Hepatitis A virus monitoring in wastewater: A

Q1 complementary tool to clinical surveillance

i The corrections made in this section will be reviewed and approved by a journal production editor.

Anabella Fantillia,b,⁎, anabellafantilli@gmail.com, Guadalupe Di Colaa,b, Gonzalo Castroc, Paola Siciliac, Ariana

Mariela Cachid,e, María de los Ángeles Marinzaldad,e, Gustavo Ibarraf, Laura Lópezg, Celina Valduvinog, Gabriela

Barbásh, Silvia Natesa, Gisela Masachessia,b,1, María Belén Pisanoa,b,1, Viviana Réa,b

a
Instituto de Virología “Dr. J. M. Vanella”, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba,
Enfermera Gordillo Gómez s/n, Ciudad Universitaria, Córdoba X5000, Argentina
b
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, CABA C1425FQB,
Argentina
c
Departamento Laboratorio Central, Ministerio de Salud de la Provincia de Córdoba, T. Cáceres de Allende 421,
Córdoba ´ X5000HVE, Argentina
d
Instituto Nacional de Medicina Aeronáutica y Espacial, FAA, Av. Fuerza Aérea Argentina Km 6 1/2 S/N B.0
Cívico, Córdoba X5010, Argentina
e
Facultad de la Fuerza Aérea, Universidad de la Defensa Nacional, Av. Fuerza Aérea Argentina 5011, Córdoba
X5000, Argentina
f
Planta Municipal de tratamiento de efluente cloacales Bajo Grande-Laboratorio de análisis fisicoquímicos,
bacteriológicos EDAR Bajo Grande, Cam. Chacra de la Merced 901, Córdoba X5000, Argentina
g
Área de Epidemiología, Ministerio de Salud de la Provincia de Córdoba, Av. Vélez Sarsfield 2311 Ciudad
Universitaria, Córdoba X5016 GCH, Argentina
h
Ministerio de Salud de la Provincia de Córdoba, Av. Vélez Sarsfield 2311 Ciudad Universitaria, Córdoba X5016
GCH, Argentina. Ministerio de Salud de la Provincia de Córdoba, Argentina Av. Vélez Sarsfield 2311 Ciudad
Universitaria, Córdoba X5016 GCH, Argentina

⁎ Corresponding author.

1These authors contributed equally to this work

Abstract
Monitoring wastewater is an effective tool for tracking information on trends of enteric viral dissemination.
This study aimed to perform molecular detection and genetic characterization of HAV in wastewater and to
correlate the results with those obtained from clinical surveillance.

Wastewater samples (n=811) of the second most populous city in Argentina were collected from the main
wastewater treatment plant (BG-WWTP, n=261), and at 7 local neighborhood collector sewers (LNCS,
n=550) during 2017–2022. Clinical samples of acute hepatitis A cases (HA, n=54) were also analyzed.
HAV molecular detection was performed by real time RT-PCR, and genetic characterization by RT-Nested
PCR, sequencing and phylogenetic analysis.

RNA-HAV was detected in sewage samples throughout the entire period studied, and detection frequencies
varied according to the location and year (2.9% - 56.5%). In BG-WWTP, 23% of the samples were RNA-
HAV+. The highest detection rates were in 2017 (30.0%), 2018 (41.7%) and 2022 (56.5%), which
coincides with the highest number of HA cases reported. Twenty-eight (28) sequences were obtained (from
clinical and sewage samples), and all were genotype IA. Two monophyletic clusters were identified: one
that grouped clinical and wastewater samples from 2017–2018, and another with specimens from 2022,
evidencing that environmental surveillance might constitute a replica of viral circulation in the population.

