Nucleic acid amplification tests (NAAT) are currently the gold standard for the diagnosis of SARS-CoV-2 infection. Within weeks of the first cases in Wuhan, NAAT tests – specifically real-time Polymerase Chain Reaction (RT-PCR) tests – were developed by laboratories of the World Health Organisation (WHO) as well as the China and US centres for disease control (CDC) public health agencies.
This was possible because of the dramatic achievement of quickly sequencing the genetic material of SARS-CoV-2, a new virus in humans. This allowed the development of tests using specific probes or primers that recognise unique sections of the genetic material of the virus. Primers are either directed at essential viral particle structures like the envelope (E gene) or nucleocapsid (N gene) or enzymes that the virus uses to replicate (R or RNAse dependent RNA polymerase gene), or to nonstructural sections of the genetic material of the virus, like the section coding the open reading frame (ORF).
If one or more of these genes are found in a sample, this confirms the presence of SARS-CoV-2.
These essential RT-PCR tests have some drawbacks however, which make them an imperfect gold standard test for pandemic control.
The detection of viral genetic material by RT-PCR tests depends both on the stage of the illness when the sample is taken, as well as on where and how the sample is obtained. This is because the viral load in a respiratory sample is too low both during early infection and late after the infection started, giving a false negative result – even though the person is infected.
The issue of sample type is also critical. The ideal sample for which RT-PCR tests for respiratory viruses are designed to use is a nasophyngeal swab sample. This procedure requires skilled staff, is relatively uncomfortable for the patient and can cause gagging and aerosol generation, necessitating extensive personal protective equipment (PPE) for the healthcare worker taking the sample to mitigate exposure risk.
Most importantly however, is that RT-PCR tests are complex and expensive, requiring skilled laboratory staff with the relevant experience and equipment to conduct them. Each of the processes necessary to amplify the detected viral material in the lab is vitally important. The steps needed involve extraction of viral genetic material from the patient sample, conversion of that viral genetic material from RNA to DNA so that it can be detected by the primers, and then amplified sufficiently to give a signal.
Each step is a potential bottleneck and makes these types of tests difficult to scale up to the number of tests needed and to complete within the turn arounds times necessary for critical public health intervention of identifying infected individuals, isolating them, identifying contacts and isolating them as well.
The approach to RT-PCR testing for SARS-CoV-2 in South Africa was initially limited to patients who fulfilled criteria that classified them as “patients under investigation” (PUI). These criteria included travel to places that already had epidemics of Covid-19, as well as symptoms that were generic; common symptoms experienced with respiratory viruses – sore throat, shortness of breath, cough, fever.
RT-PCR resources were in demand worldwide, making access to these reagents difficult, so there was a lot of scrambling by labs to procure sufficient reagents for the ever-increasing demands for this test. Nevertheless, clinical laboratories going to extraordinary lengths, managed to significantly ramp up testing capacity in both private and public laboratories to meet this insatiable and ever-increasing demand for more and more RT-PCR testing.
Changing protocols for Covid-19 testing
What happened next, though, was chaotic from a laboratory and public health perspective.
There was a push to do community-based testing using teams that visited and tested entire households regardless of contacts or symptoms. The volumes and logistics required to do this was completely unsustainable and not useful as a public health intervention.
Then the criteria for testing access changed – as the epidemic started to take hold in South Africa there was no longer a need for a travel history.
The pressure on RT-PCR resources became unbearably difficult, with widespread RT-PCR testing being demanded for repatriation and quarantine of South Africans returning from other countries where they had worked; outbreaks occurring in the workplace; and with the closure of schools, supermarkets and police stations.
People became fearful of having been exposed and infected and demanded tests for themselves and to protect their families.
From a public health perspective, this type of testing also skewed the number of cases that could be identified. Cases were only equal to the number of people that managed to get a test, shifting the whole focus of the classic public health intervention of testing to identify cases, isolate and trace and test their contacts, and erroneously defining the number of tests done as the number of cases.
