COVID-19 and Viral Testing

This COVID-19 pandemic has killed more than 210,000 people in the United States and million around the world. Our elderly and the most underprivileged populations have paid a heavy price because of their co-morbidities. We have refined the way to test for the disease. Tests results are based on the reverse transcription of the polymerase chain reaction to confirm COVID-19 like illness in the emergency rooms or hospitals. We noticed in the last 3 months that the median age of COVID-19 has shifted down in the United States from 45 and up to 35 and less in the southern states especially.

A similar trend based on different age group (below19, 20 to-39, 40 to 59 and above 60 was demonstrated. The median age showed a similar trend for decreased median age and the incidence was higher among the second age group with more than 20% of confirmed cases suggesting that this younger group contributed to the transmission of the disease to others. It was found that social distancing and mask wearing in this age-group was a less common practice increasing the risk factor for severe illness and death to others. The young population also returned on the campus at the universities to their classes or to their jobs on the assembly lines increasing the rate of catching and transmitting the Virus SARS-CoV-2.

Let us review the trend in testing.

Specimen are taken from each nostril or from the saliva with nasopharyngeal swabs to test for a chain reaction SARS-CoV-2 specific polymerase as the preferred method. The specimen can be self-collected or taken by some healthcare personnel. It is preferable to take the specimen from the anterior aspect of the nasal region (ANS). Patients were also seen at a drive through testing facility using the same technique. In a study, a cohort of 354 patients participated with symptoms of fever, sore-throat, shortening of breath, loss of smell, malaise, chills etc. 47% were female. Patients were asked to self-swabbed both nostrils and to spit the saliva in a tube while the healthcare personnel was also taking specimen from the anterior aspect of the nostrils to be examined following the Hologic Aptima Sars-CoV-2 test.

Results: 66 patients (18.6%) were found to be positive for SARs-CoV-2 and 268 (75.7%) were tested negative. The higher positivity rate was found when a combined analysis of nasal swabs and saliva on the same patient were examined. These tests lack in sensitivity and specificity. The self-performed (ANS) swabbing’s detected the fewest cases and is now discouraged. saliva is more easily collected from children and can also avoid the exposure of the healthcare personnel. There is a limited stability for the viral RNA and these specimens should be refrigerated at 4 degrees Centigrade.

In this time when the SARS-CoV-2 virus is mutating and perhaps getting weaker, one has to ask who should be tested for the disease and what kind of test should be requested. The universal precautions in wearing masks, washing hands as often as possible remain essential in the prevention of the COVID-19. There are two kind of tests available for the disease: a viral test to assure that you do have a current infection and an antibody test to search or admit that you may have had a past infection.

There are reasons to be tested for the COVID-19 especially if you have symptoms related to the disease like a dry cough, shortness of breath, high temperature etc. It is recommended that people who had closed contact with someone who was diagnosed or confirmed to have the disease should be tested. Close contact is still considered in the vicinity of the infected person around 6 feet for a certain period of time at least 15 minutes by example at work or at home or in a public place. Recent episode of contamination at the Rose garden of the white house in Washington DC has perhaps confirmed that the virus can be carried airborne at more than 6 feet in social distancing. Finally, people can be asked to perform the test especially when recently many countries governments are requiring a “passport of health” to visit, assuring that you are COVID-19 free of disease. Finally, people can be referred by a healthcare provider who has performed a physical exam on a suspicious patient.

Not everybody needs to be tested but if you get tested at the request of a healthcare provider, you should remain in a self-quarantine until the results are known officially and the healthcare provider have discussed the results with you. If you have symptoms of COVID-19 as mentioned and want to get tested, you can call your healthcare provider first. But, if your test is positive, you should prevent other from catching the virus from you by wearing a mask. If you tested negative, you probably were not infected although the test may mean that you were not infected at the time the specimen was collected.

As discussed earlier, COVID-19 testing centers on the assessment of a current or past presence of SARS-CoV-2 in an individual either by looking for the virus itself, either by looking for the antibodies. The test searching for the virus will allow health authorities to trace or contain an outbreak while the antibody test will demonstrate that somebody was in contact with the virus or has recuperated from the disease. Antibodies take time to generate and can be used to assess the disease prevalence or often helping in an estimation of the infection-fatality rate. There may be an urgent need for frequent surveillance because the transmission of the virus occurs 4 to 5 days after exposure. A positive viral test means an active infection while a positive antibody test to COVID-19 means that the individual has recuperated from a previous infection.

The virus replicates through a polymerase chain reaction (PCR) which is a process used to amplify a defined segment of DNA which can then be extracted by a reverse transcription (RT-PCR) responsible for the conversion of RNA into DNA. This test has a high sensitivity as well as a high specificity to study samples from nasopharyngeal swabs / suction catheter or sputum. It seems that the specimen from sputum have more RNA material than the nasopharyngeal swabs meaning that the virus may escape detection. Sensibility in clinical samples studied by RT-PCR is 63% for nasal swabs but only 32% from pharyngeal swabs, but 75% from the sputum samples.

