Environmental Pollution 290 (2021) 118003

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

SARS-CoV-2 in a stream running through an underprivileged, underserved,

urban settlement in São Paulo, Brazil: A 7-month follow-up☆
Maria Tereza Pepe Razzolini a, b, *, Mikaela Renata Funada Barbosa b, c, Ronalda Silva de Araújo c,
Ivo Freitas de Oliveira c, Maria Cássia Mendes-Correa d, Ester C. Sabino d, Suzi Cristina Garcia c,
Anderson V. de Paula d, Lucy S. Villas-Boas d, Silvia Figueiredo Costa d, Milena Dropa a,
Denise Brandão de Assis e, Beatriz S. Levin f, Antonio Carlos Pedroso de Lima g, Anna S. Levin d
a
School of Public Health of Universidade de São Paulo, Brazil
b
NARA - Center for Research in Environmental Risk Assessment, Brazil
c
CETESB - Environmental Company of São Paulo State, Brazil
d
Department of Infectious Diseases and Instituto de Medicina Tropical, Faculdade de Medicina, Universidade de São Paulo, Brazil
e
Sao Paulo State Health Department, São Paulo, Brazil
f
Guttman Community College, City University of New York, New York, USA
g
Instituto de Matemática e Estatística, Universidade de São Paulo, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: COVID-19 pandemic has led to concerns on the circulation of SARS-CoV-2 in the environment, its infectivity from
Environmental monitoring the environment and, the relevance of transmission via environmental compartments. During 31 weeks, water
Contaminated water samples were collected from a heavily contaminated stream going through an urban, underprivileged community
SARS-CoV-2
without sewage collection. Our results showed a statistically significant correlation between cases of COVID-19
COVID-19
Virus infectivity
and SARS in the community, and SARS-CoV-2 concentrations in the water. Based on the model, if the concen­
trations 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.

1. Introduction
During the COVID-19 pandemic, questions have arisen about circu­
lation of SARS-CoV-2 in the environment and its relevance to the
transmission of the disease. The presence of the virus in sewage raises
the question of whether it is necessary to establish sanitary barriers in
order to control the pandemic.
Monitoring of community wastewater is an effective tool to track
diseases and to guide public health responses. Wastewater-based
epidemiology (WBE) has been demonstrated as a complementary
approach for spatial tracking of infectious diseases (Sims and
Horden-Kasprzyk, 2020; Randazzo et al., 2020), including coronavirus
disease 2019 cases (COVID-19), as well as early warning of the occur­
rence of infected populations (Medema et al., 2020; Prado et al., 2021).
There has been extensive discussion on the role of aerosol generated by
cough, talking and singing in the transmission of COVID-19. However,
environmental-generated aerosols may also have a role. Giacobbo et al.
(2021) state that aerosols generated from sewage and contaminated
waters deserve a deeper discussion on the possible fecal-nasal trans­
missions. Likewise, Usman et al. (2020) brought the issue of the po­
tential of the aerosols generated by contaminated waters could
potentially be a route of transmission of the virus if present. The same
was discussed by Al-Gheethi et al. (2020) whose literature review re­
veals that larger droplets can travel through aerosols at a short distance

This paper has been recommended for acceptance by Da Chen.

☆

* Corresponding author. Environmental Health, Department, School of Public Health, University of Sao Paulo, Avenida Doutor Arnaldo, 715, Cerqueira César, São
Paulo, SP, Brazil.
E-mail address: razzolini@usp.br (M.T. Pepe Razzolini).

