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The continuous introduction of cleaning products containing benzalkonium chloride (BAC) from household discharges can mold the microbial communities in wastewater treatment plants (WWTPs) in a way still poorly understood. In this study, we performed an in vitro exposure of activated sludge from a WWTP in Costa Rica to BAC, quantified the changes in intI1, sul2, and qacE/qacEΔ1 gene profiles, and determined alterations in the bacterial community composition. The analysis of the qPCR data revealed elevated charges of antibiotic resistance genes in the microbial community; after BAC's exposure, a significant increase in the qacE/qacEΔ1 gene, which is related to ammonium quaternary resistance, was observed. The 16S rRNA gene sequences’ analysis showed pronounced variations in the structure of the bacterial communities, including reduction of the alpha diversity values and an increase of the relative abundance of Alphaproteobacteria, particularly of Rhodospseudomonas and Rhodobacter. We confirmed that the microbial communities presented high resilience to BAC at the mg/mL concentration, probably due to constant exposure to this pollutant. They also presented antibiotic resistance-related genes with similar mechanisms to tolerate this substance. These mechanisms should be explored more thoroughly, especially in the context of high use of disinfectant. HIGHLIGHTS In vitro BAC's exposure enhances qacE/qacEΔ1 gene presence in bacterial communities from AS.; AR related genes in AS's microbial communities from tropical countries are reported.; The BAC exposure can alter the AS microbial community composition.; Putative nitrifiers are enhanced by BAC's exposure.; The presence of intI-1 gene in AS could indicate the anthropogenic spread of microbial resistance into the environment.

Resumen Introducción: la enfermedad diarreica es una de las principales causas de muerte en niños menores de cinco años. Uno de los agentes etiológicos bacterianos más reconocido es Escherichia coli, que puede causar infecciones a través de diversos mecanismos asociados a la adquisición de genes de virulencia. Metodología: se recolectaron muestras de aguas residuales en los afluentes y los efluentes de cinco plantas de tratamiento de la Gran Área Metropolitana, durante tres días consecutivos, en los meses de marzo, mayo, octubre y diciembre del año 2013, a fin de cuantificar la concentración de E. coli, mediante la técnica de fermentación en tubos múltiples, y evaluar la presencia de los genes de virulencia aatA, bfpA, eaeA, ipaH, lt, st, stx1 y stx2, vinculados con patotipos de E. coli diarrogénica, con la técnica de PCR punto final. Resultados: el estudio reveló niveles de E. coli similares a los encontrados en afluentes de otras regiones del mundo, en cambio, los efluentes presentaron concentraciones superiores a los 4 Log10 NMP/100 mL. La circulación anual de genes de virulencia fue relacionada con los patotipos enterotoxigénica, productores de toxina Shiga/enterohemorrágica, enteroagregativa y enteropatógena. No se detectaron genes referentes a la variedad enteroinvasora. Además, se determinó que no todas las plantas de tratamiento remueven significativamente la carga de E. coli. Conclusiones: el flujo de E. coli diarrogénica es común en nuestro país y su diseminación en el ambiente es posible cuando los efluentes se descargan en cuerpos de aguas superficiales Abstract Introduction. Diarrheic disease is one of the most important diseases causes in children under five years. One of the most recognized etiological agents is Escherichia coli. This bacterium has the capacity of causing infections by multiple mechanisms associated with the acquisition of different virulence genes. Methodology: water samples were collected from the affluents and effluents of five wastewater treatment plants located in the Costa Rican Great Metropolitan Area, during three sequential days, in March, May, October, and December from 2013. The aim was to quantify E. colis concentration by multiple tubes fermentation method and to evaluate the presence of aatA, bfpA, eaeA, ipaH, lt, st, stx1, and stx2 virulence genes, related with diarrheagenic E. coli, by PCR end-point method. Results: The study revealed that the levels of E. coli into the affluents are similar to those reported in other world regions. The effluents concentration presented higher than 4 Log10 NMP/100 mL level of E. coli. A year-round circulation of virulence genes was demonstrated, and it was associated with enterotoxigenic, Shiga-toxin producers/ enterohemorrhagic, enteroaggregative, and enteropathogenic pathotypes. Genes related to enteroinvasive variety did not were detected. Not all wastewater treatment plants removed E. coli efficiently. Conclusions: Diarrheagenic E. coli circulation is common in our county with the possibility of dissemination to the environment by effluents wastewater discharge in surface water bodies.

