Lo - The changing landscape of COVID testing (Adapt or perish!)
Raymundo W. Lo, MD

In the beginning, there was RT-PCR (Reverse Transcriptase Polymerase Chain Reaction). But it was very limited. The pandemic was raging, and we could not see the enemy since we had very limited testing at the start. Without the means to identify the enemy, people in desperation resorted to RATs (Rapid Antibody Tests). However, RATs are not designed to detect current infections because they are produced by the human body as an immune response one week after infection. The kits available varied wildly in accuracy, sometimes reduced to the equivalent of tossing a coin to determine the outcome. RATs were used indiscriminately by many LGUs condemning “positives” to isolation without real proof, giving antibody tests a bad reputation. Even IgG positives were branded as COVID cases, good grief!

The advent of the instrumented antibody tests (ELISA/ECLIA) showed the way to better accuracy. However, they were still not good enough to diagnose currently infected persons. Somehow, in the public mind, all antibody tests were “rapid”, thus these better tests were also tarred and feathered along with RATs. These ELISA/ECLIA antibody tests do have utility in ascertaining recovery.

Now, we have rapid antigen tests (RAgTs) which are designed to catch current infections and use the same nasopharyngeal and oral swabs as with RT-PCR. These are promising with their excellent specificity, meaning if you test positive with RAgT, you will test positive with RT-PCR. However, it is still subject to approval for use with experts currently having a clash of ideas regarding its utility. The issue is sensitivity wherein its ability to detect current infections in at least 80% compared to RT-PCR is being debated.

Studies in Germany (1259 subjects) and Brazil (400 subjects)  had 87.8%  and 91.9%% sensitivity respectively. However, the local study showed a 71% sensitivity. The local study only had 134 subjects whereas the foreign studies had bigger population sizes and are thus statistically more significant.

There are certainly some advantages to doing RAgTs. It is a point of care use meaning it can be employed in quickly diagnosing patients in triage in the Emergency Room so positive patients will not be mixed with negative ones. At entry points to less prevalent areas, airports, and seaports, it can be used as quick screening tool and decongest these places. In remote areas with little or no access to RT-PCR testing, the RAgT is the only choice for testing. For returning economy workers, it is useful in that positive cases can be quickly identified and isolated preventing spread among co-workers. Since it is easy to administer and less expensive than Rt-PCR, it can be frequently used to insure worker and customer safety.

What is holding back the more extensive deployment of RAgTs is the disagreement among the experts. Some are insisting on better equivalency with RT-PCR technology which is acknowledged as the “gold standard” in COVID testing.

Truth be told, the classic gold standard for any infectious disease testing is the ability to culture the organism from the patient’s sample (Koch’s Postulates). In this case, it is viral culture for the SARS-CoV-2 organism. However, this can only be done in highly secure and well-protected BSL3 laboratories, meaning all measures are in place to prevent the laboratory staff from being infected in their workplace. Thus, the RT-PCR has been a proxy for viral culture with its ability to detect the ribonucleic acid (RNA) of the SARS-CoV-2 in as little as 50-100 copies per microliter.

The RT-PCR’s exquisite sensitivity is both its strength and its weakness in identifying current infections. Its strength is that it will identify most current infections if the sample collection is correct and testing conditions are ideal. Its weakness is that it can also identify recovered patients who are still excreting remnants of non-culturable virus and who are no longer infectious.

Earlier in the pandemic, the criterion for recovery was that the patient should test negative with Rt-PCR on two separate days. There were patients testing positive up to 3 months from first positive tests. When it was determined that these patients were no longer infectious, this requirement was dropped for a time/symptom- based recovery. This is important to note in that though it is a gold standard, it can still come up with false positives, meaning it can be positive in those who are not currently infected. Plus, RT-PCR will still miss up to 30% of cases due to many factors as sample collection errors, poor timing etc. Thus, it can be said that RT-PCR is a “flawed” gold standard and we should look at it with a more critical eye, not “worship” it as the end-all and be-all of COVID testing.