So the way the samples are taken is important. for some tests performed on the throat or the nose by swabs are reliable for only a week. This is again why sputum or deep collections are preferred. Collecting saliva may decrease the risk of exposure for the health workers while this process becomes easier for the patient. A new test developed at Yale University appears to give results in hours.

There are tests that amplify the virus genome which are faster than the PCR are being studied like the “Isothermal nucleic acid amplification test” to detect DNA using fluorescent tags like by the CRISPR gene editing technology. This test will amplify RNA directly without using the route RNA-to-DNA step. It is expected to be cheaper.

There is also an antigen test to identify part of the pathogen that illicit the immune response. It looks at the surface antigens from the viral surface spikes. The virus of SARS-CoV-2 antigens can be detected even before the onset of the symptoms. They may show less sensitivity than the PCR. RT-PCR may be more accurate but require too much time. Swabs collected from nostrils or saliva may lack antigen material especially if the patient is asymptomatic. The WHO reports a sensitivity as low as 34% to 80% by these tests which can easily miss half of the COVID-19 affected patients. Other argue that the higher the viral load the more sensitive is the antigen test. placing the best time to perform this test to  the moment a patient becomes contagious.

Lung CT Scans have also been used to show bilateral multilobar “ground-glass opacities” with peripheral or posterior distribution. CT can diagnose COVID-19 with a higher precision than RT-PCR.

The body react to an antigen by producing antibodies to help neutralize the virus and a serology test will detect them. This can help to assess the percentage of a population that has been in contact with the virus and subsequently one can figure the mortality rate in the population. We are not too sure how many days the antibodies will circulate in a patient recovering from the COVID-19 but so far studies have shown that antibodies have been found during three to four weeks after treatment of the infected persons. We are not too sure if a positive antibody test provides any immunity to the SARS-CoV-2 nor if it prevents the individual who recovered from the disease to be contaminated for a second time. It is certainly not like other diseases where antibodies persist for longtime. We believe that cases of re-infection have been reported around the world and in the United Stated. For some the re-infection was fatal and for others, it was a milder form of the disease. In these cases, it was proven that the SARS-CoV-2 virus has shown mutation.

Antibodies like IgM and IgG are detected several days after the initial infection but mainly the IgG are detectable after two weeks of the initial infection while the IgM can be as well seen earlier in a week after the beginning of the disease. There is generally a high specificity rate of 99.5% but a lesser sensitivity at 55%. while this test is performed.

There are also genetic tests which can verify even before the antibody tests that the individual has suffered a viral infection. A “rapid diagnostic test (RDT) uses blood or saliva or nasal swabs samples on a flow assay to exhibit a positive or a negative result which is noted by discolored line.

There are serologic tests; An Enzyme-linked immunosorbent assay (ELISA) uses blood, plasma or serum samples which are incubated allowing the antibodies to bind the viral protein SARS-CoV-2. The antibody-protein complex can be the detected in a color /fluorescent readout.

A Neutralization assay will assess if the antibodies prevent the viral infection in the cell. This test uses sample of blood, plasma or serum. Then a culture of the cell will demonstrate viral reproduction (VeroE6 cells). Researchers have shown then how many test antibodies block the virus reproduction.

A chemiluminescent immunoassay quantitative test can be performed on samples of blood, plasma and sputum. The samples are mixed with a known viral protein, with buffer reagents and enzyme labeled antibodies. A chemical immunoassay detects luminescent proteins-coated micro-particles then the antibodies react to the viral proteins and form a complex. Enzyme-labeled antibodies are added to allow them bind to these complexes. The radiance is used to calculate the number of antibodies like IgG, IgM and IgA.

A neutralizing antibody (NAb) test which looks for binding antibodies able defend a cell from infectious particles by neutralizing its biological effect. A binding antibody bind to the infectious pathogen while this pathogen remains infective. It also flags the pathogen for destruction by the immune system. Others believe that it may also enhance infectivity by interacting with the receptors on the microphages. Binding antibodies imply the presence of Nabs which may last for 2 years as demonstrated in the original SARS virus but can persist after six years. Unfortunately, it has not yet been established for COVID-19 yet but memory cells (B cells and T cells) can last even longer.

After recovery most patients show no longer have viral RNA in the upper respiratory specimen and there is no clear correlation between the length of the illness and the duration of the post recovery shedding of the viral RNA.

Testing strategies vary by country and over time. Some will test widely and others will test only the seriously ill (confirmed cases). Country that test more have a lower estimated case fatality rate and a younger age distribution of cases while the one testing only confirmed cases will have a higher confirmed/million people.

Maxime Coles MD (10-6-2020)


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