Prolonged mask wearing does not alter the oral microbiome, salivary flow rate or gingival health status - A pilot study.

The COVID-19 pandemic has resulted in the widespread use of N95 respirators and surgical masks, with anecdotal reports among healthcare providers and the public of xerostomia, halitosis, and gingivitis, a consortium of symptoms colloquially termed "mask mouth". However, this has not been scientifically verified. The aim of this study was to assess changes in salivary flow rate, gingival health status and oral microbiome associated with prolonged mask use. A total of 25 dental students (mean age = 26.36 ± 1.58) were included in the study and evaluated at three time points: T1, at the end of at least 2 months of full-day mask wear (7.26 ± 1.56 hours/day); T2, at the end of a period of minimal mask use (1.13 ± 1.13 hours/day); and T3, at the end of 2-3 weeks of resuming full-day mask wear (6.93 ± 1.80 hours/day). Unstimulated whole saliva (UWS) flow rate, xerostomia (on a quantitative scale of 10), gingival index (GI) and plaque index (PI) were assessed at each time point. The salivary microbiome was characterized using 16S rRNA gene sequencing. Overall, UWS flow rates were normal (mean of 0.679 ml/min) and xerostomia, PI and GI scores were low (Mean of 3.11, 0.33 and 0.69, respectively) with no significant differences as a result of prolonged mask wearing. Similarly, there were no significant microbial changes at a false discovery rate (FDR) ≤ 0.05. However, some trends were identified using a nominal p-value cut-off of ≤ 0.01, namely Gemella sanguinis, Streptococcus sp. Oral taxon 066 and Oral taxon 058 were associated with prolonged mask wear. Trends were also seen by gender, race and age, for example an increase in P. gingivalis and P. intermedia with age. In conclusion, we found no evidence that prolonged mask wear adversely affects oral health. The findings support that the oral microbiome of healthy individuals is resilient.

Mutation profile of SARS-CoV-2 genome in a sample from the first year of the pandemic in Colombia.

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is the etiopathogenic agent of COVID-19, a condition that has led to a formally recognized pandemic by March 2020 (World Health Organization -WHO). The SARS-CoV-2 genome is constituted of 29,903 base pairs, that code for four structural proteins (N, M, S, and E) and more than 20 non-structural proteins. Mutations in any of these regions, especially in those that encode for the structural proteins, have allowed the identification of diverse lineages around the world, some of them named as Variants of Concern (VOC) and Variants of Interest (VOI), according to the WHO and CDC. In this study, by using Next Generation Sequencing (NGS) technology, we sequenced the SARS-CoV-2 genome of 422 samples from Colombian residents, all of them collected between April 2020 and January 2021. We obtained genetic information from 386 samples, leading us to the identification of 14 new lineages circulating in Colombia, 13 of which were identified for the first time in South America. GH was the predominant GISAID clade in our sample. Most mutations were either missense (53.6%) or synonymous mutations (37.4%), and most genetic changes were located in the ORF1ab gene (63.9%), followed by the S gene (12.9%). In the latter, we identified mutations E484K, L18F, and D614G. Recent evidence suggests that these mutations concede important particularities to the virus, compromising host immunity, the diagnostic test performance, and the effectiveness of some vaccines. Some important lineages containing these mutations are the Alpha, Beta, and Gamma (WHO Label). Further genomic surveillance is important for the understanding of emerging genomic variants and their correlation with disease severity.

Genomic characterization of SARS-CoV-2 and its association with clinical outcomes: a 1-year longitudinal study of the pandemic in Colombia.

