Patrones de resistencia a antibióticos de Acinetobacter baumannii en un hospital de Colombia

Objetivo: Identificar patrones de resistencia en los aislamientos de A. baumannii obtenidos en una unidad de cuidados intensivos de Cali, Colombia. Diseño: Estudio descriptivo, prospectivo de corte transversal. Institución: Clínica Universitaria Rafael Uribe Uribe, Cali, Colombia. Materiales: Los aislamientos se obtuvieron de cultivos de muestras de sangre, heridas quirúrgicas, secreción nasal, orina, secreción uretral y puntas de catéter. Intervenciones: Se recolectaron 52 aislamientos durante los años 2009 y 2010. Mediante el análisis del antibiograma se identificaron patrones de resistencia (antibiotipos), se realizó antibiograma cuantitativo y se construyó un cladograma basado en el agrupamiento por el método de promedios aritméticos de grupos apareados no ponderados (conocido en inglés como UPGMA). Principales medidas de resultados: Medida de la concentración mínima inhibitoria (CMI) y el coeficiente de similitud generado por las distancias de los diámetros de los halos de inhibición entre dos aislamientos (antibiograma cuantitativo). Resultados: Se identificaron 5 antibiotipos; el 50% de los aislamientos se agruparon en el antibiotipo 1, con resistencia a todos los antibióticos y sensibilidad a tigeciclina y sulperazona; el antibiotipo 4 agrupó los aislamientos con resistencia a todos los antibióticos (19,3%). En el antibiograma cuantitativo se identificó dos clados con 5 y 47 aislamientos, respectivamente. Conclusiones: Los aislamientos de Acinetobacter baumannii tuvieron pocas diferencias fenotípicas y es probable que presenten alguna de las β-lactamasas tipo OXA

PCR para la confirmación de transmisión experimental de Leishmania chagasi a hámster sano por picadura de Lutzomyia longipalpis (Diptera: Psychodidae).

The use of PCR (polymerase chain reaction) was evaluated for its effectiveness as a tool in the detection of transmission of Leishmania chagasi to a hamster host, Mesocricetus auratus, by insect vector bite. Two pairs of uninfected and anesthetized hamsters were introduced into cages containing infected females of the typical phlebotomine sand fly vector, Lutzomyia longipalpis. The flies were experimentally infected with Leishmania chagasi and the infection was verified by dissection of subsamples. At 37 and 51 days after exposure to the infected flies, biopsies of each hamster's liver and spleen were subjected to direct histopathological and PCR examination. DNA was extracted with Chelex 100; for PCR amplification, primers specific to Leishmania minicircle DNA were used. PCR product was separated on agarose gels and visualized with UV. A band of approximately 120 base pairs was observed in 3 of the 4 biopsies, corresponding to the expected minicircle size. PCR was the only method that detected presence of the parasite. The results demonstrated that the sensitivity of PCR greatly expedites the confirmation process of a particular phlebotomine species as a vector of leishmaniasis.Se evaluó la efectividad de la PCR como herramienta en la detección de la transmisión experimental de Leishmania chagasi a hámster, Mesocricetus auratus, por picadura del insecto vector. Dos pares de hámsteres sanos y anestesiados fueron colocados en jaulas que contenían hembras de Lutzomyia longipalpis. Previamente, las hembras se infectaron experimentalmente con Leishmania chagasi y la infección se confirmó por disección en una submuestra. A los 37 y 51 días después de la exposición a los insectos infectados, las biopsias de hígado y bazo de cada hámster se sometieron a examen directo al microscopio, histopatología y PCR. El ADN se extrajo con Chelex 100®; en la amplificación se utilizó un par de iniciadores específicos para la región conservada de los minicírculos del ADN de Leishmania. El producto amplificado se separó en geles de agarosa y se visualizó bajo luz UV. En tres de las cuatro biopsias se observó una banda de 120 pares de bases, aproximadamente, correspondiente al tamaño esperado de la fracción del minicírculo. La técnica de PCR fue el único método que detectó la presencia del parásito. Estos resultados demostraron que la sensibilidad de la PCR acelera los procesos de incriminación vectorial de las especies vectoras de leishmaniasis

