Swabs, serological tests and rapid tests: how to assess the epidemiologic trend of COVID-19 pandemic

Monitoring the epidemiologic trend of the pandemic by performing tests is the elective method to fight the new Coronavirus.

However, there are different situations to be faced in an emergency like this: a limited availability of tests at international level, impossible safe storage of samples, a workload for laboratory personnel.

Following the recommendations published by the European Commission (EUCOMM) and the World Health Organisation (OMS), last 3 April the Ministry of Health issued a memorandum letter (no. 11715) with the purpose of “adopting a strategy identifying priorities for the performance of diagnostic tests for SARS-CoV-2, to ensure an optimal use of resources and relieve pressure on laboratories as far as possible”.

The tests used fall into the category of in vitro diagnostics (or IVDs), whose reference directive (98/79/EC) establishes that they have to be designed and manufactured in order to comply with the specified intended use, taking account of the generally acknowledged state of the art. Therefore, in a situation like this, with the state of the art rapidly evolving, the use of safe tests, able to provide the performance they are intended for, must be ensured.

From a scientific point of view, there are two main types of tests used for Covid-19 and widely under discussion: those detecting the virus, and those detecting a previous exposure to it. Clearly, this substantial difference results in a variety of technical aspects, especially in terms of sensitivity, specificity, repeatability, reproducibility and limits of detection. The selection of the different parameters is justified by the intended use attributed to the device: rapid screening, diagnosis, confirmation.

A further distinction can be made between the tests detecting the virus: those detecting the genetic viral material (by reverse transcriptase-polymerase chain reaction, or RT-PCR as Covid-19 is a RNS virus – molecular test) and those detecting components of the virus such as the proteins on its surface (antigen test). These tests are performed on nasal or throat secretions, withdrawn by using the so called swabs.

Tests detecting a previous exposure to the virus are based on the identification of the antibodies present in the blood of the infected person, and therefore on the ability of his/her immune system to respond. As they are performed on the blood serum, they are called serological tests.

Before being used, tests have to be validated: inadequate tests can lead to erroneous conclusions further hindering the efforts to control the pandemic. The validation allows to know the technical characteristics of the tests such as their sensitivity, specificity and therefore the reliability of their results.

RT-PCR tests are the test currently recommended by the WHO and the European Centre for Disease Prevention and Control (EDCD) for the diagnosis of Covid-19.

Annex 1 of the memorandum letter issued by the Ministry of Health on 3 April includes a first list of diagnostic kits, with CE marking, registered in the Italian database, that were validated only by the manufacturing companies under their own responsibility.

In the Communication of the European Commission of 15 April it is “recommended to carry out additional validation of the clinical performance of tests for COVID-19 by comparison with a reference method in a sufficiently large number of target population subjects before introducing the devices into the clinical routine.”

A peer review of the scientific results is also recommended: validation studies are made difficult by the current shortage of reference methods and materials.

The WHO started a validation procedure (“Emerging Use Listing Procedure”) for IVDs detecting genetic material of SARS-CoV-2 and stressed the importance of the surveillance data (PMS): based on the information from these activities, the WHO will in fact be able to decide whether to maintain or remove a product from the list.

Considering the emergency situation, CE-IVD and/or EUA/FDA rapid molecular tests (Point of Care tests, POCT) were also developed. These are based on the detection of viral genes directly in the respiratory secretions in a short time. According to the Technical Scientific Committee (CTS), these tests could be useful in the cases where the diagnosis of Covid-19 infection is urgent.

A type of rapid diagnostic test (RDT) detects the presence of viral proteins (antigens) expressed by the COVID-19 virus in a sample from the respiratory tract. If the target antigen is present in sufficient concentration in the sample, it will bind to specific antibodies, usually in 30 minutes. The functioning of these rapid tests depends on various factors, including the time from the onset of the disease, the concentration of the virus in the sample, the quality of the collected sample, how it is processed. As the detected antigens are only expressed when the virus is under active replication, these test could be used to identify early or acute infection.

False positive outcomes could also occur if the antibodies do not recognise the antigens of viruses other than COVID-19, such as those of other human coronaviruses.

With the data currently available, the WHO does not recommend the use of rapid diagnostic tests for antigen detection for clinical diagnostic purposes, although research on their performance is encouraging and their use could lead to a decrease in the high costs of the confirming molecular tests.

Although there are many on the market, the reliability of serological tests for the diagnosis is also limited: several studies suggest that, in most patients, immune response only occurs in the second week after the onset of symptoms, and that the production of antibodies starts after 6-15 days.

Moreover, the immune system response depends on different factors such as age, nutrition state, concomitant diseases, treatment with specific drugs. Therefore, the lack of antibodies detection does not rule out a possible ongoing infection at its early stage or with no symptoms and the relevant risk that the person is contagious. Moreover, due to a possible cross-reactivity with other similar pathogens such as other human coronaviruses, the detection of antibodies could be non specific for the SARS-CoV-2 infection, leading to false positive results.

This means that, until new supporting evidence is available, at the current stage serological tests can help in the evaluation of the epidemiologic trend of the pandemic, but they cannot be used for diagnostic purposes.

High commitment and information shared between WHO and other agencies, regulatory authorities, research groups, and Member States is essential for the development and correct interpretation of the data on the different types of tests. These, depending on their characteristics and intended use, can be useful during the current pandemic for the clinical management, diagnosis and control of the contagion, as well as for epidemiological research.

Written by: Ilaria Perretti