Effective wastewater management is crucial to ensure the safety of water reuse projects and effluent discharge into surface waters. Multiple studies have demonstrated that municipal wastewater treatment with conventional activated sludge processes is inefficient for the removal of a wide spectrum of viruses in sewage. In this study, a well-accepted statistical approach was used to investigate the relationship between viral indicators and human enteric viruses during wastewater treatment in a resource-limited region. Influent and effluent samples from five urban wastewater treatment plants (WWTPs) in Costa Rica were analyzed for somatic coliphage and human enterovirus, hepatitis A virus, norovirus genotypes I and II, and rotavirus. All WWTPs provide primary treatment followed by conventional activated sludge treatment prior to discharge into surface waters that are indirectly used for agricultural irrigation. The results revealed a statistically significant relationship between the detection of at least one of the five human enteric viruses and somatic coliphage. Multiple logistic regression and receiver operating characteristic curve analysis identified a threshold of 3.0 × 103 (3.5 log10) somatic coliphage PFU per 100 ml, which corresponded to an increased likelihood of encountering enteric viruses above the limit of detection (>1.83 × 102 virus targets/100 ml). Additionally, quantitative microbial risk assessment was executed for farmers indirectly reusing WWTP effluent that met the proposed threshold. The resulting estimated median cumulative annual disease burden complied with World Health Organization recommendations. Future studies are needed to validate the proposed threshold for use in Costa Rica and other regions. IMPORTANCE Effective wastewater management is crucial to ensure safe direct and indirect water reuse; nevertheless, few countries have adopted the virus log reduction value management approach established by the World Health Organization. In this study, we investigated an alternative and/or complementary approach to the virus log reduction value framework for the indirect reuse of activated sludge-treated wastewater effluent. Specifically, we employed a well-accepted statistical approach to identify a statistically sound somatic coliphage threshold value which corresponded to an increased likelihood of human enteric virus detection. This study demonstrates an alternative approach to the virus log reduction value framework which can be applied to improve wastewater reuse practices and effluent management.

Two ether and one ester derivatives of the 4-nitro-3-hydroxybenzoic acid were synthesized and characterized. The in vitro antimicrobial and cytotoxic activities of the three novel compounds were also evaluated. The aromatic derivatives showed antibacterial activity against one of the four microorganisms tested and two compounds (C8 and NOBA) had a lower IC50 in HeLa cells. Ministerio de Ciencia y Tecnología/[MAT2013-45657-P]/MICITT/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA) UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Química UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicina

Giardia cysts stained with hot carbolfuchsin were used as internal controls in a concentration method for surface water samples. The morphological integrity of stained cysts and the stain's stability and intensity were tested with each of the chemical reagents used in the aluminum sulfate flocculation method. No alterations in morphology or color were noted. The stained cyst preparation has a low cost, high stability, and suitability for both light and immunofluorescent microscopy, making it affordable to researchers in low- and middle-income countries. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA)

This study used the Canadian Water Quality Index (CWI) to characterize water sampled at three points within the Purires River micro basin, Costa Rica. The first sampling point is located in a high zone with domestic agricultural activities, the second point around the mid-point of the flow of the river, and the third point at the lowest zone with extensive agricultural activities mainly centered on the production of fresh vegetables. Eleven physicochemical parameters (As, Cd, Cr, biochemical oxygen demand (BOD), chemical oxygen demand (COD), NH4+, NO3−, Pb, pH, percent saturation of dissolved oxygen (PSO), and total suspended solids (TSS)) and two microbiological parameters (fecal coliforms and enterococci) were evaluated. We evaluated three different Canadian Water Quality Indexes (CWIs): CWI-1 included only physicochemical parameters, CWI-2 included CWI-1 parameters plus fecal coliforms, and CWI-3 included CWI-2 in addition to enterococci. Statistical analysis of individual parameters showed significant differences between sampling sites. CWI-1 was unable to discriminate between the three sampling points, and characterized the water quality as ‘fair’. CWI-2 was only able to discriminate when the water contained high levels of chemical and microbiological contaminants, while CWI-3 adequately discriminated water quality at each of the sampling points. The evaluation of enterococci together with more traditional water quality parameters enabled better categorization of surface water quality. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