Distribución de los vectores de Leishmania infantum (Kinetoplastida: Trypanosomatidae) en Colombia

Introduction. Since entomological surveillance is the main control strategy for visceralleishmaniasis, updated information on the distribution and ecology of involved vector species isnecessary for planning preventive measures.Objective. To present the updated and geo-referenced distribution of L. longipalpis and L.evansi, vectors of visceral leishmaniasis in Colombia, considering their relationship with their habitat.Materials and methods. Distribution was estimated from records of the sand fly specimenscollected since 1967.The information was organized in a database from which the localitieswere selected and geographically analyzed with Arc view in order to develop the distributionmaps.Results. 40 localities were established for L. longipalpis along the upper (24), middle (11) andlower (5) Magdalena river valley. L. evansi was recorded in 19 localities of the middle (5) andlower (14) Magdalena valley.Conclusions. Both species showed consistent association with dry tropical forest (sensuHoldridge 1967), confirming the epidemiological risk for visceral leishmaniasis in these areas.Introducción. Debido a la importancia que tiene la vigilancia entomológica como principalmedida de control en el manejo de la leishmaniasis visceral, es necesario contar con informaciónactualizada acerca de la distribución y ecología de los insectos involucrados en la transmisiónpara optimizar las estrategias de prevención.Objetivo. Presentar la distribución actualizada geo-referenciada de L. longipalpis y L. evansi,vectores de los parásitos que causan leishmaniasis visceral en Colombia, teniendo en cuentala asociación de los insectos con su hábitat.Materiales y métodos. Los registros de distribución se obtuvieron a partir de los ejemplaresrecolectados en Colombia desde 1967. La información obtenida se organizó en una base dedatos a partir de la cual se tomaron las localidades que, posteriormente, fueron sometidas aanálisis geográficos por medio de Arc View que se utilizaron para realizar los mapas dedistribución.Resultados. Para L. longipalpis se obtuvieron 40 localidades todas distribuidas a lo largo delvalle del río Magdalena: Alto (24), Medio (11) y Bajo (5) Magdalena. L. evansi fue registradoen 19 localidades también ubicadas en el mismo valle: cinco en el Magdalena Medio y 14 elMagdalena Bajo.Conclusiones. Ambas especies demostraron una consistente asociación con regionesclasificadas principalmente como bosque seco tropical según las zonas de vida de Holdridgelo que confirma el riesgo epidemiológico de leishmaniasis visceral en estas áreas.Palabras clave: Psychodidae, leishmaniasis visceral, Colombia

Definición de las condiciones de temperatura y almacenamiento adecuadas en la detección de ADN de Leishmania por PCR en flebotominos.

