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 stratified 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.
To be included in the study patients will have been admitted to hospital with symptoms matching the PHE case definition for testing,2 and have an accurate history recorded of at least 5 days of symptoms.
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 definition symptoms or those with vague onset of symptom duration.
Initial study (n=15)
For each patient identified a unique identifier, 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.
15 SARS-CoV-2 CE marked split IgM/IgG antibody tests (Core tests®) will be paired alongside 15 SARS-CoV-2 CE marked total antibody rapid tests (Wantai), 15 COVID-19 CE marked split IgM/IgG Rapid Tests (VivaDiag), 15 COVID-19 CE marked split IgG/IgM Rapid Test Cassettes (OrientGene), and 15 SARS-CoV-2 CE marked split IgM/IgG One Step Rapid Test Devices (Encode).
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 reflect 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 verified 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 stratified 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 difficulties. 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 fill the pipette to the indicated line and one test cassette will be completed with too much blood, by over-filling 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 specific patient populations.
Each of the kits in this protocol can be performed using whole blood (including EDTA, sodium citrate or heparin samples), serum or plasma specimens.
1. Testing should be performed immediately following collection if possible.
2. Samples can be stored for a limited period of time (Encode, Core tests® 2-8oC for 5 days or long term storage at -20oC; Wantai 2-8oC for 7 days or long term storage at -15oC; OrientGene 2-8oC for 3 days or for long term storage at less than -20oC VivaDiag™ no comment made).
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.
1. Ensure you are wearing the correct PPE for carrying out the test in the extraction hood in the laboratory.
2. Gather the materials required
3. Double check the name, hospital number, and date of birth for each patient included in the study.
4. Cross check the blood sample bottle with the day since symptom onset data for each patient to ensure the sample is the correct date.
5. Ensure the test kit is within date and not damaged, record LOT number and date of expiry for each test used.
6. Remove the test cassette from the packaging and label it with serial number of the patient.
7. Place horizontally on a flat surface.
Performing the Test
Familiarise yourself with the manufacturers guidelines for the brand of RDT being used. Test read time is specific for each cassette but typically ranges from 10-15 minutes. The following details that are particular to each RDT used in this study.
1. Each RDT has its own plastic pipette for transferring sample (whole blood/plasma/serum) from the collection vial to the specimen well. Use the RDT specific cassette for each test.
2. Having completed your preparations, remove the cap from the identified collection vial.
3. Using the RDT specific pipette, transfer the requisite amount of sample from the vial to the designated specimen window. Place cap back on vial and return to storage rack. Discard plastic pipette into a biohazard waste container.
4. Immediately add the requisite amount of buffer solution to the appropriate well (see RDT specific instructions/RDT instruction leaflet) from a vertical position above the well.
5. Start the timer.
6. Read and record the result (serial number recorded on the cassette).
7. Discard the cassette into a biohazard waste container.
8. Do not read cassettes beyond the designated time period.