How do we diagnose SARS CoV-2 infection? There are three tests available for diagnosing SARS CoV-2, RT-PCR, Rapid Antigen Tests, and IgG and IgM antibody tests. Diagnosis is made primarily by reverse transcriptase-polymerase chain reaction, abbreviated RT-PCR, sometimes referred to as the molecular test. Within a week of identifying the outbreak in Hunan China, Chinese scientists published the full sequence of the virus, shown schematically at the bottom of this slide. Using this sequence short probe spanning parts of the RNA messages for the production of the spike protein arrow, the nucleocapsid envelope, RNA polymerase protein were all created. The sample is first treated with reverse transcriptase RT to synthesize the complimentary DNA sequence from the viral RNA. As compared to RNA which you can quickly degrade, DNA is stable and will not break down during repeated heating and cooling. Each heat and cool cycle doubles the amount of DNA since synthesis exhibition DNA for detection usually takes 2-3 hours and 15-30 cycles. The higher the virus concentration, the fewer cycles are required to reach the detectable levels. If no viral RNA is present the reactions will not take place and no DNA will be produced and the test will be negative. Samples of virus can be collected by swabbing of the posterior pharynx and throat and more recently by collecting saliva samples, which approve to be equally sensitive because special PCR machines, multiple reagents, and highly skilled technicians are required. This test is expensive, 75 to $120 per test. The results of this test are often delayed. Test values usually do not return for 24 hours because you need viral nucleic acid primer sequence are used, the test is highly specific, 99-100 percent specificity. The best RT-PCR test can detect as few as 10 copies of the virus. However, the sensitivity is difficult to estimate because of variability of the sampling method. Nasal pharyngeal swab or saliva samples appear to be equally sensitive. Shedding of the virus varies during the course of disease, explaining the relatively low sensitivity of 70-80 percent. This means that in situations where the likelihood of infection is high, because RT-PCR sensitivity is low, false-negative tests can occur and a single test should not be trusted to exclude disease. Originally, many countries had a shortage of tests, including the US. Now with improved variability, many health systems are screening all patients admitted to the hospital to exclude asymptomatic carriers. Aggressive testing combined with contact tracing and isolation are the ideal approach for controlling an outbreak once the number of daily cases becomes manageable. This is the approach being used by South Korea as shown in this epidemiology curve, plotting the number of daily cases per million over time, this country has maintained a low level of infection, 37 per million or 3.7 per 100,000 through September 23rd, 2021. Although many states in the US are administering similar numbers of tests, per capita, contact tracing and quarantining are often delayed and the opening of public spaces has continued to encourage disease spread. As a consequence, our level of infection is over 10 times higher, 380 per million or 38 per 100,000 as of September 23rd of this year. The second very useful test is the Antigen Test. This test detects a protein on the surface of the virus, most tests using an antibody that binds the spike protein. When the antibody binds to the virus spike protein it attracts a fluorescent dye and results in a fluorescent light signal, or the antibody is immobilized on a plate, using the same principles as a pregnancy test. When the bar turns black after exposure to the sample, instead of being pregnant, you are infected with the SARS CoV-2 virus. Because results can come back in 15 minutes rather than several hours or days, this is the ideal screening tool. These tests detect 120-150 viral particles as compared to PCR, which can detect 10 viral particles. Or as described earlier most infected individuals have millions, even billions of viral particles in their nasal secretions the saliva. The specificity and sensitivity of the antigen tests, are slightly lower than PCR. These tests, however, are one half to one third the cost of RT-PCR and do not require laboratory expertise. As I mentioned, they can be conducted in 15 minutes and are called point-of-care tests because of their rapid turnaround. Antigen tests should be the preferred method for screening. In industrialized countries, antigen tests are now available for home use. Finally, a word about antibody testing. This test will be helpful to an epidemiology tool. But because IgG and IgM antibodies usually take 7-14 days to rise, this test is not recommended for diagnosis. As shown in this graph, the vertical axis is the quantity of antibody and the horizontal axis is time in days. The lower blue line shows the antibody response of someone with mild disease as compared to someone with severe disease, the green line. Which results in the production of higher levels of antibody. This test can be used to determine the prevalence disease in a specific location. Now let's look at the time course of viral shedding and how this relates to test positivity. This