Point-of-care serological assays for SARS-CoV-2 in a UK hospital population: potential for enhanced case nding

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic, causing over 3,600,000 reported cases and 250,000 deaths worldwide. 1 Case identication has predominantly been made by real-time polymerase chain reaction (PCR) during the acute phase and largely restricted to healthcare laboratories. Serological assays are emerging but independent validation is urgently required to assess their utility. Where a plurality of point-of-care (POC) SARS-CoV-2 antibody test kits have become available, we will therefore aim to evaluate a range of kits against the current available gold-standard diagnostic test of PCR in an initial, exploratory study. We will then proceed to carry out testing with 200 hospital inpatients using the OrientGene COVID-19 IgG/IgM Rapid Test Cassette against PCR in order to undergo a preliminary evaluation of POC serological test performance characteristics within a hospital inpatient cohort.


Introduction
Following the spread of SARS-CoV-2 to cause a global pandemic in early 2020, several serological tests have been developed for the detection of SARS-CoV-2 antibody. Rapid diagnostic tests (RDT) for SARS-CoV-2 are typically small plastic cassettes to which is added patient whole blood or serum followed by buffer solution and qualitative results made available via a chromotographic lateral ow assay. Positive tests from different manufacturers are designed to be read as either a total antibody reaction (single line) or as an IgM, IgG split antibody reaction (two separate lines). In addition, a control line must be apparent to ensure validity of result read.
This protocol provides instruction to carry out rapid diagnostic, point-of-care (POC) serology testing for coronavirus disease-19 (COVID-19) caused by the pathogen Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2). It aims to enable evaluation of point-of-care (POC) antibody test kits against the current gold-standard of PCR. Where this has been conducted speci cally in a UK based-inpatient cohort, results are therefore not intended for extrapolation to the general population. Consecutive inpatients presenting to hospital and requiring admission, that subsequently test positive with real-time PCR for SARS-CoV-2 (AusDiagnostics®, Australia), will be selected for testing and strati ed based on duration of symptoms. All samples will be tested using patient whole blood by the same dedicated research team. Product inserts report whole blood, serum or plasma as suitable samples for testing.

Inclusion criteria
To be included in the study patients will have been admitted to hospital with symptoms matching the PHE case de nition for testing, 2 and have an accurate history recorded of at least 5 days of symptoms.

Exclusion criteria
The following patients will not be eligible to be included in the study: 1) all patients under 16 years old; 2) mild infection not requiring admittance to hospital; 3) patients who are unable to provide an accurate history of PHE case de nition symptoms or those with vague onset of symptom duration.

Initial study (n=15)
For each patient identi ed a unique identi er, general demographics (including age, sex), symptoms reported at presentation, days since symptom onset, day of PCR testing, result of PCR test, and day of POC testing are all to be recorded.
10 PCR positive that meet inclusion criteria are to be selected. Each POC kit will be run on the same blood sample at the same time, taken on the day of testing. Positive cases will be selected to re ect an array of times since symptom onset from 5-14 days in order to inform as a screen for a minimum time point at which reaction POC testing in known PCR positive patients appears to become concordant. False positive reactivity will be evaluated by testing true negative patients (estimate 5) using all available kits in the same manner.
The same research team will test each patient, as per each product protocol. Test kits will be read at the designated time period as per individual protocol and both the appearance of a control line and the presence or absence of antibody test reactivity lines will be recorded. All test readings will be veri ed independently by two of the researchers.

Further study (n=200)
A larger study is to then be conducted utilising a single test kit. In this case the COVID-19 CE marked split IgG/IgM Rapid Test Cassettes (OrientGene) is to be used. Patients are to be strati ed based on consecutive PCR positive results to one of three time interval groups since symptom onset: days 5-9, 10-14, and more than 14 days (150). A further 50 patients are to be tested that are considered negative for SARS-CoV-2 infection, either secondary to negative PCR testing or radiological imaging demonstrating no signs indicative of COVID-19 and an alternative primary febrile diagnosis.
Five test cassettes will be used to pilot and evaluate possible anticipated end-user di culties. Three test cassettes will be used to retest a patient with a positive result to comment on reproducibility. One test cassette will be completed with too little blood, failing to ll the pipette to the indicated line and one test cassette will be completed with too much blood, by over-lling the well.
Prevalence calculations have been estimated in the general population for the UK during the same testing period. 3 Hospital inpatient prevalence should be calculated for the appropriate time period and used as a marker of prevalence for the testing cohort across the same period of testing. Prevalence calculations are to be used to inform positive and negative predictive values in the general population and when utilised in speci c patient populations.

Sample collection
Each of the kits in this protocol can be performed using whole blood (including EDTA, sodium citrate or heparin samples), serum or plasma specimens. 3. If delayed testing is conducted, samples should be brought back to room temperature for 15 minutes prior to testing.

Preparing the Test
Each manufacturer instructions must be followed for the individual test kit used. The overall technique remains the same regardless of manufacturer. 6. Remove the test cassette from the packaging and label it with serial number of the patient. 7. Place horizontally on a at surface. solution. Read time for VivaDiag cassette results is at 15 minutes after the buffer solution is added to the specimen well.

4.
OrientGene: This is dual specimen well cassette requiring the placement of 5µl of sample into the specimen well (labelled 'S'). The specimen well is located closest to the chromatography paper. After the sample is loaded, 2 drops (up to 80µl) of OrientGene supplied buffer solution is to be added to the lower buffer well (labelled 'B). Read time for OrientGene cassette results is set at 10-15 minutes after the buffer solution is added to the specimen well. Instructions note that if sample is clearly visible within the specimen well at 2 minutes, the operator is to add an additional drop of buffer solution.
5. Core tests: This is single specimen well cassette requiring the placement of 10µl of sample into the well followed by the immediate addition of 2 drops of (80-100µl) of Core tests supplied buffer solution. Read time for Core tests cassette results is at 15 minutes after the buffer solution is added to the specimen well.

Anticipated Results
Results are qualitative and the number of test lines are particular to the manufacturer design. Refer to the test instruction lea et for full details on interpretation of results. Example interpretation of tests that employ detection of separate IgM and IgG lines is shown in Figure 1.
Results are recorded at 15 minutes and entered on to the data collection sheet. Comparison will be made against day since symptom onset and PCR result. Failed tests will be repeated with a new cassette. 3. Failure of buffer/sample solution to progress. Some RDTs were found to routinely require additional buffer solution to cause an advance of the buffer/sample solution. The OrientGene in particular often required a 3 drops. of buffer solution as standard. The kit provides suitable volume of buffer to expend 3 drops per cassette.
4. Insu cient sample: If insu cient sample has been collected then the cassette should not be run with this sample. A further sample collection must be made prior to testing.

Time Taken
Each individual test should take a total of 25 minutes. 10 minutes is given for preparation of materials, checking of patient details on sample and recording of results. 15 minutes is the maximum designated time taken to await result as per manufacturer instruction lea ets and tests should not be examined after this time period.

Anticipated Results
Results are qualitative and the number of test lines are particular to the manufacturer design. Refer to the test instruction lea et for full details on interpretation of results. Example interpretation of tests that employ detection of separate IgM and IgG lines is shown in Figure 1.
Results are recorded at 15 minutes and entered on to the data collection sheet. Comparison will be made against day since symptom onset and PCR result. Failed tests will be repeated with a new cassette.