Challenges detecting SARS-CoV-2 in Costa Rican domestic wastewater and river water.

This study presents the development of a SARS-CoV-2 detection method for domestic wastewater and river water in Costa Rica, a middle-income country in Central America. Over a three-year period (November to December 2020, July to November 2021, and June to October 2022), 80 composite wastewater samples (43 influent and 37 effluent) were collected from a Wastewater Treatment Plant (SJ-WWTP) located in San José, Costa Rica. Additionally, 36 river water samples were collected from the Torres River near the SJ-WWTP discharge site. A total of three protocols for SARS-CoV-2 viral concentration and RNA detection and quantification were analyzed. Two protocols using adsorption-elution with PEG precipitation (Protocol A and B, differing in the RNA extraction kit; n = 82) were used on wastewater samples frozen prior to concentration, while wastewater (n = 34) collected in 2022 were immediately concentrated using PEG precipitation. The percent recovery of Bovine coronavirus (BCoV) was highest using the Zymo Environ Water RNA (ZEW) kit with PEG precipitation executed on the same day as collection (mean 6.06 % ± 1.37 %). It was lowest when samples were frozen and thawed, and viruses were concentrated using adsorption-elution and PEG concentration methods using the PureLink™ Viral RNA/DNA Mini (PLV) kit (protocol A; mean 0.48 % ± 0.23 %). Pepper mild mottle virus and Bovine coronavirus were used as process controls to understand the suitability and potential impact of viral recovery on the detection/quantification of SARS-CoV-2 RNA. Overall, SARS-CoV-2 RNA was detected in influent and effluent wastewater samples collected in 2022 but not in earlier years when the method was not optimized. The burden of SARS-CoV-2 at the SJ-WWTP decreased from week 36 to week 43 of 2022, coinciding with a decline in the national COVID-19 prevalence rate. Developing comprehensive nationwide surveillance programs for wastewater-based epidemiology in low-middle-income countries involves significant technical and logistical challenges.

Occurrence of pathogenic microorganisms in small drinking-water systems in Costa Rica.

This study describes the quality of drinking water sampled over 2 years (2018 and 2019) from 20 ASADAS (Spanish acronym for Administrative Associations for Water and Sewer Systems) in Costa Rica. The analysis included Rotavirus (RV), somatic coliphages, fecal coliforms, and Escherichia coli. The ASADAS were categorized into three regions as temperate rainy (region 1), tropical rainy (region 2), and tropical rainy and dry (region 3) according to biogeographic classification. The concentrations of fecal coliforms and E. coli were higher in samples from surface water sources from the ASADAS in region 3 compared to regions 1 and 2. RV-positive samples (24/296) were detected in drinking-water samples from regions 2 and 3 during dry and transition seasons, with higher concentrations more frequently in the dry season. In addition, somatic coliphages were detected in samples from the three regions, with higher concentrations in region 2. Furthermore, a statistically significant relationship was found between somatic coliphages and diarrheal cases, classified as outbreaks or alerts in the region. Thus, the results confirmed that somatic coliphages are a good indicator of the presence of diarrhea cases in a specific region.

Wastewater-Based Epidemiology of Enteric Viruses and Surveillance of Acute Gastrointestinal Illness Outbreaks in a Resource-Limited Region.

Acute gastrointestinal illness (AGI) continues to be an important cause of morbidity and mortality among all ages. This study applied the principles of wastewater-based epidemiology for the preventive identification of potential outbreaks of acute viral gastroenteritis and hepatitis A by analyzing the presence of human enteric viruses in influents of small municipal wastewater treatment plants (WWTPs) handling domestic sewage, together with public health reports of acute diarrheal and hepatitis A disease in Costa Rica during 2013. Raw wastewater samples were collected during four seasonal periods with different rainfall levels. The presence of five human enteric viruses (rotavirus A, norovirus GI, norovirus GII, enterovirus, and hepatitis A virus) was studied by endpoint and real-time polymerase chain reaction in influents of five WWTPs. Cases of AGI were analyzed using historical public health reports of endemic levels and quartile ranges for each administrative and territorial area where the WWTPs are located and for its surrounding counties. A tendency for a higher rate of positive viral tests was present 1 week before an increase of AGI cases. Epidemiological weeks categorized as Outbreak (above the 75th percentile) and Success (below the 25th percentile) showed statistically significant differences in terms of positive viral test rates (Wilcoxon test, P = 0.05). Virological monitoring of wastewater in small WWTPs is an appropriate model for epidemiological surveillance of diarrheal and hepatitis A diseases in low- and middle-resource countries.

