Relationship between SARS-COV-2 full genome analysis and clinical findings in patients diagnosed with COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Coronavirus Disease-2019 (COVID-19) caused by it, emerged in Wuhan at the end of December 2019 and quickly spread worldwide. SARS-CoV-2 variants that emerged shortly after the pandemic began have changed the course of the pandemic by causing changes in epidemiological and clinical findings, as well as in vaccination and treatment options. Sequencing, a technique used to determine the genomic sequence of the SARS-CoV-2 virus, is an important tool for the detection and characterization of new variants and mutations. Determining changes in the RNA sequence contributes to understanding factors such as the virus's evolution, spread, infectivity, clinical findings of the disease, and effectiveness of vaccines, and plays an important role in controlling the epidemic. In this study, genomic sequencing was performed on SARS-CoV-2 samples obtained from COVID-19 patients with certain characteristics followed during different periods, and the frequency of structural changes in the virus and their effects on clinical findings of the disease were analyzed. Between January and November 2022, 21 patients from the Department of Infectious Diseases and Clinical Microbiology at Pamukkale University Hospital were included in the study. Nasopharyngeal and oropharyngeal swab samples were obtained from COVID-19 patients, and their viral RNA was extracted and subjected to whole-genome sequencing analysis. The patients were grouped as having mild, moderate, or severe clinical courses based on their clinical status, and the relationship between their clinical status, laboratory results, radiological findings, PCR positivity duration, and genetic analysis results was investigated. Of the 21 patients included in the study, 13 (61.9%) were male, and their mean age was 65.3±20.7 (26-97). Eighteen (85.7%) patients were hospitalized, and three (14.3%) patients were followed up as outpatients. In the study, Omicron BA.2 subvariants were detected in 12 patients, Omicron BA.1 subvariants in four patients, Omicron BA.5 subvariants in four patients, and Delta variant in one patient. The mutations c.1841A>G (D614G), c.425G>A (G142D), c.9764C>T, NSP4 (T492I), and c.14144C>T (NSP12 P323L) were detected in all isolates analyzed. When the mutations detected in the samples taken from the patients were compared with the frequencies of these mutations in other sequences worldwide, a high positive correlation was found between these variables (r(163)=0.81, p<0.001). Additionally, some missense mutations observed for the first time in samples from Turkey were identified. Obesity was statistically significantly more common in patients with severe clinical courses than in those with mild or moderate clinical courses (p=0.042). When the laboratory findings were examined, significantly higher levels of C-reactive protein (p=0.014), aspartate aminotransferase (p=0.001), BUN and (p=0.018), creatinine (p=0.003), and lower hemoglobin levels (p=0.037) were detected in patients with severe clinical courses than in those with other clinical courses. Prolonged PCR positivity (over four weeks) was detected more frequently in immunosuppressed patients (malignancy (p=0.016), lymphoma (p=0.006)). At the same time, it was found that most patients had a moderate to severe clinical course (p=0.019). In patients with immunodeficiency, statistically significant increased hospitalization rates (p=0.026) and more severe disease course were observed (p=0.018). When the mutations detected in the patients included in the study were evaluated, the Spike Q493R mutation was detected at higher rates in patients vaccinated with any of the vaccines with 4 doses or more (p=0.043). Looking at the rare mutations in our study, Spike A67V, del211, and ins214EPE mutations were found in Omicron BA.1 subvariant cases in a manner consistent with the literature, associated with increased viral infectivity and resistance to immune response. It was suggested that the clinical effect of Spike R346K and the Spike A263T mutation, which was first reported in Turkey and detected in our patient despite four doses of vaccination in the last three months, could be related to escape from the antibody response of this variant. It has been concluded that the Spike R21T mutation detected in our patient, consistent with the literature, may be responsible for severe clinical course and gastrointestinal symptoms by increasing the virus's entry into human and intestinal cells, and may cause a decrease in antibody neutralization due to the patient's history of four doses of vaccination. It is thought that the Spike L452M mutation for the Omicron BA.2 subvariant, in synergy with the D155Y detected in ORF3a, may trigger excessive immune response and hyperinflammation, as there was a significant increase in inflammatory parameters in our patient. The NSP1 H110Y mutation was observed to escape the immune system by inhibiting host gene expression. The Spike A222V, T323I, and NS7a A106T mutations were associated with decreased infectivity, increased immune evasion, and host-virus binding, which could explain the mild clinical course and prolonged PCR positivity detected in our patient. The NSP4 T114I mutation was observed to cause prolonged PCR positivity in our patient, consistent with the literature, by increasing viral replication or decreasing viral clearance. The Spike A27S, K417N, N440K, Q493R, T19I, and G142D mutations, detected in our patients, were observed to have a common effect by causing resistance to the antibody response generated by the vaccine, consistent with the literature. It has been suggested that the presence of Spike mutations G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, and N501Y in our patient who has received four doses of vaccine and has high antibody titers may lead to immune escape. In a case with the Delta (B.1.617.2) variant detected in our study, it was thought that the combination of Spike protein NTD-specific E156G/del157-158 and L452R may have led to increased infectivity, resistance to polyclonal antibodies generated by the vaccine, and increased cell-to-cell fusion effect, which was also observed in our patient. The presence of Spike P681R in our patient was concluded to be associated with severe clinical course and prolonged PCR positivity, as observed in our case. For the Omicron BA.5 variant, the NSP13 A389V, NSP14 M315I, and NSP16 T140I mutations were considered to be able to cause exaggerated inflammatory response and lung immunopathology, similar to the literature, due to the hyperinflammatory clinical presentation in our patient. The N E136D mutation, as observed in our patient with a travel history and contact history with a person who had a travel history, demonstrated the importance of travel history in our case and its contribution to the epidemiology and distribution of the variants, as it may cause more severe and prolonged upper respiratory tract symptoms. This study evaluated the clinical impact of sequencing results for the first time in Turkey. However, this study was limited to a small group of patients and a single center, and therefore does not represent all country data. There is a need for further investigation into the clinical impact of these results in a larger population, with more centers and sequencing studies. The tracking of the virus's spread at national and global levels, comparison of viral genomes, and creation of molecular phylogenetic trees can help with this investigation. The detection of genetic changes, emergence of variants, and understanding of mutation rates can have a positive impact on effective treatment and vaccine development.