By Dr Edward Nazareth
Aug 3: Recently there has been a lot of adverse comments and anguish expressed in social media when inaccurate results of COVID 19 infection were reported.During the ongoing pandemic, there has been a lot of buzz regarding the possible errors in diagnoses with the current tests for COVID 19 infection all over the world. To understand how serious these errors might be during a pandemic, we need to understand the basics of the tests and the nature of different types of errors.
Two types-three tests
At present we have two main types of tests: diagnostic tests (molecular and antigen) and antibody test (serological). Diagnostic tests detect active infection with the virus, whereas antibody tests check if an individual has had a previous infection with the virus.
RT-PCR test: (Reverse-transcription polymerase chain reaction test or RT-PCR test). This is the most common diagnostic test used to identify people currently infected with COVID19. In this test the presence of viral genetic material in a sample is detected. The specific technique that is used is called reverse transcription polymerase chain reaction, or RT-PCR. In this technique the nucleic acid of the virus is amplified by subjecting it to different temperature which is usually automated and can be detected in the form of graph after a stipulated time. It works by detecting the RNA of COVID 19 virus in the infected person’s cells—most often collected from the upper part of the throat that joins the nose-known as nasopharynx.
Rapid antigen test: The rapid antigen test has been recently introduced in our state. It is a test done on nasal swabs to detect antigens that are found on the surface of the COVID19 virus. This test can be done in small set ups, clinics or small hospitals and it gives quick results. In India, the ICMR has allowed the use of antigen detection kits developed by the South Korean company S D Biosensor, which has a manufacturing unit in Manesar in Haryana. The kit iscommercially known as Standard Q COVID-19 Ag detection kit.
Antibody test: The third test is the serological or antibody test. This test detects the antibodies produced by a person to eliminate the COVID 19 virus. The blood is used to detect the antibodies. This test is not useful to detect who might have the disease, because it can be positive even if one was infected and cured. This test also identifies if a person has had the novel corona virus at some point in the past and has produced antibodies to fight it. This test will help to estimate what percentage of the population got exposed to the virus and developed antibodies to fight the virus.
Accuracy of medical tests
Two terminologies are used to determine the accuracy of a medical test-‘sensitivity’ and ‘specificity’.
A sensitive test will correctly identify people with the disease. The sensitivity of a test is the measure of correct positive results or positive accuracy.Most of the tests is not 100 percent sensitive. Suppose a test is 90 percent sensitive, it will correctly identify 90 percent of people who are infected—termed as a true positive. However, 10 percent of people who are infected and tested would get a wrong result—they have the virus, but the test says they don’t.
A specific test will accurately identify people without the disease but the tests are not 100 percent specific either. The specificity of a test is the measure of correct negatives or negative accuracy. If a test is 90 percent specific, it will correctly identify 90 percent of people who are not infected—registering a true negative. However, 10 percent of people who are not infected will test positive for the virus and receive a wrong report.
False positive and false negative
Two more terminologies are also in use while considering the accuracy of a test report- ‘false positive’ and ‘false negative’.
A false positive test is when the test results show that a person is infected with COVID19 virusbut in reality, that person is not infected with the virus. Similarly a false negative result means that the person’s report shows that he does not have the COVID 19 virus but in reality he does. False negative tests can be extremely dangerous as it allows a person with COVID19 virus to live a normal life infecting their contacts and risking their own health as the treatment cannot be started because the person tested negative for the virus while they actually have it.
Recent data reportedly suggests that about 15% of all tests conducted in the United States are returning false negatives. It means for every 100 individuals infected with COVID-19, 15 of them are told they don't have it.