HIV/AIDS transmission among drug users is associated with education, sex practices and substance use. This study examined 159 drugs users’ knowledge, beliefs and sex behavior related to HIV/AIDS risk in Costa Rica. Results showed considerable use of marihuana, alcohol, crack and cocaine and a very low lifetime incidence of other drugs. All substance use patterns were higher than national averages. Respondents showed a high level of knowledge about HIV/AIDS transmission and prevention. However, there did not seem to be a relationship between knowledge and practice. Unprotected sex was common and having an HIV/AIDS test was not a regular practice. Knowledge about HIV/AIDS is not a determinant factor for condom use among this group. It is concluded that having the proper knowledge about transmission and prevention does not guarantee safe sex practices. Further research and public health evidence based practices for HIV/AIDS prevention should target drug user population

Los métodos de recuento en placa capa doble y capa simple de agar, para la cuantificación de colifagos so-máticos en aguas, fueron implementados utilizando como base metodologías estándar. Diferentes variables fueron ensayadas, lo cual permitió la precisión en algunos pasos no incluidos en metodologías estándares. De los hallazgos de mayor importancia, se exponen las consecuencias de utilizar un cultivo de Escherichia coliexcesivamente concentrado y se describe la obtención de un cultivo en fase logarítmica en solo 4 horas de incubación, ajustando la concentración a una densidad óptica de 0,3 a 600nm (3,1 x 10 8 UFC/ mL), o a un McFarland 1 (3,0 x108 UFC/ mL). Se determinó que los controles de colifagos deben ser almacenados a -70 °C para reducir su degradación y que se deben evitar cantidades superiores a 20 mL de mezcla de reacción por plato de Petri, para reducir las burbujas que pueden interferir con la lectura de unidades formadoras de placas (UFP). Se demostró que los colifagos de las muestras de agua pueden almacenarse 48 horas a 4 °C sin que sufran degradación y que en las muestras con altas concentraciones de colifagos no se observa UFP porque se da una lisis confluente de la capa bacteriana. No se encontraron diferencias significativas en la recuperación de colifagos al utilizar un método u otro, pero dichos métodos deben ser evaluados por medio de controles, antes de aplicarlos directamente en el análisis de muestras de agua.Two plate count methods, double layer and single layer of agar for quantification of somatic coliphages in water, were implemented using standard methodologies. Several variables were tested and provided valuable information that was not included in standard methodologies. The most important findings are described, such as the effect of using an excessively concentrated culture of E. coliand production of a log phase culture in only 4 hours of incubation, adjusting the concentration to an optical density of 0.3 at 600 nm (3.1 x 10 8 CFU / mL), or to McFarland 1 (3.0 x 108 CFU / mL). It was determined that coliphages controls must be stored at -70 °C to reduce its degradation. Quantities of reaction mixture exceeding 20 mL per Petri dish must be avoided to prevent interfere with bubbles during the counting of plate forming units (PFU). It was demonstrated that coliphages isolated from water samples can be stored for 48 hours at 4 °C without any degradation, and PFU are not observed in samples with high concentrations of coliphages, because a confluent lysis of the bacte-rial layer. There was no significant difference in the recovery of coliphages using doble layer or single layer methods, but such methods should be evaluated by means of controls, before applying them directly in the analysis of water samples.