https://doi.org/10.1016/j.envpol.2021.118003
Received 4 May 2021; Received in revised form 15 August 2021; Accepted 17 August 2021
Available online 19 August 2021
0269-7491/© 2021 Elsevier Ltd. All rights reserved.
M.T. Pepe Razzolini et al.
and, settle down very quickly on surfaces.
On January 31, 2020, the WHO declared the outbreak of COVID-19
to be a Public Health Emergency of International Concern (WHO, 2020).
In Brazil the first COVID-19 case was confirmed in São Paulo on 26
February 2020 (Jesus et al., 2020).
SARS-CoV-2 was reported in sewage samples (Medema et al., 2020;
Wu et al., 2020; Gonzalez et al., 2020) indicating that the virus circu­
lates in the environment. However, these findings do not mean there is
infectious virus in the environment. The finding of viable virus may pose
a threat to populations who live in areas with poor sanitation, with a
disproportionate exposure driven by inequality and social exclusion.
Due to the importance to establish control strategies pandemic,
SARS-CoV-2 viability has been investigating. Oliveira et al. (2021)
demonstrated SARS-Cov-2 persistence in river water and wastewater
with T90 values at 4 ◦ C was 7.7 and 5.5 days, respectively, higher than
observed for temperature at 24 ◦ C.
Like other Latin American countries, Brazil presented a rapid and
intense urbanization process, which outpaced the development of
infrastructure, such as basic services and housing. Cities struggle to
overcome deficiencies in sanitation, urban mobility, and adequate
housing. The extent of informal settlements and the spatial segregation
of low-income groups are demonstrated by the growth and proliferation
of precarious and informal settlements, known as favelas (UN Habitat,
2013). In 2010, according to the Brazilian Census (IBGE, 2010) 11.4
million people lived in favelas, corresponding to 7% of the urban
population.
Excretion of SARS-CoV-2 in the feces has been documented (Xiao
et al., 2020; Fumian et al., 2021). Fumian et al. (2021) reported the
presence of SARS-CoV-2 in stool samples from 10 patients, in which N1
was present in 100% of the samples with the viral load varying from 4.7
× 103 to 4.7 × 105 genomic copies/g of the stool, whilst N2 was present
in 50% ranging from 2.6 × 103 to 1.7 × 104 genomic copies/g of stool.
However, to date, it is not possible to assure that fecal-oral transmission
of SARS-CoV-2 is a relevant route. On the other hand, as mentioned
before SARS-CoV-2 can be detected in sewage of infected communities
during outbreaks and sometimes even before the first clinical cases are
confirmed (Prado et al., 2021; Gonzalez et al., 2020; Bowmick et al.,
Fig. 1. Location of São Remo Community, in the city of São Paulo, state of São Paulo, and Brazil (23◦ 56’.29′′ S-46◦ 74’’.62′′ W). (Source: Google Earth; March
15, 2021).
2
Environmental Pollution 290 (2021) 118003
2020). Little research has been carried out on streams and rivers that run
through areas in which sanitation is poor, and may provide estimations
of the frequency of COVID-19 in a population. Guerrero-Latorre et al.
(2020) conducted a study in Quito (Ecuador) that evaluated the levels of
SARS-CoV-2 in three urban rivers and found significant concentrations
of the virus in these waters.
This study aimed to evaluate the presence of SARS-CoV-2 in a stream
that runs through a favela (slum) over a period of 7 months, and to
investigate whether it correlates with the occurrence of clinical cases in
this community.
2. Methods
2.1. Setting
The study was carried out in “São Remo Community ’’ located in
western region of São Paulo city (Fig. 1). According to a census pub­
lished in July 2019 the estimated population in the São Remo commu­
nity is approximately 8000 inhabitants (https://jornal.usp.br/
universidade/acoes-para-comunidade/censo-coleta-dados-sobre-comu­
nidades-proximas-a-usp/#:~:text=Segundo%20o%20Censo%20do%
20IBGE,Vila%20Guaraciaba%2C%20cerca%20de%20500). The entire
population of São Remo has free primary health care managed by the
municipality.
This community is connected to a water supply network and all
homes receive treated potable water. However, there is no sewer system.
All wastewater generated in São Remo is released into a stream known
as “Riacho Doce”, which runs through the community. Ultimately, the
