Summary SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2 1 , and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro , the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.

Objectives When SARS-CoV-2 prevalence is low, many positive test results are false positives. Confirmatory testing reduces overdiagnosis and nosocomial infection and enables real-world estimates of test specificity and positive predictive value. This study estimates these parameters to evaluate the impact of confirmatory testing, and to improve clinical diagnosis, epidemiological estimation and interpretation of vaccine trials. Methods Over one month, we took all respiratory samples from our laboratory with a patient’s first detection of SARS-CoV-2 RNA (Hologic Aptima SARS-CoV-2 assay or in-house RT-PCR platform), and repeated testing using two platforms. Samples were categorised by source, and by whether clinical details suggested COVID-19 or corroborative testing from another laboratory. We estimated specificity and positive predictive value using maximum likelihood-based approaches. Results Of 19,597 samples, SARS-CoV-2 RNA was detected in 107. 52 corresponded to first-time detection (0.27% of tests on samples without previous detection); further testing detected SARS-CoV-2 RNA ≥1 time (“confirmed”) in 29 (56%), and failed to detect SARS-CoV-2 RNA (“not confirmed”) in 23 (44%). Depending upon assumed parameters, point estimates for specificity and positive predictive value were 99.91%–99.98% and 61.8%–89.8% respectively using the Hologic Aptima SARS-CoV-2 assay, and 97.4%–99.1% and 20.1%–73.8% respectively using an in-house assay. Conclusions Nucleic acid amplification testing for SARS-CoV-2 is highly specific. Nevertheless, when prevalence is low a significant proportion of initially positive results fail to confirm and confirmatory testing substantially reduces false positive detections. Omitting additional testing in samples with higher prior detection probabilities focuses testing where clinically impactful and minimises delay.