OBJECTIVES: This study aimed to explore associations between the molecular characterization of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and disease severity in ambulatory and hospitalized patients in two main Colombian epicentres during the first year of the coronavirus disease 2019 pandemic. METHODS: In total, 1000 patients with SARS-CoV-2 infection were included in this study. Clinical data were collected from 997 patients, and 678 whole-genome sequences were obtained by massively parallel sequencing. Bivariate, multi-variate, and classification and regression tree analyses were run between clinical and genomic variables. RESULTS: Age >88 years, and infection with lineages B.1.1, B.1.1.388, B.1.523 or B.1.621 for patients aged 71-88 years were associated with death [odds ratio (OR) 6.048036, 95% confidence interval (CI) 1.346567-32.92521; P=0.01718674]. The need for hospitalization was associated with higher age and comorbidities. The hospitalization rate increased significantly for patients aged 38-51 years infected with lineages A, B, B.1.1.388, B.1.1.434, B.1.153, B.1.36.10, B.1.411, B.1.471, B.1.558 or B.1.621 (OR 8.368427, 95% CI 2.573145-39.10672, P=0.00012). Associations between clades and clinical outcomes diverged from previously reported data. CONCLUSIONS: Infection with lineage B.1.621 increased the hospitalization and mortality rates. These findings, plus the rapidly increasing prevalence in Colombia and other countries, suggest that lineage B.1.621 should be considered as a 'variant of interest'. If associated disease severity is confirmed, possible designation as a 'variant of concern' should be considered.

Wastewater-Based Epidemiology and Long-Read Sequencing to Identify Enterovirus Circulation in Three Municipalities in Maricopa County, Arizona, Southwest United States between June and October 2020.

We used wastewater-based epidemiology and amplicon-based long-read high-throughput sequencing for surveillance of enteroviruses (EVs) in Maricopa County, Arizona, Southwest United States. We collected 48 samples from 13 sites in three municipalities between 18 June and 1 October 2020, and filtered (175 mL each; 0.45 µm pore size) and extracted RNA from the filter-trapped solids. The RNA was converted to cDNA and processed through two workflows (Sanger sequencing (SSW) and long-read Illumina sequencing (LRISW)) each including a nested polymerase chain reaction (nPCR) assay. We subjected the ~350 bp amplicon from SSW to Sanger sequencing and the ~1900-2400 bp amplicon from LRISW to Illumina sequencing. We identified EV contigs from 11 of the 13 sites and 41.67% (20/48) of screened samples. Using the LRISW, we detected nine EV genotypes from three species (Enterovirus A (CVA4, EV-A76, EV-A90), Enterovirus B (E14) and Enterovirus C (CVA1, CVA11, CVA13, CVA19 and CVA24)) with Enterovirus C representing approximately 90% of the variants. However, the SSW only detected the five Enterovirus C types. Similarity and phylogenetic analysis showed that multiple Enterovirus C lineages were circulating, co-infecting and recombining in the population during the season despite the SARS-CoV-2 pandemic and the non-pharmaceutical public health measures taken to curb transmission.

Predominance of the SARS-CoV-2 Lineage P.1 and Its Sublineage P.1.2 in Patients from the Metropolitan Region of Porto Alegre, Southern Brazil in March 2021.

Almost a year after the COVID-19 pandemic had begun, new lineages (B.1.1.7, B.1.351, P.1, and B.1.617.2) associated with enhanced transmissibility, immunity evasion, and mortality were identified in the United Kingdom, South Africa, and Brazil. The previous most prevalent lineages in the state of Rio Grande do Sul (RS, Southern Brazil), B.1.1.28 and B.1.1.33, were rapidly replaced by P.1 and P.2, two B.1.1.28-derived lineages harboring the E484K mutation. To perform a genomic characterization from the metropolitan region of Porto Alegre, we sequenced viral samples to: (i) identify the prevalence of SARS-CoV-2 lineages in the region, the state, and bordering countries/regions; (ii) characterize the mutation spectra; (iii) hypothesize viral dispersal routes by using phylogenetic and phylogeographic approaches. We found that 96.4% of the samples belonged to the P.1 lineage and approximately 20% of them were assigned as the novel P.1.2, a P.1-derived sublineage harboring signature substitutions recently described in other Brazilian states and foreign countries. Moreover, sequences from this study were allocated in distinct branches of the P.1 phylogeny, suggesting multiple introductions in RS and placing this state as a potential diffusion core of P.1-derived clades and the emergence of P.1.2. It is uncertain whether the emergence of P.1.2 and other P.1 clades is related to clinical or epidemiological consequences. However, the clear signs of molecular diversity from the recently introduced P.1 warrant further genomic surveillance.

Mutation hotspots and spatiotemporal distribution of SARS-CoV-2 lineages in Brazil, February 2020-2021.

