Wastewater surveillance is considered a promising approach for COVID-19 surveillance in communities. In this study, we collected wastewater samples between November 2020 and February 2022 from twenty-three sites in the Bangkok Metropolitan Region to detect the presence of SARS-CoV-2 and its variants for comparison to standard clinical sampling. A total of 215 wastewater samples were collected and tested for SARS-CoV-2 RNA by real-time PCR with three targeted genes (N, E, and ORF1ab); 102 samples were positive (42.5%). The SARS-CoV-2 variants were determined by a multiplex PCR MassARRAY assay to distinguish four SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Multiple variants of Alpha–Delta and Delta–Omicron were detected in the wastewater samples in July 2021 and January 2022, respectively. These wastewater variant results mirrored the country data from clinical specimens deposited in GISAID. Our results demonstrated that wastewater surveillance using multiple signature mutation sites for SARS-CoV-2 variant detection is an appropriate strategy to monitor the presence of SARS-CoV-2 variants in the community at a low cost and with rapid turn-around time. However, it is essential to note that sequencing surveillance of wastewater samples should be considered complementary to whole genome sequencing of clinical samples to detect novel variants.

The emergence of Omicron as the fifth variant of concern within the SARS-CoV-2 pandemic in late 2021, characterized by its rapid transmission and distinct spike gene mutations, underscored the pressing need for cost-effective and efficient methods to detect viral variants, especially given their evolving nature. This study sought to address this need by assessing the effectiveness of two SARS-CoV-2 variant classification platforms based on RT-PCR and mass spectrometry. The primary aim was to differentiate between Delta, Omicron BA.1, and Omicron BA.2 variants using 618 COVID-19-positive samples collected from Bangkok patients between November 2011 and March 2022. The analysis revealed that both BA.1 and BA.2 variants exhibited significantly higher transmission rates, up to 2-3 times, when compared to the Delta variant. This research presents a cost-efficient approach to virus surveillance, enabling a quantitative evaluation of variant-specific public health implications, crucial for informing and adapting public health strategies.

Wastewater-based epidemiology is a valuable tool for population-level pathogen surveillance, complementing clinical methods. While most sampling focuses on municipal wastewater treatment plants, emerging evidence suggests wastewater collected from hospital settings can lead to targeted clinical interventions. To investigate wastewater pathogen surveillance in hospital settings further, we tracked the presence and concentration of SARS-CoV-2 RNA in wastewater across multi-scale sample sites within a large, public tertiary care hospital in Bangkok, Thailand. From July 2022 to May 2023, weekly wastewater samples (n=392) were collected from various sample sites including clinical and non-clinical facilities, as well as the hospital’s wastewater treatment plant. Influent wastewater at the hospital’s wastewater treatment center yielded the most consistent SARS-CoV-2 RNA detection across all sample sites, with detection in all 26 samples. Despite varied building usage patterns, significant moderate negative correlations were found in 90% (9/10) of sample sites between wastewater RT-PCR cycle threshold values and clinical case data from hospital and national reports. Targeting specific buildings yielded distinct data trends, indicating their potential to offer complementary insights into viral shedding and transmission among clinical and non-clinical sub-populations within a hospital campus. Our findings suggest that hospital wastewater-based epidemiology reflects broader community disease trends, which may be especially useful in regions with limited municipal wastewater treatment coverage. Large tertiary care hospitals could serve as effective and cost-efficient sentinel surveillance sites for future pathogen monitoring, guiding public health actions.

Omicron emerged as the fifth variant of concern of SARS-CoV-2 coronavirus pandemic in late 2021 and rapidly overtook the previously predominant Delta variants with a significantly faster transmission rate and unique mutations on the spike gene. Hence, the ability to identify viral variants rapidly and affordably in large number of patients, which facilitates the monitoring of the transmission and clinical impact of new variants, is needed to obtain information for updating the public health policy. In this study, we evaluated the capability of two RT-PCR and mass spectrometry-based SARS-CoV-2 variant classification platforms to distinguish Delta, Omicron BA.1, and Omicron BA.2 variants in 618 COVID-19-positive samples from patients in Bangkok collected during November 2011-March 2022. Analysis of the time-evolution pattern of SARS-CoV-2 variant profiles indicated that the BA.1 and BA.2 possess up to 2-3 times higher transmission rates than the Delta variant. Our study showcases a cost-effective virus surveillance that enables a quantitative estimation of variant-specific public health impact.

Background: The 2022 MPXV global health emergency highlighted the necessity for rapid diagnostic measures in response to mpox clade IIb outbreaks. This study investigates the mpox lineages identified in Thailand during this period and explores the transmission dynamics of dominant lineages. Methods: Using targeted next-generation sequencing from 162 individuals infected with mpox, we created a transmission network map by linking high-similarity sequences through molecular-temporal distance matrices. Results: Our findings reveal that the study population aligns with global trends of transmission almost exclusively within the community of men who have sex with men, with a majority of cases resulting from lineage C viruses as well as 8 additional sublineages. Within the observed substitutions, a majority of mutations were either G→A (n = 289, 49.74%) or C→T (n = 221, 38.04%), indicating APOBEC-3–driven deaminase activity. We also present a global transmission network for lineage C mpox genomes, illustrating domestic and international spread. Conclusions: This research underscores the significance of whole genome sequencing in diagnostics during outbreaks and the importance of sharing genomic data to enhance our understanding of disease transmission on a global scale.

Proactive surveillance of wildlife has the potential to help prevent pandemics caused by coronaviruses spilling over into human populations, but the relative value of this approach versus early outbreak investigation has been debated. This study investigated bat coronavirus zoonotic potential in a high human-wildlife interaction site in Ratchaburi, Thailand. We sampled 261 Rhinolophus pusillus bats, a known reservoir for sarbecoviruses, and detected diverse alpha- and betacoronaviruses (prevalence = 16.47%), including SARS-CoV-2-related coronaviruses, alongside paramyxoviruses at a lower prevalence (5.36%). Notably, the detected paramyxoviruses were phylogenetically distinct from previously described paramyxoviruses, suggesting the presence of previously uncharacterized viral diversity. Five complete SARS-CoV-2-related coronavirus genomes were obtained, showing significant divergence in spike and ORF8 regions and diverse recombination patterns. One identified SARS-CoV-2-related coronavirus, RpuRB21065, exhibited high spike and receptor-binding domain similarities to SARS-CoV-2. The receptor-binding domain demonstrated binding to human ACE2 and the spike protein induced syncytia in vitro. However, human sera from SARS-CoV-2 vaccinated individuals effectively neutralized RpuRB21065, suggesting pre-existing SARS-CoV-2 immunity may minimize current spillover potential. Our study is a proof-of-concept that proactive viral discovery and wildlife surveillance in EID hotspots can provide information of direct public health relevance, even in the absence of viral isolation.