Genomic epidemiology of the primary methicillin-resistant Staphylococcus aureus clones causing invasive infections in Paraguayan children.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major human pathogens. It could carry numerous resistance genes and virulence factors in its genome, some of which are related to the severity of the infection. An observational, descriptive, cross-sectional study was designed to molecularly analyze MRSA isolates that cause invasive infections in Paraguayan children from 2009 to 2013. Ten representative MRSA isolates of the main clonal complex identified were analyzed with short-read paired-end sequencing and assessed for the virulome, resistome, and phylogenetic relationships. All the genetically linked MRSA isolates were recovered from diverse clinical sources, patients, and hospitals at broad gap periods. The pan-genomic analysis of these clones revealed three major and different clonal complexes (CC30, CC5, and CC8), each composed of clones closely related to each other. The CC30 genomes prove to be a successful clone, strongly installed and disseminated throughout our country, and closely related to other CC30 public genomes from the region and the world. The CC5 shows the highest genetic variability, and the CC8 carried the complete arginine catabolic mobile element (ACME), closely related to the USA300-NAE-ACME+, identified as the major cause of CA-MRSA infections in North America. Multiple virulence and resistance genes were identified for the first time in this study, highlighting the complex virulence profiles of MRSA circulating in the country. This study opens a wide range of new possibilities for future projects and trials to improve the existing knowledge on the epidemiology of MRSA circulating in Paraguay. IMPORTANCE: The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide. The most frequent MRSA clones identified in Paraguay in previous studies (including community and hospital acquired) were the Pediatric (CC5-ST5-IV), the Cordobes-Chilean (CC5-ST5-I), the SouthWest Pacific (CC30-ST30-IV), and the Brazilian (CC8-ST239-III) clones. In this study, the pan-genomic analysis of the most representative MRSA clones circulating in invasive infection in Paraguayan children over the years 2009-2013, such as the CC30-ST30-IV, CC5-ST5-IV, and CC8-ST8-IV, was carried out to evaluate their genetic diversity, their repertoire of virulence factors, and antimicrobial resistance determinants. This revealed multiple virulence and resistance genes, highlighting the complex virulence profiles of MRSA circulating in Paraguay. Our work is the first genomic study of MRSA in Paraguay and will contribute to the development of genomic surveillance in the region and our understanding of the global epidemiology of this pathogen.

Genomic epidemiology of Staphylococcus aureus isolated from bloodstream infections in South America during 2019 supports regional surveillance.

Staphylococcus aureus remains one of the leading causes of infections worldwide and a common cause of bacteraemia. However, studies documenting the epidemiology of S. aureus in South America using genomics are scarce. We hereby report on the largest genomic epidemiology study to date of both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in South America, conducted by the StaphNET-SA network. We characterised 404 genomes recovered from a prospective observational study of S. aureus bacteraemia in 58 hospitals from Argentina, Bolivia, Brazil, Paraguay and Uruguay between April and October 2019. We show that a minority of S. aureus isolates are phenotypically multi-drug resistant (5.2%), but more than a quarter are resistant to macrolide-lincosamide-streptogramin B (MLSb). MSSA were more genetically diverse than MRSA. Lower rates of associated antimicrobial resistance in community-associated(CA)-MRSA versus hospital-associated (HA)-MRSA were found in association with three S. aureus genotypes dominating the MRSA population: CC30-MRSA-IVc-t019-lukS/F-PV+, CC5-MRSA-IV-t002-lukS/F-PV- and CC8-MRSA-IVc-t008-lukS/F-PV+-COMER+. These are historically from a CA origin, carry on average fewer antimicrobial resistance determinants, and often lack key virulence genes. Surprisingly, CC398-MSSA-t1451-lukS/F-PV- related to the CC398 human-associated lineage is widely disseminated throughout the region, and is described here for the first time as the most prevalent MSSA lineage in South America. Moreover, CC398 strains carrying ermT (largely responsible for the MLSb resistance rates of MSSA strains: inducible iMLSb phenotype) and sh_fabI (related to triclosan resistance) were recovered from both CA and HA origin. The frequency of MRSA and MSSA lineages differed between countries but the most prevalent S. aureus genotypes are high-risk clones widely distributed in the South American region without a clear country-specific phylogeographical structure. Therefore, our findings underline the need for continuous genomic surveillance by regional networks such as StaphNET-SA. This article contains data hosted by Microreact.

Analysis of antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery.