There is a critical need to balance many factors other than technical such as rapidity of results, ease of performance, availability, emerging data, and cost of testing. Thus, we in the Philippine Society of Pathologists came up with a position paper advocating pooled RT-PCR testing for asymptomatic populations in late May 2020. This caught the eye of the DOH and we were asked to provide proof of concept which we did in a research study just concluded this month. Considering the circumstances with having to secure ethics approval from 3 different institutions, this study was conducted in a record time of 4 months. The results are promising and should open more populations to be tested with this pooled approach which will reduce costs markedly by as much as 70% and use less reagents not sacrificing speed of results generation.

The research acknowledges the fact that there will be a few cases missed compared to individual RT-PCR testing. However, it is again important to note that those missed will most probably be with low viral loads who are no longer infectious. In essence, this is analogous to the RAgT in terms of being able to detect highly infectious persons and not having the “Achilles heel” of the individual Rt-PCR which can detect non-viable virus.

The added advantage of pooled RT-PCR testing is that it will identify asymptomatic persons with high viral loads who can become “super spreaders” of the virus that is  driving the spread of the pandemic among the general population. Thus, due to its reduced cost and ease of testing large numbers of subjects, pooled testing can be applied to frontline workers in the economy as well as in the health sector due to its economy of scale. Efforts are underway to roll out the pooled testing procedure in pre-qualified laboratories that will undergo a stringent process of training and validation.

Another recent innovation is the use of saliva as the specimen for RT-PCR and antigen testing. This promises to be another game-changer in the COVID testing landscape. Imagine doing away with the time-and labor- and resource-consuming effort of swabbing patients to get their samples for testing. That alone will reduce the costs and time to perform testing, not to mention the aversion of patients to an invasive and obnoxious procedure causing at the least discomfort in many and pain in some. Come to think of it, why wasn’t saliva initially thought of as the sample of choice initially? The few studies that looked into this were not successful though they were using different reagents and methods than the more recent promising researches.

To be able to use saliva as a valid specimen for antigen testing and pooled testing, we are now looking into this possibility with another research proposal to validate the concept. This again requires some time to achieve but it will be worth it since it will make testing even more accessible and cost-efficient for the companies seeking to protect their workers and customers from being infected as we open up the economy more with its attendant risk of a second wave of infections like we’re seeing in Europe.

We should emulate our Asian neighbors who have controlled the pandemic in their own lands like Taiwan, Korea, Vietnam and Thailand. They employed aggressive mass testing and contact tracing to tamp down infection hotspots. With saliva-based testing, both antigen and pooled RT-PCR, we should be able to achieve that.

A more recent newcomer to the COVID testing scene is the CRISPR Cas-12 RT-LAMP assay utilizing a nucleic acid amplification step similar to RT-PCR. It is now available in other countries and is also in the rapid test format of the lateral flow method. This method also gives out results within 30-40 minutes similar to the antigen assay but is more analogous to the classic RT-PCR test method albeit in a more simplified format since there is no repeated heating and cooling as in RT-PCR meaning the process occurs at one temperature range (isothermic). Thus, it is less cumbersome and more economical as well as faster than RT-PCR. Sensitivity is essentially similar to RT-PCR in the range of 10 copies per microliter. Indeed, a game-changer! (Pardon the cliché).

For sure, there will be more innovative test methods that will come along. It is important that we don’t lose sight of what we want to achieve, which is to control the spread of the SARS-CoV-2 virus. We must employ all the available armamentaria currently available and more so, those that will come out in the future, to minimize the effects of this pandemic on our lives and in the economy. It should not be a choice of health versus livelihood but of a co-existence of the two with the efficacious approach keeping an open mind on our options. The science behind the SARS-CoV-2 pandemic is still rapidly evolving and we learn new lessons almost every day with the seemingly fast proliferating research articles published online every day. If we keep clinging to the RT-PCR alone in dealing with COVID-19, we may doom ourselves to cycles of death and economic destruction from which we may not be able to rise above.

Indeed, adapt or perish!

      The author is an American and Philippine Board-certified pathologist who is the principal investigator of the pooled testing research of the Philippine Society of Pathologists, Inc. entitled An Evaluation of Pooling Strategies for qRT-PCR Testing for SARS-CoV-2 Infection and is a member of the DOH COVID Lab Experts Panel.

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