water from Riacho Doce ends up untreated in a main river of the São
Paulo city, called Rio Pinheiros.
2.2. Water sampling
One liter water samples (n = 31) were collected at 8 a.m. always on
Mondays (from May to November 2020) at the same location of “Riacho
Doce” in the community (Fig. 2). They were collected according to the
Standard Methods for Examination of Water and Wastewater (APHA,
M.T. Pepe Razzolini et al.
Fig. 2. Riacho Doce stream, São Remo Community, São Paulo, Brazil. A: before entering the community; B: in the community. (Photography: Yuval Co).
2017). Samples were transported to the Laboratory of the Division for
Microbiology and Parasitology of CETESB, under refrigeration (>0 ◦ C to
8 ◦ C) and analyzed within a maximum period of 24 h.
2.3. SARS CoV-2 detection and quantification by RT-qPCR
2.3.1. Ultracentrifugation
Briefly, 40 mL of sample was ultracentrifuged (110,000 g for 1 h at
4 ◦ C) to pellet all the viral particles together with any suspended ma­
terial. The sediment was resuspended in 4 mL of 0.25 N glycine buffer,
pH 9.5, on ice for 30 min. After the pellet elution, 4 mL of PBS 2x were
added and the suspended solids were removed by centrifugation (3000 g
for 15 min). The supernatant was transferred to pre-weight tubes and
ultracentrifuged at 110,000 g for 1 h at 4 ◦ C.Then the supernatant was
carefully discarded, and the pellet was eluted with the remaining vol­
ume of the supernatant solution (approximately 300 μL) before the RNA
extraction procedure. To estimate the final sample volume, the tubes
were weighed again and the difference between the weights was
calculated.
Method recovery was evaluated for each sample, with the addition of
105-106 gene copies equivalents of Bovine Coronavirus (BCoV) to the
water sample. After 1 h of incubation at 4 ◦ C, the samples were ultra­
centrifuged according to the method described in the item 2.3.1.1.
2.3.2. Ultrafiltration - Centricon® Plus-70
Water samples submitted to the SARS-CoV-2 infectivity assay in cell
culture were concentrated using the ultrafiltration method with Cen­
tricon® Plus-70 10 kDa (Millipore, USA). This procedure was carried out
until August 31, while the concentrations of SARS-CoV-2 were in the
order of 104 CG/L. The method consists of filtering approximately 100
mL of sample through a 0.22 μm PES filter (Millipore). Then, 60 mL of
the filtered sample was centrifuged at 3000 g for 20 min. The procedure
was repeated until the volume of the concentrated was reduced to
approximately 1 mL. The filter was inverted, and the concentrate was
recovered after centrifugation (1000 g for 2 min) and treated with 10 μL
of 10 mg/mL gentamicin.
2.3.3. RNA extraction and RT-qPCR
RNA was extracted using QIAamp® Viral RNA Mini Kit (QIAgen,
Germany) using 140 μL of sample. The final volume of extracted RNA
was approximately 80 μL.
Standard quantification curves for N1 and N2 SARS-CoV-2 regions
were constructed using serial dilutions of the 2019-nCoV_N_Positive
Control kit (IDT, IA, USA), which consists in 2 × 104 to 2 copies/μL of
3
Environmental Pollution 290 (2021) 118003
a plasmid cloned with the N1 and N2 sequences of SARS-CoV-2. Primers
and probes were also based on CDC protocol (https://www.cdc.gov/
coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). RT-
qPCR assays for SARS-CoV-2 were performed in 20 μL reaction vol­
ume containing 5 μL of TaqPath™ 1-Step RT-qPCR Master Mix, CG
(ThermoFisher, MA, USA), 1.5 μL of 2019-nCoV_N1 (N1) primer–probe
or 2019-nCoV_N2 (N2) primer–probe set (500 nM of primers and 125
nM of probe) (IDT, IA, USA) and 0.5 μL of the extracted RNA. To assess
the presence of PCR inhibitors in the RNA samples, an internal positive
control (0.8 μL of VetMAX Xeno Internal positive Control – VIC Assay
ThermoFisher; 0.1 μL of 10,000 copies/μL of VetMAX Xeno Internal
Positive Control RNA Thermo Fisher) was procedure in a duplex reaction
with N1 target gene.
All reactions were performed in duplicate. A StepOne Plus (ABI, CA,
USA) instrument was used for the RT-qPCR with the thermal cycling
conditions as follows: initial incubation at 25 ◦ C for 10 min and initial
denaturation at 95 ◦ C for 30 s, followed by 45 cycles od denaturation at
95 ◦ C for 5 s and primer annealing and extension reaction at 60 ◦ C for 30
s. A sample was considered positive if the cycle threshold (CT) was below
