key: cord-0981927-e825p3sg authors: Padilla-Reyes, Diego A.; Álvarez, Mario Moises; Mora, Abrahan; Cervantes-Avilés, Pabel A.; Kumar, Manish; Loge, Frank J.; Mahlknecht, Jürgen title: Acquired insights from the long-term surveillance of SARS-CoV-2 RNA for COVID-19 monitoring: The case of Monterrey Metropolitan Area (Mexico) date: 2022-02-18 journal: Environ Res DOI: 10.1016/j.envres.2022.112967 sha: 09d59c7ec1a7a3d93ef72e0750d0cdc2bd2e723c doc_id: 981927 cord_uid: e825p3sg Wastewater-based epidemiology offers a time- and cost-effective way to monitor SARS-CoV-2 spread in communities and therefore represents a complement to clinical testing. WBE applicability has been demonstrated in a number of cases over short-term periods as a method for tracking the prevalence of SARS-CoV-2 and an early-warning tool for predicting outbreaks in the population. This study reports SARS-CoV-2 viral loads from wastewater treatment plants (WWTPs) and hospitals over a 6-month period (June to December 2020). Results show that the overall range of viral load in positive tested samples was between 1.2 × 10(3) and 3.5 × 10(6) gene copies/L, unveiling that secondary-treated wastewaters mirrored the viral load of influents. The interpretation suggests that the viral titers found in three out of four WWTPs were associated to clinical COVID-19 surveillance indicators preceding 2–7 days the rise of reported clinical cases. The median wastewater detection rate of SARS-CoV-2 was one out of 14,300 reported new cases. Preliminary model estimates of prevalence ranged from 0.02 to 4.6% for the studied period. This comprehensive statistical and epidemiological analysis demonstrates that the applied wastewater-based approach to COVID-19 surveillance is in general consistent and feasible, although there is room for improvements. 119 120 Table 1 summarizes the characteristics of the four main wastewater treatment facilities 121 in the MMA. The treatment process for Dulces Nombres and Norte WWTPs included 122 pretreatment, primary treatment, secondary treatment using activated sludge and disinfection 123 using chloride. Primary treatment is not included in the Noreste and Cadereyta WWTPs. 124 In response to the sudden surge of severe COVID-19 cases during March 2020, the state 128 health authority increased the physical capacity by converting five hospitals into COVID-19 129 hospitals (Gobierno de Nuevo Leon, 2020). The most relevant of these were Hospital General 130 de Zona 4 (HGZ 4). Hospital General de Zona 6 (HGZ 6), Hospital Metropolitano (HM), and 131 g; 400 μl of the centrifuge supernatant were discarded. The remaining 100 μl was added to 20 190 equivalent gene copies per milliliter (gc/ml). For this purpose, we used commercial synthetic 213 genetic material that contained the complete N gene from SARS-CoV-2 (Integrated DNA 214 Technologies, Iowa, USA). Samples containing different concentrations of synthetic nucleic 215 acids of SARS-CoV-2, in the range of 10 2 to 10 5 genome copies ml −1 were prepared by successive 216 dilutions from stocks. This plasmid has been used before as a positive control in amplification 217 assays of SARS-CoV-2 genetic material (González-González et al., 2020). The estimated lower 218 limit of detection was ~1 copy/reaction of the N gene of SARS-CoV-2 per ml of water. 219 The prevalence of SARS-CoV-2 infection within a catchment was estimated by employing a 237 mass balance approach using the total number of viral RNA copies in wastewater for a given 238 day, as measured in wastewater by RT-qPCR, and the number of SARS-CoV-2 RNA copies shed 239 in stool by an infected individual (Ahmed et al., 2020a) (Eq.1): 240 The viral load in wastewater as gene copies per liter (gc/L) was determined by the RT-242 qPCR analyses performed on each sample. The daily wastewater flow rate (L/day) was 243 obtained by direct flow readings at each WWTP (SADM, 2021). The daily stool production (g 244 stool/person-day) was simulated as a normal distribution with a mean of 149 and standard 245 deviation of 95, with data reported for high-income countries (Rose et al., 2015) . Monterrey is 246 one of the wealthiest cities in Mexico qualifying as a high-income area. Finally, the viral load in 247 stool (gc/g of feces) in log10 was modeled as a log-normal distribution from 5. 79 Oracle©) Excel add-on. The Monte Carlo simulation is an iterative mathematical technique that 251 is used to approximate the likelihood of outcomes by running thousands of "trial" scenarios. 