This blog shares a brief overview of the testing process of people who may be infected with Coronavirus disease 2019 (COVID-19). As COVID-19 has already infected nearly 250,000 people and killed more than 10,000 as of last Friday afternoon, a cure or vaccine continues to elude researchers worldwide. However, tests to determine whether or not an individual is affected do exist. But why isn’t everyone being tested? And how do we test for COVID-19?
What is COVID-19?
Chances are high that this is not the first time you hear this but for the sake of being complete, here it is once more. This particular virus is part of a large family of coronaviruses (CoV), that entails diseases like the common cold and Severe Acute Respiratory Syndrome (SARS-CoV). CoV are zoonotic, meaning that they circulate among animals and some of these CoV have the capability of transmitting between animals and humans. For example, SARS-CoV was passed on from civet cats to humans.
There are a couple of steps proceeding the confirmation of the virus that will be explained into detail as well as the question of why the result is taking so long while time is of the essence.
Who are we sampling for COVID-19?
Clinicians should determine if a patient has signs and symptoms compatible with COVID-19 and whether the patient should be tested. Most patients have developed a fever and/or have symptoms such as a cough or difficulty breathing. Based on the pre-triage at home, through remote consultations or in the emergency care, the need for testing is determined. Factors that promote immediate testing and possible hospitalization include age of the patient, medical condition and history (chronic medical and/or imminocompromised state), severness of the symptoms and contact with a confirmed COVID-19 patient.
How are we sampling for COVID-19?
For proper diagnostic testing for COVID-19, the preffered collection method is an upper respiratory nasopharyngeal swab (NP). Suggested but not a priority is the collection of oropharyngeal swabs (OP). Sputum, endotracheal aspirate or bronchoalveolar lavage can also be obtained and collected in a sterile container if the patient suffers from a cough. The quality of the swabs is particularly important. The use of synthetic fibre swabs with plastic shafts is advised since swabs containing calcium alginate and swabs with wooden shafts may contain substances that inactivate some viruses and thus inhibit PCR testing. The collected specimens should be stored and shipped at 2-8°C up to 72 hours. In case of a delay, the sampling should be stored at -70°C.
How do we test for COVID-19?
The specimens should be tested in qualified laboratories, with specific trained personnel and at least in a biosafety level 2 facility. Additional testing for other respiratory pathogens such as influenza is advised but should not delay the COVID-19 testing.
Routine confirmation of COVID-19 is based on the detection of unique sequences of virus RNA by nucleic acid amplification tests (NAAT) such as a real-time reverse-transcription polymerase chain reaction (rRT-PCR). Specifically designed rRT-PCR panels are molecular in vitro diagnostic tests resulting in the detection and determination of the COVID-19 virus.
The test contains oligonucleotide primers and dual-labelled hydrolysis probes (TaqMan®) and control material used in rRT-PCR for the in vitro qualitative detection of COVID-19 RNA in respiratory specimens.
What is RT-PCR?
The RT-PCR is a technique that is actually a combination of two other techniques used together to identify a specific genetic sequence (currently, the only way to determine if someone has COVID-19):
SARS-CoV-2 is made up out of (single-stranded) RNA so, first, you have to turn its genome into DNA with an enzyme called reverse transcriptase in order to be able to use it in DNA sequencing. (RT)
Polymerase chain reaction, is a DNA amplification technique that is routinely used in the lab to turn tiny amounts of DNA into large enough quantities that they can be analyzed. It's actually pretty old as it was invented in the '80s already. (PCR)
The summarized process for RT-PCR :
1. The virus contains single-stranded RNA which needs to be extracted.
2. An oligo dT primer is added that binds to the poly A tail of the RNA strand.
3. Afterwards, the enzyme reverse transcriptase and deoxyribose nuleotide triphospates (dNTPs) are added.
4. After binding to the oligo dT primer the reverse transcriptase sysnthesizes the cDNA by adding the dNTPs.
5. Finally, the cDNA is complemented by adding additional primers and TAQ polymerase and can be amplicfied to levels that are sufficient to allow detection in an assay.
How do we analyze test results?
Laboratory confirmation of cases by NAAT depends on whether or not a geographic area has a known COVID-19 virus circulation among the population or not. To consider a case as laboratory-confirmed by NAAT in an area with no COVID-19 virus circulation, one of the following conditions needs to be met:
A positive NAAT result for at least two different targets on the COVID-19 virus genome, of which at least one target is preferably specific for COVID-19 virus using a validated assay (as at present no other SARS-like coronaviruses are circulating in the human population it can be debated whether it has to be COVID-19 or SARS-like coronavirus specific); OR
One positive NAAT result for the presence of beta coronavirus, and COVID-19 virus further identified by sequencing partial or whole genome of the virus as long as the sequence target is larger or different from the amplicon probed in the NAAT assay used.
When there are discordant results, the patient should be resampled and, if appropriate, sequencing of the virus from the original specimen or of an amplicon generated from an appropriate NAAT assay, different from the NAAT assay initially used, should be obtained to provide a reliable test result. Laboratories are urged to seek confirmation of any surprising results in an international reference laboratory.
In areas where COVID-19 virus is widely spread, a screening by rRT-PCR of a single discriminatory target is considered sufficient. One or more negative results do not rule out the possibility of COVID-19 virus infection. A number of factors could lead to a negative result in an infected individual, including:
poor quality of the specimen, containing little patient material (as a control, consider determining whether there is adequate human DNA in the sample by including a human target in the PCR testing)
the specimen was collected late or very early in the infection
the specimen was not handled and shipped appropriately
technical reasons inherent to the test, e.g. virus mutation or PCR inhibition.
If a negative result is obtained from a patient with a high index of suspicion for COVID-19 virus infection, particularly when only upper respiratory tract specimens were collected, additional specimens, including from the lower respiratory tract if possible, should be collected and tested.
Why does the COVID-19 testing process take so long?
Taking into account that all COVID-19 tests have to be conducted in a lab with the appropriate quality standards, performed by trained technicians, specific qualified PCR equipment and their specific reagents (RNA extraction kits, primer, probes & standards), it becomes obvious that we are facing a capacity issue. In addition, sample collection, storage and transport to the testing facility is difficult as the clinicians or health professionals require additional measures for their own safety.
Both governments, regulatory agencies, life sciences companies and suppliers of in vitro diagnostic equipment are working hard to overcome the challenges we face today by developing, validating and authorizing new test procedures to multiply the capacity.
Blog by Stefanie Pauwels
(5) Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases, WHO