40. The detection limit was determined to be around 1000 genomic
copies/L. Results were expressed as copies of SARS-CoV-2 RNA (N1 or
N2) per liter of water sample.
BCoV RT-qPCR reactions were performed using same conditions as
described above with primers and probes described by Decaro et al.
(2008). BCoV titers were estimated using standard curves prepared by
five 10-fold dilutions (2.5 × 105 to 2.5 copies/μL) of 183 bp DNA
fragment containing the targeted BCoV (GeneArt, Invitrogen, Thermo
Fisher Scientific). To calculate the recovery efficiency of the total
method, the number of RNA copies detected in 40 mL of water sample
were divided by the number of RNA copies spiked and the results were
expressed as a percentage. The mean recovery efficiency was 22.34%
(±14.35%).
2.4. Virus identification: RNA extraction, PCR amplification and viral
culture
All specimens were handled according to laboratory biosafety
guidelines.
Samples were subjected to total nucleic acid extraction with the
QIAamp viral RNA kit (QIAGEN, Hilden, Germany), according to the
manufacturer instructions. Samples were then subjected to RT-PCR
(RealStar® SARS-CoV-2 RT-PCR Kit 1.0, Altona Diagnostics) followed
by DNA amplification (Roche LightCycler® 96 System).
Only samples confirmed to be positive by the two different RT-qPCR
M.T. Pepe Razzolini et al.
assays, performed at Division for Microbiology and Parasitology of
CETESB (N1 and N2 target regions) and at the Virology Laboratory at the
Institute of Tropical Medicine of São Paulo (E and S genes) were sent for
virus isolation.
Viral culture for SARS-CoV-2, conducted in a biosafety level-3 fa­
cility, utilized Vero CCL81 cells (ATCC® CCL-81™) in Dulbecco minimal
essential medium supplemented with 10% heat inactivated fetal bovine
serum and antibiotics/antimycotics. Samples were inoculated into a
Vero cell culture in plastic bottles (Jet biofilm, 12,5 cm2 area, 25 mL
capacity) and incubated in a 37 ◦ C incubator in an atmosphere of 5%
CO2. Cultures were maintained for at least 2 weeks and observed daily
for evidence of cytopathic effects (CPEs). At least two subcultures were
performed on each sample. The detection of CPEs was investigated using
an inverted microscope (Nikkon, Japan) and the presence of virus in
supernatants from cultures showing CPEs was determined by specific
RT-PCR, as described above. RT-PCR analysis was performed using RNA
extracted from culture supernatants obtained two passages after the
initial inoculation.
2.5. Data on respiratory infections in São Remo
During the same period, the cases of confirmed COVID-19, and the
cases of Severe Acute Respiratory Syndrome (SARS) by any cause, that
occurred in people residing in the zip codes referring to São Remo, were
evaluated. The data was extracted from the SIVEP-Gripe database
(available at: https://opendatasus.saude.gov.br/dataset/bd-srag-2021).
The ZIP codes for the cases were obtained from the State of Sao Paulo
notification system.
2.5.1. Data analysis
Based on the notification system from the State of São Paulo, weekly
confirmed COVID-19 and Severe Acute Respiratory Syndrome (SARS)
cases were combined - for each epidemiological week.
For statistical analysis, we adopted the logarithmic transformation
for the concentration of N1 and N2 gene fragments. For cases in which
the viral load was equal to the limit of detection, we assigned a value of
1000 genomic copies/L. In cases in which the viral load was below the
limit of detection we could not quantify the load a value of 500 genomic
copies/L was assigned.
In order to study the relationship between the number of cases and
the RNA concentrations, we considered univariate Poisson regression
models (Agresti, 1990) for N1 and N2 target regions. The number of
Fig. 3. Concentration of SARS-CoV-2 (genomic copies per liter of N1 and N2) in ultracentrifuged water samples and weekly number of cases of Severe Acute
Respiratory Syndrome (SARS) and COVID-19 in the São Remo community.
4
Environmental Pollution 290 (2021) 118003
SARS and COVID 19 cases was taken as the response variable and the
logarithm of N1 and N2 concentrations as independent variables.
Goodness-of-fit tests and residual analyses were performed showing that
the model fits were adequate (not shown in this paper).
3. Results
From May to November 2020, 31 water samples were collected, on