These findings evidence that WBE, in a centralized and decentralized sewage monitoring, might be an
effective strategy to track HAV circulation trends over time, contributing to the knowledge of HAV in the
new post-vaccination epidemiological scenarios in Argentina and in Latin America.
Keywords:

Hepatitis A virus, HAV, Wastewater-based epidemiology, Clinical surveillance, Argentina

Abbreviations

No keyword abbreviations are available

1 Introduction
More than 100 enteric viruses are excreted in human feces. Among these, adenovirus, hepatitis A virus (HAV),
norovirus, rotavirus, and enterovirus are the most frequently detected viruses in the environment (Metcalf et al., 1995,
Joseph et al., 1980, Strubbia et al., 2019). During the last few years, more attention has been focused on the sewage
virological quality, the risk of virus-associated waterborne illness, the need for routine monitoring of viral contamination
and the environmental viral surveillance through the analysis of sewage (Kokkinos et al., 2011). Wastewater-based
epidemiology (WBE) serves as an important tool for tracking enteric virus circulation in a community, providing
opportunities to estimate its prevalence and geographic distribution (Sinclair et al., 2008, Xagoraraki and O'Brien, 2020
), including both symptomatic and asymptomatic infected individuals (Carducci et al., 2006, La Rosa and Muscillo,
2013). WBE approach is useful to complete the epidemiological data in populations with little or questionable clinical
surveillance; when the persistent circulation of a virus is suspected, or a reintroduction/re-emergence of others is
perceived; to track the appearance of new variants, monitor viral circulation under a vaccination program; and, as
described for SARS-CoV-2, it might predict a possible increase in clinical cases (Larsen and Wigginton, 2020,
Masachessi et al., 2022). Previous local studies from Argentina focused on WBE have been carried out showing that
this methodological tool can be used to track viruses excreted in a population and, in some cases, predict the
consequent increase in clinical cases. Nevertheless, little data regarding the use of this tool for HAV monitoring have
been reported in South America, particularly in Argentina.

The epidemiology of HAV in several geographic regions is changing worldwide. It is estimated that more than 100
million HAV infections occur annually worldwide (World Health Organization (WHO) 2019). In Argentina, since the
introduction of the single-dose-scheme vaccination among children of 12 months in 2005, notification of clinical cases
of hepatitis A (HA) has decreased drastically and liver transplants related to HAV have not been recorded since 2007 (
Dirección Nacional de Epidemiología y Análisis de Salud del Ministerio de Salud y Desarrollo Social de Argentina
2019, OPS (Organización Panamericana de la Salud) 2018). In addition, age-specific HAV-vaccination coverage is
reported to be high (>75%) (Dirección de Control de Enfermedades inmunoprevenibles M de S de A 2021, Dirección
de Control de Enfermedades inmunoprevenibles M de S de A 2020) and subsequent studies have demonstrated the
presence of long-term immune memory in both seroprotected and unprotected individuals after 12 years of
immunization (Urueña et al., 2022). As a consequence, the epidemiology of hepatitis A in Argentina has shown a
change in pattern in recent years from high endemicity to current low endemicity with lower incidence, sporadic cases
and limited outbreaks in unvaccinated individuals over 20 years of age (Andani et al., 2020). More recently, clinical
cases have been registered among young adults between 20 and 39 years of age, in whom increased susceptibility to
HAV infection has been reported (Dirección Nacional de Epidemiología y Análisis de Salud del Ministerio de Salud y
Desarrollo Social de Argentina 2019, Andani et al., 2020, Yanez et al., 2014, Angeleri et al., 2019). In this context,
between mid-2016 and early 2018, several hepatitis A outbreaks that disproportionately affected unvaccinated young
adult men, mainly men who have sex with men (MSM), were described in Europe, the US and South America,
including central Argentina (Mariojoules et al., 2019).

The presence of HAV in sewage has been well-documented and extensively studied (Elkana et al., 1983, Graff et al.,
1993, Morace et al., 2002, Tsai et al., 1994). In Europe, as seroprevalence of hepatitis A and the number of acute HAV
infections have declined, WBE has provided a better understanding of epidemiological changes in disease trends (
Bisseux et al., 2018). Thus, varied frequencies of HAV detection in urban wastewater have been reported in different
years and European countries, ranging from 3.1% to 60% (Kokkinos et al., 2011, Bisseux et al., 2018, Rodriguez-
Manzano et al., 2010, Pina et al., 2001, Pintó et al., 2007, Iaconelli et al., 2015, La Rosa et al., 2014, Pellegrinelli et al.,
2019). Higher frequencies of detection (>60%) have been found in countries with lower resources and higher
endemicity such as South Africa, Cairo and Tunisia (Pintó et al., 2007, Béji-Hamza et al., 2014, Rachida and Taylor,
2020, Ouardani et al., 2015).
While monitoring HAV in environmental samples is not a common practice in South America, the circulation of HAV
in wastewater has also been previously demonstrated in some Latin American countries, albeit to a lesser extent. Thus,
in Colombia, a HAV detection frequency of 13.3% was reported (Baez et al., 2017). In Brazil, in highly urbanized
cities (Rio de Janeiro and São Paulo), HAV detection frequencies in wastewater were greater than 50%, increasing
between 2017–2018, when a HAV outbreak in MSM was described (Prado et al., 2012, Prado et al., 2021).