The COVID-19 pandemic has already reached more than 110 million people and is associated with 2.5 million deaths worldwide. Brazil is the third worst-hit country, with approximately 10.2 million cases and 250 thousand deaths. International efforts have been established to share information about Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemiology and evolution to support the development of effective strategies for public health and disease management. We aimed to analyze the high-quality genome sequences from Brazil from February 2020-2021 to identify mutation hotspots, geographical and temporal distribution of SARS-CoV-2 lineages by using phylogenetics and phylodynamics analyses. We describe heterogeneous sequencing efforts, the progression of the different lineages along time, evaluating mutational spectra and frequency oscillations derived from the prevalence of specific lineages across different Brazilian regions. We found at least seven major (1-7) and two minor clades related to the six most prevalent lineages in the country and described its spatial distribution and dynamics. The emergence and recent frequency shift of lineages (P.1 and P.2) carrying mutations of concern in the spike protein (e. g., E484K, N501Y) draws attention due to their association with immune evasion and enhanced receptor binding affinity. Improvements in genomic surveillance are of paramount importance and should be extended in Brazil to better inform policy makers about better decisions to fight the COVID-19 pandemic.

Isolation and characterization of an early SARS-CoV-2 isolate from the 2020 epidemic in Medellín, Colombia / [Aislamiento y caracterización de una cepa temprana de SARS-CoV-2 durante la epidemia de 2020 en Medellín, Colombia].

Introduction: SARS-CoV-2 has been identified as the new coronavirus causing an outbreak of acute respiratory disease in China in December, 2019. This disease, currently named COVID-19, has been declared as a pandemic by the World Health Organization (WHO). The first case of COVID-19 in Colombia was reported on March 6, 2020. Here we characterize an early SARS-CoV-2 isolate from the pandemic recovered in April, 2020. Objective: To describe the isolation and characterization of an early SARS-CoV-2 isolate from the epidemic in Colombia. Materials and methods: A nasopharyngeal specimen from a COVID-19 positive patient was inoculated on different cell lines. To confirm the presence of SARS-CoV-2 on cultures we used qRT-PCR, indirect immunofluorescence assay, transmission and scanning electron microscopy, and next-generation sequencing. Results: We determined the isolation of SARS-CoV-2 in Vero-E6 cells by the appearance of the cytopathic effect three days post-infection and confirmed it by the positive results in the qRT-PCR and the immunofluorescence with convalescent serum. Transmission and scanning electron microscopy images obtained from infected cells showed the presence of structures compatible with SARS-CoV-2. Finally, a complete genome sequence obtained by next-generation sequencing allowed classifying the isolate as B.1.5 lineage. Conclusion: The evidence presented in this article confirms the first isolation of SARSCoV-2 in Colombia. In addition, it shows that this strain behaves in cell culture in a similar way to that reported in the literature for other isolates and that its genetic composition is consistent with the predominant variant in the world. Finally, points out the importance of viral isolation for the detection of neutralizing antibodies, for the genotypic and phenotypic characterization of the strain and for testing compounds with antiviral potential.

Introducción. El nuevo coronavirus causante de un brote de enfermedad respiratoria aguda en China en diciembre de 2019 se identificó como SARS-CoV-2. La enfermedad, denominada COVID-19, fue declarada pandemia por la Organización Mundial de la Salud (OMS). El primer caso de COVID-19 en Colombia se reportó el 6 de marzo de 2020; en este estudio se caracterizó un aislamiento temprano del virus SARS-CoV-2 de una muestra ecolectada en abril de 2020. Objetivos. Describir y caracterizar una cepa temprana a partir de un aislamiento de SARSCoV-2 durante la pandemia en Colombia. Materiales y métodos. Se obtuvo una muestra de un paciente con COVID-19 confirmada por qRT-PCR; la muestra fue inoculada en diferentes líneas celulares hasta la aparición del efecto citopático. Para confirmar la presencia de SARS-CoV-2 en el cultivo, se utilizó la qRT-PCR a partir de los sobrenadantes, la inmunofluorescencia indirecta (IFI) en células Vero-E6, así como microscopía electrónica y secuenciación de nueva generación (nextgeneration sequencing). Resultados. Se confirmó el aislamiento de SARS-CoV-2 en células Vero-E6 por la aparición del efecto citopático tres días después de la infección, así como mediante la qRT-PCR y la IFI positiva con suero de paciente convaleciente positivo para SARS-CoV-2. Además, en las imágenes de microscopía electrónica de trasmisión y de barrido de células infectadas se observaron estructuras compatibles con viriones de SARS-CoV-2. Por último, se obtuvo la secuencia completa del genoma, lo que permitió clasificar el aislamiento como linaje B.1.5. Conclusiones. La evidencia presentada en este artículo permite confirmar el primer aislamiento de SARS-CoV-2 en Colombia. Además, muestra que esta cepa se comporta en cultivo celular de manera similar a lo reportado en la literatura para otros aislamientos y que su composición genética está acorde con la variante predominante en el mundo. Finalmente, se resalta la importancia que tiene el aislamiento viral para la detección de anticuerpos, para la caracterización genotípica y fenotípica de la cepa y para probar compuestos con potencial antiviral.