PURPOSE: To analyze antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery with the focus on coagulase-negative staphylococci (CNS) during cataract surgery and discuss the implications of this colonization as a potential risk of acquiring endophthalmitis. METHODS: After approval of the institutional review board and informed consent from patients had been obtained, conjunctival swabs for culture from 59 patients undergoing cataract surgery were taken of the fellow eye at baseline (C0) and from the eye to be operated before (T0) and after (T1) irrigation with povine-iodine 5%, and at the end of surgery (T2). Genes responsible for virulence (mecA, ica and atlE) and antibiotic profile were determined; strain clonality of persistent colonizing Staphylococcus epidermidis strains was established by the Multi-locus sequence typing (MLST). RESULTS: The frequency of CNS was significantly reduced in T1 (13.6%) from 81.4% in T0 and 86.4% in C0. The frequency of mecA, ica and atlE genes was 34.4%, 37.5% and 61.4%, respectively; and methicillin phenotypic resistance was 35.4%. S. epidermidis was the most frequent species isolated in every time point. MLST revealed in 7 patients 100% coincidence of the seven alleles of the S. epidermidis isolated previous to povine-iodine 5% disinfection and at the end of the surgery. CNS isolates from T1 or T2 corresponded to the same species, antibiotic and virulence profile as those isolates from C0 or T0. CONCLUSION: Povidone-iodine 5% prophylaxis before surgery significantly reduced conjunctival contamination; in those that persisted, the source of contamination was mostly the patient's microbiota confirmed by the MLST system.

The evolutionary outcomes of climate-change-induced hybridization in insect populations.

Rapid range shifts are one of the most frequent responses to climate change in insect populations. Climate-induced range shifts can lead to the breakdown of isolation barriers, and thus, to an increase in hybridization and introgression. Long-term evolutionary consequences such as the formation of hybrid zones, introgression, speciation, and extinction have been predicted as a result of climate-induced hybridization. Our review shows that there has been an increase in the number of published cases of climate-induced hybridization in insects, and that the formation of hybrid zones and introgression seems to be, at the moment, the most frequent outcomes. Although introgression is considered positive, since it increases species' genetic diversity, in the long term, it could lead to negative outcomes such as species fusion or genetic swamping.

Terpenes Combinations Inhibit Biofilm Formation in Staphyloccocus aureus by Interfering with Initial Adhesion.

The biofilm is a conglomerate of cells surrounded by an extracellular matrix, which contributes to the persistence of infections. The difficulty in removing the biofilm drives the research for new therapeutic options. In this work, the effect of terpenes (−)-trans-Caryophyllene, (S)-cis-Verbenol, (S)-(−)-Limonene, (R)-(+)-Limonene, and Linalool was evaluated, individually and in combinations on bacterial growth, by assay with resazurin; the formation of biofilm, by assay with violet crystal; and the expression of associated genes, by real-time PCR, in two clinical isolates of Staphyloccocus aureus, ST30-t019 and ST5-t311, responsible for more than 90% of pediatric infections by this pathogen in Paraguay. All combinations of terpenes can inhibit biofilm formation in more than 50% without affecting bacterial growth. The most effective combination was (−)-trans-Caryophyllene and Linalool at a 500 µg/mL concentration for each, with an inhibition percentage of 88%. This combination decreased the expression levels of the sdrD, spa, agr, and hld genes associated with the initial cell adhesion stage and quorum sensing. At the same time, it increased the expression levels of the cap5B and cap5C genes related to the production of capsular polysaccharides. The combinations of compounds tested are promising alternatives to inhibit biofilm formation in S. aureus.

Litiasis vesical gigante recurrente en el adulto. Reporte de un caso / Recurrent giant bladder stones in the adult.Case report

RESUMEN La ocurrencia de casos de cálculos urinarios considerados como gigantes ha disminuido sensiblemente en su frecuencia desde el advenimiento de los antibióticos y el control apropiado de las infecciones urinarias. El diagnóstico de los mismos se da por sintomatología y estudios de imágenes. Se presenta el caso clínico de un hombre de 59 años de edad con litiasis vesical gigante recurrente. El paciente fue diagnosticado de dos litiasis de origen vesical tras realización de una tomografía axial computarizada del aparato urinario y posteriormente se le realizó una cistolitotomía para extracción de los cálculos de hasta 1435 g. El análisis morfoconstitucional de los cálculos reveló a la Brushita, Carbapatita y Estruvita como componentes principales. La evaluación metabólica señaló presencia de hipocitraturia e hiperoxaluria. Es el primer reporte en el país acerca de una litiasis gigante en que la composición de los cálculos indicaría procesos infecciosos como posibles causas de formación.

ABSTRACT The occurrence of cases of urinary stones considered as giant has decreased significantly in its frequency since the advent of antibiotics and the appropriate control of urinary tract infections. The diagnosis of them is based on symptomatology and imaging studies. We present a case of a 59-year-old man with recurrent giant bladder stones. The patient was diagnosed with two vesical stones after performing a computerized axial tomography of the urinary system and later a cystolithotomy was performed to remove stones of up to 1,435 g. The morphoconstitutional analysis of the calculus revealed brushite, carbapatite and struvite as main components. The metabolic evaluation indicated the presence of hypocitraturia and hyperoxaluria.This is the first report in the country of a giant lithiasis in which the composition of the stones would indicate infectious processes as the possible causes of formation.