For epidemiological studies and control programs of leishmaniasis, taxonomic identification of the etiologic agent of the disease in the insect vector is of critical importance. The implementation of molecular techniques such as the polymerase chain reaction (PCR) has permitted great advances in the efficacy and sensitivity of parasite identification. Previously, these investigations involved labor-intensive dissections and required expert personnel. The present work evaluates the effects of storage methods of phlebotomine samples in the optimization of PCR identification of Leishmania. Females of Lutzomyia longipalpis, from the colony of the Instituto Nacional de Salud, were experimentally infected with Leishmania chagasi (= L. infantum), from the upper Magdalena Valley (Quipile, Cundinamarca, Colombia). The infected insects were preserved in three solutions: 100% ethanol, 70% ethanol, and TE; subsamples of each class were stored at -80 degrees C, -20 degrees C and room temperature. To determine infection rates, samples were dissected and screened microscopically. Chelex 100 was used for extraction of total Leishmania DNA. For PCR amplification, the kinetoplastic minicircle DNA primers OL1 and OL2 of Leishmania were used, and the products were visualized by electrophoresis in 1% agarose gels. For each of the 3 storage conditions, amplifications were successful, producing a approximately 120 base pair product unique to Leishmania. The results demonstrated the advantage of PCR as a routine screening method for detecting infected flies in endemic foci of visceral leishmaniasis. Since storage method did not affect PCR amplification success, the most cost effective method -70% ethanol at room temperature--is the option recommended for storing entomological samples in vector incrimination studies.Para estudios epidemiológicos y programas de control de las leishmaniasis es importante la determinación taxonómica del insecto vector y del agente etiológico causante de esta enfermedad. Por su eficacia y sensibilidad, la utilización de técnicas moleculares, como la reacción en cadena de la polimerasa (PCR) ha permitido avances en entomología médica. La metodología tradicional usada para la búsqueda de infección en los flebótomos es un método dispendioso que requiere mucho tiempo y capacitación para emitir un diagnóstico acertado. En el presente trabajo se evaluaron las condiciones y prácticas adecuadas para el almacenamiento de flebótomos con el propósito de aplicar la PCR. Hembras de Lutzomyia longipalpis de la colonia del Instituto Nacional de Salud se infectaron experimentalmente con una cepa de Leishmania chagasi del valle alto del río Magdalena (Quipile, Cundinamarca). Los insectos infectados se preservaron en tres soluciones: etanol al 100%, etanol al 70% y en buffer Tris- EDTA (TE); submuestras de cada una se almacenaron a -80 °C, -20 °C y a temperatura ambiente. Para determinar los porcentajes de infección, algunas de las hembras se disecaron para buscar las formas flageladas al microscopio. La extracción del KADN se realizó con Chelex 100. Para la amplificación se utilizaron los iniciadores OL1 y OL2 de Leishmania con electroforesis en geles de agarosa al 1%. En cada una de las condiciones descritas se logró la amplificación de un fragmento de »120 pb, correspondiente a Leishmania spp. Estos resultados muestran la ventaja de incorporar como técnica de rutina la PCR para detectar flebótomos infectados de zonas endémicas de leishmaniasis visceral. Por costo-efectividad, se concluyó que las muestras entomológicas para estudios de incriminación vectorial utilizando la PCR, se pueden preservar a temperatura ambiente en etanol al 70%

Caracterización fenotípica de aislamientos de Acinetobacter baumannii en una institución de salud de alta complejidad de Cali

Introduction: Phenotypic characterization of the Acinetobacter genus bacteria through biochemical and microscopic tests is possible. Studies have shown that the isolates from health-care associated infections show high resistance to first-line antibiotics.Objective: To describe the resistance patterns of the A. baumannii isolates obtained in a health care institution, the phenotypic characteristics of the isolates, and the possible resistance mechanisms.Materials and methods: A descriptive cross-sectional study was conducted with 28 reports of samples taken from patients hospitalized with infection by A. baumannii. Susceptibility testing for ceftazidime, cefepime, meropenem, amikacin, and ciprofloxacin was performed with the Vitek™ automated system and the susceptible, intermediate, and resistant classification was based on the protocol established by the Clinical and Laboratory Standards Institute for 2007.Results: The highest percentage of isolates corresponded to males (53.6 %), to the infectology ward (28.5 %), and to the month of September (21.4 %); the most frequent sample site were endotracheal secretions (53.6 %). From the profile patterns for susceptibility to antibiotics used, 13 phylotypes were obtained.Conclusion: Acinetobacter baumannii is a pathogen resistant to multiple antimicrobial agents involved in outbreaks of health-care associated infections. The resistance profile patterns allow inferring that the possible resistance mechanisms present in the majority of the isolates are: Production of extended-spectrum β-lactamases, antibiotic modifying enzymes, and target site modification.Introducción. La caracterización fenotípica de las bacterias del género Acinetobacter mediante pruebas bioquímicas y microscópicas es posible. Varios estudios han demostrado que los aislamientos provenientes de infecciones asociadas a la atención en salud presentan una elevada resistencia a los antibióticos de primera elección.Objetivo. Describir los patrones de resistencia de los aislamientos de Acinetobacter baumannii obtenidos en una institución de salud, así como sus características fenotípicas y los posibles mecanismos de resistencia.Materiales y métodos. Se realizó un estudio descriptivo de corte transversal con 28 informes de muestras tomadas a pacientes hospitalizados con infección por A. baumannii. Las pruebas de sensibilidad para ceftazidime, cefepime, meropenem, amikacina y ciprofloxacina se realizaron con el sistema automatizado Vitek® y la clasificación de sensible, intermedia y resistente se hizo con base en el protocolo establecido por el Clinical and Laboratory Standards Institute para el año 2007.Resultados. El mayor porcentaje de aislamientos correspondió al sexo masculino (53,6 %), a la sala de infectología (28,5 %) y al mes de septiembre (21,4 %); el tipo de muestra más frecuente fue el de secreción endotraqueal (53,6 %). A partir de los patrones de los perfiles de sensibilidad a los antibióticos empleados se obtuvieron 13 filotipos.Conclusión. Acinetobacter baumannii es un agente patógeno resistente a múltiples antimicrobianos, involucrado en brotes de infecciones asociadas a la atención en salud. Los patrones de los perfiles de resistencia permiten inferir que los posibles mecanismos de resistencia presentes en la mayoría de los aislamientos son la producción de betalactamasas de espectro extendido, las enzimas modificadoras del antibiótico y la modificación del sitio diana