Previous environmental virology studies, based on a small number of wastewater samples, have shown the presence of
RNA-HAV in central (20.8%) and west (39%) Argentina, showing its continuous circulation among the population,
becoming a potential risk to exposed individuals (Yanez et al., 2014, Masachessi et al., 2018). Since HAV is an enteric
transmission virus under a vaccination program, and considering that currently, due to the pandemic caused by SARS-
CoV-2, vaccination coverage has declined (Dirección de Control de Enfermedades inmunoprevenibles M de S de A
2021), it is essential to perform effective surveillance and continuous monitoring of the virus and its variants.

Thus, the aim of the present study was to perform the molecular detection and genetic characterization of HAV in
wastewater, and to correlate the results with those obtained from clinical surveillance.

2 Material and methods

2.1 Study Area: Córdoba city

Córdoba city is the capital of the province of Córdoba, located in the central region of Argentina (31°25′ 00′′S 64°11′
00′′O), and has 1329,604 inhabitants distributed in 39 neighborhoods, with a population density of 2319.5
inhabitants/km2 (INDEC, 2010) (Fig. 1). The main wastewater treatment plant (WWTP), which is called "Bajo
Grande" (BG-WWTP), receives about 50% of the sewage system coverage. Additionally, seven local neighborhood
collector sewers (LNCS-1 to LNCS-7) located upstream of BG-WWTP were also included in the study (Fig. 1). These
neighborhoods were selected for being the most populated ones. The collector sewer pipe of each neighborhood
sampled receives the discharge of 100% of its entire population, except for LNCS-7.

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alt-text: Fig 1

Fig. 1

Map of the study area in Córdoba city, located in the province of Córdoba, in the central region of Argentina. The study area includes
the main wastewater treatment plant (BG-WWTP), and the 7 local neighborhood collector sewers (LNCS): Alberdi-Páez (LNCS-1);
General Bustos (LNCS-2); San Vicente (LNCS-3); Pueyrred ón (LNCS-4); Villa Libertador (LNCS-5); Don Bosco (LNCS-6); and
Marqu és de Sobremonte (LNCS-7).

2.2 Wastewater samples

Raw sewage specimens (n=811) were collected as follows: 1) from BG-WWTP (n=261), samples were monthly
collected from January 2017 to November 2019, and weekly collected from mid-May 2020 to the end of July 2022; 2)
at the 7 LNCS (n=550) samples were weekly collected from the end of January 2021 to the end of July 2022. Periods
and frequencies of wastewater sample collection are shown in Table 1.

alt-text: Table 1

Table 1

i The table layout displayed in this section is not how it will appear in the final version. The representation below is solely
 purposed for providing corrections to the table. To view the actual presentation of the table, please click on the
located at the top of the page.

Periods of sample collection, sampling frequency and number of samples collected from each sampling site included in the study.

Sampling location Period of Sample Collection Sampling Frequency Number of Samples Collected

January 2017-November 2019 Once per month 33

BG-WWTP
mid-May 2020- end of July 2022 Once per week 228

LNCS End of January 2021-end of July 2022 Once per week 550

From BG-WWTP, 0.5 L grab samples of wastewater were taken, and at LNCS, 0.25 L samples were collected, both
on weekday mornings by the grab collection method described in the WHO Guidelines for Environmental Surveillance
of Poliovirus Circulation (World Health Organization (WHO) 2003). Each sample was transported within 6 h at 4°C to
8°C to the Institute of Virology, National University of Córdoba, Córdoba City, for further processing and analysis.

2.3 Clinical samples

During the same period of time as the wastewater sampling (2017 – 2022), the official number of confirmed cases of
HA reported to the notification system of Córdoba city, and their demographic data (age, gender and address) were
provided by the Direction of Epidemiology of the Government of the Province of Córdoba.