graph is adapted from a publication from the Journal of the American Medical Association, JAMA. As depicted by the red line, which represents live virus, it generally takes 3-4 days after exposure for sufficient viral growth for RT-PCR to detect the virus, shown in the blue line. RT-PCR will detect virus earlier than the antigen test, which is also depicted by the red line. But, the problem with molecular tests, they often remain positive for several weeks after live virus is no longer present. The RT-PCR is detecting residual RNA released from the dead virus. The antigen test, on the other hand, detects intact live virus, closely approximating the same red line. Finally, the concentration of anti IgG-green line and anti IgM antibody-purple line are shown. You can see they both begin to increase within 10-14 days of onset of symptoms. IgM comes down over the first one to two months. IgG can persist for months to years. Is there a better way to identify early cases of COVID-19. Can a rise in core temperature be used as a pre-screen prior to ordering a PCR or antigen test. Could each of us take our oral temperature one to two times every day to establish a basal core temperature and to identify the early onset of fever and COVID-19. To answer this question, let's go back to the pathogenesis of the virus. When the virus binds to H2 receptors, it stimulates danger detectors that identify foreign virus and bacteria called Toll-like receptors, specifically TLR-2 and TLR-4. Also when the virus replicates its own RNA in the cytoplasm of cells, this foreign RNA stimulates two other TLRs, TLR-7 and TLR-8. TLRs activate a signal cascade that induces the production and release of the cytokines, TNF, IL6, and IL1 Beta, all known to stimulate the rise in body temperature inducing fever. Therefore, based on this understanding the prediction is that when the virus infects respiratory epithelial cells and macrophages, it should induce fever. This would be expected to be the first sign of disease. We have tested this prediction using the smartphone thermometer, Kinsa. This thermometer downloads temperature measurements to a phone allowing monitoring core temperature over time. These temperatures are also collected in a central databank. Here are some samples of my personal temperature monitoring. My base or core temperature averages 97.5 degrees Fahrenheit. A fever is defined as a rise in core temperature, 1.8 degrees Fahrenheit or 1 degree centigrade. Then if my core temperature reach 99.3, I would have a fever. As compared to a set temperature, defined as fever as 100.4 degrees Fahrenheit or 38 degrees centigrade, this approach increases the ability to detect fever by approximate 25 percent. The company provide us with temperatures readings from 1,180 patients in order to assess the sensitivity fever for detecting possible COVID-19. As show in this table, if fever was monitored for over 72 hours, they've always detected in 73 percent of test positive patients. However, if only one temperature was taken, fever was detected in only 30 percent of patients. Emphasizing that a single temperature determination is a poor COVID-19 screening tool. Monitoring for fever for 72 hours has a comparable sensitivity to antigen testing. I recommend that everyone monitor their temperature at home and immediately quarantine if you detect a fever and then obtain a more specific PCR or antigen test. Finally, a few words about test sensitivity and specificity. Sensitivity of a test of patients infected with the virus, what percentage are detected by the test? If 100 individuals are SARS-CoV-2 viral culture positive, how many of this culture positive individuals will be detected as a positive by the antigen test? Any individual who is culture positive but antigen test negative is defined as a false-negative test. Higher sensitivity is preferred because the higher the sensitivity, the lower the percentage of false- negative test. In situations where there's a high probability of disease, both PCR and antigen test have a high risk of being falsely negative. Specificity. How accurate does it test? Of patients uninfected, how many test positive? If you test 100 individuals who are SARS-CoV-2 culture negative, what percentage will be detected as positive by the antigen test? Any individual who is culture negative and antigen test positive is defined as a false positive test. Higher specificity is preferred. The higher the specificity, the lower the percent of false positive test. In situations where there is a low probability of disease, antigen tests are at moderate risk of being falsely positive. In summary, RT-PCR is the most specific and also the most expensive test. It has a slow turnaround time and should be used predominantly for diagnosis and treatment. Antigen testing is less expensive. It has a rapid turnaround time, but it is less sensitive and specific, it should be used for screening. Antibody testing shows a delay in increase in IgG and IgM and should be used to determine the number of cases in an area or the prevalence. It should be used for epidemiologic studies. Finally, core temperature monitoring for fever is equivalent in sensitivity to antigen testing and can be used as the first step for detecting the onset of disease.