Mondays, and analyzed for the presence of SARS-CoV-2 RNA, of which
29 were positive. Fig. 3 presents the results of RT-qPCR of N1 and N2
concentrations in ultracentrifuged samples and the cases of SARS and
COVID-19 in the São Remo community.
Among the 31 samples analyzed, only 1 showed a discordant result
between the N1 and N2 assays (only N1 was detected at a concentration
of 2.56 × 103 CG/L). Furthermore, the results with N1 showed higher
concentrations in 64.5% of the samples, demonstrating that, for the US
CDC assay, the 2019-nCoV_N1 (N1) primer–probe set was more sensitive
than the 2019-nCoV_N2 (N2) primer–probe set in this study. The higher
sensitivity of the N1 assay when compared to N2 has already been
observed in other studies (Vogels et al., 2020; Pecson et al., 2021), but
also with non-significant differences between the concentration values.
There is a visible linear relationship between the number of new
disease cases and the logarithm of SARS-CoV-2 RNA concentrations (N1
and N2) found in the water samples (Fig. 4), motivating further analyses
based on Poisson regression models.
For the logarithm of N1 and N2 gene fragments concentrations,
separated Poisson regression models were fit and showed statistical
significance on the relationship with the number of COVID-19 and Se­
vere Acute Respiratory Syndrome (SARS) cases (p < 0.001 for both
genes). In addition, for the first model fit, relating the number of cases
with the log-concentration of N1, the estimated regression coefficient
resulted 0.71 (standard error 0.12). This result can be interpreted in the
following way: for an increase of one unit in the log-concentration of N1
the expected number of cases of COVID-19 will be multiplied by e2.04 =
2.04. In other words, if we multiply the concentration of N1 by 10 we
will end up with an increase of 104% in the expected number of cases,
with an associated confidence interval CI (95%) = [62%, 157%].
Similarly, from the model fit relating the number of cases with N2 log-
concentrations, the estimated regression coefficient is 0.65 (0.12)
meaning that an increase of one unit in the log-concentration of N2
implies the expected number of cases of COVID-19 will be multiplied by
e0.65 = 1.92; alternatively, it is estimated an increase of 92% in the
M.T. Pepe Razzolini et al.
Fig. 4. Scatterplot relating the number of cases of COVID-19 and Severe Acute Respiratory Syndrome (SARS) according to the logarithm of N1(a) and N2 (b)
concentrations.
expected number of cases of COVID-19 when the concentration of N2 is
multiplied by 10 with CI (95%) = [51%, 143%]. Table 1 shows the
descriptive statistics for the concentrations of the fragments genes N1
and N2 obtained from water samples. The missing data (Table 1) refers
to samples from which the method applied detected these fragments, but
the actual value was not registered.
Results of SARS-CoV-2 RT-qPCR and isolation in Vero cells assays are
presented in Table 2.
Out of the 13 samples to evaluate viability and infectivity only seven
confirmed to be positive by a different RT-qPCR assays (E and S genes).
All seven samples were sent for virus isolation. In two samples we
observed cytopathic effect (CPE), during the first passage in the Vero
cells. However, we were not able to isolate culture-competent virus from
any of the seven samples sent for virus isolation, after a second passage
in Vero cells.
4. Discussion
During a 31-week serial evaluation of water from a stream running
through an underprivileged, underserved, urban settlement (favela or
slum), we observed a correlation between counts of SARS-CoV-2 in the
water and the number of cases of COVID-19 and of Severe Acute Res­
piratory Syndrome (SARS) in the community. In this community
wastewater runs directly into the stream as there is no sewage collection.
Guerrero-Latorre et al. (2020) analyzed samples (n = 3) from three
locations from Quito’s river obtaining concentrations ranging from 2.84
× 105 to 3.19 × 106 genomic copies/L and from 2.07 × 105 to 2.23 ×
106genomic copies/L for genes N1 and N2, respectively, which is the
same level of concentration found in this study (Table 1). The qualitative
study conducted by Rimoldi et al. (2020) detected the presence of
SARS-CoV-2 RNA in wastewater and, rivers that received sewage from