The strict countrywide quarantine or lockdown measures at the end of March relieved the pressure on the RT-PCR resource that had mounted in laboratories. The restrictions of movement and gatherings of people socially and in the workplace eased the need for testing significantly, allowing labs to more rationally limit testing resources where they were most needed in hospitals for sick people, and to screen essential healthcare workers who may have been exposed while working with sick people.
Yet strict countrywide restrictions were just not possible for long, given the scale of human restrictions and economic loss. The only measurements available to persuade politicians that we had “flattened the curve”, buying the necessary time to prepare hospitals and the country for Covid-19, were sophisticated mathematical models. These models were used to predict various scenarios about the number of infected people in the country based only on one measurement that did not reflect the actual burden of infections – the number of RT-PCR tests.
Antibody tests and how they could help
This is exactly where laboratory scientists and clinicians could have made significant contributions. The missing weapon was another type of laboratory test, a blood test that measures evidence of infection or exposure to a virus. These are serology tests that analyse blood to detect SARS-CoV-2 antibodies that are formed by the immune system in response to infection with this virus.
This type of test is affordable and easily scalable on robotic instruments that already exist in most laboratories, giving results for thousands of tested people within a few hours. Exactly this type of test is needed to define the important public health question of what is the real number of people infected with SARS-CoV-2, and to better estimate how serious this infection is by allowing the calculation of the case fatality rate (CFR).
We don’t know this yet for Covid-19, despite the millions tested already with RT-PCR – we can not calculate the CFR without a proper denominator. The only way to derive a proper denominator is to know how many people have been infected with SARS-CoV-2, and the only way to do that is to make testing cheaper, easier and available to all with serology tests.
Clinical laboratories are familiar with high throughput immunoassays, and they are used all the time to assist with the diagnosis, management and public health interventions for viral infectious diseases.
A familiar example is an HIV ELISA test. HIV serology tests have a window period of 21 days – that is, they only detect antibodies 21 days after exposure to HIV. The antibody they detect is of different classes (IgM, IgG and IgA) and the antibodies detected are not protective and do not indicate that the person will get better or protected from reinfection.
Similarly, SARS-CoV-2 antibody tests have a window period of 14 days, and only become positive two weeks after the person has been exposed to Covid-19. They measure either IgG or combinations of IgG and IgM antibodies. Detecting these antibodies does not necessarily indicate immunity or protection from reinfection with SARS-CoV-2.
At this stage only high throughput tests done in clinical and diagnostic laboratories by accredited and highly trained laboratory professionals are the tests that have some utility. Point of care, finger prick home tests for antibodies are not good enough yet to be supported for widespread use.
Like RT-PCR tests, reputable manufacturers of immunoassays throughout the world rushed to make SARS-CoV-2 serology tests to high standards for high throughput instruments in clinical laboratories. These tests are designed to detect antibodies to different parts of the virus’ outside coat (the S or spike protein) or the internal coat covering the genetic material (the nucleocapsid).
Antibody tests were made using purified proteins to detect SARS-CoV-2 antibodies of various types (IgA, IgM and IgG). Usually with viral infections, IgM antibodies can be detected by tests early after viral infections, followed by IgG antibodies that signify recovery and immunity. But this pattern has not been seen with SARS-CoV-2, and both IgM and IgG appear to occur simultaneously after infection becoming detectable on day 14.
We also know that recurrence of Covid-19 illness is uncommon, suggesting that the presence of antibodies gives some form of immunity to SARS-CoV-2 infection. Experimental evidence in primate models supports that, and the presence of SARS-CoV-2 antibodies provides immunity to reinfection. In infected humans, SARS-COV-2 viral load in the respiratory tract samples has been shown to decrease as antibody levels increase, effectively decreasing infectiousness.
Antibody tests reveal evidence of previous SARS-CoV-2 infection and they are versatile because they can be used in contact tracing, weeks or longer, after a suspected infection. Antibody tests can be used to inform policymakers about the prevalence of infection in communities, specific outbreaks and in giving a clear total number of those who were infected including those who were asymptomatic.
Serology tests can help to confirm suspected cases in those who tested late after symptom onset or had mild symptoms.
