Rotavirus y campylobacter fetus jejuni asociados a un brote de diarrea en terneros

artículo -- Universidad de Costa Rica. Instituto de Investigaciones en Salud, 1984Rotaviruses and Campylobacter fetus jejuni are ubiquitous agents of diarrheal disease in animals and humans. Under natural conditions they do not seem to cross inter-species barriers; a zoonosis has not been documented for man. However, animal rotaviruses might contribute to the emergence of new reassortment strains in view of their segmented genome, and thus, produce new antigenic variants. On the contrary, Campylobacter fetus jejuni produces a true zoonosis. Man acquires bacilli by ingesting water and foodstuffs contaminated with feces from infected animals. In an outbreak of diarrhea in 22 calves, rotavirus was detected in 8 (36%) and Campylobacter in 6 (27%). Three (14%) calves experienced double infection. There were no human cases involved in this outbreak.Universidad de Costa Rica. Instituto de Investigaciones en SaludUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA

Diarrhea associated with rotaviruses, enterotoxigenic Escherichia coli, Campylobacter, and other agents in costa rican children, 1976-1981

artículo -- Universidad de Costa Rica. Instituto de Investigaciones en Salud, 1983Rotaviruses, enterotoxigenic Escherichia coil, Salmonella, Shigella, and parasites were investigated in outpatient diarrheic children, and in hospitalized diarrheic and nondiarrheic children, between January 1976 and June 1979. In outpatient cases studied within 4 days of onset of symptoms, rotaviruses were the most common agents (45.3%); E. coil heatstable enterotoxin ranked second (13.4%); Shigella was third (8.1%); Salmonella was fourth (7.3%). In 63.2%, one or more enteric agents were detected. In hospitalized non-diarrheic children, asymptomatic shedding of pathogens was rarely observed. A later survey of outpatient diarrheic children revealed Campylobacter fetus jejuni in 8%. In 5.5 years of observation rotaviruses were endemic with excess frequency in the dry and cooler months of December and January. The excess occurrence of bacterial pathogens did not coincide with that of rotaviruses.Universidad de Costa Rica, Instituto de investigaciones en Salud.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA

Emergence of an Outbreak-Associated Clostridium difficile Variant with Increased Virulence

The prevalence of Clostridium difficile infections has increased due to the emergence of epidemic variants from diverse genetic lineages. Here we describe the emergence of a novel variant during an outbreak in a Costa Rican hospital that was associated with severe clinical presentations. This C. difficile variant elicited higher white blood cell counts and caused disease in younger patients than did other strains isolated during the outbreak. Furthermore, it had a recurrence rate, a 30-day attributable disease rate, and disease severity as great as those of the epidemic strain NAP1. Pulsed-field gel electrophoresis genotyping indicated that the outbreak strains belong to a previously undescribed variant, designated NAPCR1. Whole-genome sequencing and ribotyping indicated that the NAPCR1 variant belongs to C. difficile ribotype 012 and sequence type 54, as does the reference strain 630. NAPCR1 strains are resistant to fluoroquinolones due to a mutation in gyrA, and they possess an 18-bp deletion in tcdC that is characteristic of the epidemic, evolutionarily distinct, C. difficile NAP1 variant. NAPCR1 genomes contain 10% more predicted genes than strain 630, most of which are of hypothetical function and are present on phages and other mobile genetic elements. The increased virulence of NAPCR1 was confirmed by mortality rates in the hamster model and strong inflammatory responses induced by bacteria-free supernatants in the murine ligated loop model. However, NAPCR1 strains do not synthesize toxin A and toxin B at levels comparable to those in NAP1 strains. Our results suggest that the pathogenic potential of this emerging C. difficile variant is due to the acquisition of hypothetical functions associated with laterally acquired DNA.Universidad de Costa Rica/[803-B1-654]/UCR/Costa RicaUniversidad de Costa Rica/[803-B1-602]/UCR/Costa RicaConsejo Nacional de Ciencia y Tecnología/[FV-0004-13]/CONICIT/Costa Rica. Fondo gestionado a través de FORINVESPrograma de Cooperación Internacional/[130621650]/FA0/BrasilWellcome Trust/[098051}//Reino UnidoWellcome Trust/[086418]//Reino UnidoWellcome Trust/[098051]//Reino UnidoUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