Accuracy and limitation of RT-PCR test
At present all over the world RT-PCR test is considered as ‘gold standard test’ to detect COVID 19 infection. It is a nucleic acid (RNA) detection test, that means it detects the ‘real virus’ and the method is most accurate. Therefore real time RT-PCR assay can be considered as a main method to be applied to detect the causative agent-COVID19 virus.While RT-PCR is the gold standard due to its accuracy, the slow speed and difficulty of sample collection are its drawbacks.An important issue with the real-time RT-PCR test is the risk of eliciting false-negative and false-positive results. It is reported that many ‘suspected’ cases with typical features of COVID-19 are not diagnosed by this test. The treating physician is almost sure that the patient treated, has COVID19 infection, but the test says ‘no’! Thus, a negative result does not exclude the possibility of COVID-19 infection.
‘Though at present RT-PCR is considered as ‘gold standard test’ in the real sense it is not’-reported the British Medical Journal in May 2020. A systematic review of the accuracy of RT-PCR to detect COVID19 virus reported false negative rates of between 2% and 29%; that means the sensitivity was 71-98%, based on negative RT-PCR tests which were positive on repeat testing.
In a pre-print article in Researchgate in May 2020, the false positive results of RT-PCR are reported in a range of 0-16.7%. Such rates would have large impacts on test data when prevalence is low.
Virus and the test kits
Several factors have been proposed to be associated with the inconsistency of real-time RT-PCR test. Excluding the human errors, there are two important factors for consideration.
The most important factor is that the virus may be changing its genetic code itself. The viruses are known for this and the process is known as mutation. To understand this let me give an analogy: Let us presume that the COVID 19 virus has a core with a red colour but has now changed itself to pink by mutation. Our test was designed to identify a red coloured core; however it will fail to identify the one with pink colour. The result given will be negative-‘false negative’. (This is only an analogy-core is not dependent on colour).
Second factor is many of the test kits are not standardized. There has not been enough time to trial the tests repeatedly to get an accurate method. The infection is spreading rapidly and the nations are in a hurry to detect the infected people. Several types of COVID 19 real-time RT-PCR kit have been developed and approved rapidly, but with differing quality. As a result the sensitivity and specificity of the real-time RT-PCR test is not 100%. When a new test is rapidly created and deployed, as in the case of the current COVID 19 virus test, its accuracy cannot be exactly predicted beforehand.
A test developed under controlled conditions might behave in a different way when applied in the real world, and this might enhance the likelihood of errors due to many unforeseen events.Even if the test is standardized at a research center, it may not maintain the same standard in the laboratory setting. For example researchers at the ‘Foundation for Innovative New Diagnostics’ a nonprofit research center in Geneva, Switzerland tested five COVID-19 RT-PCR test kits and found that all five were 100% sensitive and 96% specific. In the laboratories the results were far from perfect- the clinical sensitivity was from 66% to 80%. That means nearly one in three infected people who are tested will receive false negative results.
‘The current CDC nucleic acid test kits for COVID19 (RT-PCR test kits) generate 30% false-positive and 20% false-negative results in the best state public health laboratory’ reported Dr. Sin Hang Leeon July 17, 2020, in a peer-reviewed article published in the International Journal of Geriatrics and Rehabilitation, an online journal based in Japan.(The CDC -Centers for Disease Control and Prevention-is an agency of the U.S. Department of Health and Human Service).
Collecting good samples
Most experts believe that the methods of collecting the samples through swabs are the main culprit behind inaccurate reports. False negative results are likely because the swabs do not have enough viruses. It can happen if the person who is collecting the sample does not insert the swab deep enough in the nose or does not collect enough of the sample.It is recommended to use nasopharyngeal (NP) swabs for molecular testing because in most patients, the nasopharynx, that is the space above the soft palate at the back of the nose, appears to have the highest concentration of virus. However, nasopharyngeal swab samples are technically challenging to obtain, and a suboptimal collection may reduce the test sensitivity and increase the likelihood of obtaining a false-negative result in a patient with the virus. Testing a swab from the oropharynx(back of the mouth) or nose is also likely to reduce the sensitivity further.
The sensitivity of the nasopharyngeal specimen for the RT-PCR test is reported between 63% and 78%. Few studies report the specificity, but when reported specificity has been cited as high as 98.8%. Sensitivity changes depending on the site of collection.With oropharyngeal swabs which are collected from the part where the mouth meets the throat, sensitivity is about 72%, but sputum and samples from the lungs (taken during a procedure called bronchoscopy) have sensitivities reported as high as 93%. It is important to note that these numbers may differ between and even within institutions due to the wide variety of testing platforms that have different molecular targets for the virus particle.
