Wastewater-based epidemiology: A Brazilian SARS-COV-2 surveillance experience.

Since 2020, developed countries have rapidly shared both publicly and academically relevant wastewater surveillance information. Data on SARS-CoV-2 circulation is pivotal for guiding public health policies and improving the COVID-19 pandemic response. Conversely, low- and middle-income countries, such as Latin America and the Caribbean, showed timid activities in the Wastewater-Based Epidemiology (WBE) context. In these countries, isolated groups perform viral wastewater monitoring, and the data are unevenly shared or accessible to health agencies and the scientific community. This manuscript aims to highlight the relevance of a multiparty effort involving research, public health, and governmental agencies to support usage of WBE methodology to its full potential during the COVID-19 pandemic as part of a joint One Health surveillance approach. Thus, in this study, we explored the results obtained from wastewater surveillance in different regions of Brazil as a part of the COVID-19 Wastewater Monitoring Network ANA (National Water Agency), MCTI (Ministry of Science, Technology, and Innovations) and MS (Ministry of Health). Over the epidemiological weeks of 2021 and early 2022, viral RNA concentrations in wastewater followed epidemiological trends and variations. The highest viral loads in wastewater samples were detected during the second Brazilian wave of COVID-19. Corroborating international reports, our experience demonstrated usefulness of the WBE approach in viral surveillance. Wastewater surveillance allows hotspot identification, and therefore, early public health interventions. In addition, this methodology allows tracking of asymptomatic and oligosymptomatic individuals, who are generally underreported, especially in emerging countries with limited clinical testing capacity. Therefore, WBE undoubtedly contributes to improving public health responses in the context of this pandemic, as well as other sanitary emergencies.

Sampling strategies for wastewater surveillance: Evaluating the variability of SARS-COV-2 RNA concentration in composite and grab samples.

The shedding of SARS-CoV-2 RNA titers by infected individuals, even asymptomatic and oligosymptomatic ones, allows the use of wastewater monitoring to track the COVID-19 spread in a community. This approach is interesting especially for emerging countries with limited clinical testing capabilities. However, there are still important methodological aspects that need validation so that wastewater monitoring data become more representative and useful for public health. This study evaluated the between-day and within-day variability of SARS-CoV-2 RNA concentrations in 24-hour composite and grab samples from three different sampling points, including two wastewater treatment plants (WTTP) and a sewer manhole. In the between-day evaluation (17 weeks of monitoring), a good agreement between the SARS-CoV-2 RNA concentration of each sampling method was observed. There were no significant differences between the mean concentrations of the grab and composite samples (p-value > 0.05), considering N1 and N2 gene assays. The strong relationship between composite and grab samples was proven by correlation coefficients: Pearson's r of 0.83 and Spearman's rho of 0.78 (p-value < 0.05). In within-day evaluation, 24-hour cycles were analyzed and low variability in hourly viral concentrations was observed for three sampling points. The coefficient of variation (CV) values ranged from 3.0% to 11.5%. Overall, 24-hour profiles showed that viral RNA concentrations had less variability and greater agreement with the mean values between 8 a.m. and 10 a.m, the recommended time for grab sampling. Therefore, this study provides important information on wastewater sampling techniques for COVID-19 surveillance. Wastewater monitoring information will only be useful to public health and decision-makers if we ensure data quality through best practices.

First case of SARS-CoV-2 RNA detection in municipal solid waste leachate from Brazil.

This work presents the first case of SARS-CoV-2 RNA detection in leachate collected from a transfer station in the city of São Paulo, Brazil. After calibration of the viral detection method already used for wastewater samples with a pilot leachate sample and virus fragments in laboratory, twelve polyethylene glycol concentrated leachates samples were tested by RT-qPCR. The results confirmed the presence of N1 gene in 9 of the 12 analyzed samples between epidemiological weeks 33 and 38 of the year 2021 (08/15/2021 to 09/19/2021). The occurrence of the N2 gene was only observed in 5 of the 12 samples. The concentration values for N1 and N2 genes varied between 3.1 and 4.6 log10.GC·L-1, which are values close to those measured in sanitary wastewater. This method showed to be a promising procedure to verify the presence of viral RNA in municipal solid waste leachate, being especially useful where there is no treatment system and sanitation infrastructure, which makes the conventional wastewater surveillance unfeasible.

Alternative SARS-CoV-2 detection protocol from self-collected saliva for mass diagnosis and epidemiological studies in low-incoming regions.

Until mass vaccination befalls, control of the new betacoronavirus-associated severe acute respiratory syndrome pandemic (SARS-CoV-2) is based on decreasing virus circulation by social distancing and blocking transmission foci after diagnosis. Globally adopted SARS-CoV-2 diagnostic criteria embrace viral RNA detection by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) on nasopharynx secretions, which requires healthcare facilities and specialized personnel for sample collection. To develop an alternative protocol, hydrophilic cotton as the material and saliva as the source for biological sample collection in qRT-PCR/RT-endpoint-PCR SARS-CoV-2 diagnostic methods prepared with local consumables were evaluated using 99 archived nasopharynx samples previously diagnosed as positive for SARS-CoV-2 and 111 prospective saliva samples pared with nasopharynx samples from patients attending the local reference ABC Medical School diagnostic laboratory. The kappa agreement coefficient between the SARS-CoV-2 qRT-PCR and RT-endpoint-PCR was k = 0.97 (95 % CI 0.92-1.00) and k = 0.90 (95 % CI 0.81-0.99), respectively, on SARS-CoV-2-positive archived samples, with the initial qRT-PCR CT under 25. The agreement coefficient of the SARS-CoV-2 alternative saliva diagnostic protocol, when used to test the paired nasopharynx samples, was k = 0.79 (95 % CI 0.56-1,00). These data support that the SARS-CoV-2 diagnostic assay based on self-collected saliva on cotton represents an alternative protocol for mass diagnosis and epidemiological studies in low-income regions.

