Tracking SARS-CoV-2 in rivers as a tool for epidemiological surveillance.

The aim of this work was to evaluate if rivers could be used for SARS-CoV-2 surveillance. Five sampling points from three rivers (AR-1 and AR-2 in Arenales River, MR-1 and MR-2 in Mojotoro River, and CR in La Caldera River) from Salta (Argentina), two of them receiving discharges from wastewater plants (WWTP), were monitored from July to December 2020. Fifteen water samples from each point (75 in total) were collected and characterized physico-chemically and microbiologically and SARS-CoV-2 was quantified by RT-qPCR. Also, two targets linked to human contributions, human polyomavirus (HPyV) and RNase P, were quantified and used to normalize SARS-CoV-2 concentration, which was compared to reported COVID-19 cases. Statistical analyses allowed us to verify the correlation between SARS-CoV-2 and the concentration of fecal indicator bacteria (FIB), as well as to find similarities and differences between sampling points. La Caldera River showed the best water quality; FIBs were within acceptable limits for recreational activities. Mojotoro River's water quality was not affected by the northern WWTP of the city. Instead, Arenales River presented the poorest water quality; at AR-2 was negatively affected by the discharges of the southern WWTP, which contributed to significant increase of fecal contamination. SARS-CoV-2 was found in about half of samples in low concentrations in La Caldera and Mojotoro Rivers, while it was high and persistent in Arenales River. No human tracers were detected in CR, only HPyV was found in MR-1, MR-2 and AR-1, and both were quantified in AR-2. The experimental and normalized viral concentrations strongly correlated with reported COVID-19 cases; thus, Arenales River at AR-2 reflected the epidemiological situation of the city. This is the first study showing the dynamic of SARS-CoV-2 concentration in an urban river highly impacted by wastewater and proved that can be used for SARS-CoV-2 surveillance to support health authorities.

Propuestas para el control y la mitigación de la diseminación de COVID-19: un manejo estratégico de la enfermedad

Resumen La velocidad de diseminación del COVID-19 en el mundo llevó a que los países afectados cerraran sus fronteras y tomaran medidas de distanciamiento social. Después de seis meses de que la enfermedad fuera declarada pandemia, muchos países están tomaron medidas de flexibilización del aislamiento, aunque sin una vacuna o un medicamento capaz de enfrentar la infección por el SARS-CoV-2, la situación podría revertirse en cualquier momento. El objetivo del presente trabajo fue proponer un algoritmo de decisión tendiente a optimizar las detecciones de casos asintomáticos y administrar la cuarentena de una manera estratégica, para así evitar la diseminación del virus y tender hacia una normalidad administrada. Se elaboró una propuesta tentativa de optimización y ordenamiento de pruebas de detección del SARS-CoV-2, basada en el análisis de muestras compuestas reunidas a partir de aquellas tomadas de manera individual a personas asintomáticas que integran cohortes de interés. Se definieron cohortes según su función en la sociedad o grado de vulnerabilidad. El algoritmo contempla variables como la prioridad de la cohorte, el número de integrantes de los grupos de análisis dentro de cada cohorte, el contacto intragrupal e intergrupal, la vulnerabilidad al contagio por la actividad desarrollada y el tiempo transcurrido desde que se realizó la prueba por última vez. Se ilustró la propuesta con cohortes hipotéticas definidas, con un único grupo de análisis para simplificar, y se comprobó que la aplicación de la herramienta permite establecer de una manera racional un orden de prioridad para realizar las pruebas en grupos críticos de la sociedad. Esta herramienta permitirá optimizar recursos y disminuir el impacto de la enfermedad en la salud, la sociedad y la economía de una región.