the Milano Metropolitan Area. In a comprehensive review carried out by
Langone et al. (2021) addressed the detection of SARS-CoV-2 in envi­
ronmental compartments showed several studies demonstrating the
presence of the virus in Europe, USA, and Australia.
Some studies reported SARS-CoV-2 RNA presence in feces, including
from asymptomatic people, and anal/rectal swabs (Holshue et al., 2020;
Jiehao et al., 2020, Tang et al., 2020; Wang et al., 2020). Regarding the
persistence of the virus, Casanova et al. (2009) carried out a study based
Table 1
Descriptive statistics for the measures of number of cases and for N1 and N2
fragments.
Cases Gene N1 Gene N2
Minimum 0 0 0
Maximum 5.00 3,280,000 2,440,000
Mean 1.16 223,434 197,302
Standard Deviation 1.24 695,280 611,874
Median 1.00 26,900 17,450
Missing Data 0 0 1
5
Environmental Pollution 290 (2021) 118003
on surrogates for coronavirus (TGEV – transmissible gastroenteritis
coronavirus and MHV – mouse hepatitis) and were able to demonstrate
that the virus can remain in water and sewage from days to weeks (nine
to 49 days at 25 ◦ C in pasteurized settled sewage and seven to 70 days at
4 ◦ C). According to Gundy et al. (2009) the human coronavirus is less
stable remaining from 1.57 to 2.36 days. Studies using seeding of
SARS-CoV-2 RNA in order to assess the persistence of this virus in water
matrices were carried out by Bivins et al. (2020) and Oliveira et al.
(2021), and both studies showed the presence of gene markers of
SARS-CoV-2 and, some level of persistence in the environmental samples
analyzed. Rimoldi et al. (2020) demonstrated the presence of
SARS-CoV-2 RNA (orf1ab, N, E genes) in sewage and rivers but did not
find cytopathic effect in none of the samples analyzed. Although virus
had been detected in feces (Foladori et al., 2020; Chen et al., 2020),
growth in culture is a rare event (Van Doorn et al., 2020; Amirian, 2020;
Fumian et al., 2021). Viability of the virus in environmental matrices is
still unknown. Studies carried out by several researchers in various
countries (Medema et al., 2020; Ahmed et al., 2020; La Rosa et al., 2020;
Randazzo et al., 2020; Guerrero-Latorre et al., 2020; Rimoldi et al.,
2020) found SARS-CoV-2 in wastewater or river and streams samples
but did not report if the virus was viable, which still remains
undetermined.
Analyzing the water from a stream contaminated with human waste
is probably a different scenario from evaluating sewage, because this
natural environment probably receives none or very little of products
such as chlorine and other disinfectants or other substances with po­
tential inhibitory activity. Furthermore, it receives high levels of organic
material. Both of these conditions may allow the virus to survive for
longer periods of time in rivers and streams, which raises the question of
whether SARS-CoV-2 in these waters can be a source of infections and
thus contribute to the spread of COVID-19. The implications of this in
communities such as Sao Remo would be tremendous as fecal-oral
spread of COVID-19 would deapen the socioeconomic inequalities
already observed for this disease (Costa et al., 2020; Wu et al., 2021).
Because of this, we attempted viral culture. We found positive RT-qPCR
only after the first passage in Vero cells as well as cytopathic effect. A
positive PCR after the first passage in culture may represent viral frag­
ments, other virus such as enterovirus, and not necessarily viable
SARS-CoV-2. Thus, we considered a culture positive if PCR were positive
after the second passage or later. This did not occur in our samples. As
discussed by Jefferson et al. (2020) a positive culture would be the
strongest evidence of potential for transmission. Cytopathic effect is
considered to be a step below viral culture as evidence of transmission.
In our study, 2 samples presented cytopathic effect during the first
passage. We believe that this should not be taken into account, as we
used extremely contaminated water. Cytopathic effect can be caused by
a variety of enteric viruses (Sylvestre et al., 2021) and is not necessarily
evidence of viable SARS-CoV-2.
However, the occurrence of this new virus in the environment is used
as an early warning for surveillance systems, either national or
M.T. Pepe Razzolini et al. Environmental Pollution 290 (2021) 118003

