Diagnóstico etiológico en meningitis y encefalitis por técnicas de biología molecular

ResumenIntroducciónEl estudio etiológico de las infecciones del sistema nervioso central se ha realizado tradicionalmente con cultivos bacterianos y con reacción en cadena de la polimerasa (PCR) para virus herpes simple (VHS). Los cultivos bacterianos pueden disminuir su rendimiento en pacientes que hayan usado antibióticos previos a la toma de muestra, y el solicitar PCR solo para virus VHS reduce el diagnóstico etiológico a un solo agente. El objetivo de este trabajo fue determinar las causas infecciosas en meningitis y encefalitis en niños, utilizando conjuntamente la microbiología convencional y la biología molecular, con el fin de mejorar el diagnóstico etiológico de estas enfermedades.Pacientes y métodoSe estudiaron 19 pacientes con sospecha de meningitis y encefalitis, de manera prospectiva, hospitalizados en el hospital Luis Calvo Mackenna en Santiago de Chile, entre el 1 de marzo de 2011 y el 30 de marzo de 2012. Luego de obtener el consentimiento informado, a las muestras de LCR se les realizó examen citoquímico, cultivo, PCR múltiple bacteriana (N. meningitidis, S. pneumoniae, H. influenzae) y PCR en tiempo real para HSV-1 y 2, VVZ, VEB, CMV, VHH-6 y enterovirus. Se recabaron datos clínicos y epidemiológicos desde la ficha clínica del paciente.ResultadosDe los 19 pacientes analizados 2 (10%) fueron diagnosticados por métodos microbiológicos convencionales y 7 (37%) al adicionar biología molecular (p=0,02). Tres pacientes presentaron meningitis por S. pneumoniae, uno por Enterobacter cloacae, 2 pacientes meningoencefalitis por VHS-1 y uno meningitis por VVZ.ConclusionesLa adición de la PCR a los métodos microbiológicos convencionales de diagnóstico en las infecciones del sistema nervioso central aumenta significativamente la probabilidad de detectar el agente causal. La incorporación rutinaria del diagnóstico molecular permitiría un manejo más oportuno y racional.AbstractIntroductionThe aetiological study of infections of the central nervous system has traditionally been performed using bacterial cultures and, more recently, using polymerase chain reaction (PCR) for herpes simplex virus (HSV). Bacterial cultures may not have good performance, especially in the context of patients who have received antibiotics prior to sampling, and a request for HSV only by PCR reduces the information to only one aetiological agent. The aim of this study is to determine the infectious causes of meningitis and encephalitis, using traditional microbiology and molecular biology to improve the aetiological diagnosis of these diseases.Patients and methodA prospective study was conducted on 19 patients with suspected meningitis, admitted to the Luis Calvo Mackenna Hospital in Santiago, Chile, from March 1, 2011 to March 30, 2012. After obtaining informed consent, the CSF samples underwent cytochemical study, conventional culture, multiplex PCR for the major producing bacterial meningitis (N. meningitidis, S. pneumoniae, H. influenzae), real-time single PCR for HSV-1 and 2, VZV, EBV, CMV, HHV-6 and enterovirus. Clinical and epidemiological data were also collected from the clinical records.ResultsOf the 19 patients analysed, 2 were diagnosed by conventional methods and 7 by adding molecular biology (increase to 37%). Three patients had meningitis due to S. pneumoniae, one due to Enterobacter cloacae, 2 patients meningoencephalitis HSV-1, and one VZV meningitis.ConclusionsThe addition of PCR to conventional diagnostic methods in CNS infections increases the probability of finding the causal agent. This allows a more adequate, timely and rational management of the disease

TLR4 Participates in the Inflammatory Response Induced by the AAF/II Fimbriae From Enteroaggregative Escherichia coli on Intestinal Epithelial Cells

Enteroaggregative Escherichia coli (EAEC) infections are one of the most frequent causes of persistent diarrhea in children, immunocompromised patients and travelers worldwide. The most prominent colonization factors of EAEC are aggregative adherence fimbriae (AAF). EAEC prototypical strain 042 harbors the AAF/II fimbriae variant, which mediates adhesion to intestinal epithelial cells and participates in the induction of an inflammatory response against this pathogen. However, the mechanism and the cell receptors implicated in eliciting this response have not been fully characterized. Since previous reports have shown that TLR4 recognize fimbriae from different pathogens, we evaluated the role of this receptor in the response elicited against EAEC by intestinal cells. Using a mutual antagonist against TLR2 and TLR4 (OxPAPC), we observed that blocking of these receptors significantly reduces the secretion of the inflammatory marker IL-8 in response to EAEC and AAF/II fimbrial extract in HT-29 cells. Using a TLR4-specific antagonist (TAK-242), we observed that the secretion of this cytokine was significantly reduced in HT-29 cells infected with EAEC or incubated with AAF/II fimbrial extract. We evaluated the participation of AAF/II fimbriae in the TLR4-mediated secretion of 38 cytokines, chemokines, and growth factors involved in inflammation. A reduction in the secretion of IL-8, GRO, and IL-4 was observed. Our results suggest that TLR4 participates in the secretion of several inflammation biomarkers in response to AAF/II fimbriae