Abstract The rapid spread of COVID-19 throughout the world, has led most of the affected countries to close their borders and implement some form of lockdown. Six months after the pandemic started, many countries made decisions tending to relax the lockdown, although wit-hout a vaccine or treatment capable of confronting SARS-CoV-2 infection, the situation could be reversed at any time. In this context, the aim of this work was to propose a decision algorithm that will allow to optimize asymptomatic case detections and strategically manage quarantine to prevent the spread of the virus and drive the transition to a managed new normal. This tentative proposal was developed for optimizing and ordering the number of tests for the detection of SARS-CoV-2, analyzing composite samples (group analysis) combining with those samples individually taken from asymptomatic members of cohorts of interest. Cohorts were defined according to their critical role in society and/or their vulnerability. The algorithm includes variables such as cohort priority, number of cohort members in the analysis groups, intra-and intergroup contact, vulnerability to contagion due to the activity performed, and time elapsed since last testing. The proposed tool was illustrated with defined hypothetical cohorts, in which, for the sake of simplification, only one analysis group was considered. The application of this tool allowed to establish in a rational way a priority order to test critical groups in society. Furthermore, this tool would help to optimize resources, reducing the impact on a region's health, society, and economy.

[Proposal for the control and mitigation of COVID-19 dissemination: strategic management of the disease]. / Propuestas para el control y la mitigación de la diseminación de COVID-19: un manejo estratégico de la enfermedad.

The rapid spread of COVID-19 throughout the world, has led most of the affected countries to close their borders and implement some form of lockdown. Six months after the pandemic started, many countries made decisions tending to relax the lockdown, although without a vaccine or treatment capable of confronting SARS-CoV-2 infection, the situation could be reversed at any time. In this context, the aim of this work was to propose a decision algorithm that will allow to optimize asymptomatic case detections and strategically manage quarantine to prevent the spread of the virus and drive the transition to a managed new normal. This tentative proposal was developed for optimizing and ordering the number of tests for the detection of SARS-CoV-2, analyzing composite samples (group analysis) combining with those samples individually taken from asymptomatic members of cohorts of interest. Cohorts were defined according to their critical role in society and/or their vulnerability. The algorithm includes variables such as cohort priority, number of cohort members in the analysis groups, intra-and intergroup contact, vulnerability to contagion due to the activity performed, and time elapsed since last testing. The proposed tool was illustrated with defined hypothetical cohorts, in which, for the sake of simplification, only one analysis group was considered. The application of this tool allowed to establish in a rational way a priority order to test critical groups in society. Furthermore, this tool would help to optimize resources, reducing the impact on a region's health, society, and economy.

Sediments quality must be considered when evaluating freshwater aquatic environments used for recreational activities.

Although water quality from freshwater recreational aquatic environments (RAEs) has been long analyzed worldwide, little information is available about their sediments. The aim of this work was to study the physicochemical and bacteriological quality of water and sediment under different seasonal events. For that, Wierna River (WR) and General Belgrano reservoir (GB) were used as freshwater RAEs models. A total of 33 water and 33 sediment samples (15 from WR and 18 from GB from each phase) were collected and analyzed. Physicochemical variables in water (pH, turbidity, dissolved oxygen, temperature, conductivity, alkalinity, hardness) and sediments (organic matter, humidity, ash, and conductivity) were measured. For the bacteriological characterization, total aerobic mesophiles, total and thermotolerant coliforms, E. coli, enteroccocci, Salmonella spp., and Pseudomonas aeruginosa were evaluated using culture-based methods. Universal and human Bacteroides were also quantified by real-time PCR. Univariate (Kruskall-Wallis), bivariate (Spearman correlation), and multivariate (cluster analysis, principal component analysis) statistical techniques were applied for data analysis. All bacterial indicators were almost two-logs higher in sediments than in water, for both RAEs. Also, due to rainfall events and recreational activities, sediments were resuspended in surface water exceeding in most cases the limit values established by international regulation for bacteria. Significant correlation was observed between culturable bacteria and turbidity (p < 0.05) supporting this. We found that while physicochemical variables clustered samples by geographical location in water and sediments, microbiological aggrupation in water was mostly driven by seasonal events. No aggrupation was observed when using microbiological variables in sediments. Thus, geographical location, type of water and sediments, and seasonal events influenced on RAEs quality. Including sediment analysis during RAEs monitoring campaigns is essential as it will allow knowing the real health risk to which bathers are exposed and proposing solutions to mitigate it.

