ABSTRACT
Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.
Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/epidemiology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , Genome, Viral , Humans , Infection Control/methods , Limit of Detection , Mass Screening/methods , RNA, Viral/genetics , RNA, Viral/isolation & purification , RNA-Directed DNA Polymerase/genetics , Reproducibility of Results , Reverse Transcription/genetics , Sensitivity and SpecificityABSTRACT
To study host-virus interactions after SARS coronavirus-2 (SARS-CoV-2) infection, genetic virus characteristics and the ensued humoral immune response were investigated for the first time. Fifty-five SARS-CoV-2-infected patients from the early pandemic phase were followed up including serological testing and whole genome sequencing. Anti-spike and nucleocapsid protein (S/N) IgG and IgM levels were determined by screening ELISA and IgG was further characterized by reactivity to S-subunit 1 (anti-S1), S-subunit 2 (anti-S2) and anti-N. In 55 patients, 90 genetic SARS-CoV-2 changes including 48 non-synonymous single nucleotide variants were identified. Phylogenetic analysis of the sequencing data showed a cluster representing a local outbreak and various family clusters. Anti-S/N and anti-N IgG were detected in 49 patients at an average of 83 days after blood collection. Anti-S/N IgM occurred significantly less frequently than IgG whereas anti-S2 was the least prevalent IgG reactivity (P < 0.05, respectively). Age and overweight were significantly associated with higher anti-S/N and anti-S1 IgG levels while age only with anti-N IgG (multiple regression, P < 0.05, respectively). Anti-S/N IgG/IgM levels, blood group A + , cardiovascular and tumour disease, NSP12 Q444H and ORF3a S177I were independent predictors of clinical characteristics with anti-S/N IgM being associated with the need for hospitalization (multivariate regression, P < 0.05, respectively). Anti-SARS-CoV-2 antibody generation was mainly affected by higher age and overweight in the present cohort. COVID-19 traits were associated with genetic SARS-CoV-2 variants, anti-S/N IgG/IgM levels, blood group A + and concomitant disease. Anti-S/N IgM was the only antibody associated with the need for hospitalization.
Subject(s)
COVID-19 , Antibodies, Viral , Humans , Immunoglobulin G , Immunoglobulin M , Phylogeny , SARS-CoV-2 , Spike Glycoprotein, CoronavirusABSTRACT
Since the end of 2019 a new coronavirus, SARS-CoV-2, first identified in Wuhan, China, is spreading around the world partially associated with a high death toll. Besides hygienic measurements to reduce the spread of the virus vaccines have been confected, partially based on the experiences with Ebola virus vaccine, based on recombinant human or chimpanzee adenovirus carrying the spike protein and its ACE2 receptor binding domain (RBD). Further vaccines are constructed by spike protein coding mRNA incorporated in lipid nano vesicles that after entry in human cells produce spike protein. Both vaccine types induce a strong immune response that lasts for months possibly for T-cell immunity a few years. Due to mutations in the coronavirus genome in several parts of the world variants selected, that were partially more pathogenic and partially easier transmissible - variants of concern (VOC). Until now vaccinees are protected against the VOC, even when protection might be reduced compared to the Wuhan wild virus.An open field is still how long the vaccine induced immunity will be sufficient to prevent infection and/or disease; and how long the time period will last until revaccination will be required for life saving protection, whether a third vaccination is needed, and whether revaccination with an adenovirus-based vaccine will be tolerated.