ABSTRACT
During the Coronavirus Disease 2019 (COVID-19) pandemic, residual SARS-CoV-2 genome and subgenomic RNA fragments were observed in recovered COVID-19 patients. The presence of such RNAs in the absence of live virus leads to incorrectly positive RT-qPCR results, potentially delaying medical procedures and quarantine release. We here propose a simple modification to turn commercial COVID-19 RT-qPCR protocols into long-range RT-qPCR assays that can differentiate between infectious and non-infectious influenza and coronavirus RNA levels. We find that the long-range RT-qPCR method has a sensitivity that is indistinguishable from a commercial Taq-Path COVID-19 RT-qPCR assay when tested on clinical samples taken withing 5 days of the onset of symptoms. In clinical samples taken at least 15 days after the onset of symptoms when patients had recovered from COVID-19, the modified RT-qPCR protocol leads to significantly fewer positive diagnoses. These findings suggest that the long-range RT-qPCR method may improve test-to-release protocols and expand the tools available for clinical COVID-19 diagnosis. ImportanceVarious molecular tests can detect viral RNA in clinical samples. However, these molecular tests cannot differentiate between RNA from infectious viruses or residual viral genome fragments that are not infectious. In several percent of COVID-19 patients, such residual viral RNAs can be detected long after recovery and the disappearance of infectious SARS-CoV-2. These "persistently-positive" RT-qPCR results are different from false-positive RT-qPCR results, which can be generated due to in vitro cross-reactivity or contaminations. However, the detection of RNA fragments leads to incorrect conclusions about the status of a COVID-19 patient and an incorrect diagnosis. We here modified the commercial Taq-Path COVID-19 RT-qPCR kit to make this test less sensitive to residual viral RNA genome fragments, reducing the likelihood that incorrect RT-qPCR results affect the treatment or quarantine status of recovered COVID-19 patients.
ABSTRACT
Many high-income countries have met the SARS-CoV-2 pandemic with overwhelming sequencing resources and have identified numerous distinct lineages, including some with notably altered biology. Over a year into the pandemic following unprecedented reductions in worldwide human mobility, distinct introduced lineages of SARS-CoV-2 without sequenced antecedents are increasingly discovered in high-income countries as a result of ongoing SARS-CoV-2 genomic surveillance initiatives. We here describe one such SARS-CoV-2 lineage, carrying many mutations and deletions in the spike protein shared with widespread variants of concern (VOCs), including E484K, S477N and deletions HV69{Delta}, Y144{Delta}, and LLA241/243{Delta}. This lineage - designated B.1.620 - is known to circulate in Lithuania and has now been found in several European states, but also in increasing numbers in central Africa owing to important recent increases in genome sequencing efforts on the continent. We provide evidence of likely ongoing local transmission of B.1.620 in Lithuania, France, Germany, Spain, Belgium and the Central African Republic. We describe the suite of mutations this lineage carries, its potential to be resistant to neutralising antibodies, travel histories for a subset of the European cases, and evidence of local B.1.620 transmission in Europe. We make a case for the likely Central African origin of this lineage by providing travel records as well as the outcomes of carefully crafted phylogenetic and phylogeographic inference methodologies, the latter of which is able to exploit individual travel histories recorded for infected travellers having entered different European countries.