How long can culturable bacteria and total DNA persist in environmental waters? The role of sunlight and solid particles.

In this work, sunlight inactivation of two indicator bacteria in freshwater, with and without solid particles, was studied and the persistence of culturable cells and total DNA was compared. Environmental water was used to prepare two matrices, with and without solid particles, which were spiked with Escherichia coli and Enterococcus faecalis. These matrices were used to prepare microcosm bags that were placed in two containers: one exposed to sunlight and the other in the dark. During one month, samples were removed from each container and detection was done by membrane filter technique and real-time PCR. Kinetic parameters were calculated to assess sunlight effect. Indicator bacteria without solid particles exposed to sunlight suffered an immediate decay (<4h) compared with the ones which were shielded from them. In addition, the survival of both bacteria with solid particles varied depending on the situation analyzed (T99 from 3 up to 60days), being always culturable E. coli more persistent than E. faecalis. On the other side, E. faecalis DNA persisted much longer than culturable cells (T99>40h in the dark with particles). In this case active cells were more prone to sunlight than total DNA and the protective effect of solid particles was also observed. Results highlight that the effects caused by the parameters which describe the behavior of culturable microorganisms and total DNA in water are different and must be included in simulation models but without forgetting that these parameters will also depend on bacterial properties, sensitizers, composition, type, and uses of the aquatic environment under assessment.

Strategies to optimize monitoring schemes of recreational waters from Salta, Argentina: a multivariate approach.

Several recreational surface waters in Salta, Argentina, were selected to assess their quality. Seventy percent of the measurements exceeded at least one of the limits established by international legislation becoming unsuitable for their use. To interpret results of complex data, multivariate techniques were applied. Arenales River, due to the variability observed in the data, was divided in two: upstream and downstream representing low and high pollution sites, respectively, and cluster analysis supported that differentiation. Arenales River downstream and Campo Alegre Reservoir were the most different environments, and Vaqueros and La Caldera rivers were the most similar. Canonical correlation analysis allowed exploration of correlations between physicochemical and microbiological variables except in both parts of Arenales River, and principal component analysis allowed finding relationships among the nine measured variables in all aquatic environments. Variable's loadings showed that Arenales River downstream was impacted by industrial and domestic activities, Arenales River upstream was affected by agricultural activities, Campo Alegre Reservoir was disturbed by anthropogenic and ecological effects, and La Caldera and Vaqueros rivers were influenced by recreational activities. Discriminant analysis allowed identification of subgroup of variables responsible for seasonal and spatial variations. Enterococcus, dissolved oxygen, conductivity, E. coli, pH, and fecal coliforms are sufficient to spatially describe the quality of the aquatic environments. Regarding seasonal variations, dissolved oxygen, conductivity, fecal coliforms, and pH can be used to describe water quality during dry season, while dissolved oxygen, conductivity, total coliforms, E. coli, and Enterococcus during wet season. Thus, the use of multivariate techniques allowed optimizing monitoring tasks and minimizing costs involved.

Cuantificación y evaluación de la estacionalidad de elementos parasitarios en ambientes acuáticos recreativos de la provincia de Salta, Argentina / Quantification of parasites in aquatic environments in the Province of Salta, Argentina

