Coronavirus (SARS-CoV-2) Rapid Antigen/Near-Patient/PoC Tests for COVID-19 - Buyer's Guide
- Rapid, point of care tests for SARS-CoV-2 antigen, using saliva or swab samples are now widely available
- These POC tests are less sensitive than the gold-standard RT-PCR but do have high specificity
- Easy to use and interpret, time to result can be between 10 minutes to 40 minutes depending on the type of assay
- Isothermal amplification can be combined with other detection methods, i.e. CRISPR, to improve accuracy.
- Kit manufacturers performance claims are often generated from high viral load samples that may not be representative of real-life samples.
COVID-19 Antigen Detection Tests for Screening
Antigen detection kits on the market target SARS-CoV-2 protein antigens, mainly the nucleoprotein (NP) antigen. There are several different types of antigen detection kits:
- chemiluminescence immunoassay (CLIA)
- fluorescent (FIA) immunoassays
- lateral flow immunoassays (LFIA)
- lateral flow fluorescent immunoassays
The World Health Organization currently (Nov 2020) state that rapid antigen detection tests (RADTs) should only be used if RT-PCR is not available and must be ≥ 90% sensitivity and 99% specificity. The reason for this precautionary principle toward RADTs is due to reports of a high false-negative rate with the first generation of COVID-19 RADTs.There are also reports of false positives from the cross-reactivity of the test-kit antibodies with other coronaviruses in circulation. These false-positives are mainly associated with tests that target the N-protein (nucleocapsid protein) as that antigen is highly conserved among other human coronaviruses. However, the spike protein (of which many COVID-19 vaccines are designed upon) has a subunit (S1) that is highly conserved in SARS-CoV-2 only. Thus, any antigen test kits that target this protein are less likely to give false-positives. (The recent Mink outbreak in Nov,2020 showed a SARS-CoV-2 variant, with a mutation in the S-protein, and has the potential to invalidate diagnostic kits targeting this protein/antigen).
However, tests have improved recently, with many certified RADTs now using saliva samples and more sophisticated reading devices been developed that increase sensitivity of the test. Although some countries have been hesitant to adopt rapid testing, RADTs have been deployed at many international airports to screen individuals for SARS-CoV-2.
Immunochromatographic/Lateral-Flow Immunoassays (LFIA):
This is based on the design of the common pregnancy test. The test is a strip containing immobilised reagents; specially-coated antibodies, negative and positive controls, and a window to view the result. The result shows in the form of a coloured line, that must be viewed at a specified time-point.
There are also commercialized automatic reading devices, that the diagnostic strip is inserted into, thus avoiding human error. These readers have performed better in validation studies than visual reading. These readers can give a semi-quantitative or quantitative result. These tests can be performed by unskilled operators in near-patient settings and turnaround times are usually 15 minutes. This makes them ideal for ports but only if passenger levels are reduced, as there is a chance of false-negatives due to low viral load in the patient not been detected.
COVID-19 Nucleic-Acid/Molecular Near-Patient Testing/Point-of-Care Testing:
COVID-19 tests that use isothermal nucleic acid amplification technology to detect SARS-CoV-2 RNA are available. Loop-mediated isothermal amplification (LAMP) is the most well-known version of this molecular tool, due to its use during the zika virus epidemic LAMP testing does not require thermal cycles, but needs only a simple heat-block to supply a constant temperature (ranging from (60-65°C) to activate its two enzymes, one for converting RNA to DNA and another to copy DNA. A set of primers is also added to bind to the targets on the viral genome. 'Loop' structures are formed that allow rapid interpretation for a result (i.e.changes in turbidity, colour or fluorescence).
This method has a low limit of detection (LoD), but studies have shown it to be less accurate than RT-PCR. However, this lower level of sensitivity may be due to improper procedure; using viral transport media instead of inserting swabs directly, while other evidence shows increased accuracy with an extra RNA extraction step before inserting swab.
Other variations of this technology include nicking enzyme-assisted reaction (NEAR), recombinase polymerase amplification (RPA) and Insulated Isothermal Polymerase Chain Reaction (RT-iiPCR).
CRISPR & isothermal amplification:
CRISPR SARS-CoV-2 tests on the market use the isothermal amplification method. But its use of proprietary molecular tools; SHERLOCK and DETECTR tools to amplify the sequence in combination with the CRISPR Cas 12/13 system for detection have demonstrated similar results to that of PCR. Its sensitivity depends on the type of readout, with fluorescence in the lab being superior to lateral-flow readouts.
Sequencing & Isothermal amplification:
One problem with isothermal amplification techniques is that substances present in the sample might interfere with the color change or turbidity, which is interpreted into a result. The use of nano-sequencing to read the results can increase sensitivity and specificity. An example of this test is the LAMPORE assay developed at Oxford University.
Several rapid PCR tests are available (FDA-EUA and CE-IVD) and are designed for near-patient settings, with some already in use at airports. These PCR cyclers have a small footprint and typically weigh 1Kg, they analyze swab samples and automate the nucleic acid extraction and amplification, with only basic skills needed to operate. Average turnaround time to result is 40 minutes.