Serum and/or stool samples from these notified cases were referred to the Institute of Virology, National University of
Córdoba, for molecular detection and phylogenetic analysis.

2.4 Concentration of HAV in wastewater samples

Virus in wastewater samples were concentrated by the polyethylene glycol 6000 (PEG-6000) precipitation method,
according to the WHO guideline for environmental surveillance of poliovirus circulation (WHO, Guidelines for
environmental surveillance of poliovirus circulation, 2003), using the method described by Lewis & Metcalf (Lewis
and Metcalf, 1998), and previously implemented in our Institute (Masachessi et al., 2022). Briefly, a volume of 0.5 L
(BG-WWTP) or 0.25 L (LNCS) of the sample was concentrated 100-fold to 15 mL by high-speed centrifugation (two
centrifugation steps at 10,700 g for 20 to 25 min) in a Beckman J2–21 refrigerated centrifuge, elution (two steps at
room temperature for 1 h) and PEG precipitation overnight at 4°C and centrifuged at 10,000 x g for 25 min. The PEG-
containing supernatant was discarded, and the pellet was suspended in 5 mL PBS (Gibco, Invitrogen Corporation,
New York, USA) (pH 7.2), adjusted to pH 8.0, incubated for 1 h with occasional vortex, and centrifuged at 10,000 x g
for 20 min. The supernatant was stored at − 80 °C, until processing.

2.5 Genomic RNA extraction

RNA was extracted from all samples (500 µL) using the MagNA Pure 96 DNA and Viral NA Large Volume Kit in
MagNA Pure 96 system (Roche Diagnostics GmbH – Mannheim, Germany), according to the manufacturer's
instructions. The extracted RNA was eluted in a final volume of 50 µL of a 60 mM Tris-HCl buffer. A negative control
was included during the RNA extraction to monitor any possible cross contamination during sample processing.

2.6 HAV genome detection and molecular analysis

Molecular detection of RNA-HAV in clinical and wastewater samples was performed by real time RT-PCR (Pintó et
al., 2009) using the TaqMan® Fast Applied Biosystems single-step RT-qPCR Kit (Carlsbad, CA, USA) performed in a
StepOne™ Real-Time PCR equipment (Applied Biosystems) (Carlsbad, CA, USA). Process controls, real time RT-
PCR controls, primers and hydrolysis probes sequences, and cycling conditions used for HAV detection are described
in Supplementary material. Positive samples were subjected to RT-Nested PCR using the GoTaq® DNA Polymerase
(Promega, Madison, USA) to amplify a genomic fragment encoding the C terminus of VP3 to the N terminus of 2A
proteins (1022 bp) using a protocol previously implemented (Mbayed et al., 2002). Positive PCR products were
purified using PureLink® Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA) and sequenced in both directions
by an ABI automatic sequencer (3500xL Genetic Analyzer - Applied Biosystems) in the Molecular Biology
Department of the Central Laboratory of the Province of Córdoba.

Sequences obtained were deposited at GenBank under the accession numbers OP796670 to OP796679, and
OP796681 to OP796697, and were used to perform phylogenetic analysis. For this, a dataset including sequences
obtained in this study and reference sequences from each HAV genotype (HAV IA: AB020565, X83302, EU131373;
HAV IB: AF314208, NC001489, DQ646426; HAV IIA: AY644676; HAV IIB: AY644670; HAV IIIA: DQ991030,
AJ299464, AB279733; HAV IIIB: AB258387, AB279735, AB425339; HAV V: EU140838, D00924) was
constructed (Mariojoules et al., 2019). Mega v.11.0.1 software (Tamura et al., 2021) was used for the alignment and for
the inference of the maximum likelihood (ML) phylogenetic consensus tree (mid-point rooted). To evaluate the
reliability of the groups and branches obtained in trees, the bootstrap approximation (1000 replicates) method was used.

2.7 Ethics approval

The study was conducted according to the guidelines of the Declaration of Helsinki (1964, amended most recently in
2008) of the World Medical Association, and in accordance with specific local ethical regulations (Legislature of the
Province of Córdoba 2009). The Research Evaluation Committee of the Institute of Virology “Dr. José María Vanella”
approved to perform this study.