La contaminación microbiológica de aguas recreativas es un problema preocupante, ya que las personas que las utilizan pueden contraer enfermedades que podrían afectar su bienestar general. Para evaluar la calidad del agua, las legislaciones existentes solo establecen límites de indicadores bacterianos, los cuales no predicen con exactitud la presencia de parásitos. Además, la cantidad de parásitos presentes en el agua, aunque suficiente para producir enfermedad, suele ser pequeña, por lo que, se necesita una etapa previa de concentración para poder detectarlos. En este trabajo se monitorearon trimestralmente durante un año tres ambientes acuáticos de usos recreativos de la provincia de Salta, realizando la concentración de las muestras y la posterior preparación para la búsqueda de elementos parasitarios por microscopía. Adicionalmente, en cada ambiente se midieron mensualmente variables fisicoquímicas in situ y variables bacteriológicas por técnicas microbiológicas tradicionales. En cada ambiente se encontraron como mínimo 9 de los 14 parásitos detectados en conjunto. La presencia de los elementos parasitarios no presentó correlación con indicadores bacterianos en ningún ambiente ni en ninguna de las estaciones (p > 0,05). Mientras que en invierno la contaminación bacteriológica disminuyó entre un 76 % y un 99 %, los elementos parasitarios no presentaron disminución estacional. Los resultados permiten sugerir al género Entamoeba como indicador anual de contaminación parasitaria, ya que este fue encontrado en todos los ambientes con mínimas variaciones estacionales. Estos resultados poseen relevancia epidemiológica, dado que permitirán a los tomadores de decisiones proponer medidas para mejorar el bienestar de la población.(AU)

Microbiological pollution of recreational waters is a major problem for public health as it may transmit waterborne diseases. To assess water quality, current legislation only requires limits for bacterial indicators; however, these organisms do not accurately predict the presence of parasites. Small number of parasites is usually present in water and although they are capable of causing disease, they may not be high enough to be detected. Detection therefore requires water samples to be concentrated. In this work three recreational aquatic environments located in the province of Salta were monitored over one year. For parasite quantification, water samples were collected every three months and concentrated by ultrafiltration. Detection was performed by microscopy. In addition, monthly monitoring was carried out in each aquatic environment: physicochemical variables were measured in situ and bacteriological counts were determined by traditional microbiological techniques. Of 14 parasites identified, at least nine were detected in each aquatic environment sampled. While bacteriological contamination decreased in most cases during winter (76-99%), parasites were present year-round, becoming a continual threat to public health. Thus, we here propose that it is necessary to use specific parasitological indicators to prevent waterborne disease transmission. Our results suggest that Entamoeba would be a suitable indicator as it was found in all environments and showed minimal seasonal variation. The results obtained in this study have epidemiological relevance and will allow decision-makers to propose solutions for water protection in order to care for population health.(AU)

Cuantificación y evaluación de la estacionalidad de elementos parasitarios en ambientes acuáticos recreativos de la provincia de Salta, Argentina / Quantification of parasites in aquatic environments in the Province of Salta, Argentina

La contaminación microbiológica de aguas recreativas es un problema preocupante, ya que las personas que las utilizan pueden contraer enfermedades que podrían afectar su bienestar general. Para evaluar la calidad del agua, las legislaciones existentes solo establecen límites de indicadores bacterianos, los cuales no predicen con exactitud la presencia de parásitos. Además, la cantidad de parásitos presentes en el agua, aunque suficiente para producir enfermedad, suele ser pequeña, por lo que, se necesita una etapa previa de concentración para poder detectarlos. En este trabajo se monitorearon trimestralmente durante un año tres ambientes acuáticos de usos recreativos de la provincia de Salta, realizando la concentración de las muestras y la posterior preparación para la búsqueda de elementos parasitarios por microscopía. Adicionalmente, en cada ambiente se midieron mensualmente variables fisicoquímicas in situ y variables bacteriológicas por técnicas microbiológicas tradicionales. En cada ambiente se encontraron como mínimo 9 de los 14 parásitos detectados en conjunto. La presencia de los elementos parasitarios no presentó correlación con indicadores bacterianos en ningún ambiente ni en ninguna de las estaciones (p > 0,05). Mientras que en invierno la contaminación bacteriológica disminuyó entre un 76 % y un 99 %, los elementos parasitarios no presentaron disminución estacional. Los resultados permiten sugerir al género Entamoeba como indicador anual de contaminación parasitaria, ya que este fue encontrado en todos los ambientes con mínimas variaciones estacionales. Estos resultados poseen relevancia epidemiológica, dado que permitirán a los tomadores de decisiones proponer medidas para mejorar el bienestar de la población.

