One-year surveillance of SARS-CoV-2 in wastewater from vulnerable urban communities in metropolitan São Paulo, Brazil.

The current COVID-19 pandemic has emphasized the vulnerability of communities living in the urban outskirts and informal settlements. The lack of reliable COVID-19 case data highlights the importance and application of wastewater-based epidemiology. This study aimed to monitor the COVID-19 trends in four vulnerable urban communities (slums and low-income neighborhoods) in metropolitan São Paulo by assessing the SARS-CoV-2 RNA viral load in wastewater. We analyzed 160 samples from May 2020 to June 2021 with weekly or fortnightly samplings. The samples were ultracentrifuged with glycine elution and quantified by N1/N2 SARS-CoV-2 RT-qPCR. The results of positivity were 100% (Paraisópolis, Heliópolis and Cidade Tiradentes) and 76.9% (Vila Brasilândia). The new case numbers of COVID-19, counted from the onset of symptoms, positively correlated with SARS-CoV-2 N1 viral loads from the two largest communities (p<0.001). SARS-CoV-2 infectivity was tested in Vero E6 cells after concentration with the two techniques, ultrafiltration (Centricon® Plus-70 10 kDa) and sucrose cushion ultracentrifugation, but none of the evaluated samples presented positive results. Next-generation sequencing (NGS) analysis from samples collected in March and August 2021 revealed the presence of the clade 20 J (lineage P.1) belonging to the most prevalent circulating variant in the country. Our results showed that wastewater surveillance data can be used as complementary indicators to monitor the dynamics and temporal trends of COVID-19. The infectivity test results strengthened the evidence of low risk of infection associated with SARS-CoV-2 in wastewater.

Hepatitis A Outbreaks and Environmental Circulation of Genotype IA Strains in the São Paulo City, 2017-2018.

Hepatitis A virus (HAV) is the major cause of enterically transmitted infectious hepatitis. Between 2016 and 2017, the number of confirmed cases of hepatitis A virus (HAV) increased from 64 to 786 in São Paulo affecting mainly adults aged between 18 and 39 years (80%) and males (88%). To support epidemiological surveillance, the present study monitored the presence of HAV in urban sewage samples collected bimonthly for 1 year (November 2017-November 2018) in the central region of the city, where most of cases were detected. Sewage samples were concentrated by polyethylene glycol precipitation and HAV RNA was quantified by RT-qPCR. Nucleotide sequencing targeting the VP1/2A junction region was carried out to genotype the HAV strains. HAV was detected in 76.9% (40/52) of the samples, with a geometric mean viral load of 5.09 × 104 (± SD 4.51 × 105) genome copies (GC/L) (Mauá Street) and 5.27 × 104 (± SD 1.26 × 106) GC/L (Prestes Maia Avenue). Of the 40 positive samples, 8 were typed as HAV subgenotype IA [100% nucleotide (nt) identity with HAV strain VRD_521_2016]. Highest homology was obtained with sequences from European countries (Italy, Spain) and Israel, all of which had reported recent HAV outbreaks associated with men who have sex with men. Our results highlight that wastewater surveillance is a useful tool to support investigating HAV outbreaks in the community, including circulating genotypes.

SARS-CoV-2 in a stream running through an underprivileged, underserved, urban settlement in São Paulo, Brazil: A 7-month follow-up.

COVID-19 pandemic has led to concerns on the circulation of SARS-CoV-2 in the environment, its infectivity from the environment and, the relevance of transmission via environmental compartments. During 31 weeks, water samples were collected from a heavily contaminated stream going through an urban, underprivileged community without sewage collection. Our results showed a statistically significant correlation between cases of COVID-19 and SARS in the community, and SARS-CoV-2 concentrations in the water. Based on the model, if the concentrations of SARS-CoV-RNA (N1 and N2 target regions) increase 10 times, there is an expected increase of 104% [95%CI: (62-157%)] and 92% [95%CI: (51-143%)], respectively, in the number of cases of COVID-19 and SARS. We believe that differences in concentration of the virus in the environment reflect the epidemiological status in the community, which may be important information for surveillance and controlling dissemination in areas with vulnerable populations and poor sanitation. None of the samples were found infectious based cultures. Our results may be applicable globally as similar communities exist worldwide.

Performance of wastewater reclamation systems in enteric virus removal.