A Clostridium difficile Lineage Endemic to Costa Rican Hospitals Is Multidrug Resistant by Acquisition of Chromosomal Mutations and Novel Mobile Genetic Elements

The antimicrobial resistance (AMR) rates and levels recorded for Clostridium difficile are on the rise. This study reports the nature, levels, diversity, and genomic context of the antimicrobial resistance of human C. difficile isolates of the NAPCR1/RT012/ST54 genotype, which caused an outbreak in 2009 and is endemic in Costa Rican hospitals. To this end, we determined the susceptibilities of 38 NAPCR1 isolates to 10 antibiotics from seven classes using Etests or macrodilution tests and examined 31NAPCR1 whole-genome sequences to identify single nucleotide polymorphisms (SNPs) and genes that could explain the resistance phenotypes observed. The NAPCR1 isolates were multidrug resistant (MDR) and commonly exhibited very high resistance levels. By sequencing their genomes, we showed that they possessed resistance-associated SNPs in gyrA and rpoB and carried eight to nine acquired antimicrobial resistance (AMR) genes. Most of these genes were located on known or novel mobile genetic elements shared by isolates recovered at different hospitals and at different time points. Metronidazole and vancomycin remain the first-line treatment options for these isolates. Overall, the NAPCR1 lineage showed an enhanced ability to acquire AMR genes through lateral gene transfer. On the basis of this finding, we recommend further vigilance and the adoption of improved control measures to limit the dissemination of this lineage and the emergence of more C. difficile MDR strains.Ministerio de Ciencia, Tecnología y Telecomunicaciones de la República/[803-B4-510]MICITT/Costa RicaUniversidad de Costa Rica/[803-B4-652]/UCR/Costa RicaUniversidad de Costa Rica/[803-B5-600]/UCR/Costa RicaUniversidad de Costa Rica/[803-B5-770]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Molecular detection of Clostridium difficile on inert surfaces from a Costa Rican hospital during and after an outbreak

Background: Hospital transmission of Clostridium difficile is fostered by contamination of surfaces and medical equipment with spores highly resistant to disinfectants and regular cleaning procedures. Despite the outbreaks and fatalities that C difficile causes, its epidemiology has not been studied in hospitals from middle- and low-income countries. To tackle this knowledge gap, the detection frequency of C difficile DNA on inert surfaces of a major Costa Rican hospital during and after an outbreak was compared. Methods: We used a presence-absence real-time polymerase chain reaction to detect a fragment of the tpi gene of C difficile on 21 surface samples collected during an outbreak and 54 surface samples taken 2 years later at the same hospital. Results: C difficile DNA was detected in 40% of the 75 environmental samples analyzed. Whereas 71% of the samples collected during the outbreak were positive, only 28% of the samples obtained 2 years after the outbreak gave the same result. This 2.5× ratio was maintained when the comparison was restricted to the wards that were sampled both during and after the outbreak (72% vs 35%, P = .016). Conclusions: Our results show that environmental surfaces in the hospital analyzed are continuously being contaminated with C difficile DNA and that their level of contamination is higher during an outbreak than after itUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Genes that encode botulism neurotoxins A, B, E and F in Neotropical bee honey identified with the Polymerase Chain Reaction