Antibody testing is useful in specific Covid-19 syndromes that occur late after infection when RT-PCR detection of the virus is not likely. These syndromes include multisystem inflammatory disease and SARS-CoV-2 encephalitis that can only be diagnosed with the detection of antibodies in the blood or cerebrospinal fluid (CSF).
Finally, SARS-CoV-2 antibody testing is the only end-point measurement to confirm that SARS-CoV-2 vaccines work. Without these tests the trials that have started throughout the world and with much fanfare in South Africa will be a waste of time and money. Also, SARS-coV-2 antibody testing is crucial to identify donors of convalescent plasma that is used to assist severely ill Covid individuals who have not responded to other treatments.
Evaluating the efficacy of antibody tests
As with all new tests, clinical laboratories in South Africa have done careful evaluations of these high throughput serology tests in their labs with blood samples from people with Covid-19. This is standard practice and is always done on introducing a new test into a clinical laboratory. Laboratory professionals need to know how to use these tests and what are their limitations.
Test performance depends on the test sensitivity and specificity. Clinical laboratory scientists and doctors need to be sure that these tests do not falsely call a patient infected when they are not – false positive results for SARS-CoV-2 infection have a cascade of negative personal and public health outcomes for a patient that must be avoided.
Another performance characteristic that is very important is that these tests must not cross-react with other respiratory viruses, again to avoid a false positive result, and they must be sensitive enough to identify an infection in an individual most of the time.
What is already known in the published evidence base, is that these tests work best in outbreaks or communities where there are high numbers of infections, for example, in a community or workplace or school or factory where there are infections already occurring, and are of less use in areas where there are no infections.
So why don’t we have these tests yet in South Africa?
The main reason is the regulatory authority for in vitro diagnostic testing (IVDs), the South African Health Products Regulatory Authority (SAHPRA) is being overly cautious in not allowing these tests to be used by professional clinical laboratories. They argue that there is still no definitive proof that antibodies are protective against reinfection with SARS-CoV-2, or how long those antibodies will last and what concentration exactly is needed to get this protection in humans.
They also fear misuse of these tests for “immunity passports”, and this has been a major stumbling block in accessing these tests for all the other important uses that are necessary for properly managing the Covid epidemic.
SAHPRA has been frightened by the mistakes of some countries that bought home-testing point of care (POC), finger-prick tests to use as epidemic control tests. There are hundreds of these tests, from various suppliers that have hit the markets, and SAHPRA’s caution here is well founded for POC and home testing, which have not been properly evaluated or proven to be not sufficiently good yet.
But, we believe, this caution has unfortunately paralyzed SAHPRA’s consideration and approval of properly evaluated tests to be used by professional certified and accredited clinical laboratories.
For two months, established clinical laboratories with significant expertise in serological testing of viral infections have engaged with SAHPRA to do the best evaluations possible for high throughput SARS-CoV-2 serology tests. Every time evaluation data is presented to SAHPRA, they reject it, they don’t trust the data, it is too good, they are always asking for more data, better data, data from other labs in the public sector.
Local clinical virologists have reviewed every published study that has evaluated these particular tests and submitted it to SAHPRA as evidence that the tests work, and this was still not accepted by them.
At this point of the stalemate, it is not clear if they just don’t have the necessary expertise and resources to review evaluation data and are refusing to accept local and international scientific evaluation data based on allegations of data manipulation by laboratories, or if they are just misinformed.
In contrast, the FDA has reviewed evaluation data from US laboratories, and has granted Emergency Use Authorisation (EUA) to many high throughput tests already. These same tests have shown good performance in South African clinical laboratories.
Discussions and debates about the reluctance of the SAHPRA to accept evaluations by local laboratories with the relevant experience has been debated at the Covid Ministerial Advisory Committee (MAC) and, despite an advisory to the Minister of Health that these tests should be used, the regulator remains unmovable.
Serological tests for SARS-CoV-2 can play an important role in the diagnosis and management of Covid-19, and are essential for public health policies and measurement of virus epidemiology.
These tests will provide critical information in the fight against Covid in South Africa, especially as Gauteng enters a sustained community epidemic. DM/MC
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