Epidemiología de bacterias nosocomiales resistentes a los antimicrobianos

Nosocomial infections are a major challenge for public health because of the high rates of morbidity and mortality generated. It was considered that the excessive or inappropriate use of antibiotics triggers the emergence of resistant strains. Among the clinically important bacteria that most commonly cause nososcomial infections, Gram positive multiresistant pathogens stand out such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus spp (VRE), and the Gram negative strains of Klebsiella pneumoniae, Escherichia coli, Pseudomonas spp. and Acinetobacter baumannii producing expanded spectrum β-lactamases (ESβL). This review describes the behavior of the main bacterial pathogens resistant to antibiotics that cause infections in Europe, United States, and Latin America, emphasizing studies of molecular epidemiology on a global scale, including the major epidemiological studies in Colombia. The genetic structure of S. aureus and Enterococcus spp strains shows a clonal characteristic favored by the predominance of a small number of clones with the capacity to spread globally, due probably to cross-infection. However, the introduction of MRSA strains from the community encourages genetic diversity, tending to establish a genetic polyclonal endemic structure in places like the United States. In Gram negative bacteria, the high genetic diversity among isolates, mainly in Latin American countries, indicates that the polyclonal spread is influenced by horizontal transfer of plasmids, by excessive exposure to antibiotics, and prolonged hospital stays. In Colombia, there is information on nosocomial resistant pathogens, but molecular epidemiological information is still scarce. Las infecciones nosocomiales constituyen un gran desafío para la salud pública por las altas tasas de morbilidad y mortalidad que generan. Se ha considerado que el uso inapropiado o excesivo de antibióticos desencadena la aparición de cepas resistentes. Entre las bacterias de importancia clínica que con mayor frecuencia causan infecciones nososcomiales, se destacan los patógenos Gram positivos multiresistentes como Staphylococcus aureus con resistencia a meticilina (SARM) y Enterococcus spp. resistentes a vancomicina (ERV). En los Gram negativos, hay resistencia sobre todo con las cepas de Klebsiella pneumoniae, Escherichia coli, Pseudomonas spp. y Acinetobacter baumannii productoras de β-lactamasas de espectro extendido (BLEEs, en inglés: ESβL expanded spectrum β-lactamases). Esta revisión tiene como finalidad realizar una decripción del estado de la resistencia bacteriana a los antibióticos en los principales patógenos que causan infecciones nosocomiales en países de Europa, Estados Unidos y de Latinoamérica, destacando los estudios de epidemiología molecular a escala global e incluyendo los principales estudios epidemiológicos realizados en Colombia. La estructura genética de las cepas de Staphylococus aureus y Enterococcus spp. evidencia una característica clonal favorecida por el predominio de un número pequeño de clones con capacidad de diseminarse en forma global, debida probablemente a infecciones cruzadas. Sin embargo, la introducción de cepas SARM desde la comunidad está favoreciendo la diversidad genética, tendiendo a establecerse una estructura genética policlonal en lugares endémicos como los Estados Unidos. En las bacterias Gram negativas, se destaca una alta diversidad genética entre los aislados, sobre todo en países de Latinoamérica, indicando que la diseminación sigue una estructura genética policlonal, influida por la transferencia horizontal de plasmidos, por la excesiva exposición a antibióticos y una estancia hospitalaria prolongada. En Colombia se dispone de información sobre los patógenos nosocomiales resistentes, pero la información epidemiológica molecular aún es escasa