La miel de abeja es un producto que podría ser utilizado en el tratamiento de heridas, abrasiones y quemaduras de piel; no obstante, podría estar contaminada con esporas de C. botulinum. Con el fin de evaluar muestras de miel de origen costarricense, se detectó las secuencias de genes productores de neurotoxina correspondientes a C. botulinum tipos A, B, E y F utilizando la técnica de PCR (reacción de polimerasa en cadena). 64 diferentes muestras de miel, provenientes de diversos sitios costarricenses, fueron analizadas. Con el fin de evaluar la efectividad del método, se utilizó cepas de referencia tipos A (ATCC 19397), B (ATCC 7949), E (ATCC 17786) y F (ATCC 25764). El procedimiento consistió en cultivar las muestras de miel en caldo triptona peptona glucosa levadura prerreducido, por cinco días. Luego de esto, se obtuvo un lisado bacteriano, el cual fue utilizado para la técnica de PCR. Los amplicones producto de la reacción fueron visualizados utilizando geles de azarosa al 2%. De las 64 muestras de miel analizadas, ninguna produjo resultados positivos en el PCR. No obstante, todas las cepas de referencia usadas como controles produjeron bandas, que demuestran la efectividad del método de extracción utilizado y de la técnica en sí. Los resultados obtenidos demuestran la ausencia de contaminación por C. botulinum de la miel de abeja de origen costarricense, no obstante, deben realizarse evaluaciones adicionales para garantizar la seguridad del producto

Bacterias anaerobias aisladas en muestras clínicas de pacientes de un hospital regional de adultos de Costa Rica

Introduction. Anaerobic bacteria are important etiological agents of infections linked to human morbidity and mortality. Nevertheless, the limited development of most Costa Rican health care facilities with respect to the isolation and manipulation of anaerobic bacteria prevents the compilation of precise data on the prevalence of this sort of bacteria in the country. Objective. To assess the occurrence of anaerobic bacteria in clinical samples collected in a regional hospital in Costa Rica. Material and Methods. During 18 months, anaerobic bacteria were cultivated from diverse aspirates following a standardized protocol for sample collection and isolation developed for this study.Results. Forty seven anaerobic bacteria were isolated from 22 of 46 samples analyzed (48% of prevalence). While 25 of these organisms exhibited Gram-negative staining (53%), 22 isolates were characterized as Gram-positive (47%). Most bacteria were allocated to the genus Bacteroides (42%), followed by Eggerthella (13%) and Propionibacterium (11%). A limited number of isolates was identified as Prevotella sp., Bifidobacterium sp., Porphyromonas sp., or Fusobacterium sp. Discussion. Although traditional procedures for sample collection and isolation of anaerobic bacteria are painstaking, a diversity of anaerobic species, including very strict and fastidious species, was isolated with the practical method applied in the current investigation. On account of this remark, we urge others to implement our protocol in their health care facilities, as this would likely help to define more precisely the role that anaerobic bacteria play in clinical infections.Introducción. Las bacterias anaerobias se han implicado en algunos procesos infecciosos que son causa importante de morbilidad y mortalidad en el humano. Sin embargo, el limitado desarrollo de la bacteriología anaerobia en algunos centros médicos de Costa Rica no permite obtener datos precisos sobre su prevalencia. Objetivo. Evaluar la presencia de bacterias anaerobias en muestras clínicas de pacientes en un hospital regional de adultos en Costa Rica. Material y Métodos. Durante 18 meses se recolectaron, por aspiración, 46 muestras provenientes de diferentes sitios anatómicos. Se implementó un protocolo de toma de muestra en el hospital y de aislamiento en el laboratorio. Resultados. De las 46 muestras recibidas 22 (48%) fueron positivas para bacterias anaerobias, a partir de las cuales se aislaron 47 cepas: 25 (53%) Gram negativas y 22 (47%) Gram positivas. Bacteroides fue el género más frecuente (42%), seguido de Eggerthella (13%) y Propionibacterium (11%) en tanto que Prevotella, Bifidobacterium, Porphyromonas y Fusobacterium fueron los menos frecuentes. Discusión. Los procedimientos de toma de muestra y aislamiento en bacteriología anaerobia siempre van a ser difíciles de realizar; sin embargo, en el presente estudio se aislaron una diversidad de especies e incluso anaerobios considerados como muy estrictos y exigentes. Lo anterior respalda los protocolos implementados, por lo que consideramos importante aplicarlos en otros centros médicos en Costa Rica, a fin de obtener un conocimiento más preciso acerca de los agentes anaerobios de mayor importancia

Caracterización molecular y resistencia antimicrobiana de aislamientos de Clostridium perfringens de diferentes orígenes en Costa Rica

