departments (EDs), and clinics are overwhelmed by an influenza pandemic. The University of Michigan Hospital System (UMHS), a large tertiary care center in southeast Michigan, has been developing a model for offsite care of patients during an influenza pandemic. This article summarizes our planning efforts and the lessons learned from 2 functional exercises over the past 3 years.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63368/1/bsp.2008.0030.pd

Herpes Simplex Virus Infection in Infants Undergoing Meningitis Evaluation

BACKGROUND:Although neonatal herpes simplex virus (HSV) is a potentially devastating infection requiring prompt evaluation and treatment, large-scale assessments of the frequency in potentially infected infants have not been performed. METHODS:We performed a retrospective cross-sectional study of infants ≤60 days old who had cerebrospinal fluid culture testing performed in 1 of 23 participating North American emergency departments. HSV infection was defined by a positive HSV polymerase chain reaction or viral culture. The primary outcome was the proportion of encounters in which HSV infection was identified. Secondary outcomes included frequency of central nervous system (CNS) and disseminated HSV, and HSV testing and treatment patterns. RESULTS:Of 26 533 eligible encounters, 112 infants had HSV identified (0.42%, 95% confidence interval [CI]: 0.35%-0.51%). Of these, 90 (80.4%) occurred in weeks 1 to 4, 10 (8.9%) in weeks 5 to 6, and 12 (10.7%) in weeks 7 to 9. The median age of HSV-infected infants was 14 days (interquartile range: 9-24 days). HSV infection was more common in 0 to 28-day-old infants compared with 29- to 60-day-old infants (odds ratio 3.9; 95% CI: 2.4-6.2). Sixty-eight (0.26%, 95% CI: 0.21%-0.33%) had CNS or disseminated HSV. The proportion of infants tested for HSV (35%; range 14%-72%) and to whom acyclovir was administered (23%; range 4%-53%) varied widely across sites. CONCLUSIONS:An HSV infection was uncommon in young infants evaluated for CNS infection, particularly in the second month of life. Evidence-based approaches to the evaluation for HSV in young infants are needed

Impact of Enteroviral Polymerase Chain Reaction Testing on Length of Stay for Infants 60 Days Old or Younger

ObjectiveTo determine the impact of a cerebrospinal fluid enterovirus polymerase chain reaction (PCR) test performance on hospital length of stay (LOS) in a large multicenter cohort of infants undergoing evaluation for central nervous system infection.Study designWe performed a planned secondary analysis of a retrospective cohort of hospitalized infants ≤60 days of age who had a cerebrospinal fluid culture obtained at 1 of 18 participating centers (2005-2013). After adjustment for patient age and study year as well as clustering by hospital center, we compared LOS for infants who had an enterovirus PCR test performed vs not performed and among those tested, for infants with a positive vs negative test result.ResultsOf 19 953 hospitalized infants, 4444 (22.3%) had an enterovirus PCR test performed and 945 (21.3% of tested infants) had positive test results. Hospital LOS was similar for infants who had an enterovirus PCR test performed compared with infants who did not (incident rate ratio 0.98 hours; 95% CI 0.89-1.06). However, infants PCR positive for enterovirus had a 38% shorter LOS than infants PCR negative for enterovirus (incident rate ratio 0.62 hours; 95% CI 0.57-0.68). No infant with a positive enterovirus PCR test had bacterial meningitis (0%; 95% CI 0-0.4).ConclusionsAlthough enterovirus PCR testing was not associated with a reduction in LOS, infants with a positive enterovirus PCR test had a one-third shorter LOS compared with infants with a negative enterovirus PCR test. Focused enterovirus PCR test use could increase the impact on LOS for infants undergoing cerebrospinal fluid evaluation

An apicomplexan parasite-specific anti-actin antibody.

<p><b>A</b>) Sequence comparison between human non-muscle actin amino acids 237–251 (the basis of anti-Gly₂₄₅<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188-Varma1" target="_blank">[39]</a>) and apicomplexan actin I orthologues over the amino acids 239–253 (the basis for anti-Act_239–253). <b>B</b>) Surface representation of the structures of rabbit G-actin (PDB:1J6Z; A) and a protomer in rabbit F-actin (PDB:3G37; B) showing anti-Gly₂₄₅ epitope. Residues in yellow indicate polymorphisms between mammalian and <i>P. falciparum</i> actin. <b>C</b>) Representative immunoblot showing reactivity of rabbit® anti-Act_239–253 serum with human erythrocytes (hRBC), asexual <i>P. falciparum</i> (3D7), mouse erythrocytes (mRBC), asexual <i>P. berghei</i> (ANKA), human foreskin fibroblasts (HFF) and <i>T. gondii</i> tachyzoites (RH). Lower panel shows same hRBC and 3D7 sample probed with mouse (m) anti-Act_239–253 serum. <b>D</b>) As C but using generic anti-actin monoclonal C4.</p

Concentration of actin labelling in the nucleus and around the nuclear periphery.

<p>Widefield IFA of representative <i>P. berghei </i><b>A</b>) ookinetes and <b>B</b>) sporozoites that show pronounced nuclear labelling using rabbit anti-Act_239–253 (Green) surface markers Pbs28 or PbCSP (Red) and DAPI (Blue). Scale bar = 5 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188.s009" target="_blank">Movie S5</a>. <b>C</b>) Widefield IFA of <i>P. falciparum</i> rings labelled with rabbit anti-Act_239–253 (Red) and DAPI (Blue). <b>D</b>) As <b>C</b> but following 6 hour JAS treatment. <b>E</b>) Two colour widefield IFA using rabbit anti-Act_239–253 (Red), rat anti-ERD2 (Green) and DAPI (Blue) in absence or presence of 1 µM JAS. All scale bars = 5 µm.</p

The tight junction is composed of dynamic actin filaments that localise posterior to the junction during invasion.

<p><b>A</b>) Widefield IFA with deconvolution and <b>B</b>) 3D reconstruction of <i>P. berghei</i> merozoites incubated with and without 1 µM JAS and labelled with anti-Act_239–253 (Green) and DAPI (Blue). Scale bar = 2 µm. Arrows show direction of invasion. <b>C</b>) Graphic representation of actin labelling in <i>P. berghei</i> merozoites with and without the addition of JAS. <i>n</i> = 124 merozoites for each of three replicates, mean is shown. <b>D</b>) 3D structured illumination microscopy (3D SIM) of three separate invading <i>P. falciparum</i> merozoites labelled with rabbit (upper row) and mouse (lower row) anti-Act_239–253. Labelling shows actin (Red), RON4 (Green) and DAPI (Blue). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188.s010" target="_blank">Movie S6</a>. Gamma settings were altered in 3D reconstructions.</p