2.8 Consent to participate

Written informed consent was obtained from all individual participants included in the study and from parents or legal
guardians of minor participants (age<18).

3 Results
3.1 HAV detection in wastewater and clinical samples

Out of 261 wastewater samples analyzed from BG-WWTP, 60 were RNA-HAV+ (23%) by RT-real time PCR. RNA-
HAV was detected throughout the entire period studied, obtaining varied detection frequencies according to the year
(between 2.9% and 56.5%). Thus, the overall detection rates per year in BG-WWTP were: 30% (3/10) in 2017, 41.7%
(5/12) in 2018, 9.1% (1/11) in 2019, 7.8% (5/64) in 2020, 10.8% (11/102) in 2021, and 56.5% (35/62) in 2022. The
years with highest detection rates were 2017, 2018 and 2022, which coincides with the highest number of HA clinical
cases in the city of Córdoba that were officially reported to the notification system of the Province of Córdoba (n=16,
n=22 y n=8, respectively) (Fig. 2.a).

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alt-text: Fig 2

Fig. 2

a. HAV detection rates in wastewater from BG-WWTP, and number of reported HA cases from January-2017 to July-2022; b. HAV
detections in BG-WWTP, and HA clinical cases reported per month in 2017, 2018 and 2019. a No sample was available for these
months; c. HAV detections in BG-WWTP, at LNCS, and HA clinical cases reported per epidemiological week (EPI W) in 2021 and
2022.

Additionally, in 2017 and 2018 the first RNA-HAV detection in wastewater corresponded to the month of August,
almost two months before the unusual increase in the number of HA clinical cases was reported to the notification
system that originated the outbreak in MSM in Córdoba (Fig. 2.b). Moreover, a similar situation could be recognized in
2022, when a continuous HAV circulation in sewage (BG-WWTP) preceded the rise of the HAV-positive cases
notified by up to 6 epidemiological weeks (Fig. 2.c).

The presence of RNA-HAV was detected at all LNCS (Fig. 2.c). The overall HAV detection rates at the LNCS were
2.9% (10/341) in 2021 and 9.1% (19/209) in 2022, and there were slight differences between different locations. HAV
detection per epidemiological week in each neighborhood are shown in Fig. 2.c. Positive samples were repeatedly
identified during 2022 at LNCS 3, 4, 6 and 7, even though there were no HA cases reported during the same
epidemiological weeks.

As it is shown in Fig. 2.c, there are some discrepancies in HAV detections in BG-WWTP and at the LNCS. While in
some epidemiological weeks viral RNA signals are detected in BG-WWTP, it was not detected at the 7 LNCS included
in the study. Similarly, although RNA-HAV was detected in some epidemiological weeks at some of the LNCS, non-
detection of RNA-HAV was observed in BG-WWTP during those weeks.

Out of the 54 clinical samples from reported HA cases derived to the Institute of Virology “Dr. J. M. Vanella”, 35
(64.8%) tested RNA-HAV positive by real time RT-PCR, and 87.0% were males (47/54). The mean age of these
individuals was 33.3 years old, 96.3% (52/54) were adult patients from 16 to 66 years old, and 3.7% (2/54) were
unvaccinated children, one from Peru and the other one from Bolivia, both living in the province of Córdoba.

3.2 Genotyping and phylogenetic analysis

Out of all the positive samples obtained (both from patients and sewage), 28 could be amplified by RT-nested PCR and
sequenced afterward. Epidemiological data from the sequenced samples recovered from HA clinical cases are detailed
in Table 2, and data from sequenced wastewater samples in Supplementary Table 1.

alt-text: Table 2

Table 2

i The table layout displayed in this section is not how it will appear in the final version. The representation below is solely
purposed for providing corrections to the table. To view the actual presentation of the table, please click on the
located at the top of the page.

Epidemiological data from the sequenced samples recovered from HA clinical cases.