252 The technique numerically quantifies and graphically depicts potential results (along with their 253 associated likelihoods) based on provided uncertain inputs. Temporal dynamics of SARS-CoV-2 in wastewater 256 We collected and processed 422 wastewater samples from four municipal WWTPs and 257 159 effluent samples from four hospitals. In June, an average of 19 hospitalized COVID patients per day were reported, and viral 302 loads on the order of 10 5 gc/L were detected. During July and August, the number of patients 303 decreased to an average of 12 per day and viral loads in the wastewater also showed a decrease, 304 being on the order of 10 3 -10 4 gc/L. In September, viral loads increased to 10 4 -10 5 gc/L, while 305 the daily average number of hospitalized patients was 15. In October, there was an increase in 306 the number of patients in the hospitals, averaging 20, and in this month the highest viral load 307 was reached, which was on the order of 10 5 -10 6 gc/L. During November, the average number 308 of hospitalized patients remained at 20, while the viral loads decreased to 10 4 -10 5 gc/L. Finally, 309 during December, the daily average number of patients were 25 and the viral loads were in the 310 order of 10 3 -10 4 gc/L. 311 It is possible to investigate the detection rate and accuracy of wastewater surveillance by 313 comparing wastewater detection data to clinically reported case counts from the health 314 authority (Wu et al., 2021) . The reported daily incidence of COVID-19 cases is used to calculate 315 the percentage of positive wastewater samples for different incidence rates. The detection of 316 virus titers increases exponentially with clinical incidence rate. At a detection rate of 80%, the 317 clinical incidence lies on the order of 8 cases per 100,000 people. For all positive wastewater 318 samples at the metropolitan level, the incidence rates of daily new cases ranged from 3.6 to 9.9 319 cases per 100,000 people (median 7.0). This means that the surveillance was capable to detect 320 SARS-COV-2 for one new reported case out of ~14,300 people. It must be noted that this 321 number does not consider unreported infections. 322 A weak positive correlation was found between the incidence of daily new cases at the 323 metropolitan level, and the wastewater viral titers at the sewershed level (Fig. 5a) . The 324 catchment size may influence the probability of SARS-CoV-2 detection in wastewater samples. 325 2.1x10-6.7x10 3 (see also Table 5) . 351 The detection of SARS-CoV-2 titers in treated wastewaters from WWTPs before and after 352 disinfection of MMA were directly related to the viral loads in the influents, especially when the 353 influents showed a concentration equal to or higher than 10 4 gc/L. The viral loads found in 354 treated wastewater remain on the same order of magnitude as those found in the influents 355 is no longer considered an active case. 512 Table 4 shows a selection of recent studies which attempted to calculate the prevalence 513 of COVID-19 cases based on the viral loads in wastewater according to a mass balance approach. 514 Similar to this study, the results show a wide variation in the number of infected persons. 515 While WBE has proven to be a valuable tool for health monitoring at population-level, 573 one of the main challenges to implement this method for COVID-19 surveillance, is the 574 establishment of standardized methods and procedures for a successful extraction of viral 575 material from wastewater, as well as its analysis and concentration in the laboratory 576 (Saththasivam et al., 2021) . This standardization will also improve comparability between 577 studies. In addition, the emergence of new SARS-CoV-2 variants in the early 2021 represents 578 an opportunity to improve COVID-19 surveillance. One of the burning questions is whether the 579 measurement of variants and its frequency through sequencing will provide a tool to check the 580 precision of environmental surveillance of COVID-19. 581  For all days with new clinical cases, only 34% were detected in wastewater. This is probably 597 due to the sampling strategy applied to the case. However, the median wastewater 598 detection rate of SARS-CoV-2 is one out of 14,300 new reported cases. 599  Simultaneous measurements of viral concentrations in influents and treated wastewater in 600 WWTPs were found similar, probably owing to a low effectivity of treatment processes or 601 differences in the sampling procedure from authorities. In any case, the viral loads in 602 receiving rivers were lower by three orders of magnitude. 603  The COVID-19 point prevalence according to WBE ranged from 0.02 to 4.6%. Model 604 estimates of infected individuals are highly capricious and significantly higher than active 605 cases reported by authorities due to combination of shortcomings in clinical testing and 606 large uncertainties in the model parameter estimation. 607  Although the WBE is highly time-and cost-effective for COVID-19 surveillance of 608 communities in comparison to clinical testing, there is still a lack of reproducibility and 609 repeatability across studies. In other words, there is still room for improving the 610 understanding and standardization of best practices including tracking of variants. 611 and inspiration. 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