In vitro antibacterial activity, biofilm formation inhibition and chromatographic profile of methanolic extracts of two Pterocaulon species against MRSA.

The methanolic extracts of Pterocaulon alopecuroides and Pterocaulon angustifolium were assayed for antibacterial activity and biofilm formation inhibition of four community-acquired-MRSA isolates representative of ST30 t975, ST30 t021, ST5 t311, and ST4335 t008 clones that are responsible for invasive infections in Paraguayan children. Both Pterocaulon extracts showed significant antibacterial activity with a minimum inhibitory concentration of 200 µg/mL against the four isolates. P. angustifolium showed inhibition of biofilm formation for the four isolates, whereas P. alopecuroides showed inhibition for three of them. The chemical constituents were identified by liquid chromatography coupled to tandem mass spectrometry. Phenolic compounds were detected in the two species as well as coumarins. These results showed that these plants are sources of compounds with activity against MRSA.

Next-generation sequencing, isolation and characterization of 14 microsatellite loci of Canthon cyanellus (Coleoptera: Scarabaeidae).

BACKGROUND: We used Illumina paired-end sequencing to isolate and characterize microsatellites of Canthon cyanellus, a Neotropical roller dung beetle, encompassing several lineages within its distribution range. METHODS AND RESULTS: We examined C. cyanellus specimens collected at eight different localities in Mexico (two or three specimens per locality). We initially performed amplification tests with 16 loci, but two of which were unsuccessful. The 14 remaining microsatellites were polymorphic, with 2-16 alleles each. The expected and observed heterozygosity ranged from 0.11 to 0.76 and from 0.20 to 0.78, respectively. CONCLUSIONS: These microsatellites will help to assess structure at the population and lineage levels, identify zones of potential hybridization between lineages, and draw a more precise geographic delimitation of C. cyanellus lineages.

Estandarización del análisis multi-locus de número variable de repeticiones en tándem automatizado para tipificación de Staphylococcus aureus Resistentes a Meticilina causantes de infecciones invasivas en población pediátrica paraguaya / Standardization of automated multi-locus variable number tandem repeats analysis for Methicillin-resistant Staphylococcus aureus typing isolated from invasive infections in paraguayan pediatric population

Introducción: Staphylococcus aureus es considerado uno de los patógenos humanos más importantes a nivel mundial y sus niveles de resistencia a meticilina han aumentado incluso en cepas aisladas de personas sin factores de riesgo nosocomial, por lo que la tipificación genética de los clones circulantes es fundamental para comprender los patrones de diseminación. Objetivo: Obtener la tipificación de SARM que causaron infecciones invasivas a niños mediante el empleo de la técnica de análisis multi-locus de número variable de repeticiones en tándem (MLVA) automatizada. Materiales y Métodos: Estudio observacional, descriptivo de corte transverso. Resultados: Se analizaron 25 cepas SARM que representan más de 700 aislamientos de S. aureus colectados en los años 2010, 2012 y 2013 de 4 hospitales de referencia nacional. La automatización de la técnica MLVA incluyó la tipificación del 88% (22/25) de los aislamientos en estudio, resultando 3 perfiles diferentes, cada uno asociado a un "spa tipo" distinto, siendo el perfil 1-t019 el predominante (86%), seguido por el perfil 3-t002 (9%), arrojando 100% de concordancia con el método MLVA manual, así como una alta concordancia con el método estándar de oro, PFGE. Conclusiones: La inclusión de un método de análisis de fragmentos automatizado permitió llevar a cabo la caracterización de aislamientos mejorando el tiempo de respuesta y manteniendo una alta sensibilidad en comparación con el método manual.

Introduction: Staphylococcus aureusis considered one of the most critical human pathogens worldwide, and its levels of methicillin resistance have increased even in strains isolated from people without nosocomial risk factors. Therefore the genetic typing of circulating clones is essential to understand dissemination patterns. Objective: Obtain the MRSA typing that caused invasive infections in children by using the automated multi-locus variable number of tandem repeats (MLVA) analysis technique. Materials and methods: Observational, descriptive, cross-sectional. Results: 25 strains MRSA representing more than 700S. aureusisolates collected in 2010, 2012, and 2013 from 4 national reference hospitals were analyzed. The MLVA automation included the typing of 88% (22/25) isolates, resulting in 3 different profiles, each one associated with a different spa type, being the 1-t019 the predominant (86%), followed by the 3-t002 profile (9%), yielding 100% concordance with the MLVA manual, as well as high concordance with the standard gold method, PFGE. Conclusions: The inclusion of an automated fragment analysis method led to the characterization of isolates, improving response time, and maintaining high sensitivity compared to the manual process.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) clones from Paraguayan children.