The errors can also happen if the samples are not properly stored before testing. The current guidelines are to store the specimens at 2-8°C for up to 72 hours after collection. If delay in testing or shipping is expected, the specimens need to be stored at -70°C or below. If this is not followed the viral RNA may break down and false negative result can occur.
Result and time of testing
False negatives could occur if a person is tested too early for their infection and there is not a lot of virus in their cells. A person who has been a primary contact and had got infected about two to three days prior to testing may not have enough viruses. The report may be negative. If the same person develops symptoms after five days and gets tested, the result may be positive.
The researchers estimated that those tested with RT-PCR test for COVID19 infection, four days after the infection were 67 percent more likely to test negative, even if they had the virus. When the individual began displaying symptoms of the virus, the false-negative rate was 38 percent. The test performed best, eight days after infection (on average, three days after symptom onset), but even then had a false negative rate of 20 percent, meaning one in five people who had the virus had a negative test result.
In many of the clinical situations, if an individual is displaying features of a particular disease, despite their negative report, the report is considered false negative and the patient is treated. Many of the doctors are following the same logic in managing symptomatic COVID 19 infected individuals-if a person presents with classic symptoms of COVID-19 and is in an area with an outbreak, doctors will often diagnose a person with the disease in spite of a negative test. At times repeat tests or some other tests like CT scan of the chest are done to confirm the diagnosis.
Sample pooling
Sample pooling allows multiple samples to be tested at the same time.This was introduced to increase the COVID19 testing capacity in order to meet the high demand for testing.This technique allows a testing facility to mix several samples together in a “batch” or pooled sample and then test the pooled sample with a diagnostic test. For example, five samples may be tested together, using only the resources needed for a single test. If the pooled sample is negative, it can be deduced that all five patients are negative. If the pooled sample comes back positive, then each sample needs to be tested individually to find out which was positive. Because samples are pooled together, ultimately fewer tests are run overall, meaning fewer testing supplies are used, and results can be given more quickly in most cases. However, because samples are diluted, which could result in less viral genetic material available to detect, there is a greater likelihood of false negative results, particularly if not properly validated. This method works well when there is a low prevalence of cases, meaning more negative results are expected. ICMR has allowed 5 samples to be pooled and tested in our country when the prevalence rate is between 2% to 5%. This is better in community survey or surveillance among asymptomatic individuals.
No virus but, test positive
In RT-PCR test, the result may remain positive for several weeks after the symptoms have disappeared and the individual is disease free. Initially it was thought to be ‘reinfection’, now it is known that the RT-PCR can detect a fragment of the RNA in the cell which may reflect only the lack of complete elimination of the nucleic acid from the tissues. Such people may be having what is known as ‘dead RNA’ of the virus, they may be positive but not infective-they may not spread the disease.The virus infectivity depends on the presence of the whole virus, not just its RNA. In many other viral diseases, such as Zika, it is well known that its RNA can be detected long after the clearance of the infectious virus. RT-PCR is not able to differentiate infective virus from non-infectious RNA.
RT-PCR test: no other choice?
Different values of the sensitivity of RT-PCR tests to COVID-19 have been reported in different parts of the globe. According to a report from China, up to 29% of people with the COVID 19 infection were tested negative. Studies in the US returned multiple values – sometimes it was 95%, sometimes 85%, and even 75%. If the sensitivity of one RT-PCR test kit is 90%, for example, and two successive negative test results are used to declare someone free of disease, there is still a 1% chance that a person with the disease would be declared negative. And if that is so, about 200,000 COVID-19 patients could have been wrongly diagnosed among the more than 20 million tests worldwide! This may be sufficient ground for quarantining patients with ‘negative’ test results for the recommended period (say, 14 days) in order to restrict the virus’s spread.
