Sewage surveillance can be used as an effective complementary tool for detecting pathogens in local communities, providing insights into emerging threats and aiding in the monitoring of outbreaks. In this study using qPCR and whole genomic sewage surveillance, we detected the Mpox virus along with other viruses, in municipal and hospital wastewaters in Belo Horizonte, Brazil over a 9-month period (from July 2022 until March 2023). MPXV DNA detection rates varied in our study, with 19.6% (11 out of 56 samples) detected through the hybrid capture method of whole-genome sequencing and 20% (12 out of 60 samples) through qPCR. In hospital wastewaters, the detection rate was higher, at 40% (12 out of 30 samples) compared to 13.3% (4 out of 30 samples) in municipal wastewaters. This variation could be attributed to the relatively low number of MPXV cases reported in the city, which ranged from 106 to 341 cases during the study period, and the dilution effects, given that each of the two wastewater treatment plants (WWTP) investigated serves approximately 1.1 million inhabitants. Additionally, nine other virus families were identified in both hospitals and municipal wastewaters, including Adenoviridade, Astroviridae, Caliciviridae, Picornaviridade, Polyomaviridae, Coronaviridae (which includes SARS-CoV-2), Herspesviridae, Papillomaviridae and Flaviviridae (notably including Dengue). These findings underscore the potential of genomic sewage surveillance as a robust public health tool for monitoring a wide range of viruses circulating in both community and hospitals environments, including MPXV.

This study evaluated and compared molecular methods (Whole Genome Sequencing-WGS, MinION, and RT-qPCR) for the detection of arboviruses Dengue (DENV), Chikungunya (CHIKV), and Zika (ZIKV) in 63 hospital and municipal wastewater samples collected from July 2022 to May 2023 in the region of Belo Horizonte, Brazil. Detection rates varied substantially across the methods (WGS, MinION, and RT-qPCR). DENV was identified in 24% (15/63) of samples using a hybrid capture method of WGS and MinION sequencing and in 66.6% (20/30) using only WGS but was not detected using the CDC Trioplex RT-PCR Assay Kit or ZDC (IBMP). CHIKV was detected in 19.0% (12/63) of the samples by WGS and MinION and in 85.7% (12/14) using only MinION sequencing. Using the RT-qPCR kit to detect CHIKV yielded a rate of 4.7% (3/63) in false positives. ZIKV was found in only one sample (1/63) by WGS, while RT-qPCR yielded a high false positive rate (65.1%, 41/63). These findings highlight the operational advantage of these methods (WGS and MinION) for enhancing early-warning surveillance where standard RT-qPCR might underperform in low-prevalence settings. This is the first study that has compared these methods to detect and genetically characterize DENV, CHICK, and ZIKV in wastewater in Brazil and has indicated that hospital wastewater can be used as a sentinel system for arbovirus surveillance. The relative effectiveness of genomic wastewater surveillance for arboviruses was demonstrated, and it was found that diagnostic RT-qPCR kits used for clinical samples were not directly suitable for environmental surveillance. The feasibility of arbovirus wastewater surveillance as an epidemiological tool was demonstrated, although absolute quantifications were not performed.

Sewage surveillance can be used as an effective complementary tool for detecting pathogens in local communities, providing insights into emerging threats and aiding in the monitoring of outbreaks. In this study using qPCR and whole genomic sewage surveillance, we detected Mpox virus along with other viruses, in municipal and hospital wastewaters in Belo Horizonte, Brazil over a 9 months period (from July 2022 till March 2023). MPXV DNA detection rates varied in our study, with 19.6% (11 out of 56 samples) detected through the hybrid capture method of whole genome sequencing and 20% (12 out of 60 samples) through qPCR. In hospital wastewaters, the detection rate was higher at 40% (12 out of 30 samples), compared to 13.3%(4 out of 30 samples) in municipal wastewaters. This variation could be attributed to the relatively low number of MPXV cases reported in the city, which ranged from 106 to 341 cases during the study period, and to dilution effects, given that each of the two wastewater treatment plants (WWTP) investigated serves approximately 1.1 million inhabitants. Additionally, nine other virus families were identified in both hospitals and municipal wastewaters including Adenoviridade, Astroviridae, Caliciviridae, Picornaviridade, Polyomaviridae, Coronaviridae (which includes SARS-CoV-2), Herspesviridae, Papillomaviridae and Flaviviridae (notably including Dengue). These findings underscore the potential of genomic sewage surveillance as a robust public health tool for monitoring a wide range of viruses circulating in both community and hospitals environments including MPXV.

Airports are strategic targets for wastewater-based epidemiology because they concentrate highly mobile populations and may provide early signals of pathogen circulation. However, metagenomic investigations of airport wastewater remain limited, particularly in South America. Here, we present one of the first hybrid-capture target-enriched metagenomic investigations of airport wastewater in Brazil, integrating the detection of human-associated viruses and bacteriophage-derived host signatures to evaluate airports as sentinel surveillance sites. Seven untreated wastewater samples collected from a major Brazilian airport between December 2021 and March 2023 were concentrated, subjected to nucleic acid extraction, and analyzed using hybrid-capture target-enriched next-generation sequencing. Taxonomic analysis identified 615 viral and bacteriophage-associated taxa, including 440 viruses and 175 bacteriophages. Among the viral fraction, 21 human-associated viral taxa representing eight viral families were selected for detailed analysis. Norovirus GII was detected in all samples, while Mamastrovirus 1 and JC polyomavirus were detected in six of seven samples. SARS-CoV-2 and dengue virus type 1 were simultaneously detected in the March, 2023 sample. The bacteriophage fraction comprised 47 host-associated phage groups, with Streptococcus-associated phages predominating across samples. These findings demonstrate that airport wastewater can capture diverse human viral and bacteriophage-derived signatures associated with population mobility, supporting its application in environmental genomic surveillance and early-warning systems for emerging and circulating pathogens.

This study evaluated and compared molecular methods for the detection of arboviruses Dengue (DENV), Chikungunya (CHIKV), and Zika (ZIKV) in wastewaters collected from July 2022 to May 2023 in the region of Belo Horizonte, Brazil. Detection rates varied substantially across the methods. DENV was identified in 24% (15/63) of samples using hybrid capture method of whole-genome sequencing (WGS) and MinION sequencing, in 66.6% (20/30) using only WGS, but was not detected using the CDC Trioplex RT-PCR Assay Kit and ZDC (IBMP). CHIKV was detected in 19.0% (12/63) of the samples by WGS and MinION, in 85.7% (12/14) using only MinION sequencing, and in 4.7% (3/63) by RT-PCR. ZIKV was found in only one sample (1/63) by WGS, while RT-qPCR yielded a high false-positive rate (65.1%, 41/63). The potential of genomic wastewater surveillance for arboviruses was demonstrated and that diagnostic RT-qPCR kits used for clinical samples were not directly suitable for environmental surveillance.