Sampling Year Age Sex Relevant epidemiological data Sample ID GenBank accession numbers

Travel history MSM

2017 33 M No MSM H-CbaArg-1A-2017 a OP796681

2017 38 F Yes (Ecuador) No H-CbaArg-2A-2017 a OP796682

2018 54 M No MSM H-CbaArg-3A-2018 a OP796683

2018 37 M No MSM H-CbaArg-4A-2018 a OP796684

2018 26 M No MSM H-CbaArg-5A-2018 a OP796685

2018 53 M No MSM H-CbaArg-6A-2018 a OP796686

2018 23 M N/A N/A H-CbaArg-7A-2018 a OP796687

2019 28 M N/A N/A H-CbaArg-8A-2019 a OP796688

2019 34 M Yes (Venezuela) N/A H-CbaArg-9A-2019 a OP796689

2019 24 M No N/A H-CbaArg-10A-2019 a OP796690

2019 4 M N/A No H-CbaArg-11A-2019 a b OP796691

2021 19 M No N/A H-CbaArg-12A-2021 OP796692

2022 30 M N/A MSM H-CbaArg-13A-2022 OP796693

2022 22 M N/A N/A H-CbaArg-14A-2022 OP796694

2022 43 M N/A N/A H-CbaArg-15A-2022 OP796695

2022 25 M N/A N/A H-CbaArg-16A-2022 OP796696

2022 N/A N/A N/A N/A H-CbaArg-17A-2022 OP796697

MSM: Men who have sex with men; N/A: Not available. a Samples that clustered with the VRD 521 –2016 strain, responsible of
causing outbreaks mostly in MSM in Europe since mid-2016 (OPS (Organizaci ón Panamericana de la Salud) 2018). bThis sample
belonged to an unvaccinated child from Bolivia.
All the sequences analyzed were identified as HAV genotype IA (Fig. 3). The phylogenetic analysis showed that the
sequences recovered from wastewater specimens obtained between 2017–2018 held a close identity with sequences of
HA clinical cases obtained in the same period of time (Fig. 3, blue shading), when the outbreak among MSM described
in Córdoba occurred (Mariojoules et al., 2019). In addition, the same homology pattern could be identified for the
sequences obtained from clinical and environmental samples corresponding to 2022, which formed a monophyletic
clade separated from the one previously mentioned (Fig. 3, red shading). On the other hand, four sequences recovered
from clinical samples in 2017, 2019 and 2021 (Fig. 3, yellow shading), were segregated into separated groups. Two of
them belonged to cases imported from Venezuela and Ecuador, and another sequence belonged to an unvaccinated
child from Bolivia.

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alt-text: Fig 3

Fig. 3
 Maximum Likelihood phylogenetic tree (mid-point rooted) obtained for the coding region encoding the C terminus of VP3 to the N
terminus of 2A proteins (1022 bp) of representatives of all genotypes and subtypes together with the sequences obtained during this
study. The bootstrap values higher than 80% are indicated in the tree branches.

4 Discussion
The present study describes a large-scale environmental HAV surveillance in central Argentina, involving a parallel
environmental/clinical surveillance. The results of this research show that untreated wastewaters from Córdoba city
constitute silent sentinels of the HAV circulation trend in the region, being a sensitive indicator that reports if HAV
circulates among the population and if its transmission is increasing or decreasing.
As previously reported, in countries with low endemicity for HAV infections, wastewater monitoring has revealed
sustained virus circulation in the community, and when clinical surveillance misses mild, asymptomatic or subclinical
infections, WBE reflects the real magnitude of circulating virus (La Rosa et al., 2014). In this sense, Bisseaux et al. (
Bisseux et al., 2018) found high detection rates in raw wastewater (60%) in France, despite few symptomatic acute
infections being notified. In this study, despite the almost complete absence of clinical case notifications, HAV was still
detected in wastewater during 2020 and 2021, with detection rates of 7.8% and 10.8%, respectively. The lack of
clinical case reports and low HAV detection rates registered in wastewater may be explained probably by the low
circulation of other viruses during the ongoing pandemic in those years. A similar situation occurred in Chile, where
Plaza-Garrido et al. (Plaza-garrido et al., 2023) reported that HAV was not detected among the studied WWTPs during
2021.