Clostridium perfringens es un bacilo Gram positivo, esporulado, anaerobio, ampliamente distribuido en la naturaleza, que produce cuatro toxinas principales α, β, ε y ι, las cuales permiten su clasificación en cinco toxinotipos (A-E). Algunas cepas producen una enterotoxina (CPE), codificada por el gen cpe, que causa diarrea en seres humanos y en algunos animales. La presencia de los genes de estas toxinas y la sensibilidad a los antibióticos se determinó en 81 cepas de C. perfringens previamente aisladas y que habían sido mantenidas a -80°C; 20 de suelos, 20 de origen animal, 20 de origen humano y 21 de alimentos cocidos no relacionados con brotes alimentarios. De acuerdo con los resultados de PCR, todas las cepas fueron clasificadas como C. perfringens tipo A, debido a que solo se les detectó el gen de la toxina α, mientras que el gen de la enterotoxina (cpe) se detectó en dos cepas (2.5%) aisladas de alimentos, tal como ha sido descrito en otras regiones del mundo. El 44% de las cepas fue resistente a algún antibiótico; clindamicina (41%), cloranfenicol (25%), penicilina (22%) y metronidazol (20%). En general, las cepas provenientes de suelos presentaron los mayores porcentajes de resistencia a casi todos los antibióticos. El 40% de las cepas de suelo presentó multiresistencia (a tres o más grupos de antibióticos), el 30% de las de origen humano, el 14% de las de alimentos y el 5% de las de origen animal. Las altas tasas de resistencia encontradas podrían deberse al amplio uso de antibióticos como promotores de crecimiento de plantas y animales y esas cepas resistentes podrían actuar como reservorio de genes de resistencia que pueden transferirse entre bacterias de diversos ambientes

Maggot therapy with lucilia eximia (Dipteria: Calliphoridae) of Costa Rica in an experimental model

Con el fin de analizar la aceleración en la solución de heridas dérmicas mediante el tratamiento con larvas de Lucilia eximia (Diptera: Calliphoridae), se generaron úlceras en piel en parejas de ratas mediante inoculación subcutánea de veneno de Bothrops asper (4,7 mg/0,2 mL). En cada ensayo una rata fue sometida a terapia (rata de prueba) y la otra no (rata control). Se probaron larvas de segundo estadio (L2) y larvas de tercer estadio temprano (L3). Los recambios larvales se hicieron cada 3 a 5 días hasta la solución de la úlcera. Se documentó, en cada caso, cuál rata (prueba o control) resolvió primero. Se efectuaron 17 ensayos, 10 con L2 y 7 con L3. En 8 (47,0%), la rata de prueba solucionó la úlcera primero; en 7 (41,2%) hubo una solución simultánea y en 2 (11,8 %) la rata control solucionó primero. De acuerdo al estadio, al usar L2, el 50,0% de las ratas tratadas resolvió primero, mientras que cuando se emplearon L3, sólo un 42,0% lo hizo. El análisis estadístico no permitió evidenciar un efecto acelerador asociado con la terapia larval en el modelo implementado (c2 =0,12; p=0,943; gl=2). En varios experimentos se observaron abscesos miásicos. El tipo de úlcera generado, de carácter agudo, pudo constituir una limitante en la evaluación de la terapia. El hallazgo de abscesos miásicos supone la necesidad mayor investigación antes de promover la idoneidad de L. eximia como agente para terapia larval.In order to analyze the accelerating healing effect of the treatment of dermal wounds with maggots of Lucilia eximia (Diptera: Calliphoridae), skin ulcers were generated by subdermal inoculation of venom from Bothrops asper (4.7 mg/0.2 mL) on couples of rats. For each experiment one rat was treated (test rat) and the other was untreated (control rat). Second instar larvae (L2) and early third instar larvae (L3) were applied. The maggots were substituted each third to fifth day until the resolution of the ulcer. In each experiment, the rat that resolved the ulcer first (test or control) was documented. Seventeen experiments were performed, 10 utilizing L2 and 7 with L3. In 8 (47.0 %), the test rat resolved the ulcer first; in 7 (41.2 %) there was a simultaneous resolution, and in 2 assays (11.8 %) the control rat resolved first. According to the instar, when L2 were applied, 50.0 % of the treated rats resolved the ulcer before control, while only 42.0 % resolved the wound first when L3 were used. The statistical analysis did not demonstrate an accelerator effect of maggot therapy in the implemented model (c2 =0.12; p=0.943; gf=2). In several experiments, myiasic abscesses were observed. The type of ulcer generated, which is acute, could be a limitation for evaluating larval therapy. The finding of myiasic abscesses must be investigated prior to recommending L. eximia as a secure agent in maggot therapyUniversidad de Costa Rica/[803-B1-067]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Etiología anaerobia de la diarrea nosocomial asociada a antibióticos en adultos mayores