A Somatic Coliphage Threshold Approach To Improve the Management of Activated Sludge Wastewater Treatment Plant Effluents in Resource-Limited Regions.

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.

Draft Genome Sequence of an Escherichia coli Strain Harboring bla CTX-M-115, bla CMY-2, Aminoglycoside, Tetracycline, and Sulfonamide Resistance Genes, Isolated from a Costa Rican Wastewater Treatment Plant.

We report the draft genome sequence of the multidrug-resistant Escherichia coli strain PTA A1517-5, isolated from a wastewater treatment plant in Costa Rica. The genome consists of 4,927,375 bp with a GC content of 50.57% and a total of 4,853 genes. This strain harbors bla CTX-M-115, bla CMY-2, aminoglycoside, tetracycline, and sulfonamide resistance genes.

Evaluación de la influencia de la estacionalidad climática en calidad del agua de consumo humano en un sistema de abastecimiento en San José, Costa Rica, periodo 2017-2018 / Evaluation of climatic seasonal influence on the quality of water for human consumption in a San José (Costa Rica) supply system, 2017-2018

Resumen Objetivo: Analizar el estado y la influencia de la variabilidad climática en la calidad del agua de un sistema de abastecimiento para consumo humano en San José, Costa Rica, captado en cuatro microcuencas del cantón Vásquez de Coronado, en el periodo 2017-2018, para brindar recomendaciones al operador y promover la protección de la población abastecida Métodos: Estudio descriptivo de los parámetros de calidad básicos de agua para consumo humano (Escherichia coli, coliformes fecales, turbidez, conductividad, pH, color, temperatura y cloro residual). Los análisis se realizaron siguiendo el Standard Methods for the Examination of Water and Wastewater. Se delimitaron las zonas de captación utilizando sistemas de información geográfica. Se utilizó estadística descriptiva e inferencial para describir la calidad del agua y su variabilidad espacial y estacional. Resultados: La concentración de coliformes fecales en el agua superficial fue > 103 NMP/100 mL y la turbidez >5 UTN en el 76% de las muestras. En la red de distribución se encontraron concentraciones de cloro residual y turbidez (>5 UTN) por encima de los límites recomendados para agua de consumo humano. Se encontró diferencias significativas en todos los parámetros básicos de calidad de agua entre las distintas zonas de captación (p<0,05 en todos los casos), así como entre épocas climáticas por zona de captación. Conclusión: Estos resultados indican que se está utilizando agua de calidad inadecuada para uso y consumo humano, por lo cual se recomienda un monitoreo intensivo y sectorizado de las fuentes para localizar puntos de contaminación.

Abstract Objective: Analyze the water quality and the influence of the climatic variability in supply system for the human consume in San José, Costa Rica, in four catchment areas in Vasquez of Coronado in the period 2017-2018 to give recommendations to the operator and promote the protection of the supplied population. Methods: Descriptive study of the basic parameters of the drinking water (Escherichia coli, fecal coliforms, turbidity, conductivity, pH, color, temperature, and residual chlorine). The analyses were done following Standard Methods for the examination of Water and Wastewater. The catchment areas were delimited using geographic information systems (GIS). Descriptive and inferential statistic was used to describe the water quality, spatial and seasonal variability. Results: The concentration of fecal coliforms in the catchment areas was >10 3 NMP/100 mL and the turbidity >5 UTN in 76% of the samples. In the distribution system were found concentrations of the residual chlorine and turbidity (>5 UTN) above according to recommended limits for drinking water in the country. The results showed significant differences in all of the basic parameters of the water quality between catchment areas (p<0, 05 in all cases) and between climatic periods for catchment areas. Conclusion: These results indicate that water of inadequate quality is being used for human use and consumption, for which reason intensive and sectorized source monitoring is recommended to locate contamination points.