According to a study, when patients were tested immediately after the infection, typically before symptoms occurred, the false-negative rate was 100%. On the first day of symptoms, the false-negative rate was 38%. After three days of symptoms, false-negatives dropped to 20%, then began to increase again, 66% on day 21. The false-negative rate was minimized 8 days after exposure—that is, 3 days after the onset of symptoms on average. The rate began getting worse after five days, suggesting a narrow window for the most accurate results.
Rapid but not sensitive: Rapid antigen test
Most RT-PCR tests can take anywhere from a few hours to a few days, but new rapid diagnostic tests for COVID-19 show promise of results in less than an hour. (At present in Government facilities it takes more than five days and in private laboratories it takes minimum two days to get the report.) Antigen tests tend to cost lower for manufacturers to make due to their simpler design, allowing testing to scale up more quickly.
While RT-PCR tests detect viral genetic material, antigen tests detect specific proteins on the surface of the virus. Antigen test samples are collected just like RT-PCR tests, using nasal or throat swabs. The swab is then placed into a tube with special chemicals to expose the proteins, and the sample is dispensed into a cassette that is placed into the testing device to get the results.Compared to RT-PCR tests, antigen tests are useful due to their speed but are not as sensitive as RT-PCR, meaning that there is a greater chance of a false negative result. However, they are very specific for the virus so positive results are going to be highly accurate. It means if the antigen test is positive, it is very likely that the individual has the virus. If the test is negative and if there is suspicion of infection then RT-PCR test has to be done. The negative report is not conclusive.
As per ICMR guidelines, symptomatic persons who test negative in the rapid antigen tests should be followed up with an RT-PCR test. So a person who is declared negative can be positive in rapid antigen test.
The ‘Antibody test’ - reveals the past
Unlike diagnostic tests, the antibody tests (or serological tests) can identify thepeople who were infected and have recovered from COVID19 virus. The antibody test is done using blood, usually obtained through a simple finger prick. These tests do not require special equipment to process the results, which allows them to be used in small laboratories or at clinics.
When a person is exposed to the virus, the body develops antibodies to fight the virus. These are specific to a particular virus. For example the antibodies against the common cold virus can not eliminate COVID19 virus, though both these are corona viruses.
It may take several days to over a week to develop the antibodies in an individual who is infected. There are two types of antibodies- the immunoglobulin M (IgM) antibodies indicate recent exposure to COVID19, while the immunoglobulin G (IgG) antibodies indicate laterstage of infection. The rapid response serological tests typically use a technique called (ELISA- enzyme linked immunological assay) which detects the presence of these antibodies to COVID 19 virus.
As it takes several days to weeks for the body to develop an antibody response to the virus, serological test is not useful in identifying the presence of an infection in the early stage or when the person has symptoms of the infection. It only identifies the individuals who have had the infection.
It is suggested that timing of the test is also important for the accuracy of results, with the highest accuracy being 3 weeks after the onset of symptoms. However the accuracy of antibody tests for people who are asymptomatic or have mild symptoms is yet to be determined as their immune response may be different from those with severe disease. A study from China has suggested that asymptomatic people may have a weaker immune response to the virus, with 40% testing negative for IgG antibodies (those that develop in later-stage infection) 8 weeks after discharge from the hospital, compared to 12.9% of the symptomatic group.
Case Scenarios
1. I spent a day in the office with my colleague who was tested positive and as I was the primary contact I too went to a private hospital for testing after a day. I paid Rs.4000 for the test. The report was negative. After about 4 days of negative report I had fever and headache. My doctor advised me to go to the fever clinic at the government hospital and the swab was taken. The report was positive. Unfortunately by this time I had infected my entire family because of wrong report from the private hospital.
** Immediately after exposure to the virus, the report can be negative. Ideally one has to go for testing if they have symptoms or at least 5 days after the possibility of infection. The result of the test also depends on the amount of the viruses that have entered into the system. If the amount (the viral load) is less, the report may be negative for at least 5 days after infection.