INTRODUCTION: Staphylococcus aureus is considered one of the most important human pathogens, and its levels of resistance to methicillin have increased even in strains isolated from people without nosocomial risk factors. Molecular analysis is essential for understanding the patterns of dissemination. The objective of this study was to identify community-acquired methicillin-resistant S. aureus (CA-MRSA) clones that infected Paraguayan children patients in two periods of time. METHODOLOGY: An observational, descriptive study was designed to determine the genetic variability of 115 isolates of CA-MRSA recovered from children who attended four reference centers in Paraguay between 2009-2010 and 2012-2013. RESULTS: The combined use of Pulsed Field Gel Electrophoresis (PFGE), Multi-Locus Sequencing Typing, Multi-Locus Variable Analysis (MLVA) and Spa typing techniques allowed the identification of two dominant clones: ST30-IV-t019 (77%) and ST5-IV-t311 (10%), and the establishment of the former as the leading cause of CA-MRSA infections in children during the study period. CONCLUSIONS: This is the first study that provides epidemiological information as well as microbiological and molecular characteristics of CA-MRSA isolates recovered from children from Asunción and the Central Department of Paraguay.

Insect responses to heat: physiological mechanisms, evolution and ecological implications in a warming world.

Surviving changing climate conditions is particularly difficult for organisms such as insects that depend on environmental temperature to regulate their physiological functions. Insects are extremely threatened by global warming, since many do not have enough physiological tolerance even to survive continuous exposure to the current maximum temperatures experienced in their habitats. Here, we review literature on the physiological mechanisms that regulate responses to heat and provide heat tolerance in insects: (i) neuronal mechanisms to detect and respond to heat; (ii) metabolic responses to heat; (iii) thermoregulation; (iv) stress responses to tolerate heat; and (v) hormones that coordinate developmental and behavioural responses at warm temperatures. Our review shows that, apart from the stress response mediated by heat shock proteins, the physiological mechanisms of heat tolerance in insects remain poorly studied. Based on life-history theory, we discuss the costs of heat tolerance and the potential evolutionary mechanisms driving insect adaptations to high temperatures. Some insects may deal with ongoing global warming by the joint action of phenotypic plasticity and genetic adaptation. Plastic responses are limited and may not be by themselves enough to withstand ongoing warming trends. Although the evidence is still scarce and deserves further research in different insect taxa, genetic adaptation to high temperatures may result from rapid evolution. Finally, we emphasize the importance of incorporating physiological information for modelling species distributions and ecological interactions under global warming scenarios. This review identifies several open questions to improve our understanding of how insects respond physiologically to heat and the evolutionary and ecological consequences of those responses. Further lines of research are suggested at the species, order and class levels, with experimental and analytical approaches such as artificial selection, quantitative genetics and comparative analyses.

[Inducible resistance to clindamycin in methicillin-resistant Staphylococcus aureus isolated from Paraguayan children]. / Resistencia inducible a clindamicina en Staphylococcus aureus resistentes a meticilina aislados de pacientes pediátricos en Paraguay.

BACKGROUND: The double disc diffusion method is an alternative diagnostic that allows the identification of Staphylococcus aureus isolates apparently susceptible to clindamycin but that may develop resistance due to an induction phenomena, mainly asociated to the increase in resistance to methicillin, thus increasing the possibility of failure in the treatment. AIM: To determine the frequency of induced clindamycin resistance in methicillin-resistant S. aureus (MRSA) isolated from Paraguayan children. MATERIALS AND METHODS: In this cross sectional study, we collected 145 S. aureus isolates that caused skin and soft tissue and osteoarticular infections in pediatric patients of the Central Hospital I.P.S. in the period from December-2012 to November-2013. Resistance to clindamycin was determined by automated methods and double disc diffusion. PCR was performed for ermA, ermB, ermC and msrA genes from representative isolates. RESULTS: The global resistance to methicillin and clindamycin was 67 and 13%, respectively (11% attributable to the inducible mechanism). The ermC and msrA genes were detected individually in 25 and 17% of the isolates respectively while an isolate presented both genes simultaneously. DISCUSSION: The frequency of inducible resistance to clindamycin indicates the importance of double disc diffusion methods in microbiological practice, as well as being within the cut off points considered acceptable for the use of this antibiotic for skin infections. and osteoarticular caused by MRSA.