artículo (arbitrado)--Universidad de Costa Rica.Instituto de Investigaciones en Salud, 2010 A Multiplex Polymerase Chain Reaction (PCR) assay to be used as an alternative to the conventional culture method in detecting Shigella and enteroinvasive Escherichia coli (EIEC) virulence genes ipaH and ial in lettuce was developed. Efficacy and rapidity of the molecular method were determined as compared to the conventional culture. Lettuce samples were inoculated with different Shigella flexneri concentrations (from 10 CFU/ml to 107 CFU/ml). DNA was extracted directly from lettuce after inoculation (direct-PCR) and after an enrichment step (enrichment PCR). Multiplex PCR detection limit was 104 CFU/ml, diagnostic sensitivity and specificity were 100% accurate. An internal amplification control (IAC) of 100 bp was used in order to avoid false negative results. This method produced results in 1 to 2 days while the conventional culture method required 5 to 6 days. Also, the culture method detection limit was 106 CFU/ml, diagnostic sensitivity was 53% and diagnostic specificity was 100%. In this study a Multiplex PCR method for detection of virulence genes in Shigella and EIEC was shown to be effective in terms of diagnostic sensitivity, detection limit and amount of time as compared to Shigella conventional culture. Universidad de Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA)

ResumenLa calidad del agua de consumo es una de las preocupaciones más grandes a las que se enfrentan los entes de salud. En Costa Rica, a pesar de la buena calidad de las fuentes de agua y del agua de consumo, se siguen registrando numerosos casos de diarrea anualmente. En este estudio se realizó un análisis de indicadores de contaminación virales y bacterianos, en dos comunidades del Valle Central con acueducto propio. Se analizaron en total 24 muestras de agua por cada comunidad, durante un año, 12 en la fuente de agua y 12 en el agua tratada. La frecuencia de diarreas en cada comunidad fue comparada con la calidad microbiológica del agua consumida, encontrándose ausencia de indicadores bacterianos en muestras de agua tratada, mientras que la presencia de indicadores virales en el agua de consumo (con cloro residual) se relaciona con un incremento en el número de casos de diarreas, adicionalmente se analizó el comportamiento de las diarreas de acuerdo a la precipitación promedio con el fin de observar posibles patrones de estacionalidad. Estos hallazgos respaldan la necesidad de ampliar el monitoreo de calidad microbiológica del agua con otro tipo de indicadores de contaminación que correlacionen mejor con otros agentes patógenos de transmisión hídrica, principalmente en época seca, periodo en que se observan más casos de diarrea. AbstractThe quality of potable water is one of the most important issues of public health. In Costa Rica, in spite of having good sources of water and good quality of water for consumption, there continues to be a significant number of diarrhea cases annually. In this investigation, viral and bacterial analyses of indicators of contamination were conducted in two rural communities with their own aqueducts in the Central Valley of Costa Rica. Twenty-four samples were taken from each community during the year; 12 from the water sources and 12 from the treated waters. The frequency of diarrhea in each community was compared to the microbiological quality of the water being consumed, noting an absence of bacterial indicators in samples of treated water, while the presence of viral indicators in the water (with residual chlorine) were associated with an incremental increase in the number of cases of diarrhea. The analysis also included measurements of average monthly rainfall with the aim of observing possible associations between seasonal conditions and the incidence of diarrhea cases. These findings indicate the need to increase the monitoring of the microbiological quality of the water with other types of indicators of contamination that correlate better with other pathogens transmitted by water, especially in the dry seasons when a greater number of cases of diarrhea are observed.

Artículo científico -- Universidad de Costa Rica, Instituto de Investigaciones en Salud. 2005. Este documento es privado debido a limitaciones de derechos de autor. The Comprehensive Drug Research Center (CDRC) at the University of Miami was established in the early 1970s. Through the decades, investigators from the CDRC have worked with investigators from several countries to establish joint research efforts. Countries often do not have the infrastructure or monetary resources to carry out research on their own. Collabo rating with institutions in these countries to build a sustainable capacity for research is a worthwhile and satisfying endeavor, and it presents a method for initiating research and building the necessary research structures. However, working with other countries presents a unique set of challenges and ethical dilemmas. this article presents some of the specific challenges encountered in these research efforts and describes what we have done to resolve the problems and work more effectively and efficiently with foreign investigators. 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)

