Reducing false-positive SARS-CoV-2 diagnoses using long-range RT-qPCR (preprint)

Quantitative polymerase chain reaction (qPCR) is a sensitive molecular method for the detection of genetic material and regarded as the gold-standard for diagnostic testing. To detect respiratory RNA virus infections, a reverse transcription (RT) step is implemented to create cDNA molecules that can serve as template in the qPCR step. However, positive RT-qPCR results can be found long after patient recovery, in part because the RT-qPCR can detect residual viral RNA genome fragments. To minimize the detection of such fragments, we here modified the RT-qPCR assay by replacing the routinely used random hexamers with an oligonucleotide that binds to the 3' end of the viral genome. We demonstrate that this method allows us to distinguish between infectious and non-infectious samples. Moreover, in clinical samples obtained over 15 days after the onset of symptoms, we observe that the modified RT-qPCR protocol yields significantly fewer positive results compared to a commercial RT-qPCR test. No significantly different results were found compared to the commercial test when SARS-CoV-2 clinical samples were tested within 5 days of the onset of symptoms, suggesting that the modification has a similar sensitivity for detecting infectious viral RNA. Overall, these findings may help differentiate between incorrectly-positive, persistently positive, and reinfection cases in COVID-19 patients.

Travel-driven emergence and spread of SARS-CoV-2 lineage B.1.620 with multiple VOC-like mutations and deletions in Europe (preprint)

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 HV69del, Y144del, and LLA241/243del. 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.