Situación del diagnóstico de Queratitis Amebiana en Paraguay / Status of the diagnosis of Amebic Keratitis in Paraguay

Las amebas de vida libre (AVL) existen ampliamente distribuidas en la naturaleza, donde el género Acanthamoeba es la más frecuentemente aislada en diversos ambientes del suelo, aire y agua y está asociada a enfermedades en humanos. Su capacidad para vivir en ambientes adversos se debe a su baja demanda de alimentación y a que en su ciclo biológico tiene un estadio en forma de quiste, lo que la hace muy resistente. El hombre, como hospedero puede desarrollar infecciones en el sistema nervioso central, en la piel y los pulmones. A nivel ocular es capaz de afectar la córnea y producir queratitis. Por lo tanto es considerado un importante agente etiológico de patologías humanas. En esta revisión se aborda la biología, patogénesis y los mecanismos de defensa del ser humano frente a la infección por Acanthamoeba. Además a esto, de los factores de riesgo por el uso de lentes de contacto y malos hábitos de higiene del usuario y por último el abordaje diagnóstico microbiológico y molecular. Esta herramienta ha ido mejorando con el avance de la tecnología, indispensable para la temprana identificación y el logro de una oportuna y eficaz intervención clínica y terapéutica. Finalmente exponemos la situación actual en relación a los métodos de diagnóstico con que se cuentan y los escasos reportes clínicos existentes en Paraguay. Palabras clave: Acanthamoeba sp., Lentes de contacto, Queratitis.

Free-living amoeba (FLA) exist widely in nature, where the genus Acanthamoeba is the most frequently isolated in various environments of soil, air and water. It is associated with diseases in human. Its ability to live in adverse environments is due to the low food demand and that in its biological cycle it has a cyst-shaped stage, which makes it very resistant. Man, as a host, can acquire infections in the central nervous system and in the skin and lungs. On the ocular level, it can affect the cornea and cause keratitis. Therefore, it is considered an important etiological agent of human pathologies. This review addresses the biology, pathogenesis and human defense mechanisms against Acanthamoeba infection. In addition to this, the review explores the risk factors related to the use of contact lenses and poor hygienic behavior of the users, and finally the microbiological and molecular diagnosis. This particular tool has been improved with the advancement of technology and is essential for timely identification and the achievement of a good clinical and therapeutic intervention. Finally, we present the current situation in Paraguay regarding the few existing clinical reports and the diagnostic methods available. Key words: Acanthamoeba sp., Contact lenses, Keratitis.

Alteraciones urinarias en niños litiásicos paraguayos según estado nutricional / Urinary alterations in Paraguayan lithiasic children according to nutritional status

La urolitiasis es una enfermedad multifactorial. En los últimos años se ha observado un incremento a nivel mundial de la incidencia de la litiasis urinaria tanto en adultos como en niños. Los cambios en los hábitos alimentarios y un aumento en la prevalencia de exceso de peso podrían asociarse a este fenómeno. El objetivo del trabajo fue describir la frecuencia de alteraciones urinarias presentes en niños con urolitiasis según estado nutricional. En este trabajo observacional transversal se incluyeron 104 niños litiásicos de ambos sexos divididos en dos grupos según estado nutricional: 68 niños con peso adecuado y 36 niños con sobrepeso u obesidad considerando criterios de la OMS. Se determinaron en orina de 24 horas calcio, fósforo, sodio, ácido úrico, citrato y magnesio. Del total de pacientes participantes 54 (51.9%) fueron niñas y 50 (48,1%) fueron niños. El 65,4% de los niños presentó peso normal y el 34,6% de los niños sobrepeso u obesidad con edades medias de 10±4 años y 8±4 años respectivamente. El 80% de los niños presentó al menos una alteración urinaria, siendo las más frecuentes en ambos grupos la hipocitraturia e hiperuricosuria. Es llamativa la elevada frecuencia de alteraciones en las concentraciones de promotores e inhibidores de cristalización, en ambos grupos, indicando un riesgo aumentado de recidivas(AU)

Urolithiasis is a multifactorial disease. Recently the incidence of urolithiasis in adults and children is increasing worldwide. Changes in eating habits and an increase in the prevalence of overweight could be associated with this phenomenon. The objective was to describe the frequency of urinary alterations according to nutritional status in children with urolithiasis. This cross-sectional observational study included 104 lithiasic boys and girls divided into two groups: 68 children with normal weight and 36 children with overweight or obesity considering WHO criteria. Calcium, phosphorus, sodium, uric acid, citrate and magnesium levels were determined in 24-hour urine. The frequency of boys and girls was 51.9% and 48.1% respectively. Sixty five percent of the children presented normal weight and 34.6% of the children were overweight or obese with mean ages of 10±4 years and 8±4 years respectively. Eighty percent of the children had at least one urinary disorder, the most frequent being hypocitraturia and hyperuricosuria in both groups. The high frequency of alterations in the concentrations of urinary promoters and inhibitors of crystallization in both groups was remarkable, indicating an increased risk of recurrence(AU)

Queratitis por Acanthamoeba sp. en usuarios de lentes de contacto: casos clínicos / Keratitis by Acanthamoeba sp. in users of contact lenses: clinical cases