Enterococci as a key parameter for water quality index: Purires River, Costa Rica.

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.

The importance of integrons for development and propagation of resistance in Shigella: the case of Latin America

Abstract In Latin America, the disease burden of shigellosis is found to coexist with the rapid and rampant spread of resistance to commonly used antibiotics. The molecular basis of antibiotic resistance lies within genetic elements such as plasmids, transposons, integrons, genomic islands, etc., which are found in the bacterial genome. Integrons are known to acquire, exchange, and express genes within gene cassettes and it is hypothesized that they play a significant role in the transmission of multidrug resistance genes in several Gram-negative bacteria including Shigella. A few studies have described antibiotic resistance genes and integrons among multidrug resistant Shigella isolates found in Latin America. For example, in Brazil, Bolivia, Chile, Costa Rica and Peru, class 1 and class 2 integrons have been detected among multidrug resistant strains of Shigella; this phenomenon is more frequently observed in S. flexneri isolates that are resistant to trimethoprim, sulfamethoxazole, streptomycin, ampicillin, chloramphenicol, and tetracycline. The gene cassette sul2, which is frequently detected in Shigella strains resistant to the sulfonamides, suggests that the sulfonamide-resistant phenotype can be explained by the presence of the sul2 genes independent of the integron class detected. It is to be noted that sul3 was negative in all isolates analyzed in these studies.The high frequency of sulfonamide (as encoded by sul2) and trimethoprim resistance is likely to be a result of the recurrent use of trimethoprim sulfamethoxazole as a popular regimen for the treatment of shigellosis. The observed resistance profiles of Shigella strains confirm that ampicillin and trimethoprim-sulfamethoxazole are ineffective as therapeutic options. In-depth information regarding antibiotic resistance mechanism in this pathogen is needed in order to develop suitable intervention strategies. There is a pressing need for regional and local antimicrobial resistance profiling of Shigella to be included as a part of the public health strategy.

The importance of integrons for development and propagation of resistance in Shigella: the case of Latin America.

In Latin America, the disease burden of shigellosis is found to coexist with the rapid and rampant spread of resistance to commonly used antibiotics. The molecular basis of antibiotic resistance lies within genetic elements such as plasmids, transposons, integrons, genomic islands, etc., which are found in the bacterial genome. Integrons are known to acquire, exchange, and express genes within gene cassettes and it is hypothesized that they play a significant role in the transmission of multidrug resistance genes in several Gram-negative bacteria including Shigella. A few studies have described antibiotic resistance genes and integrons among multidrug resistant Shigella isolates found in Latin America. For example, in Brazil, Bolivia, Chile, Costa Rica and Peru, class 1 and class 2 integrons have been detected among multidrug resistant strains of Shigella; this phenomenon is more frequently observed in S. flexneri isolates that are resistant to trimethoprim, sulfamethoxazole, streptomycin, ampicillin, chloramphenicol, and tetracycline. The gene cassette sul2, which is frequently detected in Shigella strains resistant to the sulfonamides, suggests that the sulfonamide-resistant phenotype can be explained by the presence of the sul2 genes independent of the integron class detected. It is to be noted that sul3 was negative in all isolates analyzed in these studies. The high frequency of sulfonamide (as encoded by sul2) and trimethoprim resistance is likely to be a result of the recurrent use of trimethoprim sulfamethoxazole as a popular regimen for the treatment of shigellosis. The observed resistance profiles of Shigella strains confirm that ampicillin and trimethoprim-sulfamethoxazole are ineffective as therapeutic options. In-depth information regarding antibiotic resistance mechanism in this pathogen is needed in order to develop suitable intervention strategies. There is a pressing need for regional and local antimicrobial resistance profiling of Shigella to be included as a part of the public health strategy.