Los métodos de recuento en placa capa doble y capa simple de agar, para la cuantificación de colifagos so-máticos en aguas, fueron implementados utilizando como base metodologías estándar. Diferentes variables fueron ensayadas, lo cual permitió la precisión en algunos pasos no incluidos en metodologías estándares. De los hallazgos de mayor importancia, se exponen las consecuencias de utilizar un cultivo de Escherichia coliexcesivamente concentrado y se describe la obtención de un cultivo en fase logarítmica en solo 4 horas de incubación, ajustando la concentración a una densidad óptica de 0,3 a 600nm (3,1 x 10 8 UFC/ mL), o a un McFarland 1 (3,0 x108 UFC/ mL). Se determinó que los controles de colifagos deben ser almacenados a -70 °C para reducir su degradación y que se deben evitar cantidades superiores a 20 mL de mezcla de reacción por plato de Petri, para reducir las burbujas que pueden interferir con la lectura de unidades formadoras de placas (UFP). Se demostró que los colifagos de las muestras de agua pueden almacenarse 48 horas a 4 °C sin que sufran degradación y que en las muestras con altas concentraciones de colifagos no se observa UFP porque se da una lisis confluente de la capa bacteriana. No se encontraron diferencias significativas en la recuperación de colifagos al utilizar un método u otro, pero dichos métodos deben ser evaluados por medio de controles, antes de aplicarlos directamente en el análisis de muestras de agua., Two plate count methods, double layer and single layer of agar for quantification of somatic coliphages in water, were implemented using standard methodologies. Several variables were tested and provided valuable information that was not included in standard methodologies. The most important findings are described, such as the effect of using an excessively concentrated culture of E. coliand production of a log phase culture in only 4 hours of incubation, adjusting the concentration to an optical density of 0.3 at 600 nm (3.1 x 10 8 CFU / mL), or to McFarland 1 (3.0 x 108 CFU / mL). It was determined that coliphages controls must be stored at -70 °C to reduce its degradation. Quantities of reaction mixture exceeding 20 mL per Petri dish must be avoided to prevent interfere with bubbles during the counting of plate forming units (PFU). It was demonstrated that coliphages isolated from water samples can be stored for 48 hours at 4 °C without any degradation, and PFU are not observed in samples with high concentrations of coliphages, because a confluent lysis of the bacte-rial layer. There was no significant difference in the recovery of coliphages using doble layer or single layer methods, but such methods should be evaluated by means of controls, before applying them directly in the analysis of water samples.

Salmonella spp. es un patógeno bacteriano muy importante causante de diarreas, que es transmitido tanto por la vía fecal-oral, como por alimentos y agua contaminados. En este trabajo se estandarizó una técnica de PCR en lechuga para la detección del gen invA de Salmonella spp.; dicho gen se relaciona con el proceso de invasión al epitelio intestinal. Con la PCR desarrollada en este trabajo se logró estandarizar un método que permite la amplificación del gen invA con una detección de 10² UFC/25 g. Este método acorta los tiempos de respuesta de los resultados presuntivos y brinda información complementaria al cultivo tradicional del patógeno. El estudio del gen invA establece el potencial patógeno del microorganismo presente en la muestra, lo que puede ser de utilidad para la salud pública. Salmonella spp. is a very important bacterial pathogen that causes diarrhea and which is transmitted both through the fecal-oral pathway, as by contaminated food and water. In this study we standardized a PCR method in lettuce for the detection of the Salmonella spp. invA gene. This gene is related to the invasion of the intestinal epithelium process. With the PCR method developed in this study we were able to standardize a method which permits the amplification of the invA gene with a 10² CFU/25 g detection. This method shortens the response times of the presumptive results and gives complementary information to the traditional culture of the pathogen. The study of the invA gene establishes the pathogenic potential of the microorganism present in the sample, which can be useful for public health purposes.