Introducción.La queratitis amebiana que con frecuencia se presenta en usuarios de lentes de contacto cursa con mucho dolor y comprometen la visión. La sintomatología es inespecífica y el diagnóstico se apoya en estudios laboratoriales basados en métodos de cultivo y moleculares para identificación específica de Acanthamoeba. Presentación de los casos.Se presentan tres casos de queratitis amebiana en usuarios de lentes de contacto, dos mujeres y un varón con cuadros que varían de lesiones leves a neoplasia, todos usuarios de lentes de contacto y con antecedentes de exposición al agua ya sea en la ducha o por almacenamiento de lentes de contacto con agua de canilla. La lesión se caracteriza por presentar infiltrados multifocales, epiteliopatía, pseudodendritas centrales y peurineritis redial. Los métodos microbiológicos detectaron al parásito en cultivo en una muestra, mientras que el método molecular fue positivo en dos casos. El tratamiento se basó en isetionato de propamidina (Brolene), polihexametilbiguanida 0,02% y clorhexidina 0,02%, inhibidores de la síntesis de ADN y la formación de la pared celular. La evolución de los cuadros fue de total recuperación en dos casos, mientras que en el tercer caso la queratitis fue una infección agregada de una neoplasia que precisó de métodos quirúrgicos para tratamiento. Conclusiones.Las queratitis amebianas pueden simular cuadros infecciosos de carácter viral herpético o micótico, y por su baja frecuencia y difícil confirmación por falta de métodos laboratoriales apropiados, no se considera a la Acanthamoeba como agente etiológico, y en consecuencia hay retraso en el diagnóstico ytratamiento.

Introduction.Amebickeratitis that often occurs in contact lens wearers are in a lot of pain and compromise vision. Symptomatology is non-specific and diagnosis is supported by laboratory studies based on culture and molecular methods for specific identification of Acanthamoeba. Case presentation.Three cases of amebickeratitis occur in contact lens wearers, two women and one male with pictures ranging from mild lesions to neoplasm, all contact lens wearers and a history of water exposure either in the shower or by storage contact lenses with cane water. The lesion is characterized by multifocal infiltrates, epithelopathy, central pseudodendrites and redial peurineritis. Microbiological methods detected the cultured parasite in a sample, while the molecular method was positive in two cases. Treatment was based on propamidine isethionate (Brolene), polyhexamethylbiguanide 0.02% and chlorhexidine 0.02%, inhibitors of DNA synthesis and cell wall formation. The evolution of the tables was of complete recovery in two cases, while in the third case keratitis was an added infection of a neoplasm that required surgical methods for treatment. The importance of this report is that amebickeratitis can simulate infectious tables of an inherited or fungal nature, and because of its low frequency and difficult confirmation for lack of proper laboratory methods, the Acanthamoeba as an etiological agent, and consequently there is a delay in diagnosis and treatment. Conclusions.Amebic keratitis can simulate infectious conditions of a herpetic or fungal viral nature, and due to its low frequency and difficult confirmation due to lack of appropriate laboratory methods, Acanthamoeba is not considered as an etiologic agent, and consequently There is a delay in diagnosis andtreatment.

Resistencia inducible a clindamicina en Staphylococcus aureus resistentes a meticilina aislados de pacientes pediátricos en Paraguay / Inducible resistance to clindamycin in methicillin-resistant Staphylococcus aureus isolated from Paraguayan children

Resumen Introducción: El método de difusión de doble disco se presenta como una alternativa diagnóstica que permite identificar aislados de Staphylococcus aureus susceptibles a clindamicina, ante el aumento de resistencia a meticilina, reduciendo así la posibilidad de fallo en el tratamiento. Objetivo: Determinar la frecuencia de resistencia a clindamicina inducida por eritromicina en S. aureus resistentes a meticilina (SARM) aislados de niños paraguayos. Materiales y Métodos: Estudio observacional, descriptivo, de corte transversal. Se colectaron 145 aislados S. aureus que causaron infecciones de piel y tejidos blandos y osteo-articulares en pacientes pediátricos del Hospital Central del Instituto de Previsión Social en el período de diciembre-2012 a noviembre-2013. La resistencia a clindamicina se determinó por métodos automatizados y de difusión de doble disco. Se realizó reacción de polimerasa en cadena para genes ermA, ermB, ermC y msrA de aislados representativos. Resultados: La resistencia global a meticilina y clindamicina fue de 67 y 13%, respectivamente (11% atribuible al mecanismo de resistencia a clindamicina inducible). Los genes ermC y msrA fueron detectados individualmente en 25 y 17% de los aislados, respectivamente, mientras que un aislado presentó ambos genes en simultáneo. Discusión: La frecuencia de mecanismo de resistencia inducible a clindamicina señala la importancia de los métodos de difusión de doble disco en la práctica microbiológica, así como se encuentran en los límites de puntos de cortes considerados como aceptables para el uso de este antimicrobiano para infecciones cutáneas y osteo-articulares causadas por SARM.