Analysis of virus removal by tertiary or advanced sewage treatment processes is an emerging topic due to importance of reusing water on a global level. This study aimed to monitor important human viral pathogens: the human adenovirus (HAdV), JC polyomavirus (JCV) and Species A rotaviruses (RVA) in urban sewage, secondary effluents and reclaimed water from metropolitan São Paulo (MSP), Brazil. Four large wastewater treatment plants (WWTPs) in MSP were sampled monthly during a one-year period (April 2015 to March 2016). The viruses were quantified by quantitative PCR (qPCR), and HAdV viability was tested by the integrated cell culture (ICC)-qPCR assay. WWTPs are composed of activated sludge processes and different tertiary treatments (coagulation/sedimentation, sand-anthracite filters, membrane bioreactors (MBRs)/reverse osmosis (RO) and disinfection by chlorination). Physicochemical parameters were also evaluated to verify association with density of viruses detected in different treatment stages. HAdV, JCV and RVA were consistently detected (100%) in the sewage influent samples (range: 106-108 genome copies GC/L). In the secondary effluent, HAdV was detected in 100% (48/48) of the analysed samples, JCV in 85.4% and RVA in 97.9% (range: 104-107 GC/L for all viruses tested). HAdV was the most frequently detected virus in the tertiary effluent (62.2%) (28/45), exhibiting a viability between 0 and 44% of the tested samples in the wastewater reclamation systems. The MBR/RO systems demonstrated better virus removal efficiencies (range: 2.3-2.9 log10). Temperature, pH, turbidity and total organic carbon presented association with the viral density in the reclaimed water samples. Presence of viruses in treated effluents can indicates health risks depending on uses of recovery water. Further risk assessment studies should be conducted to better assess health risks under different exposure scenarios for water recovery in urban settings.

Noroviruses in raw sewage, secondary effluents and reclaimed water produced by sand-anthracite filters and membrane bioreactor/reverse osmosis system.

The importance of noroviruses (NoVs) in the epidemiology of waterborne diseases has increased globally in the last decades. The present study aimed to monitor genogroup I and II noroviruses in different treatment stages of four wastewater treatment plants (WWTPs) in the metropolitan São Paulo. WWTPs consist of secondary (activated sludge) and tertiary treatments (coagulation, sand-anthracite filters, membrane bioreactor (MBR)/reverse osmosis (RO) and chlorination). Raw sewage (500mL) and treated effluents (1L) were concentrated by celite and reclaimed water (40L) by hollow-fiber ultrafiltration system. Quantitative (qPCR) and nested PCR with nucleotide sequencing were used for quantification and molecular characterization. NoVs were widely distributed in raw wastewater samples (83.3%-100% NoV GI and 91.6%-100% NoV GII) and viral loads varied from 3.8 to 6.66log10gcL-1 for NoV GI and 3.8 to 7.3log10gcL-1 for NoV GII. Mean virus removal efficiencies obtained for activated sludge processes ranged from 0.3 to 0.8 log10 for NoV GI and 0.4 to 1.4 log10 for NoV GII. NoVs were not detected in the reuse water produced by MBR/RO system, while sand-anthracite filters resulted in a NoV GI and GII decay of 1.1-1.6 log10 and 0.7-1.6 log10, respectively. A variety of genotypes (GI.2, GI.3a, GI.3b, GI.5, GII.1, GII.4 Sydney 2012, GII.5, GII.6, GII.17) was observed, with a predominance of GI.2 and GII.17 in the different genogroups. These results corroborate with recent data about the entry and dissemination of the emerging genotype GII.P17-GII.17 Kawasaki 2014 in the country, and may indicate a change in the epidemiological patterns of norovirus strains circulation in this region. This is the first large-scale study to evaluate burden and genotypes of noroviruses in WWTPs in Brazil, providing a rapid diagnosis of viruses circulating in the population.

Distribution of human fecal marker GB-124 bacteriophages in urban sewage and reclaimed water of São Paulo city, Brazil.

Bacteriophages infecting Bacteroides fragilis GB-124 have been described as potential markers of human fecal contamination in water sources. The aim of this study was to evaluate the occurrence of GB-124 phages in raw sewage, secondary effluents and reclaimed water of the São Paulo city using a low-cost microbial source tracking method. Samples were collected monthly from April 2015 to March 2016 in four municipal wastewater treatment plants that operate with activated sludge processes followed by different tertiary treatments (sand-anthracite filtration, membrane bioreactor/reverse osmosis) and final chlorination. GB-124 phages were detected in 100% of the raw sewage samples, with viral loads varying from 7.5 × 103 to 1.32 × 106 PFU/L. Virus removal efficiency in activated sludge processes ranged from 1.89 to 2.31 log10. Frequencies of phage detection were lower in reclaimed water samples (0-22.2%). The results indicated that GB-124 phage could be a complementary low-cost viral marker for the detection of human fecal pollution in waters impacted with urban sewage in this region. However, the datasets of tertiary effluents resulted in several samples with concentrations below the detection limit (DL ≤1 PFU/mL) suggesting the need to obtain analytical methods with lower DL for greater accuracy of negative results.