Furthermore, wastewaters reflect the dynamic turnover of circulating virus variants (Ranta et al., 2001). Therefore,
conducting a genetic comparison between HAV strains identified in sewage and clinical samples may deepen our
understanding of the epidemiology of this virus [(Pintó et al., 2007), 50]. Similar reports have highlighted the crucial
role of WBE in supporting research on HAV outbreaks within the community (La Rosa et al., 2014, Pellegrinelli et al.,
2019, Prado et al., 2012, Prado et al., 2021). In this sense, the highest RNA-HAV detection rates registered in this
study in sewage were associated with a simultaneous increase in the notification of HA clinical cases. This was
recorded in 2017 and 2018, when a higher detection of viral excretion in sewers was observed during an outbreak of
HA. The HAV strains detected, both in clinical and wastewater samples, clustered with sequences obtained during the
2017–2018 outbreak that occurred in Córdoba, caused by the HAV-IA strain VRD 521–2016 (Mariojoules et al., 2019
).

Additionally, these findings show that the detection of the same HAV strain in environmental samples before and at the
beginning of its spread among the population demonstrates that this outbreak might have been predicted by sewage
samples monitoring, as it has also been previously reported (La Rosa et al., 2014, Pellegrinelli et al., 2019, Hellmér et
al., 2014). Consistent with these results, Hellmer et al. (Hellmér et al., 2014) showed that one of the two strains causing
an HA outbreak in Sweden was detected in sewage in weeks 7 and 9, together with samples from three patients
infected with these strains who were diagnosed with acute hepatitis A in weeks 10 and 19. La Rosa et al. (La Rosa et
al., 2014) also documented HAV circulation in sewage during July-December 2012, before the HA outbreak reported
in January 2013 in Italy. Accordingly, we detected a slight rise in reported HA cases in 2022 coincided with a
continuous and increased detection of RNA-HAV in the central sewer pipe of BG-WWTP, even some weeks before
the first clinical case reported.

These findings suggest that the implementation of the weekly sampling strategy at this site would be a useful tool to
provide data on HAV circulation as a complement to clinical surveillance. This information, added to the phylogenetic
correlation between the sequences detected in clinical and wastewater samples from the same period of time, evidence
once again that environmental surveillance might constitute a replica of the clinical-epidemiological scenario of viral
circulation in the population.

Phylogenetic analysis in this research showed that select samples (from 2017, 2019, and 2021) grouped outside the 2
main monophyletic clusters. Two of them belonged to travelers coming from other parts of South America (Venezuela
and Ecuador), and other sequence belonged to an unvaccinated child from Bolivia, which reveals new viral
introductions with the potential to spread to the local population. In this sense, environmental surveillance becomes a
valuable tool for monitoring different HAV variants, to verify whether new viral strain introductions become the most
dominant ones in a community throughout time. These approaches are necessary in order to review public policies in
the context of a changing epidemiological scenario in Latin America, particularly due to the HAV vaccine introduction
in nine countries from the region in the last few years (Andani et al., 2020). However, in this study, these different
strains could not be detected in wastewater samples, probably due to their low representativeness in the number of
infected individuals excreting these variants into the sewer collection system. In this sense, it is important to highlight
that we performed Sanger sequencing that mainly detects the majority variants of a viral population; thus, this is a
limitation of the study. As NGS has higher sensitivity to detect low-frequency variants, it might be a more effective
methodology to capture more of the variability that might be present in the wastewater samples to identify new viral
introductions from abroad, which could represent a relevant contribution to posterior molecular epidemiology studies.

Most of the reported clinical cases from this study were among young adults, aged between 25 and 37 years, indicating
that this age group is highly susceptible to HAV infection. As a consequence of the recent implementation of socio-
economic and sanitary improvements in our region, there has been an increase in the number of young individuals who
have never been infected during childhood and, therefore, lack natural immunity. Moreover, despite the availability of
pediatric immunization programs since their introduction in Argentina in 2005, many of these young adults may not
have been eligible for inclusion in HAV vaccine programs. According to previous local reports (Dirección de Control
de Enfermedades inmunoprevenibles M de S de A 2021, Dirección de Control de Enfermedades inmunoprevenibles M
de S de A 2020), vaccination coverage among individuals under 17 years old (including those that were vaccinated
from 2005 to 2022) is greater than 75%, and those over 35 years old have >75% of IgG anti-HAV prevalence due to
naturally acquired immunity before vaccination was implemented (Yanez et al., 2014). However, Yanez et al. (Yanez et
al., 2014) and Angeleri et al. (Angeleri et al., 2019) reported an IgG anti-HAV prevalence of less than 59% in young
adults (under 30 years of age) from different regions of Argentina, indicating an increased risk of infection in this
specific group.