Background: The double disc diffusion method is an alternative diagnostic that allows the identification of Staphylococcus aureus isolates apparently susceptible to clindamycin but that may develop resistance due to an induction phenomena, mainly asociated to the increase in resistance to methicillin, thus increasing the possibility of failure in the treatment. Aim: To determine the frequency of induced clindamycin resistance in methicillin-resistant S. aureus (MRSA) isolated from Paraguayan children. Materials and Methods: In this cross sectional study, we collected 145 S. aureus isolates that caused skin and soft tissue and osteoarticular infections in pediatric patients of the Central Hospital I.P.S. in the period from December-2012 to November-2013. Resistance to clindamycin was determined by automated methods and double disc diffusion. PCR was performed for ermA, ermB, ermC and msrA genes from representative isolates. Results: The global resistance to methicillin and clindamycin was 67 and 13%, respectively (11% attributable to the inducible mechanism). The ermC and msrA genes were detected individually in 25 and 17% of the isolates respectively while an isolate presented both genes simultaneously. Discussion: The frequency of inducible resistance to clindamycin indicates the importance of double disc diffusion methods in microbiological practice, as well as being within the cut off points considered acceptable for the use of this antibiotic for skin infections. and osteoarticular caused by MRSA.

Estandarización de una técnica de PCR múltiple para la detección de los serogrupos O157, O104 y "big six" de Escherichia coli productora de la toxina Shiga (STEC) / Standardization of a multiplex PCR technique for the detection of Shiga toxin-producing Escherichia coli (STEC) serogroups O157, O104 and "big six"

Los serogrupos O26, O45, O103, O104, O111, O121, O145 y O157 de STEC se relacionan con un elevado número de casos de SUH a nivel mundial, por lo que están incluidos dentro de las categorías de mayor riesgo para los humanos, según los criterios de autoridades alimentarias de Estados Unidos y Europa. El método convencional de identificación de antígenos O y H se realiza por aglutinación con antisueros de conejo. Este método además de ser muy costoso y laborioso, no se encuentra disponible en el país para empleo masivo. En este contexto, el objetivo de este estudio observacional descriptivo de corte transverso ha sido la estandarización de una técnica de PCR múltiple para la detección de estos 8 serogrupos, a fin de contar con un sistema de detección eficiente, sensible y con potencial de aplicación en la industria alimentaria. Se estandarizaron reacciones de PCR empleando como controles positivos cepas E. coli de referencia correspondientes a la totalidad de los serogrupos citados. Se obtuvieron productos de tamaños esperados para cada serogrupo, no se observaron amplificaciones cruzadas o falsos positivos. Esta técnica estandarizada podría representar una herramienta rápida y menos costosa que la técnica serológica, con la capacidad de ser aplicada a diferentes matrices, permitiendo la detección de estos serogrupos en aislados STEC de ganado en pie, fuentes de agua de consumo, alimentos e incluso en aislamientos clínicos asociados a enfermedades humanas(AU)

STEC serogroups O26, O45, O103, O104, O111, O121, O145, and O157, are related to a high number of cases of HUS worldwide, so they are included in the categories of greatest risk for humans, according to the food administration criteria of the United States and Europe. The conventional method of identifying antigens O and H is carried out by agglutination with rabbit antisera. This method is very expensive and laborious and is not available in the country for massive-scale use. In this context, the objective of this cross-sectional descriptive observational study has been the standardization of a multiplex PCR technique for the detection of these 8 serogroups, in order to have an efficient and sensitive detection system with the potential for application in the food industry. PCR reactions were standardized using as positive controls reference E. coli strains to correspond to all the mentioned serogroups. Products of expected sizes were obtained for each serogroup; no cross-amplification or false positives were observed. This standardized technique could represent a quick and less expensive tool than the serological technique, with the possibility to be applied to different kind of samples, allowing the detection of these serogroups in STEC isolates of live cattle, sources of drinking water, food and even in clinical isolates associated with human diseases(AU)

Diagnóstico de endoftalmitis por método microbiológico convencional y molecular en pacientes de centros oftalmológicos de Paraguay / Diagnosis of endophthalmitis by molecular and conventional microbiological methods in patients from ophthalmologic centers of Paraguay