SARS-CoV-2 sequencing: The technological initiative to strengthen early warning systems for public health emergencies in Latin America and the Caribbean / [Secuenciación del SARS-CoV-2: la iniciativa tecnológica para fortalecer los sistemas de alerta temprana ante emergencias de salud pública en Latinoamérica y el Caribe].

The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem on a scale unprecedented in the last 100 years, as has been the response focused on the rapid genomic characterization of SARS-CoV-2 in virtually all regions of the planet. This pandemic emerged during the era of genomic epidemiology, a science fueled by continued advances in next-generation sequencing. Since its recent appearance, genomic epidemiology included the precise identification of new lineages or species of pathogens and the reconstruction of their genetic variability in real time, evidenced in past outbreaks of influenza H1N1, MERS, and SARS. However, the global and uncontrolled scale of this pandemic created a scenario where genomic epidemiology was put into practice en masse, from the rapid identification of SARS-CoV-2 to the registration of new lineages and their active surveillance throughout the world. Prior to the COVID-19 pandemic, the availability of genomic data on circulating pathogens in several Latin America and the Caribbean countries was scarce or nil. With the arrival of SARS-CoV-2, this scenario changed significantly, although the amount of available information remains scarce and, in countries such as Colombia, Brazil, Argentina, and Chile, the genomic information of SARS-CoV-2 was obtained mainly by research groups in genomic epidemiology rather than the product of a public health surveillance policy or program. This indicates the need to establish public health policies aimed at implementing genomic epidemiology as a tool to strengthen surveillance and early warning systems against threats to public health in the region.

La pandemia de COVID-19 causada por el SARS-CoV-2 es un problema de salud pública sin precedentes en los últimos 100 años, así como la respuesta centrada en la caracterización genómica del SARS-CoV-2 prácticamente en todas las regiones del planeta. Esta pandemia surgió durante la era de la epidemiología genómica impulsada por los continuos avances en la secuenciación de próxima generación. Desde su reciente aparición, la epidemiología genómica permitió la identificación precisa de nuevos linajes o especies de agentes patógenos y la reconstrucción de su variabilidad genética en tiempo real, lo que se hizo evidente en los brotes de influenza H1N1, MERS y SARS. Sin embargo, la escala global y descontrolada de esta pandemia ha generado una situación que obligó a utilizar de forma masiva herramientas de la epidemiología genómica como la rápida identificación del SARS-CoV-2 y el registro de nuevos linajes y su vigilancia activa en todo el mundo. Antes de la pandemia de COVID-19 la disponibilidad e datos genómicos de agentes patógenos circulantes en varios países de Latinoamérica y el Caribe era escasa o nula. Con la llegada del SARS-CoV-2 dicha situación cambió significativamente, aunque la cantidad de información disponible sigue siendo escasa y, en países como Colombia, Brasil, Argentina y Chile, la información genómica del SARS-CoV-2 provino principalmente de grupos de investigación en epidemiología genómica más que como producto de una política o programa de vigilancia en salud pública.

Rapid, Sensitive, Full-Genome Sequencing of Severe Acute Respiratory Syndrome Coronavirus 2.

We describe validated protocols for generating high-quality, full-length severe acute respiratory syndrome coronavirus 2 genomes from primary samples. One protocol uses multiplex reverse transcription PCR, followed by MinION or MiSeq sequencing; the other uses singleplex, nested reverse transcription PCR and Sanger sequencing. These protocols enable sensitive virus sequencing in different laboratory environments.