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Brazil's COVID-19 response has faced challenges due to the continuous emergence of variants of concern (VOCs), emphasizing the need for ongoing genomic surveillance and retrospective analyses of past epidemic waves to reassess and fine tune containment protocols. Rio Grande do Sul (RS), Brazil's southernmost state, has international borders and trades with Argentina and Uruguay, along with significant domestic connections within Brazil. The identification of source and sink transmission chains at national and international scales can identify main hubs and pathways to target future interventions. In this study we investigated the RS state role in the national and international SARS-CoV-2 transmission chains, which has not been fully explored. Nasopharyngeal samples from various municipalities in RS were collected between June 2020 and July 2022. SARS-CoV-2 whole genome amplification and sequencing were performed using high-throughput Illumina sequencing. Bioinformatics analysis encompassed the development of scripts and tools to perform subsampling taking into account epidemiological information to reduce sequencing disparities bias among the regions/countries, genome assembly, and large-scale alignment and phylogenetic reconstruction. We sequenced a total of 1,480 SARS-CoV-2 genomes from RS, covering all major regions. Sequences predominantly represented Gamma (April-June 2021) and Omicron (January-July 2022) lineages. Phylogenetic analysis revealed a regional pattern for transmission dynamics, particularly with Southeast Brazil for Gamma, and a range of inter-regional connections for Delta and Omicron within the country. On the other hand, international and cross-border transmission with Argentina and Uruguay was rather limited. We evaluated the three VOCs circulation over two years in RS using a new subsampling strategy based on the number of cases in each state during the circulation of each VOC. In summary, the retrospective analysis of genomic surveillance data demonstrated that virus transmission was less intense between country borders than within the country. These findings suggest that while non-pharmacological interventions were effective to mitigate transmission across international RS land borders, they were insufficient to contain transmission at the domestic level., Competing Interests: 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., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The multiplex molecular diagnostic assays described for severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), influenza A (IAV) and B (IBV) viruses have been mainly based on real-time reaction, which limits their access to many laboratories or diagnostic institutions. To contribute to available strategies and expand access to differential diagnosis, we describe an end-point multiplex RT-PCR targeting SARS-CoV-2, IAV and IBV with simultaneous endogenous control amplification. Initially, we looked for well-established primers sets for SARS-CoV-2, IAV, IBV and RNAse P whose amplicons could be distinguished on agarose gel. The multiplex assay was then standardized by optimizing the reaction mix and cycle conditions. The limit of detection (LoD) was determined using titrated viruses (for SARS-CoV-2 and IAV) and by dilution from a pool of IBV-positive samples. The diagnostic performance of the multiplex was evaluated by testing samples with different RNAse P and viral loads, previously identified as positive or negative for the target viruses. The amplicons of IAV (146 bp), SARS-CoV-2 (113 bp), IBV (103 bp) and RNAse P (65 bp) were adequately distinguished in our multiplex. The LoD for SARS-CoV-2, IAV and IBV was 0.02 TCID 50 /ml, 0.07 TCID 50 /ml and 10 -3 from a pool of positive samples, respectively. All samples positive for SARS-CoV-2 ( n =70, Ct 17.2-36.9), IAV ( n =53, Ct 14-34.9) and IBV ( n =12, Ct 23.9-31.9) remained positive in our multiplex assay. RNAse P from negative samples ( n =40, Ct 25.2-30.2) was also amplified in the multiplex. Overall, our assay is a timely and alternative tool for detecting SARS-CoV-2 and influenza viruses in laboratories with limited access to supplies/equipment.

The overwhelming majority of SARS-CoV-2 epidemiological studies cover a narrow time period, making general knowledge about the COVID-19 pandemic difficult. To assess COVID-19-related host aspects in the overall pandemic, we analyzed COVID-19 cases during the first two years of SARS-CoV-2 circulation in southern Brazil. Herein, 390 patients admitted in 2020-2022 to a Brazilian public referral hospital were allocated into two groups according to the COVID-19 outcome: hospital discharge (n=237) or death (n=153). In the univariate analysis, several variables, including sociodemographic, clinical and laboratory aspects (primary data), were significantly different between the analyzed groups. In multivariate logistic regression, eight of these factors remained associated with the COVID-19 outcome. In particular, we report oxygen supplementation and the need for hemodialysis as predictors of hospital discharge and death from COVID-19, respectively. To the best of our knowledge, none of these findings have been previously reported in the Brazilian or world population. In conclusion, our results contribute to current knowledge by demonstrating that factors described at different times may remain associated with COVID-19 over the pandemic and by identifying novel predictors of COVID-19 outcome., Competing Interests: 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., (Copyright © 2023 Elsevier B.V. All rights reserved.)