Significance of wastewater surveillance in detecting the prevalence of SARS-CoV-2 variants and other respiratory viruses in the community - A multi-site evaluation.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral genome in wastewater has proven to be useful for tracking the trends of virus prevalence within the community. The surveillance also provides precise and early detection of any new and circulating variants, which aids in response to viral outbreaks. Site-specific monitoring of SARS-CoV-2 variants provides valuable information on the prevalence of new or emerging variants in the community. We sequenced the genomic RNA of viruses present in the wastewater samples and analyzed for the prevalence of SARS-CoV-2 variants as well as other respiratory viruses for a period of one year to account for seasonal variations. The samples were collected from the Reno-Sparks metropolitan area on a weekly basis between November 2021 to November 2022. Samples were analyzed to detect the levels of SARS-CoV-2 genomic copies and variants identification. This study confirmed that wastewater monitoring of SARS-CoV-2 variants can be used for community surveillance and early detection of circulating variants and supports wastewater-based epidemiology (WBE) as a complement to clinical respiratory virus testing as a healthcare response effort. Our study showed the persistence of the SARS-CoV-2 virus throughout the year compared to a seasonal presence of other respiratory viruses, implicating SARS-CoV-2's broad genetic diversity and strength to persist and infect susceptible hosts. Through secondary analysis, we further identified antimicrobial resistance (AMR) genes in the same wastewater samples and found WBE to be a feasible tool for community AMR detection and monitoring.

Detecting SARS-CoV-2 variants in wastewater and their correlation with circulating variants in the communities.

Detection of SARS-CoV-2 viral load in wastewater has been highly informative in estimating the approximate number of infected individuals in the surrounding communities. Recent developments in wastewater monitoring to determine community prevalence of COVID-19 further extends into identifying SARS-CoV-2 variants, including those being monitored for having enhanced transmissibility. We sequenced genomic RNA derived from wastewater to determine the variants of coronaviruses circulating in the communities. Wastewater samples were collected from Truckee Meadows Water Reclamation Facility (TMWRF) from November 2020 to June 2021. SARS-CoV-2 variants resulting from wastewater were compared with the variants detected in infected individuals' clinical specimens (nasal/nasopharyngeal swabs) during the same period and found conclusively in agreement. Therefore, wastewater monitoring for SARS-CoV-2 variants in the community is a feasible strategy as a complementary tool to clinical specimen testing in the latter's absence.

Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene.

Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs. To determine the variants of SARS-CoV-2 circulating in the state of Nevada, specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform, which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads, without the need of culture-based amplification. High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak. We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA-dependent RNA polymerase) changed to phenylalanine (F) from proline (P), in the first reported isolate of SARS-CoV-2, Wuhan-Hu-1. This 323F variant was present at a very high frequency in Northern Nevada. Structural modeling determined this mutation in the interface domain, which is important for the association of accessory proteins required for the polymerase. In conclusion, we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations, which is important for understanding the evolution and circulation of SARS-CoV-2 variants of public health importance, while it circulates in humans.

Genomic evidence for reinfection with SARS-CoV-2: a case study.

BACKGROUND: The degree of protective immunity conferred by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently unknown. As such, the possibility of reinfection with SARS-CoV-2 is not well understood. We describe an investigation of two instances of SARS-CoV-2 infection in the same individual. METHODS: A 25-year-old man who was a resident of Washoe County in the US state of Nevada presented to health authorities on two occasions with symptoms of viral infection, once at a community testing event in April, 2020, and a second time to primary care then hospital at the end of May and beginning of June, 2020. Nasopharyngeal swabs were obtained from the patient at each presentation and twice during follow-up. Nucleic acid amplification testing was done to confirm SARS-CoV-2 infection. We did next-generation sequencing of SARS-CoV-2 extracted from nasopharyngeal swabs. Sequence data were assessed by two different bioinformatic methodologies. A short tandem repeat marker was used for fragment analysis to confirm that samples from both infections came from the same individual. FINDINGS: The patient had two positive tests for SARS-CoV-2, the first on April 18, 2020, and the second on June 5, 2020, separated by two negative tests done during follow-up in May, 2020. Genomic analysis of SARS-CoV-2 showed genetically significant differences between each variant associated with each instance of infection. The second infection was symptomatically more severe than the first. INTERPRETATION: Genetic discordance of the two SARS-CoV-2 specimens was greater than could be accounted for by short-term in vivo evolution. These findings suggest that the patient was infected by SARS-CoV-2 on two separate occasions by a genetically distinct virus. Thus, previous exposure to SARS-CoV-2 might not guarantee total immunity in all cases. All individuals, whether previously diagnosed with COVID-19 or not, should take identical precautions to avoid infection with SARS-CoV-2. The implications of reinfections could be relevant for vaccine development and application. FUNDING: Nevada IDEA Network of Biomedical Research, and the National Institute of General Medical Sciences (National Institutes of Health).