Long-term monitoring of SARS-COV-2 RNA in wastewater in Brazil: A more responsive and economical approach.

SARS-CoV-2, the novel Coronavirus, was first detected in Wuhan, China, in December 2019, and has since spread rapidly, causing millions of deaths worldwide. As in most countries of the world, in Brazil, the consequences of the COVID-19 pandemic have been catastrophic. Several studies have reported the fecal shedding of SARS-CoV-2 RNA titers from infected symptomatic and asymptomatic individuals. Therefore, the quantification of SARS-CoV-2 in wastewater can be used to track the virus spread in a population. In this study, samples of untreated wastewater were collected for 44 weeks at five sampling sites in the ABC Region (São Paulo, Brazil), in order to evaluate the SARS-CoV-2 occurrence in the sewerage system. SARS-CoV-2 RNA titers were detected throughout the period, and the concentration ranged from 2.7 to 7.7 log10 genome copies.L-1, with peaks in the last weeks of monitoring. Furthermore, we observed a positive correlation between the viral load in wastewater and the epidemiological/clinical data, with the former preceding the latter by approximately two weeks. The COVID-19 prevalence for each sampling site was estimated via Monte-Carlo simulation using the wastewater viral load. The mean predicted prevalence ranged 0.05 to 0.38%, slightly higher than reported (0.016 ± 0.005%) in the ABC Region for the same period. These results highlight the viability of the wastewater surveillance for COVID-19 infection monitoring in the largest urban agglomeration in South America. This approach can be especially useful for health agencies and public decision-makers in predicting SARS-CoV-2 outbreaks, as well as in local tracing of infection clusters.

Structural modelling and thermostability of a serine protease inhibitor belonging to the Kunitz-BPTI family from the Rhipicephalus microplus tick.

rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential. Besides that, rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of rBmTI-A was modeled. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism spectroscopy data corroborated the secondary structure found by the homology modelling. Also, in circular dichroism data it was shown a thermostability of rBmTI-A until approximately 70 °C, corroborated by inhibitory assays toward trypsin.

Neutrophil elastase inhibitor purification strategy from cowpea seeds.

Serine proteases and its inhibitors are involved in physiological process and its deregulation lead to various diseases like Chronic Obstructive Pulmonary Disease (COPD), pulmonary emphysema, skin diseases, atherosclerosis, coagulation diseases, cancer, inflammatory diseases, neuronal disorders and other diseases. Serine protease inhibitors have been described in many species, as well as in plants, including cowpea beans (Vigna unguiculata (L.) Walp). Here, we purified and characterized a protease inhibitor, named VuEI (Vigna unguiculata elastase inhibitor), from Vigna unguiculata, with inhibitory activity against HNE (human neutrophil elastase) and chymotrypsin but has no inhibitory activity against trypsin and thrombin. VuEI was obtained by alkaline protein extraction followed by three different chromatographic steps in sequence. First, an ion exchange chromatography using Hitrap Q column was employed, followed by two reversed-phase chromatography using Source15RPC and ACE18 columns. The molecular mass of VuEI was estimated in 10.99 kDa by MALDI-TOF mass spectrometry. The dissociation constant (Ki) to HNE was 9 pM. These data indicate that VuEI is a potent inhibitor of human neutrophil elastase, besides to inhibit chymotrypsin.

rBmTI-6 attenuates pathophysiological and inflammatory parameters of induced emphysema in mice.

Protease/anti-protease imbalance is the main pathogenic mechanism of emphysema and protease inhibitors have been recognized as potential molecules to treat the disease conditions. In this work the rBmTI-6 first domain (rBmTI-6-D1), a recombinant Kunitz-type serine proteinase inhibitor, was used to verify its effect in prevention or minimization of PPE-induced emphysema in mice. C57BL/6 mice were submitted to a PPE-induced emphysema model and treated with rBmTI-6-D1 before the emphysema development. We showed that the rBmTI-6-D1 treatment was sufficient to avoid the loss of elastic recoil, an effective decrease in alveolar enlargement and in the number of macrophages and lymphocytes in bronchoalveolar lavage fluid. Proteolytic analysis showed a significant increase in elastase activity in PPE-VE (induced emphysema) group that is controlled by rBmTI-6-D1. Kallikrein activity was decreased in the PPE-rBmTI6 (induced emphysema and inhibitor treated) group when compared to PPE-VE group. Although rBmTI-6-D1, did not present a neutrophil elastase (NE) inhibitory activity, the results show that the inhibitor interfered in the pathway of NE secretion in PPE-emphysema mice model. The role of rBmTI-6-D1 in the prevention of emphysema development in the mice model, apparently, is related with a control of inflammatory response due the trypsin/kallikrein inhibitory activity of rBmTI-6-D1.