Contaminación fecal del agua superficial de la microcuenca del río Purires, Costa Rica, 2010-2011 / Fecal contamination of the superficial water of the microbasin of Rio Purires, Costa Rica, 2010-2011

La contaminación fecal de las aguas superficiales es un problema importante para la salud pública, dada la transmisión de microorganismos patógenos. Se estima que las poblaciones ubicadas cerca de costas, ríos o lagos con elevada contaminación fecal, tienen mayor riesgo de desarrollar enfermedades infecciosas gastrointestinales. En esta investigación se analizó durante un año, la contaminación fecal en las aguas superficiales de la microcuenca del río Purires, ubicada en una zona de alta densidad poblacional en Costa Rica. En el 100% de las muestras se detectó contaminación fecal, siendo el punto de muestreo 3 el que mostró los niveles más altos, en promedio 2,2 x 10(4) Número Más Probable(NMP)/100()mL. Aunque los puntos 1 y 2 presentaron menor contaminación fecal, en promedio 6,4 x 10² NMP/100 mL y 6,3 x 10³ NMP/100 mL respectivamente, estos valores indican también mala calidad de las aguas. Con estos resultados se pretende llamar la atención sobre la problemática ambiental de alta contaminación fecal en las aguas superficiales de esta microcuenca. Esta información es un insumo para desarrollar acciones de control sobre las fuentes de contaminación que afectan la calidad de las aguas y por ende, la salud de las poblaciones ubicadas en su cercanía.

Fecal contamination of the superficial waters is an important public health problem due to the presence of pathogenic microorganisms- It has been estimated that populations located close to coasts, rivers of lakes with an elevated fecal contamination have a higher risk of developing communicable gastrointestinal diseases. This investigation analyzed the fecal contamination of the superficial waters of the microbasin of the Rio Purires, located in a highly populated area of Costa Rica. Fecal contamination was detected in 100% of the samples, and sample point 3 was the one that showed the highest levels, with an average of 2.2 x 10(4) Most Probable Number (MPN)/100mL. Even thought points 1 and 2 presented a lower fecal contamination, an average of 6.4 x 10² MPN/100mL and 6.3 x 10³ MPN/100mL, respectively, these values also indicate low quality water. With these results we intend to alert regarding the problem of a high fecal contamination of superficial waters of this microbasin. This information is an input for developing control actions over the contamination sources which compromise the quality of water and, therefore, the health of the populations located in its vicinity.

Calidad microbiológica y análisis de patógenos (Shigella y Salmonella) en lechuga / Microbiological quality and pathogen analysis (Shigella and Salmonella) of lettuce

Se analizó la calidad microbiológica en 37 muestras de lechuga variedad criolla (Lactuca sativa var. Capitata L.) de distintos intermediarios en las provincias de San José y Cartago, en Costa Rica. Las muestras se recolectaron mediante muestreo no probabilístico por selección intencional. Se cuantificó Escherichia coli (NMP/g) como indicador de contaminación fecal y se determinó la presencia de patógenos específicos (Shigella y Salmonella), por cultivo y por PCR-Múltiple. En el 65% de las muestras analizadas se detectó E. coli, aunque no se encontró Shigella ni Salmonella por PCR-Múltiple o cultivo. Una posible explicación es que los niveles de contaminación de Shigella y Salmonella están por debajo de los límites de detección de ambos métodos (menos de 10(4) UFC/g para Shigella y menos de 10² UFC/g para Salmonella). Estos resultados establecen una base importante para continuar con este tema de investigación y analizar otras fuentes de transmisión de Shigella y Salmonella, dado que ambos patógenos son frecuentes en la región.

The microbiological quality of 37 lettuce samples of the creole variety (Lactuca sativa var. Capitata L.) obtained from different intermediaries at the provinces of San José and Cartago, in Costa Rica was analyzed. The samples were collected through a non-probabilistic sampling with intentional selection. Escherichia coli (NMP/g) was quantified as indicator of fecal contamination and the presence of specific pathogens (Shigella and Salmonella) was determined by culture and Multiplex-PCR. In 65% of the samples analyzed we detected E. coli, even though we did not find Shigella or Salmonella by Multiplex-PCR or culture. A possible explanation is that the Shigella or Salmonella contamination levels may have been under the detection limits for both methods (less than 10(4) CFU/g for Shigella, and less than 10² CFU/g for Salmonella). These results establish an important basis for continuing with this research subject and analyzing other sources of transmission of Shigella and Salmonella contamination, since both pathogens are frequent in the region.