Improved functionalization of oleic acid-coated iron oxide nanoparticles for biomedical applications

Superparamagnetic iron oxide nanoparticles can providemultiple benefits for biomedical applications in aqueous environments such asmagnetic separation or magnetic resonance imaging. To increase the colloidal stability and allow subsequent reactions, the introduction of hydrophilic functional groups onto the particles’ surface is essential. During this process, the original coating is exchanged by preferably covalently bonded ligands such as trialkoxysilanes. The duration of the silane exchange reaction, which commonly takes more than 24 h, is an important drawback for this approach. In this paper, we present a novel method, which introduces ultrasonication as an energy source to dramatically accelerate this process, resulting in high-quality waterdispersible nanoparticles around 10 nmin size. To prove the generic character, different functional groups were introduced on the surface including polyethylene glycol chains, carboxylic acid, amine, and thiol groups. Their colloidal stability in various aqueous buffer solutions as well as human plasma and serum was investigated to allow implementation in biomedical and sensing applications.status: publishe

American College of Rheumatology Provisional Criteria for Clinically Relevant Improvement in Children and Adolescents With Childhood-Onset Systemic Lupus Erythematosus

10.1002/acr.23834ARTHRITIS CARE & RESEARCH715579-59

Long Polar Fimbriae participates in the induction of neutrophils transepithelial migration across intestinal cells infected with enterohemorrhagic E. coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) strains are causative agents of diarrhea and hemorrhagic colitis, both diseases associated with intestinal inflammation and cell damage. Several studies have correlated EHEC virulence factors to high levels of intestinal pro-inflammatory cytokines and we have previously described that the Long polar fimbriae (Lpf) is involved in the secretion of interleukin-8 (IL-8) and up-regulation of genes belonging to the NF-κB pathway using non-polarized epithelial intestinal T84 cells. In the current study, we evaluated the two EHEC O157 Lpf fimbriae (Lpf1 and Lpf2) for their ability to induce intestinal secretion of IL-8 and the activation of IL8, CCL20 and ICAM1 genes on polarized T84 cells. We also determined the participation of Lpf1 and Lpf2 in transepithelial migration of polymorphonuclear neutrophils (PMNs). Polarized T84 cells infected with EHEC revealed that both, Lpf1 and Lpf2, were required for the secretion of IL-8 and the induction of IL8, CCL20 and ICAM1 genes. Both fimbriae also played a role in the migration of PMNs trough the intestinal cells monolayer. Overall, the present work further demonstrated that the fimbriae Lpf1 and Lpf2 are important bacterial virulence factors that might be involved in the inflammatory responses associated with EHEC infections

The Role of Fibronectin in the Adherence and Inflammatory Response Induced by Enteroaggregative Escherichia coli on Epithelial Cells

Enteroaggregative Escherichia coli (EAEC) infections are still one of the most important etiologic pathogens of diarrhea in children worldwide. EAEC pathogenesis comprises three stages: adherence and colonization, production of toxins, and diarrhea followed by inflammation. Previous studies have demonstrated that EAEC strains have the ability to bind to fibronectin (FN); however, it remains unknown what role this extracellular matrix protein plays in the inflammatory response induced by EAEC. In this study, we postulated that FN-mediated adherence of EAEC strains to epithelial cells increases the expression of pro-inflammatory genes. To verify this hypothesis, we infected HEp-2 and HT-29 cells, in both the presence and absence of FN, with EAEC reference strain 042. We quantified IL-8 secretion and the relative expression of a set of genes regulated by the NF-κB pathway. Although FN increased EAEC adherence, no changes in IL-8 protein secretion or IL8 gene expression were observed. Similar observations were found in HEp-2 cells transfected with FN-siRNA and infected with EAEC. To evaluate the involvement of AAF/II fimbriae, we infected HEp-2 and HT-29 cells, in both the presence and absence of FN, with an EAEC 042aafA mutant strain transformed with a plasmid harboring the native aafA gene with a site-directed mutation in Lys72 residue (K72A and K72R strains). No changes in the IL-8 secretion were observed. Finally, SEM immunogold assay of cells incubated with FN and infected with EAEC revealed that AAF fimbriae can bind to cells either directly or mediated by FN. Our data suggest that FN participates in AAF/II fimbriae-mediated adherence of EAEC to epithelial cells, but not in the inflammatory response of cells infected by this pathogen