In Argentina, vaccination is recommended for people at risk of HAV infection, including men who have sex with men,
individuals with chronic liver disease, and others (Ministerio de Salud de Argentina 2012). Therefore, it is crucial to
emphasize HAV serological data collection derived from IgG anti-HAV antibody testing among young adults within
our region to determine immunity status. Vaccination promotion is essential for susceptible individuals, especially those
with risk factors and/or who are traveling to endemic areas without immunization programs.

To identify specific hotspots of HAV excretion in the city, this study also implemented decentralized sewage monitoring
through HAV detection at multiple local neighborhood collector sewers. This approach complemented the information
obtained from the BG-WWTP, providing a more comprehensive understanding of HAV circulation in the study area.
This surveillance strategy would allow alerting and taking particular control and preventive measures in targeted areas.
Detection of RNA-HAV at BG-WWTP in the absence of HAV detection at the LNCS could probably indicate that the
virus was circulating through one or some of the other 32 neighborhoods of Córdoba city not included in the sampling
of this study. On the contrary, detection of RNA-HAV during some epidemiological weeks at some of the LNCS with
no detection of HAV in BG-WWTP, might be due to a dilution effect. Despite these discrepancies, these results indicate
that HAV monitoring in wastewater in Cordoba is effective, and sampling the central sewer pipe of BG-WWTP is
useful as a representation of the clinical scenario in the city of Córdoba.

Finally, it is worth mentioning that the presence of RNA-HAV in sewage could have a considerable impact on other
environmental matrices. Due to the fragility of the excreta disposal system in our region, raw or inadequately treated
wastewater is often released into surface watercourses used for recreational purposes and/or for irrigation, as it has been
previously studied in the region (Yanez et al., 2014, Blanco Fernández et al., 2012). This situation leads to new
plausible environmental scenarios with the potential for viral transmission to the exposed population.

5 CONLUSIONS
• The results provide evidence that reinforces the importance of wastewater monitoring as a powerful and
effective tool for HAV surveillance in Argentina.

• Multiple advantages of it that were observed in this study can be mentioned: 1- in terms of operability
and efficiency, it is an effective method, that does not require large resources 2- it generates valuable
information in a short period of time (since a single sample summarizes the enteric viral excretion of
many viruses that can be detected simultaneously), 3- it provides an overview of the global
epidemiology of HAV in the community, independently of the report of HA clinical cases (as sewage
monitoring can detect asymptomatic individuals that might not be included in the official notification
system), 4-in some cases it might be useful to anticipate an increase in the number of HA clinical cases,
5- it allows the monitoring of the trends of HAV variants circulation in the community.

• Since there are no current environmental surveillance programs in Argentina, and the epidemiological
surveillance system of clinical cases is often uncertain and problematic, we propose that similar long-
term studies related to WBE should be undertaken in other regions of Argentina, and in other Latin
American countries.

• Offering this tool as a complement to clinical epidemiological surveillance systems would contribute to a
more thorough knowledge of HAV in new post-vaccination epidemiological scenarios.

Funding
This work was supported by the Ministry of Science, Technology and Innovation of Argentina (PICT 2021 CAT-II-
00041-; and CB-2-PFI2022- Res-2022–725-APN-MCT).

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
This study was possible thanks to the collaboration and coordination of the Municipality of the City of Córdoba and the
Ministry of Health of the Province of Córdoba.

Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.watres.2023.12
0102.

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Graphical abstract

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alt-text: Image, graphical abstract

Highlights
• Wastewater monitoring is an effective tool that complements HAV surveillance.

• In this study, WBE constituted a replica of the HAV clinical-epidemiological scenario.

• HAV variants from clinical and sewage samples were phylogenetically correlated.

• In some cases, WBE was useful to predict the rise in hepatitis A clinical reports.

• WBE might contribute to the knowledge of HAV in the post-vaccination era in LATAM.

Appendix Supplementary materials

Multimedia Component 1

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Multimedia Component 2

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Queries and Answers

Q1

Query: Please confirm that given names and surnames have been identified correctly.

Answer: Yes