Differential cytogenetic profile in advanced chronic myeloid leukemia with sequential lymphoblastic and myeloblastic blast crisis

Frequency of additional chromosomal abnormalities in chronic myeloid leukemia (CML) is estimated to be 7% in chronic phase and increases to 40–70% in advanced disease. Progression of CML from chronic phase to accelerated phase or blast crisis is often associated with secondary chromosomal aberrations. We report an exceptional case of CML as debut in lymphoblastic blast crisis and a subsequent progression in myeloblastic blast crisis with rare cytogenetic abnormalities

Factors associated with implementation of the 5A's smoking cessation model

Background: several health organizations have adopted the 5A's brief intervention model (Ask, Advise, Assess, Assist, Arrange), based on evidence-based guidelines for smoking cessation. We examine individual, cognitive, behavioral, and organizational factors associated with the 5A's performance among clinical healthcare workers in Catalonia. We also investigate how these factors interact and potentially predict the implementation of each component of the 5A's. Methods: a cross-sectional survey was conducted among clinical health workers enrolled in an online smoking cessation training course (n = 580). The survey included questions about individual characteristics as well as cognitive, behavioral, and organizational factors previously identified in research. We assessed self-reported performance of the 5A's, assessed on a scale from 0 to 10, and used Multivariate regression to examine factors associated with its performance. Results: the performance means (standard deviation) were moderate for the first 3A's [Ask: 6.4 (3.1); Advise: 7.1 (2.7); Assess: 6.3 (2.8)] and low for the last 2A's [Assist: 4.4 (2.9); Arrange: 3.2 (3.3)]. We observed a high correlation between Assist and Arrange (r = 0.704, p < 0.001). Having positive experiences and feeling competent were positively associated with performing the 5A's model and having organizational support with Assist and Arrange. Personal tobacco use among healthcare workers was negatively associated with Advice and Arrange. Conclusions: our study found that clinical healthcare workers do not perform the 5A's completely. The main barriers identified suggest the need of training and making available practical guidelines in healthcare services. Organizational support is essential for moving towards the implementation of Assist and Arrange

IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System

PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471

Dynamics of Cough Frequency in Adults Undergoing Treatment for Pulmonary Tuberculosis.

Background: Cough is the major determinant of tuberculosis transmission. Despite this, there is a paucity of information regarding characteristics of cough frequency throughout the day and in response to tuberculosis therapy. Here we evaluate the circadian cycle of cough, cough frequency risk factors, and the impact of appropriate treatment on cough and bacillary load. Methods: We prospectively evaluated human immunodeficiency virus-negative adults (n = 64) with a new diagnosis of culture-proven, drug-susceptible pulmonary tuberculosis immediately prior to treatment and repeatedly until treatment day 62. At each time point, participant cough was recorded (n = 670) and analyzed using the Cayetano Cough Monitor. Consecutive coughs at least 2 seconds apart were counted as separate cough episodes. Sputum samples (n = 426) were tested with microscopic-observation drug susceptibility broth culture, and in culture-positive samples (n = 252), the time to culture positivity was used to estimate bacillary load. Results: The highest cough frequency occurred from 1 pm to 2 pm, and the lowest from 1 am to 2 am (2.4 vs 1.1 cough episodes/hour, respectively). Cough frequency was higher among participants who had higher sputum bacillary load (P < .01). Pretreatment median cough episodes/hour was 2.3 (interquartile range [IQR], 1.2-4.1), which at 14 treatment days decreased to 0.48 (IQR, 0.0-1.4) and at the end of the study decreased to 0.18 (IQR, 0.0-0.59) (both reductions P < .001). By 14 treatment days, the probability of culture conversion was 29% (95% confidence interval, 19%-41%). Conclusions: Coughs were most frequent during daytime. Two weeks of appropriate treatment significantly reduced cough frequency and resulted in one-third of participants achieving culture conversion. Thus, treatment by 2 weeks considerably diminishes, but does not eliminate, the potential for airborne tuberculosis transmission