Table 2
Results of SARS-CoV-2 RT-qPCR and isolation in Vero cells assays in water samples from Richo Doce (May to August, 2020).
Day of sample Original sample SARS- Original sample Viral viability
collection CoV-2 RNA copies/L SARS-CoV-2 (Ct)

N1 N2 Gene Gene S CPE after the 1st passage in PCR SARS-CoV-2 Final result of viral isolation (number of culture
E culture (Ct) after 1st passages)
passage in
culture

Gene Gene S
E

May 04 2.26 × 1.44 × ND ND NP NP NP NP

106 106
May 12 3.28 × 2.25 × ND ND NP NP NP NP
106 106
May 18 1.22 × 8.51 × 31.4 30.1 Negative ND ND Negative (2)
105 104
May 25 4.45 × 6.26 × 33.0 32.5 Negative ND ND Negative (2)
104 104
June 1 2.33 × 1.61 × 33.5 ND Negative ND ND Negative (2)
105 105
June 15 1.24 × 2.05 × 35.2 35.1 Positive 38.0 ND Negative (2)
105 104
June 22 1.50 × 1.26 × 34.9 33.0 Negative ND ND Negative (2)
105 105
July 6 1.48 × 9.45 × 35.6 ND Negative ND ND Negative (2)
105 104
July 13 1.40 × 6.56 × 32.2 30.0 Positive 33.7 33.8 Negative (2)
104 103
July 20 2.45 × 2.14 × ND ND Negative NP NP Negative (2)
104 104
August 03 3.44 × 1.68 × ND ND NP NP NP Negative (2)
104 104
August 10 2.89 × 1.26 × ND ND NP NP NP Negative (2)
104 104
August 17 3.42 × 1.81 × ND ND NP NP NP Negative (2)
104 104

ND: Not Detected; NP: Not Performed; Ct: cycle threshold; CPE: Cytopathic effect.

international (Xagoraraki and O’Brien, 2020) this information is useful
for surveillance purposes and public health interventions. The usage of
wastewater-based epidemiology can be a challenge for low- and
middle-income countries due to most households are not connected to
sewer networking (Pandey et al., 2021). Regardless the challenges,
when SARS-CoV-2 RNA is detected in a impacted water by sewage, may
be a signal that there are a significant number of cases of the disease,
which urges immediate intervention measures. Monitoring the occur­
rence of SARS-CoV-2 RNA in the environment may be helpful as an
epidemiological tool for evaluating COVID-19 prevalence in a
community.
The main limitations of our study are that cultures were not done for
all the water samples. Furthermore, due to the generalized lack of
diagnostic resources we believe that COVID-19 cases in the database
were not an accurate reflection of the cases in the community. Because
of this, we decided to include cases of Severe Acute Respiratory Syn­
drome (SARS), to account for subnotification of COVID-19. Still, it is
possible that mild cases of COVID-19 were missed.
Communities such as Sao Remo are common throughout the world.
Although Sao Remo has slightly better conditions than many similar
communities, with 2–3 persons per home, brick housing, electricity, and
potable running water, our findings may be widely applicable.
5. Conclusion
Our results showed a correlation between cases of COVID-19 and
SARS in an underprivileged community, and SARS-CoV-2 concentra­
tions in the water of a stream running through this community. The
differences in concentration of the virus in the environment reflect the
epidemiological status in the community, which may be important in­
formation for tracking and preventing dissemination of the virus in areas
with vulnerable population and poor sanitation. We did not find viable
viral particles.
Contributors
MTPR and ASL: Conceptualization, writing, samples collection,
literature search; MRFB: Analytical procedure for virus concentration
(qPCR), review and editing; MCM-C, AVP and LSV-B: Analytical pro­
cedures for virus viability; MD, RSA, IFO, SCG: Analytical procedure for
virus concentration (qPCR); SFC, DBA and ECS: review and editing; BSL
and ACPL: Data analysis and writing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
Funding: This work was supported by the National Council of Sci­
entific and Technological Development (CNPq) for ASL (Number:
302,073/2017–5), and also for the support by CETESB (Companhia
Ambiental do Estado de Sao Paulo) and Instituto de Medicina Tropical,
Universidade de Sao Paulo, Brazil (IMT/USP). We are grateful to the São
Remo Community for their warm reception, especially to Ericsson
Michel Silva Magnavita, Orlando Vieira dos Santos, Carlos Alberto da
Silva, Claudio dos Santos Jr and Josefa Bezerra da Silva. We thank Flavia
Trevisan for the graphical abstract and Yuval Co for the photographs.
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