Artículo científico -- Universidad de Costa Rica, Instituto de Investigaciones en Salud. 2002 Antibodies titers against H. pylori antigens were determined in 78 patients referred to the Digestive Endoscopy Service of the San Vicente de Paul Hospital, Costa Rica. The assay was performed by enzyme immunoassay (ELISA), using commercial and non comercial methods with costarrican bacterial antigens and antigens prepared in Sweden, with the purpose of determining the diagnostic sensitivity and specificity of IgG, IgM and IgA inmunoglobulines. Material and methods. The commercial ELISA method was performed according to instructions and the two ELISA methods were prepared in the laboratory using no commercial antigens, one from Costa Rica and the other prepared in Sweden. Results. H pylori infection was present in 83% of studied patients. The determination of IgG anti H. pylori by commercial ELISA method presents the highest diagnostic sensitivity (76%) and the non commercial ELISA using costarrican antigen shows the lowest diagnostic sensitivity (57%) with the highest diagnostic specificity (92%). The non commercial ELISA method more specific was the one that uses costarrican bacterial antigen, but the antigen prepared in Sweden had the highest sensitivity (67%). Discussion. The use of antigens prepared of local strains permits the detection of the presence of H. pylori with greater confidence as compared to ntigens prepared with strains from other regions, because elevated serologic tests are high suggestive of the presence of the bacteria in the gastric mucous. The anti H. pylori inmunoglobulin with the highest diagnostic sensitivity and specificity was the IgG, but a histologic study of gastric biopsy instead of using the serologic test alone is recommended for the diagnosis of H. pylori. Introducción. El estudio se realizó en 78 pacientes referidos al Servicio de Endoscopia Digestiva del Hospital San Vicente de Paul, un estudio de anticuerpos sericos anti H. pylori tipo IgG, IgM e IgA por el método de ELISA utilizando antígenos comerciales y preparados con bacterias autóctonas, a fin de determinar la sensibilidad diagnostica y la especificidad diagnostica de cada inmunoglobulina. Material y métodos. Se utilizo un método de ELISA comercial y dos métodos de ELISA preparados en el laboratorio utilizando antígenos no comerciales, uno de origen costarricense y otro preparado en Suecia. Resultados. El 83% de los pacientes presentaron infección por H. pylori. En la determinación de IgG anti H. pylori el método de ELISA comercial presentó la sensibilidad diagnostica más altas (76%) mientras que el método de ELBA no comercial con antígeno de origen costarricense presento la sensibilidad diagnostica más baja (57%), pero con la mayor especificidad diagnóstica (92%). El método de ELISA no comercial más especifico fue el que utiliza antígeno de origen costarricense, sin embargo el antígeno de origen sueco mostro mayor sensibilidad (67%). Conclusiones. El use de antígenos autóctonos permite detectar la presencia de H. pylori con mayor confianza que los foráneos, ya que una prueba serológica aumentada es altamente sugestiva de la presencia de la bacteria en la mucosa gástrica. La inmunoglobulina anti H. pylori con mejor sensibilidad y especificidad diagnostica fue la IgG, pero se recomienda realizar un estudio. 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)

OBJECTIVE: The aim of the present work was to develop a rapid, specific and highly sensitive diagnostic method for the detection of Shigella sonnei directly from stool samples. DESIGN AND METHODS: An immunomagnetic separation-polymerase chain reaction (IMS-PCR) combined assay for diagnosis of S. sonnei was developed. For this, a monoclonal antibody (Mab) specific for the O-antigen of S. sonnei was coated to magnetic beads for capture, concentration and separation of S. sonnei from feces. Bacterial DNA, was amplified by the PCR with specific primers. The amplified products were developed by dot blot hybridization with a specific alkaline phosphatase-conjugated probe. RESULTS: The purified MASS MAb reacted specifically with the Plesiomonas shigelloides (the same O-antigen as Shigella sonnei) LPS. The primers were specific for invasive Shigella and enteroinvasive Escherichia coli. Only invasive Shigella sonnei strains gave a positive result in the IMS-PCR assay. The detection limit was 10 to 15 c.f.u. CONCLUSIONS: The availability of IMS-PCR assays provides an improved method for the diagnosis of shigellae directly from feces. The assay is rapid, highly sensitive and specific for the detection of Shigella sonnei directly from stool samples.