Metagenomic pipeline for identifying co-infections among distinct SARS-CoV-2 variants of concern: study cases from Alpha to Omicron.

Concomitant infection or co-infection with distinct SARS-CoV-2 genotypes has been reported as part of the epidemiological surveillance of the COVID-19 pandemic. In the context of the spread of more transmissible variants during 2021, co-infections are not only important due to the possible changes in the clinical outcome, but also the chance to generate new genotypes by recombination. However, a few approaches have developed bioinformatic pipelines to identify co-infections. Here we present a metagenomic pipeline based on the inference of multiple fragments similar to amplicon sequence variant (ASV-like) from sequencing data and a custom SARS-CoV-2 database to identify the concomitant presence of divergent SARS-CoV-2 genomes, i.e., variants of concern (VOCs). This approach was compared to another strategy based on whole-genome (metagenome) assembly. Using single or pairs of sequencing data of COVID-19 cases with distinct SARS-CoV-2 VOCs, each approach was used to predict the VOC classes (Alpha, Beta, Gamma, Delta, Omicron or non-VOC and their combinations). The performance of each pipeline was assessed using the ground-truth or expected VOC classes. Subsequently, the ASV-like pipeline was used to analyze 1021 cases of COVID-19 from Costa Rica to investigate the possible occurrence of co-infections. After the implementation of the two approaches, an accuracy of 96.2% was revealed for the ASV-like inference approach, which contrasts with the misclassification found (accuracy 46.2%) for the whole-genome assembly strategy. The custom SARS-CoV-2 database used for the ASV-like analysis can be updated according to the appearance of new VOCs to track co-infections with eventual new genotypes. In addition, the application of the ASV-like approach to all the 1021 sequenced samples from Costa Rica in the period October 12th-December 21th 2021 found that none corresponded to co-infections with VOCs. In conclusion, we developed a metagenomic pipeline based on ASV-like inference for the identification of co-infection with distinct SARS-CoV-2 VOCs, in which an outstanding accuracy was achieved. Due to the epidemiological, clinical, and molecular relevance of the concomitant infection with distinct genotypes, this work represents another piece in the process of the surveillance of the COVID-19 pandemic in Costa Rica and worldwide.

SARS-CoV-2 genomic surveillance in Costa Rica: Evidence of a divergent population and an increased detection of a spike T1117I mutation.

Genome sequencing is a key strategy in the surveillance of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Latin America is the hardest-hit region of the world, accumulating almost 20% of COVID-19 cases worldwide. In Costa Rica, from the first detected case on March 6th to December 31st almost 170,000 cases have been reported. We analyzed the genomic variability during the SARS-CoV-2 pandemic in Costa Rica using 185 sequences, 52 from the first months of the pandemic, and 133 from the current wave. Three GISAID clades (G, GH, and GR) and three PANGOLIN lineages (B.1, B.1.1, and B.1.291) were predominant, suggesting multiple re-introductions from other regions. The whole-genome variant calling analysis identified a total of 283 distinct nucleotide variants, following a power-law distribution with 190 single nucleotide mutations in a single sequence, and only 16 mutations were found in >5% sequences. These mutations were distributed through the whole genome. The prevalence of worldwide-found variant D614G in the Spike (98.9% in Costa Rica), ORF8 L84S (1.1%) is similar to what is found elsewhere. Interestingly, the frequency of mutation T1117I in the Spike has increased during the current pandemic wave beginning in May 2020 in Costa Rica, reaching 29.2% detection in the full genome analyses in November 2020. This variant has been observed in less than 1% of the GISAID reported sequences worldwide in 2020. Structural modeling of the Spike protein with the T1117I mutation suggests a potential effect on the viral oligomerization needed for cell infection, but no differences with other genomes on transmissibility, severity nor vaccine effectiveness are predicted. In conclusion, genome analyses of the SARS-CoV-2 sequences over the course of the COVID-19 pandemic in Costa Rica suggest the introduction of lineages from other countries and the detection of mutations in line with other studies, but pointing out the local increase in the detection of Spike-T1117I variant. The genomic features of this virus need to be monitored and studied in further analyses as part of the surveillance program during the pandemic.

Gastric Cancer in the Era of Immune Checkpoint Blockade.

Gastric cancer (GC) is one of the most important malignancies worldwide because of its high incidence and mortality. The very low survival rates are mainly related to late diagnosis and limited treatment options. GC is the final clinical outcome of a stepwise process that starts with a chronic and sustained inflammatory reaction mounted in response to Helicobacter pylori infection. The bacterium modulates innate and adaptive immunity presumably as part of the strategies to survive, which favors the creation of an immunosuppressive microenvironment that ultimately facilitates GC progression. T-cell exhaustion, which is characterized by elevated expression of immune checkpoint (IC) proteins, is one of the most salient manifestations of immunosuppressive microenvironments. It has been consistently demonstrated that the tumor-immune microenvironment(TIME)-exhausted phenotype can be reverted by blocking ICs with monoclonal antibodies. Although these therapies are associated with long-lasting response rates, only a subset of patients derive clinical benefit, which varies according to tumor site. The search for biomarkers to predict the response to IC inhibition is a matter of intense investigation as this may contribute to maximize disease control, reduce side effects, and minimize cost. The approval of pembrolizumab for its use in GC has rocketed immuno-oncology research in this cancer type. In this review, we summarize the current knowledge centered around the immune contexture and recent findings in connection with IC inhibition in GC.