SARS-CoV-2 Genome Variations in Viral Shedding of an Immunocompromised Patient with Non-Hodgkin's Lymphoma.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) is the most transmissible ß-coronavirus in history, affecting all population groups. Immunocompromised patients, particularly cancer patients, have been highlighted as a reservoir to promote accumulation of viral mutations throughout persistent infection. CASE PRESENTATION: We aimed to describe the clinical course and SARS-CoV-2 mutation profile for 102 days in an immunocompromised patient with non-Hodgkin's lymphoma and COVID-19. We used RT-qPCR to quantify SARS-CoV-2 viral load over time and whole-virus genome sequencing to identify viral lineage and mutation profile. The patient presented with a persistent infection through 102 days while being treated with cytotoxic chemotherapy for non-Hodgkin's lymphoma and received targeted therapy for COVID-19 with remdesivir and hyperimmune plasma. All sequenced samples belonged to the BA.1.1 lineage. We detected nine amino acid substitutions in five viral genes (Nucleocapsid, ORF1a, ORF1b, ORF13a, and ORF9b), grouped in two clusters: the first cluster with amino acid substitutions only detected on days 39 and 87 of sample collection, and the second cluster with amino acid substitutions only detected on day 95 of sample collection. The Spike gene remained unchanged in all samples. Viral load was dynamic but consistent with the disease flares. CONCLUSIONS: This report shows that the multiple mutations that occur in an immunocompromised patient with persistent COVID-19 could provide information regarding viral evolution and emergence of new SARS-CoV-2 variants.

End-point RT-PCR based on a conservation landscape for SARS-COV-2 detection.

End-point RT-PCR is a suitable alternative diagnostic technique since it is cheaper than RT-qPCR tests and can be implemented on a massive scale in low- and middle-income countries. In this work, a bioinformatic approach to guide the design of PCR primers was developed, and an alternative diagnostic test based on end-point PCR was designed. End-point PCR primers were designed through conservation analysis based on kmer frequency in SARS-CoV-2 and human respiratory pathogen genomes. Highly conserved regions were identified for primer design, and the resulting PCR primers were used to amplify 871 nasopharyngeal human samples with a previous RT-qPCR based SARS-CoV-2 diagnosis. The diagnostic test showed high accuracy in identifying SARS-CoV-2-positive samples including B.1.1.7, P.1, B.1.427/B.1.429 and B.1.617.2/ AY samples with a detection limit of 7.2 viral copies/µL. In addition, this test could discern SARS-CoV-2 infection from other viral infections with COVID-19-like symptomatology. The designed end-point PCR diagnostic test to detect SARS-CoV-2 is a suitable alternative to RT-qPCR. Since the proposed bioinformatic approach can be easily applied in thousands of viral genomes and over highly divergent strains, it can be used as a PCR design tool as new SARS-CoV-2 variants emerge. Therefore, this end-point PCR test could be employed in epidemiological surveillance to detect new SARS-CoV-2 variants as they emerge and propagate.

CovDif, a Tool to Visualize the Conservation between SARS-CoV-2 Genomes and Variants.

The spread of the newly emerged severe acute respiratory syndrome (SARS-CoV-2) virus has led to more than 430 million confirmed cases, including more than 5.9 million deaths, reported worldwide as of 24 February 2022. Conservation of viral genomes is important for pathogen identification and diagnosis, therapeutics development and epidemiological surveillance to detect the emergence of new viral variants. An intense surveillance of virus variants has led to the identification of Variants of Interest and Variants of Concern. Although these classifications dynamically change as the pandemic evolves, they have been useful to guide public health efforts on containment and mitigation. In this work, we present CovDif, a tool to detect conserved regions between groups of viral genomes. CovDif creates a conservation landscape for each group of genomes of interest and a differential landscape able to highlight differences in the conservation level between groups. CovDif is able to identify loss in conservation due to point mutations, deletions, inversions and chromosomal rearrangements. In this work, we applied CovDif to SARS-CoV-2 clades (G, GH, GR, GV, L, O, S and G) and variants. We identified all regions for any defining SNPs. We also applied CovDif to a group of population genomes and evaluated the conservation of primer regions for current SARS-CoV-2 detection and diagnostic protocols. We found that some of these protocols should be applied with caution as few of the primer-template regions are no longer conserved in some SARS-CoV-2 variants. We conclude that CovDif is a tool that could be widely applied to study the conservation of any group of viral genomes as long as whole genomes exist.