Introduction. C. difficile and C. perfringens are important etiological agents of antibiotic associated diarrhea in adults and children worldwide, including Costa Rica. We intend to expand on previous research by including hospitalized patients over 60 years, to evaluate their additional factors and more severe illnesses. Materials and Methods. Over an 18 month period 51 samples of diarrheic feces were screened and cultured for C. difficile and C. perfringens enterotoxins. Every isolate was evaluated for toxin production and antibiotic susceptibility. Results. C. difficile toxin A was detected in 12 samples (24%) and C. perfringens enterotoxin in 10 (20%). C. difficile was isolated from 6 samples (12%) and C. perfringens from 16 (31%). All C. difficile isolates and only two C. perfringens were toxigenic, but both agents were isolated from two samples. Every C. difficile isolate was sensitive to beta-lactam, chloramphenicol and metronidazole, but most were resistant to clindamycin. A high percentage of C. perfringens strains showed resistance to beta lactam antibiotics, chloramphenicol and metronidazole and like C. difficile, also clindamycin. Conclusions. According to toxin detection in feces, C. difficile and C. perfringens were the etiological agents in 44% of the diarrhea cases, reinforcing the need to consider both agents for diagnosis of diarrhea in hospitalized elderly patients. Clindamycin resistance shown by many isolates should be noted, since it has been implicated as a risk factor for the development of diarrhea. Metronidazole may not be the best choice, since 25% of C. perfringens strains were also resistant to this drug.Introducción. C. difficile y C. perfringens son importantes agentes causales de diarrea asociada a antibióticos en adultos y niños en el mundo, incluyendo Costa Rica. Con este estudio, pretendemos ampliar las investigaciones en pacientes hospitalizados mayores de 60 años, quienes tienen más factores de riesgo y presentan cuadros de mayor severidad. Materiales y Métodos. La presencia de C. difficile y de C. perfringens y sus enterotoxinas se determinó en 51 muestras de heces diarreicas durante 18 meses. Todos los aislamientos fueron estudiados por producción de toxinas y sensibilidad a los antibióticos. Resultados. Se detectó la toxina A de C. difficile en 12 muestras (24%) y la enterotoxina de C. perfringens en 10 (20%). Se aisló C. difficile a partir de seis muestras (12%) y C. perfringens de 16 (31%). Todos los aislamientos de C. difficile fueron toxigénicos, pero sólo dos de C. perfringens lo fueron; en dos de las muestras se aislaron ambos agentes. Todas las cepas de C.difficile fueron sensibles a beta lactámicos, cloranfenicol y metronidazol, y la mayoría resistentes contra clindamicina. Un alto porcentaje de C. perfringens mostró resistencia contra beta lactámicos, cloranfenicol y metronidazol y, al igual que C. difficile, también contra clindamicina. Conclusiones. De acuerdo con la detección de toxinas en heces, C. difficile y C. perfringens fueron los agentes etiológicos de 44% de los casos; lo que refuerza la necesidad de considerarlos en el diagnóstico de diarreas de adultos mayores hospitalizados. Es de interés resaltar la resistencia contra clindamicina en los aislamientos, puesto que se ha implicado como un factor de riesgo para el desarrollo de la diarrea. Además, el uso de metronidazol para el tratamiento de estas diarreas puede no ser adecuado si el agentes es C. perfringens, pues el 25% fue resistente contra esta droga