When there is possibility of infection because of contact with a positive person, it is recommended to isolate and follow the advice of home isolation and wait for the symptoms to develop. If there are no symptoms even after one week-there are two options, either to continue isolation for another week or to go for the test.
If there are symptoms and the person is aged above 65 years or has co-morbidities like diabetes, hypertension, cardiac diseases, hypothyroid or other illnesses, it is better to go for the test, confirm the infection and follow the guidelines. If there are no co-morbidities and the person is younger than 65 years, even when there are symptoms the test can be optional, but the guidelines of home isolation are to be followed.
The RT-PCR and rapid antigen tests may become negative once the viruses are eliminated from the body. The antibody test may be positive by second week of infection and may remain positive for many weeks.
2. I have undergone open heart surgery three years ago. When I went to my cardiologist for some chest discomfort, he admitted me to a private hospital. I had no fever, no cough, and no body ache; even then the doctor suggested COVID 19 test as per their protocol. The report was positive. I had strong doubt on this report. On my request the swab was sent to another private hospital. It came negative. I strongly suspect the hospital must be giving wrong report for commercial interest.
**As explained above, the reports can be false positive or false negative. More than 80% of the people infected with COVID19 are asymptomatic, this means they do not have any symptoms and will not become ill. In RT-PCR test false positive reports are rare, but not nil. In your case one of the two reports are wrong-it may be the first or the second.If one of your report is positive, it is ideal for you to isolate yourself and observe for symptoms.
Though your suspicion is genuine, please note that the people who do the tests are not doctors, not the management; the tests are done by the technicians. Most of the institutes are now insisting on COVID 19 tests for patients like you as a precautionary preparation for any emergency intervention if needed. This protocol is now routinely followed.
The management of such big institutes is also careful about their reputation and they may not go the level of influencing the technicians to give false reports for commercial interests.
3. As I was a primary contact of a positive individual with disease, I was advised to go for test and it came positive in a government testing facility. My house was sealed down and I was advised to stay at home. However, as I had no symptoms, I went for repeat testing to a private laboratory and the report came negative.
**As you had the contact with a proven infected person, your report at the government facility must be correct. As the RT-PCR test is not 100% sensitive or specific, one or both of your reports may be wrong.
As explained in the article above, the report may become negative within a few days of infection. As you were asymptomatic, we cannot definitely say when you were infected. By the time you went to the private laboratory your body must have overcome the infection. In such a case both the reports may be true!
In a given scenario, it is ideal to trust the report and follow the quarantine rules. With a positive report at hand, by traveling for repeat testing one can infect many people who come in contact.
Choice in uncertainties
Neither the RT-PCR nor serological tests are perfect, but they are far better than nothing and offer incredibly valuable information to medical professionalsand the people that get tested.
Even with the current uncertainties, at this time our primary challenge is not the accuracy of the tests but the fact that not enough people are being tested.Accurate diagnosis of COVID19 infection is considered crucial to identify and control infection. But, as explained above, the technology used in the tests can depend on getting an adequate sample from the patient, often with a swab that reaches deep into the nasal passages. If the sample collected doesn’t have enough viral material on it, either because of how it was collected or because of how far along the infection is, it can affect the result. Low accuracy from the tests could diminish their usefulness as a catch-all screening tool for public-health workers and medical personnel treating patients.
In his 2015 book ‘The Laws of Medicine: Field Notes From an Uncertain Science’ Siddhartha Mukherjee, the Pulitzer Prize-winning author and one of the world’s foremost cancer researchers, wrote that “a strong intuition is much more powerful than a weak test”.
Thus, a test result for a person should not depend on the accuracy of the test alone but also on the estimated risk of disease before testing.If an individual is tested negative for COVID19 infection, but there is a strong possibility of infection because of the contact or symptoms, it is strongly advisable to self-isolate and monitor the symptoms. It is to be remembered that a negative test does not always mean that one doesn’t have the disease.
Inputs from Dr Anuradha K., Professor & Head, Department of Microbiology, MMC&RI, Mysuru
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