La endoftalmitis infecciosa es una condición intraocular grave, aunque poco frecuente, que puede llevar a la pérdida de la visión. El diagnóstico etiológico precoz y el tratamiento adecuado llevan a una mejor evolución. Las técnicas microbiológicas convencionales tienen baja sensibilidad en el diagnóstico de la endoftalmitis infecciosa, en cambio la reacción en cadena de la polimerasa (PCR) es un método de mayor sensibilidad que está siendo utilizado ampliamente. El objetivo fue determinar por cultivo y PCR la etiología de la endoftalmitis infecciosa en pacientes que consultaron en centros oftalmológicos de Paraguay. Se estudiaron muestras de humor vítreo obtenidas por punción de 51 pacientes con endoftalmitis. La muestra se dividió en dos alícuotas; una para el estudio microbiológico convencional y la otra para la PCR anidada. Esta última utiliza un par de cebadores basado en secuencias conservadas del gen 16S ribosomal y cebadores específicos para bacterias gram positivas. Se estandarizó la PCR logrando una sensibilidad de detección de 3.10-5 ng/µl para S. aureus, 2,5.10-3 ng/µl para S. pneumoniae y 6.10-7 ng/µl para E. coli. De las 51 muestras, 12 (23,5%) fueron positivas por métodos microbiológicos convencionales y 26 (51,0%) por la PCR anidada. Por cultivo se identificaron predominantemente bacterias grampositivas 8/11 (73%) y un aislado de hongo filamentoso. La PCR anidada presentó muy buena sensibilidad y de gran utilidad en la identificación del origen bacteriano de la endoftalmitis, mejorando la sensibilidad del cultivo en 27,5%. La discriminación bacteriana en grampositivos y gramnegativos por la PCR se correlacionó con el cultivo en el 100% de los casos(AU)

Reporte de un caso de Cistinuria: Uso de herramientas de diagnóstico a nivel nacional / Report of a case of cystinuria: Use of diagnostic tools at national level

La cistinuria es una enfermedad genética que se engloba dentro de alteraciones congénitas del transporte de aminoácidos con formación de cálculos en las vías urinarias, si bien es poco frecuente se caracteriza por su elevada recurrencia. En este trabajo presentamos el caso de una paciente de 34 años, con antecedentes de haber perdido un riñón por episodios anteriores de litiasis y con múltiples recidivas que es diagnosticada mediante la detección de cistina por espectroscopía infrarroja como componente único de 96 fragmentos de cálculos removidos mediante nefrolitotomía percutánea. La paciente fue evaluada laboratorialmente mediante el perfil metabólico y la cristaluria. Las indicaciones de tratamiento específicas incluyeron la administración de agentes alcalinizantes, régimen nutricional, y entrenamiento para control de pH urinario. Es importante señalar la agresividad de la litiasis de cistina con las consecuencias que puede tener la calidad de vida del paciente, y por tanto la importancia de contar con capacidades instaladas a nivel país para el diagnóstico y seguimiento de litiasis genéticas como la causada por la cistinuria(AU)

Cystinuria is a genetic disease that is included among congenital defects of renal amino acids transport that causes urinary stone formation. Although it is rare, it is characterized by its high recurrence. We present the case of a 34-year-old patient that lost one of her kidney because of recurrent episodes of lithiasis, and that was diagnosed by the detection of cystine with infrared spectroscopy as the sole component of 96 stone fragments removed by percutaneous nephrolithotomy. The patient was evaluated by metabolic profile and crystalluria. The specific treatment indications included the administration of alkalinizing agents, nutritional regimen, and training for personal measurement of urinary pH. This case highlights the aggressiveness of cystine stones with the consequences that may have on the quality of the patient life, and therefore the importance of having installed proper diagnostic capacities at national level to detect and monitor treatment efficacy in genetic lithiasis such as cystinuria(AU)

Portación de fimH en aislados de Escherichia coli productor de Toxina Shiga provenientes de ganado bovino, Departamento Cordillera, Paraguay / Portability of fimH in isolates of Shiga toxin-producing Escherichia coli from cattle, Department of Cordillera, Paraguay

Ciertas cepas de Escherichia coli productoras de toxina Shiga (STEC) tienen la capacidad de formar biofilm en alimentos y otras superficies, aumentando su potencial como fuente de contaminación. El gen fimH se ha asociado a la capacidad de formación de biofilm en E.coli. Este estudio observacional descriptivo de corte transverso se realizó con el objetivo de describir la portación de fimH en aislados STEC provenientes de muestras de materia fecal de ganado bovino del Departamento de Cordillera en el 2016. Los aislados de STEC se obtuvieron por cultivos, extracción de ADN y amplificación por PCR de genes stx1 y stx2. El gen fimH se detectó por PCR convencional. Un total de 1006 aislamientos de E. coli se sometieron a extracción de ADN y amplificación por PCR para genes stx1 y stx2. De éstos, 269 se identificaron como STEC, en los que la detección del gen fimH se realizó por PCR convencional. Un producto de PCR representativo se sometió a secuenciación del gen fimH y mostró 100% de homología con secuencias de la Base de Datos de GenBank. De 269 aislamientos STEC, 129 aislamientos (48%) resultaron ser portadores del gen fimH y por tanto con potencial de formar biofilm. Esta frecuencia elevada representa un riesgo de persistencia de estos patógenos en elementos y superficies de trabajo de sitios de expendio y manipulación de productos cárnicos. Este trabajo contribuye como una herramienta esencial para seguir con la línea de investigación, obteniendo datos de suma importancia que ayuden a describir la situación de riesgo de contaminación alimentaria en que se encuentra el país(AU)