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.

Risk of Gastroenteritis from Swimming at a Wastewater-Impacted Tropical Beach Varies across Localized Scales.

Population growth and changing climate are expected to increase human exposure to pathogens in tropical coastal waters. We examined microbiological water quality in three rivers within 2.3 km of each other that impact a Costa Rican beach and in the ocean outside their plumes during the rainy and dry seasons. We performed quantitative microbial risk assessment (QMRA) to predict the risk of gastroenteritis associated with swimming and the amount of pathogen reduction needed to achieve safe conditions. Recreational water quality criteria based on enterococci were exceeded in >90% of river samples but in only 13% of ocean samples. Multivariate analysis grouped microbial observations by subwatershed and season in river samples but only by subwatershed in the ocean. The modeled median risk from all pathogens in river samples was between 0.345 and 0.577, 10-fold above the U.S. Environmental Protection Agency (U.S. EPA) benchmark of 0.036 (36 illnesses/1,000 swimmers). Norovirus genogroup I (NoVGI) contributed most to risk, but adenoviruses raised risk above the threshold in the two most urban subwatersheds. The risk was greater in the dry compared to the rainy season, due largely to the greater frequency of NoVGI detection (100% versus 41%). Viral log10 reduction needed to ensure safe swimming conditions varied by subwatershed and season and was greatest in the dry season (3.8 to 4.1 dry; 2.7 to 3.2 rainy). QMRA that accounts for seasonal and local variability of water quality contributes to understanding the complex influences of hydrology, land use, and environment on human health risk in tropical coastal areas and can contribute to improved beach management. IMPORTANCE This holistic investigation of sanitary water quality at a Costa Rican beach assessed microbial source tracking (MST) marker genes, pathogens, and indicators of sewage. Such studies are still rare in tropical climates. Quantitative microbial risk assessment (QMRA) found that rivers impacting the beach consistently exceeded the U.S. EPA risk threshold for gastroenteritis of 36/1,000 swimmers. The study improves upon many QMRA studies by measuring specific pathogens, rather than relying on surrogates (indicator organisms or MST markers) or estimating pathogen concentrations from the literature. By analyzing microbial levels and estimating the risk of gastrointestinal illness in each river, we were able to discern differences in pathogen levels and human health risks even though all rivers were highly polluted by wastewater and were located less than 2.5 km from one another. This variability on a localized scale has not, to our knowledge, previously been demonstrated.

Circulación de genes de virulencia asociados a Escherichia coli diarrogénica en aguas residuales del Gran Área Metropolitana de Costa Rica / Circulation of diarrheagenic Escherichia coli associated virulence genes in Costa Rican Great Metropolitan Area wastewaters

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.

The relationship between environmental parameters and microbial water quality at two Costa Rican beaches from 2002 to 2017.

Environmental conditions influence fecal indicator bacteria (FIB) levels, which are routinely used to characterize recreational water quality. This study examined 15 years of environmental and FIB data at Puntarenas and Jacó beach, Costa Rica. FIB relationships with sea level, wave height, precipitation, direct normal irradiance (DNI), wind, and turbidity were analyzed. Pearson's correlations identified lags between 24 and 96 h among environmental parameters and FIB. Multiple linear regression models composed of environmental parameters explained 24% and 27% of fecal coliforms and enterococci variability in Jacó, respectively. Puntarenas's models explained 17-26% of fecal coliforms and 12-18% enterococci variability. Precipitation, sea level anomalies, and wave height most frequently explained FIB variability. Hypothesis testing often identified significant differences in precipitation, wave height, daily sea level anomalies, and maximum sea level 24 h prior between days with and without FIB threshold exceedance. Unexpected FIB interactions with DNI, sea level, and turbidity highlight the importance of future investigations.

Relationships among microbial indicators of fecal pollution, microbial source tracking markers, and pathogens in Costa Rican coastal waters.

Tropical coastal waters are understudied, despite their ecological and economic importance. They also reflect projected climate change scenarios for other climate zones, e.g., increased rainfall and water temperatures. We conducted an exploratory microbial water quality study at a tropical beach influenced by sewage-contaminated rivers, and tested the hypothesis that fecal microorganisms (fecal coliforms, enterococci, Clostridium perfringens, somatic and male-specific coliphages, pepper mild mottle virus (PMMoV), Bacteroides HF183, norovirus genogroup I (NoVGI), Salmonella, Cryptosporidium and Giardia) would vary by season and tidal stage. Most microorganisms' concentrations were greater in the rainy season; however, NoVGI was only detected in the dry season and Cryptosporidium was the only pathogen most frequently detected in rainy season. Fecal indicator bacteria (FIB) levels exceeded recreational water quality criteria standards in >85% of river samples and in <50% of ocean samples, regardless of the FIB or regulatory criterion. Chronic sewage contamination was demonstrated by detection of HF183 and PMMoV in 100% of river samples, and in >89% of ocean samples. Giardia, Cryptosporidium, Salmonella, and NoVGI were frequently detected in rivers (39%, 39%, 26%, and 39% of samples, respectively), but infrequently in ocean water, particularly during the dry season. Multivariate analysis showed that C. perfringens, somatic coliphage, male-specific coliphage, and PMMoV were the subset of indicators that maximized the correlation with pathogens in the rivers. In the ocean, the best subset of indicators was enterococci, male-specific coliphage, and PMMoV. We also executed redudancy analyses on environmental parameters and microorganim concentrations, and found that rainfall best predicted microbial concentrations. The seasonal interplay of rainfall and pathogen prevalence undoubtedly influences beach users' health risks. Relationships are likely to be complex, with some risk factors increasing and others decreasing each season. Future use of multivariate approaches to better understand linkages among environmental conditions, microbial predictors (fecal indicators and MST markers), and pathogens will improve prediction of high-risk scenarios at recreational beaches.