Microbiological pollution of recreational waters is a major problem for public health as it may transmit waterborne diseases. To assess water quality, current legislation only requires limits for bacterial indicators; however, these organisms do not accurately predict the presence of parasites. Small number of parasites is usually present in water and although they are capable of causing disease, they may not be high enough to be detected. Detection therefore requires water samples to be concentrated. In this work three recreational aquatic environments located in the province of Salta were monitored over one year. For parasite quantification, water samples were collected every three months and concentrated by ultrafiltration. Detection was performed by microscopy. In addition, monthly monitoring was carried out in each aquatic environment: physicochemical variables were measured in situ and bacteriological counts were determined by traditional microbiological techniques. Of 14 parasites identified, at least nine were detected in each aquatic environment sampled. While bacteriological contamination decreased in most cases during winter (76-99%), parasites were present year-round, becoming a continual threat to public health. Thus, we here propose that it is necessary to use specific parasitological indicators to prevent waterborne disease transmission. Our results suggest that Entamoeba would be a suitable indicator as it was found in all environments and showed minimal seasonal variation. The results obtained in this study have epidemiological relevance and will allow decision-makers to propose solutions for water protection in order to care for population health.

[Comparison of nucleic acid extraction efficiency using different commercial kits and qPCR. Effect of inhibitors]. / Comparación de la eficiencia de extracción de ácidos nucleicos utilizando distintos kits comerciales y empleando la qPCR. Efecto de las sustancias inhibidoras.

The detection of specific nucleic acid (NA) sequences by PCR has revolutionized the biological and medical sciences. Real-time PCR (qPCR) opened up the possibility of obtaining quantitative results. NA extraction is a decisive step prior to qPCR since it may produce either the removal or co-extraction of inhibitory substances of the enzymatic reaction, which in turn affects the amplification efficiency. In the present work we compared the commercial NA extraction kits from Qiagen, Invitrogen and Macherey-Nagel, which were used to extract DNA from mice blood artificially infected with Trypanosoma cruzi and PP7 RNA, Pseudomonas aeruginosa bacteriophage, in spiked aqueous matrices. NA recovery efficiency in samples without inhibitors was similar for the three extraction kits. However, the Invitrogen kit was the only one that remained unaffected in the presence of inhibitors in the samples.

Towards a rational strategy for monitoring of microbiological quality of ambient waters.

Water is one of the main sources of human exposure to microbiological hazards. Although legislation establishes regulatory standards in terms of fecal indicator bacteria to assess the microbiological quality of water, these do not necessarily predict the presence of pathogens such as parasites and viruses. Better surveillance and management strategies are needed to assess the risk of pathogens' waterborne transmission. We established a baseline dataset to characterize river water quality, identify changes over time, and design a rational monitoring strategy. Data from a year-long monthly monitoring campaign of the polluted Arenales River (Argentina), were analyzed to statistically correlate physicochemical and microbiological variables, the seasonal and longitudinal variations of the water quality and determine the similarity between study sites. The measured variables (sixteen) reflected the deterioration in the river quality through the city. Different viruses and parasites found did not correlate with the concentration of total and thermotolerant coliforms. There was significant seasonal variation for temperature, turbidity, conductivity, dissolved oxygen, enterococci, and norovirus. Strong correlations between some variables were found; we selected eight variables (dissolved oxygen, conductivity, turbidity, total and thermotolerant coliforms, Enterococcus, and adenovirus and Microsporidium as viral and parasitological indicators, respectively) for future monitoring. There was similarity between the monitoring locations, which were grouped into four clusters validated by cophenetic correlation and supported by discriminant analysis. This allowed us to reduce the number of sites, from eleven down to five. Sixty seven percent of the total variance and the correlation structure between variables were explained using five principal components. All these analyses led to a new long-term systematic monitoring scheme. A rational monitoring strategy based on the selection of the most suitable monitoring points and of the most significant variables to measure, will result in optimal use of the limited resources available to adequately protect the public and environmental health.