Estandarización de una PCR para la detección del gen invA de Salmonella spp. en lechuga / Standardization of a PRC method for the detection of the Salmonella spp. invA gene in lettuce

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.

Prevalencia de parasitosis intestinal en niños de la comunidad de Los Cuadros, Goicoechea, Costa Rica: 2002-2003 / Prevalence of instestinal parasitosis in children of the community of Los Cuadros, Goicoechea, Costa Rica: 2002-2003

Se analizó la presencia de parásitos intestinales en muestras de heces de 350 niños, de una población de 1.563 niños de hasta cinco años de edad, de la comunidad urbano marginal de Los Cuadros de Goicoechea, San José, Costa Rica, durante los años 2003 y 2004. El diseño del muestreo fue aleatorio simple, para un tamaño de muestra de 350 y nivel de confianza del 95 por ciento. Las muestras se recolectaron mediante visitas a los hogares. Se obtuvo una muestra de heces de cada niño; no se repitieron muestras. La detección de parásitos se realizó mediante un examen directo (salina, lugol), concentrado de Ritchie (formol-éter) y tinción modificada de Zielh Neelsen. Las muestras fueron analizadas por profesionales de laboratorio. Del total de muestras 210 fueron positivas por al menos un parásito, protozoarios y helmintos intestinales, lo que representó una prevalencia total del 60 por ciento. Los resultados mostraron la presencia de Giardia intestinalis en el 18,2 por ciento; Entamoeba histolytica/E dispar en el 15,7 por ciento; Ascaris lumbricoides en el 15,4 por ciento y Trichuris 0,8 por ciento y Entamoeba coli en el 9,7 por ciento de los casos...

To analyze the presence of intestinal parasites, there has been developed a study with a 95% of confidence level at the urban-marginal community of Los Cuadros, Goicoechea, San José, Costa Rica, during the years 2003 and 2004, using a sample random of 350 children up to 5 years of age, from whom faecal samples were collected by visits...

Brote de diarrea asociado a Shigella Sonnei debido a contaminación hídrica, San José, Costa Rica, 2001 / Bud of associated diarrhea to Shigella Sonnei due to hydric contamination, San José, Costa Rica, 2001

En julio del 2001, un brote de diarrea asociado a transmisión hídrica afectó a más de 7000 personas residentes en el área urbana de San José. Desde su incio se especuló sobre el papel de Shigella spp., como uno de los posibles agentes etiológicos del mismo, por lo que el objetivo de este estudio fue determinar la presencia de esta bacteria en los casos de diarrea atendidos en una de las clínicas ubicadas en el área del brote y demostrar que Shigella spp. es un agente importente de diarrea en nuestro país asociado con la transmisión hídrica particularmente por la baja dosis infectante y alta virulencia. Para este fin, se analizaron especímenes fecales de 49 pacientes con diarrea que acudieron a la clínica de Alajuelita, del 17 al 31 de julio del 2001. Se obtuvo 16 aislamientos de Shigella spp. (34.7 por ciento), 15 correspondieron a Shigella sonnei en tanto solamente uno a Shigella flexneri. Los grupos etáreos más afectados fueron niños menores de 10 años y personas mayores de 60 años. El patrón de sensibilidad a los antibióticos de las cepas aisladas mostró resistencia a 8 de los 11 antibióticos probados: trimetroprim/sulfametoxazole, tetraciclina, ampicilina, eritromicina, cloranfenicol, cefalotina, amikacina y amoxicilina; y sensibilidad a norfloxacina, ciprofloxacina y gentamicina. No fue posible aislar Shigella spp. de las muestras de agua. Los resultados sugieren que este medio de transmisión estuvo relacionado con el brote de diarrea. El presente estudio demuestra la importancia de Shigella spp. como causa de diarrea sobre todo en niños pequeños, adultos mayores y su frecuente asociación con brotes relacionados con agua contaminada. Los resultados también sugieren que el tratamiento contra la shigelosis debería incluir información acerca de los perfiles de resistencia a los antibióticos y de la necesidad de más investigación y sobre las vías de transmisión por agua y alimentos en la prevención de este tipo de brotes. Palabras clave: Shigella sonnei, diarrea, brote