Virological and serological characterization of critically ill patient with COVID-19 in the UK: a special focus on variant detection (preprint)

Background. Treatment of COVID-19 patients with convalescent plasma containing neutralising antibody to SARS-CoV-2 is under investigation as a means of reducing viral loads, ameliorating disease outcomes, and reducing mortality. However, its efficacy might be reduced in those infected with the emerging B.1.1.7 SARS-CoV-2 variant. Here, we report the diverse virological characteristics of UK patients enrolled in the Immunoglobulin Domain of the REMAP-CAP randomised controlled trial. Methods. SARS-CoV-2 viral RNA was detected and quantified by real-time PCR in nasopharyngeal swabs obtained from study subjects within 48 hours of admission to intensive care unit. Antibody status was determined by spike-protein ELISA. B.1.1.7 strain was differentiated from other SARS-CoV-2 strains by two novel typing methods detecting the B.1.1.7-associated D1118H mutation with allele-specific probes and by restriction site polymorphism (SfcI). Findings. Of 1260 subjects, 90% were PCR-positive with viral loads in nasopharyngeal swabs ranging from 72 international units [IUs]/ml to 1.7x10^11 IU/ml. Median viral loads were 45-fold higher in those who were seronegative for IgG antibodies (n=314; 28%) compared to seropositives (n=804; 72%), reflecting in part the latter group's possible later disease stage on enrolment. Frequencies of B.1.1.7 infection increased from early November (<1%) to December 2020 (>60%). Anti-SARS-CoV-2 seronegative individuals infected with wild-type SARS-CoV-2 had significantly higher viral loads than seropositives (medians of 1.2x10^6 and 3.4 x10^4 IU/ml respectively; p=2x10^-9). However, viral load distributions were elevated in both seropositive and seronegative subjects infected with B.1.1.7 (13.4x10^6 and 7.6x10^6 IU/ml; p=0.18). Interpretation. High viral loads in seropositive B.1.1.7-infected subjects are consistent with increased replication capacity and/or less effective clearance by innate or adaptive immune response of B.1.1.7 strain than wild-type. As viral genotype was associated with diverse virological and immunological phenotypes, metrics of viral load, antibody status and infecting strain should be used to define subgroups for analysis of treatment efficacy.

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2 (preprint)

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, detection of seroconversion after vaccination, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests have a long history in blood typing, and general serology through linkage of reporter molecules to the red cell surface. They do not require special equipment, are read by eye, have short development times, low cost and can be applied as a Point of Care Test (POCT). We describe a red cell agglutination test for the detection of antibodies to the SARS-CoV-2 receptor binding domain (RBD). We show that the Haemagglutination Test (HAT) has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. The HAT can be titrated, detects rising titres in the first five days of hospital admission, correlates well with a commercial test that detects antibodies to the RBD, and can be applied as a point of care test. The developing reagent is composed of a previously described nanobody to a conserved glycophorin A epitope on red cells, linked to the RBD from SARS-CoV-2. It can be lyophilised for ease of shipping. We have scaled up production of this reagent to one gram, which is sufficient for ten million tests, at a cost of ~0.27 UK pence per test well. Aliquots of this reagent are ready to be supplied to qualified groups anywhere in the world that need to detect antibodies to SARS-CoV-2, but do not have the facilities for high throughput commercial tests.

Evaluation of antibody testing for SARS-Cov-2 using ELISA and lateral flow immunoassays (preprint)

BackgroundThe COVID-19 pandemic caused >1 million infections during January-March 2020. There is an urgent need for reliable antibody detection approaches to support diagnosis, vaccine development, safe release of individuals from quarantine, and population lock-down exit strategies. We set out to evaluate the performance of ELISA and lateral flow immunoassay (LFIA) devices. MethodsWe tested plasma for COVID (SARS-CoV-2) IgM and IgG antibodies by ELISA and using nine different LFIA devices. We used a panel of plasma samples from individuals who have had confirmed COVID infection based on a PCR result (n=40), and pre-pandemic negative control samples banked in the UK prior to December-2019 (n=142). ResultsELISA detected IgM or IgG in 34/40 individuals with a confirmed history of COVID infection (sensitivity 85%, 95%CI 70-94%), vs. 0/50 pre-pandemic controls (specificity 100% [95%CI 93-100%]). IgG levels were detected in 31/31 COVID-positive individuals tested [≥]10 days after symptom onset (sensitivity 100%, 95%CI 89-100%). IgG titres rose during the 3 weeks post symptom onset and began to fall by 8 weeks, but remained above the detection threshold. Point estimates for the sensitivity of LFIA devices ranged from 55-70% versus RT-PCR and 65-85% versus ELISA, with specificity 95-100% and 93-100% respectively. Within the limits of the study size, the performance of most LFIA devices was similar. ConclusionsCurrently available commercial LFIA devices do not perform sufficiently well for individual patient applications. However, ELISA can be calibrated to be specific for detecting and quantifying SARS-CoV-2 IgM and IgG and is highly sensitive for IgG from 10 days following first symptoms.