Environmental surveillance of Cryptosporidium and Giardia in surface supply water and treated sewage intended for reuse from an urban area in Brazil.

Environmental monitoring of protozoa, with the potential to trigger diseases, is essential for decision-making by managing authorities and for the control of water surveillance. This study aimed to detect and quantify Cryptosporidium oocysts and Giardia cysts in surface water for drinking water supply and treated sewage for reuse in the city of São Paulo. Samples collected bimonthly for one year were concentrated using the USEPA 1623.1 and 1693 methods for surface water and treated effluents, respectively. Immunofluorescence and nucleic acid amplification techniques were used to detect and quantify (oo)cysts. The cloning technique followed by sequencing and phylogenetic analyses were performed to characterize species and genotypes. The immunofluorescence detected Cryptosporidium spp. and Giardia spp. in 69.2% (9/13) and 100% (13/13) of the surface water samples (0.1-41 oocysts/L and 7.2-354 cysts/L, respectively). In the reuse samples, 85.7% (12/14) were positive for both protozoa and the concentrations varied from 0.4 to 100.6 oocysts/L and 1.2 and 93.5 cysts/L. qPCR assays showed that 100% of surface water (0.1-14.6 oocysts/L and 0.3-639.8 cysts/L) and reused samples (0.1-26.6 oocysts/L and 0.3-92.5 cysts/L) were positive for both protozoa. Species C. parvum, C. hominis, and C. muris were identified using the 18S rRNA gene, demonstrating anthroponotic and zoonotic species in the samples. Multilocus SSU rRNAanalyses of the SSU rRNA, tpi, and gdh genes from Giardia intestinalis identified the AII, BII, and BIV assemblages, revealing that contamination in the different matrices comes from human isolates. The study showed the circulation of these protozoa in the São Paulo city area and the impairment of surface water supply in metropolitan regions impacted by the discharge of untreated or inadequately treated sewage regarding the removal of protozoa, emphasizing the need to implement policies for water safety, to prevent the spread of these protozoa in the population.

Spread and persistence of antimicrobial resistance genes in wastewater from human and animal sources in São Paulo, Brazil.

The spread of antimicrobial resistance (AMR) through multiple reservoirs is a global concern. Wastewater is a critical AMR dissemination source, so this study aimed to assess the persistence of resistance genetic markers in wastewater using a culture-independent approach. Raw and treated wastewater samples (n = 121) from a wastewater treatment plant (WWTP), a human hospital, a veterinary hospital, and a pig farm were monthly collected and concentrated by filtration. DNA was extracted directly from filter membranes, and PCR was used in the qualitative search of 32 antimicrobial resistance genes (ARGs). Selected genes (blaCTX-M, blaKPC, qnrB, and mcr-1) were enumerated by quantitative real-time PCR (qPCR). Twenty-six ARGs were detected in the qualitative ARGs search, while quantitative data showed a low variation of the ARG's relative abundance (RA) throughout the months, especially at the human hospital and the WWTP. At the WWTP, despite significantly reducing the absolute number of gene copies/L after each treatment stage (p < 0.05), slight increases (p > 0.05) in the RAs of genes blaCTX-M, qnrB, and mcr-1 were observed in reused water (tertiary treatment) when compared with secondary effluent. Although the increase is not statistically significant, it is worth noting that there was some level of ARGs concentration after the disinfection process. No significant absolute or relative after-treatment quantification reductions were observed for any ARGs at the veterinary hospital or the pig farm. The spread of ARGs through sewage needs to be continuously addressed, because their release into natural environments may pose potential risks of exposure to resistant bacteria and impact local ecosystems.

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.

Occurrence of caffeine, fluoxetine, bezafibrate and levothyroxine in surface freshwater of São Paulo State (Brazil) and risk assessment for aquatic life protection.

The prioritization of active pharmaceutical ingredients (APIs) for monitoring programmes and/or environmental risk assessment (ERA) purposes is based on several criteria, including environmental occurrence data. However, data on API occurrence in Brazilian surface freshwaters are still scarce. The Brazilian Unified Health System (SUS) provides several medicines free-of-charge, including medications that have bezafibrate, fluoxetine and levothyroxine as the API. Thus, our objective was to investigate the occurrence of bezafibrate, fluoxetine and levothyroxine in samples collected at sampling sites included in the surface freshwater monitoring program of the São Paulo State Environmental Agency (CETESB); caffeine was also included in the analysis because it is commonly used as an anthropogenic marker of aquatic environment contamination. Monitoring results showed that levothyroxine was not found in any of the analysed samples. Caffeine was ubiquitous in the analysed samples, thus indicating anthropic contamination in the studied water bodies. Caffeine and bezafibrate presented risk quotient (RQ) < 1 for all the sampling sites and periods evaluated in this study. For fluoxetine, RQs > 1 were found in all water samples in which this API was found, indicating a potential risk for freshwater pelagic biota. Thus, fluoxetine should be regulated in São Paulo State in order to protect the aquatic biota. Additional occurrence studies in other Brazilian states are still needed to evaluate if fluoxetine is a nationwide pollutant.