ABSTRACT
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.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19 Testing , Humans , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/geneticsABSTRACT
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.
Subject(s)
COVID-19/epidemiology , COVID-19/virology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Basic Reproduction Number/statistics & numerical data , Biological Evolution , Genome, Viral , Haplotypes , Humans , Mexico/epidemiology , Mutation , Nasopharynx/virology , Phylogeny , RNA, Viral , SARS-CoV-2/classificationABSTRACT
OBJECTIVES: The aim of this study was to investigate the feasibility of saliva sampling as a non-invasive and safer tool to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to compare its reproducibility and sensitivity with nasopharyngeal swab samples (NPS). The use of sample pools was also investigated. METHODS: A total of 2107 paired samples were collected from asymptomatic healthcare and office workers in Mexico City. Sixty of these samples were also analyzed in two other independent laboratories for concordance analysis. Sample processing and analysis of virus genetic material were performed according to standard protocols described elsewhere. A pooling analysis was performed by analyzing the saliva pool and the individual pool components. RESULTS: The concordance between NPS and saliva results was 95.2% (kappa 0.727, p = 0.0001) and 97.9% without considering inconclusive results (kappa 0.852, p = 0.0001). Saliva had a lower number of inconclusive results than NPS (0.9% vs 1.9%). Furthermore, saliva showed a significantly higher concentration of both total RNA and viral copies than NPS. Comparison of our results with those of the other two laboratories showed 100% and 97% concordance. Saliva samples are stable without the use of any preservative, and a positive SARS-CoV-2 sample can be detected 5, 10, and 15 days after collection when the sample is stored at 4 °C. CONCLUSIONS: The study results indicate that saliva is as effective as NPS for the identification of SARS-CoV-2-infected asymptomatic patients. Sample pooling facilitates the analysis of a larger number of samples, with the benefit of cost reduction.