Government has decided to commission antigen based tests for Covid-19. It is high time we kept ourselves abreast of the state of the art of antigen based tests.
Companies and research laboratories are rolling out different types of Covid-19 identification tests that are faster, cheaper and more user-friendly. Some of these tests use the gene-editing tool CRISPR to get genetic fractions of the virus. Others are quicker variants of the PCR test that use different reagents, not bogged down by the present day supply-chain problems. Saliva-based PCR tests are also done in USA for students and basketball players. Some are as fascinating as spitting into a tube, soaking a piece of processed paper in it and getting the result in minutes, while some would be over the counter paper based antigen test. The problem nevertheless is, the result of some of these tests will hardly come to the notice of the public health authority, while people might get a false sense of security from tests having limited accuracy.Understanding the antigen based test procedures
The antigen or the antibody tests for Covid-19 are mirror images. In the former, antibody, and in the latter, antigen, is fixed to the surface of the testing apparatus (some apparatuses look like a small plastic box with holes, while some may be just a card or a paper strip). The sample taken from nasal or oral fluid is presumed to contain antigen, while the blood sample is presumed to contain antibody, which are dropped onto the relevant testing apparatus. For detecting antigen, the sample however, is mixed with a solution which breaks the virus to free its proteins. The sample containing antigen or antibody would get attached with the antibody or the antigen fixed earlier into the apparatus respectively and will produce a fluorescent colour or a dark band, indicating test positivity.
The rapid antigen test gives results in less than 30 minutes and is cheap. But while a PCR test can detect a single viral molecule in a microlitre of sample, antigen test needs a sample to contain thousands and thousands of virus particles per microlitre for a positive result. If the sample has low amounts of virus, the test would be false-negative.
Implications of the antigen based test
The drawback of the PCR test is, its positivity does not indicate if the sample comes from someone who is spreading the virus or not. For antigen based test, on the other hand, it is not known yet, as to what is the viral load that is the threshold when a person is not infectious. While infectiousness starts after a latent period of three days, SARS-CoV-2 viral load peaks after the appearance of clinical features gradually declining from the eighth or tenth day, after which a person is unlikely to spread the virus. Small amounts of RNA however, may stay in infected people’s nose or throat for weeks or months even in dead form.
While the rapid test for antibody can detect at least 90% of those who have antibody (called sensitivity of the test) and can detect those who do not possess antibody, with up to 95% accuracy (called specificity of the test). But sensitivity of rapid antigen test is variable while its specificity is 100%. In unspecified conditions, the antigen tests are less reliable (sensitive), with an accuracy rate in some cases as low as 50%.Some scientists opine, testing people twice a week with a relatively insensitive test, like the antigen based test, can control spreading SARS-CoV-2 better than a more-accurate test like PCR done once every two weeks. Some scientists on the other hand, point out the limitation of the antigen test, as it was originally supposed to be used in virus hotspots and healthcare settings. Proponents exhort that antigen test could control the pandemic through spotting those who are transmitting the disease the most. Its other strength is, fast rolling out of the kit in vast number. On the other hand, sample collection, its transportation, processing for PCR and churning out the results are time consuming, ending in long waiting times, as has been experienced in Bangladesh also. But again, to ensure good test result from antigen test, sample collection time should be decided cautiously. Some scientists worry that antigen tests will miss infectious people, since it is not sensitive enough and will end up in outbreaks. Others view that people who exhibit positive PCR test results may be infected, but may not spread the virus to others any longer.
In UK, the most common type of rapid test has an error margin of 20% (giving false negative results). But the rapid test kit developed by Oxford Nanoporeis said to pick up 98% positive cases. This however, needs independent checking by experts. Antigen tests used in USA are said to have a sensitivity of 71% and 81% respectively (against PCR), higher than those used in India.
FDA guidelines for approval of all such antigen based kits say that their performance has to be nearly as good as laboratory tests. FDA, in its Emergency Use Authorization (EUA) stated that negative results from an antigen test may need to be confirmed with a PCR test prior to making treatment decisions or to prevent the possible spread of the virus due to a false negative. The FDA authorisation of antigen-based tests is only for people who have had symptoms for 12 days or fewer, test has been prescribed by a physician, and that it is administered by a health-care professional. When it gave EUA for the first antigen kit, the FDA pointed out that antigen tests are very specific for the virus, but are not as sensitive as molecular PCR tests, indicating that antigen based positive results are highly accurate, but with higher chance of false negatives. The World Health Organization (WHO) and the US Food and Drugs administration have therefore advised getting a PCR test if the test is negative by rapid antigen test.
There are others who however, think that, during containment phase authorities should use tests that are more accurate, as missing even just one positive individual could lead to a steep increase in the number of cases. One sanguine point that begs contemplation is- there is no solid proof yet that testing a large number of people has reduced or halted the infection by volume or by rate.
Approval of different types of antigen based tests by FDA
The US Food and Drug Administration (FDA) authorized Quidel’s SARS Antigen FIA, named Sofia2, in May, as the first-ever antigen based rapid test for use in high and moderate complexity laboratories certified by Clinical Laboratory Improvement Amendments (CLIA), as well as for point-of-care testing by facilities operating under a CLIA Waiver Certificate. In July, FDA issued a EUA to the BD (Becton Dickinson) Veritor System for the rapid antigen based test under the conditions granted to Quidel and also for Emergency use limited to authorized laboratories using the BD Veritor Plus Analyzer Instrument.
The Indian experience with the rapid antigen test
The Indian Council of Medical Research (ICMR), has approved the use of three antigen tests developed in South Korea (SD Biosensor), Belgium and India. When independently evaluated by ICMR and the All Indian Institute of Medical Sciences (AIIMS), their accuracy in giving a true negative result (specificity) ranged between 50% and 84%. ICMR issued guidelines saying those with negative results from an antigen test should also get a PCR test if they show symptoms.
From 29 June to 28 July, 63% of tests in Delhi was antigen based. 18% of less than 1% of those who tested negative in an antigen test was positive by PCR. The authorities have now asked testing centers to conduct more PCR tests. But data shows that more than 50% tests are still antigen based. The Delhi High Court ordered that it should be used only in hotspots and healthcare settings. In the last week of July, in Kerala 38% of those who initially tested negative but had symptoms were found to be positive by PCR test. After mounting concerns about the accuracy of antigen testing, it increased PCR tests. India's worst affected state, Maharashtra, first began antigen tests in Mumbai. The city's municipal corporation reported that 65% of those who had symptoms of Covid-19 tested negative in the antigen test, but were positive in a PCR test.
ICMR has advised that the test will have to be used under medical supervision, maintaining the kit temperature between 2° and 30°C and to be used in containment zones, hotspots, or healthcare settings.
Different antigen kits should be tested first with regard to their sensitivity, compared to the RT-PCR machine. Samples should be tested for antigen on the fourth and the eight day of appearance of clinical features at upazila health complexes (UHC) or also at community clinic (CC) level to be confirmed at UHC, before regular testing is instituted at CCs. The various guidelines given by FDA (USA) and ICMR should be examined before their adoption. Prices should be negotiated based on the sensitivity of the kit. The Government should brace itself up to face shortfall of the kits in the international market.
The writer is a former director, Primary Health Care and Disease Control, former director of IEDCR, DGHS, former regional adviser of SEARO, WHO and former staff consultant, Asian Development Bank, Bangladesh.