Early Genomic, Epidemiological, and Clinical Description of the SARS-CoV-2 Omicron Variant in Mexico City.

Omicron is the most mutated SARS-CoV-2 variant-a factor that can affect transmissibility, disease severity, and immune evasiveness. Its genomic surveillance is important in cities with millions of inhabitants and an economic center, such as Mexico City. Results. From 16 November to 31 December 2021, we observed an increase of 88% in Omicron prevalence in Mexico City. We explored the R346K substitution, prevalent in 42% of Omicron variants, known to be associated with immune escape by monoclonal antibodies. In a phylogenetic analysis, we found several independent exchanges between Mexico and the world, and there was an event followed by local transmission that gave rise to most of the Omicron diversity in Mexico City. A haplotype analysis revealed that there was no association between haplotype and vaccination status. Among the 66% of patients who have been vaccinated, no reported comorbidities were associated with Omicron; the presence of odynophagia and the absence of dysgeusia were significant predictor symptoms for Omicron, and the RT-qPCR Ct values were lower for Omicron. Conclusions. Genomic surveillance is key to detecting the emergence and spread of SARS-CoV-2 variants in a timely manner, even weeks before the onset of an infection wave, and can inform public health decisions and detect the spread of any mutation that may affect therapeutic efficacy.

Automated Reverse Transcription Polymerase Chain Reaction Data Analysis for Sars-CoV-2 Detection.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is a current public health concern. Rapid diagnosis is crucial, and reverse transcription polymerase chain reaction (RT-PCR) is presently the reference standard for SARS-CoV-2 detection. OBJECTIVE: Automated RT-PCR analysis (ARPA) is a software designed to analyze RT-PCR data for SARSCoV-2 detection. ARPA loads the RT-PCR data, classifies each sample by assessing its amplification curve behavior, evaluates the experiment's quality, and generates reports. METHODS: ARPA was implemented in the R language and deployed as a Shiny application. We evaluated the performance of ARPA in 140 samples. The samples were manually classified and automatically analyzed using ARPA. RESULTS: ARPA had a true-positive rate = 1, true-negative rate = 0.98, positive-predictive value = 0.95, and negative-predictive value = 1, with 36 samples correctly classified as positive, 100 samples correctly classified as negative, and two samples classified as positive even when labeled as negative by manual inspection. Two samples were labeled as invalid by ARPA and were not considered in the performance metrics calculation. CONCLUSIONS: ARPA is a sensitive and specific software that facilitates the analysis of RT-PCR data, and its implementation can reduce the time required in the diagnostic pipeline.

The Evolutionary Landscape of SARS-CoV-2 Variant B.1.1.519 and Its Clinical Impact in Mexico City.

The SARS-CoV-2 pandemic is one of the most concerning health problems around the globe. We reported the emergence of SARS-CoV-2 variant B.1.1.519 in Mexico City. We reported the effective reproduction number (Rt) of B.1.1.519 and presented evidence of its geographical origin based on phylogenetic analysis. We also studied its evolution via haplotype analysis and identified the most recurrent haplotypes. Finally, we studied the clinical impact of B.1.1.519. The B.1.1.519 variant was predominant between November 2020 and May 2021, reaching 90% of all cases sequenced in February 2021. It is characterized by three amino acid changes in the spike protein: T478K, P681H, and T732A. Its Rt varies between 0.5 and 2.9. Its geographical origin remain to be investigated. Patients infected with variant B.1.1.519 showed a highly significant adjusted odds ratio (aOR) increase of 1.85 over non-B.1.1.519 patients for developing a severe/critical outcome (p = 0.000296, 1.33-2.6 95% CI) and a 2.35-fold increase for hospitalization (p = 0.005, 1.32-4.34 95% CI). The continuous monitoring of this and other variants will be required to control the ongoing pandemic as it evolves.