No test is better than false negative tests

by Muhammad Morshed | Published: 00:00, Apr 26,2020

 
 

Laboratory technicians wearing full PPE hold containers of test tubes containing live samples taken from people tested for the novel coronavirus, at a new Lighthouse Lab facility dedicated to the testing for COVID-19, at Queen Elizabeth University Hospital in Glasgow on April 22. — Agence France-Presse/Pool/Andrew Milligan

LABORATORY test is an essential element to diagnose any disease. Medical literature suggests that more than 75 per cent of decision-making for patient care depends on laboratory diagnosis. For most of the infectious diseases, commonly two types of tests are performed in the laboratory — the antigen test and the antibody test. Hundreds of companies market tests for COVID-19 caused by SARS CoV-2.

Antigen tests look for the organism under microscope, grow microorganism under laboratory condition or detect genetic materials of the pathogen of interest using advanced molecular technology. Electron microscopes are essential to look for virus morphology; growing virus in laboratory requires live cells, several days and a highly specialised laboratory, which is challenging and a very expensive endeavour. So, the main test is used to detect viruses these days by amplifying their genetic materials in a test tube and the procedure is called nucleic acid amplification test, commonly known as polymerase chain reaction, or PCR in short. This can be achieved in few hours and the sensitivity is much higher compared with other methods. There are different types of polymerase chain reaction and the most common one being used for SARS CoV-2 is a real-time reverse transcription polymerase chain reaction (rRT-PCR) using fluid from nasal or throat cavity as test samples. PCR tests have to go through several steps such as sample collection, RNA extraction, genetic material amplification and finally data analysis and produce a result for physician’s use.

Any flaws in the above steps will affect the overall quality of the test. As a result, we will not be able to detect genetic material from COVID-19 patients or it could yield a false positive result. Over 45 per cent of laboratory errors occur at pre-analytical phase, which includes selection of patients, collection of samples, shipping samples to the testing laboratory etc. Sample collection is utmost important. For SARS CoV-2 PCR test, it requires a cotton swab, buffer and a test tube. SARS CoV-2 rRT-PCR tests are available in different formats such as in-house developed test, manual or semi-automated and high-volume automated platforms. To have easy access, several companies make those three items in a plastic or cotton pouch as a sample collection kit. Big companies supply them with their test kits and advocate customers to use their own sample collection kits as a business strategy. But the problem is that several platforms recommend different swab types. Are all of them good or have equal quality and reproducibility? I guess, not. Many laboratories have evaluated different swabs and buffers and the outcomes have varied greatly. So, laboratory should be very careful in selecting the swabs, buffers and collection tubes, based on research data available in the scientific domain. Literature is mounting every day. So far we know, nasopharyngeal swab seems to be the best among all sample types tested regarding viral yield if collected immediately (2 to 3 days) after the onset of symptoms. Test sensitivity of rT-PCR could even be significantly low; so, samples should be collected at least 4 to 5 days after the symptom onset. Among those molecular test devices, a few of them can be done in a limited settings and can be employed in smaller town and cities; they are, however, very expensive. Some companies have also developed rapid testing device called rapid antigen test, similar to blood glucose or pregnancy test done on the street or in the park. However, I would not recommend this because this particular virus transits through aerosolization. So, it should not be encouraged. This particular test worked extremely well for the West Nile virus, or WNv, to see whether mosquitoes or bird are positive for WNv during outbreaks.

The second type of test is called antibody test. When people get infected with any microorganism, the body’s defence system tries to fight that out and as a result, human body creates a few weapons called antibody/ies. Antibody could be of different types and they are named as immunoglobulin IgA, IgM, IgG etc. It is now understood that for the SRAS-CoV-2, IGA and IgM appear first in the blood within 5 to 7 days followed by IgG, which needs at least 7 to 12 days. IgA test specificity has always been an issue because of it specificity. So, if IgM is detected in any symptomatic individual, this can be interpreted as the ‘most likely to have been exposed to SRAS-CoV-2 very recently’. Similarly, when IgG appears in the blood, it suggests that the patient was exposed to that pathogen for a while and most likely has become immune to SARS CoV-2.

So, antibody test can be used for dual purposes — when antigen test is not available or could not be performed, then IgM marker can be used for the diagnosis of an early acute infection along with patient’s clinical presentation. Second, the IgG antibody test against SARS CoV-2 can be used to see what per cent of population is exposed to COVID-19 and how of them have grown immunity. IgG test could be used as tool for making public health decisions, eg, if someone with symptoms or no symptom, however, has enough IgG level against SARS CoV-2 and then the results could be interpreted that the person is likely immune to SARS CoV-2 and can go out for work and that is extremely helpful for healthcare workers and in a long term, it could be used for phased lifting of lockdown restrictions.

What is now important is that how we choose the test that we can recommend for uses. Organisations such as the Institute of Epidemiology, Disease Control And Research or the International Centre for Diarrhoeal Disease Research, Bangladesh should play a role as gatekeepers of the tests I mentioned. They must ensure the quality of the tests and recommend their use to reputable private laboratories and tertiary healthcare hospitals. There are hundreds of companies marketing both rRT-PCR and serology, especially PoC-formatted serology, tests products. Many of these products are of extremely low quality, which will provide faulty, negative and false positive results. Both false negative (person having the disease but yielding negative results) laboratory and false positive (person not having disease but yielding positive results) tests can make more harm than good. False negative test result will accelerate more transmission of this virus and false positive results will create panic in the community for no reason.

In that case, no laboratory test is better than low-quality test available on the market. So, complete evaluation and validation process should be completed before employing any test in the laboratory and continuous monitoring on the test kits is essential. For Bangladesh, evaluating test kits may pose challenges because of the unavailability of serum samples from related diseases such as SARS and other viruses essential to determine the specificity. To overcome this situation, we should find kit evaluation performances from accredited institutions globally, analyse the global data and pick the best one that suits the country’s need. Clinical diagnosis is much better than less sensitive (failing to detect the antibody) and or less specific (showing antibody, but from other pathogens) laboratory tests. Physicians may also use other tests such as chest X-ray, computed tomography as well as routine blood enzyme tests, along with rRT-PCR or antibody tests.

In conclusion, the most practical test for COVID-19 is an antigen test, which tests the RNA in the virus. It is performed on a nose/throat swab. The test can be negative in as high as 30 per cent cases. It takes a few hours to get results. There is a rapid test being released now which will give a result in about an hour. All these test types need to be performed in reputable laboratories.

Several groups are evaluating antibody tests for COVID-19. Antibodies are proteins made by the body to fight infections and they are detected in blood tests. There are IgM antibodies which tend to indicate more recent infections and IgG antibodies that tend to reflect antibody that has been present longer which might imply immunity. These tests could be used to measure how much asymptomatic infection has been in a group of people and also in the long term, they could be used for lifting lockdown restrictions.

Diagnosing acute COVID-19 disease would depend on the clinical impression, a history of exposure, if any, appropriate tests like chest X-ray and chest computed tomography, if necessary.

 

Dr Muhammad Morshed is a clinical microbiologist and programme head, zoonotic diseases and emerging pathogens, at BC Centre for Disease Control and clinical professor, department of pathology and laboratory medicine, University of British Columbia.

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