Rapid and Frequent Testing for COVID-19

The cases of new SARS-CoV-2 infections are currently increasing at an alarming rate, suggesting that winter might bring a large healthcare crisis. Diagnostics have become a critical tool in curbing COVID-19. It is important that accurate results be obtained rapidly and that collection of samples be as fast, simple, and convenient as possible.

What tests are becoming available, which promise better accuracy and greater speed and ease of use?  Thus far, testing has been based on a real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 RNA detection and serology on enzyme-linked immunosorbent assay (ELISA) for antibody tests. Yet, PCR can show false positives due to detection of fragmented RNA of virus. This has been an obstacle in determining the presence of infectious virus particles in COVID-19 patients. A recent study showed that the presence of infectious virus may be predictable by evaluating the threshold cycle (Ct value, the number of cycles of PCR amplification) (1).  Now, there are also a variety of novel tests for SARS-CoV-2 RNA detection in development that rely on CRISPR12 or 13 cleavage of SARS-CoV-2 amplified RNA, which releases a visually readable fluorescent chromophore. These rely on isothermal amplification, either LAMP(7) or recombinase polymerase amplification (RPA)(8-10), which does not require a thermal cycler. These test all have rapid turnaround times (on the order of an hour) and sensitivities that can be well comparable with other tests. One of these tests can use a cheap chemical hand warmer as the thermal source(9). Another similar CRISPR based assay can simultaneously detect and identify 169 different human viruses(11), illustrating the potential utility and efficiency of this system. These tests all have advantages of speedy detection and user-friendly application over RT-PCR.

Serological testing is also necessary. The presence of antibodies marks an ongoing or past infection, and anti-spike protein and neutralizing antibodies are considered a possible indication of immunity, although T cell activities are also contributing to protective immunity. Antibody tests can indicate past infections but are prone to be false positive in low frequency settings and have provided a difficulty with uneven results because of wide variations in test kit quality. What serological tests are likely to be most useful? As with RNA tests, this requires sensitivity, specificity, speed, and user-friendly application.

One consideration for serological tests is that there is some degree of serologic cross-reactivity of SARS-CoV-2 proteins with those of the other coronaviruses, especially SARS-CoV-1(12). It is also possible that the S2 spike domain of SARS-CoV-2  may also have some degree of cross-reactivity with that of other human coronavirus (13). Interestingly, some of these cross-reactive antibodies also appear to cross-neutralize, suggesting that there may exist conserved epitopes for the development of a broadly effective vaccine.

Most point-of care serologic assays rely in lateral flow-type assays, in which the results are read as bands on a strip and indicate the presence of IgG and/or IgM antibodies. The regulatory processes for these tests has perhaps lacked some of the usual rigor, and their accuracy tends to vary. This can be a problem in low incidence settings, where false positives are an issue. Fortunately, a recently published  study carefully compared five different test kits to ELISA and viral neutralization tests(14). As might be expected, antibodies were more readily detected when sera were collected more than 14 days after symptom onset. Using samples collected from this time period, three lateral flow tests for IgG were comparable to ELISA, but the other two kits were inferior. Prior to 14 days post symptoms, sensitivity was only on the order of 50-60% by any test. It is clear that serological tests are mostly retrospective in nature. They are useful for diagnosing past infections and viral prevalence in populations, and will be very important in evaluating vaccine durability.

In this last aspect, it would be extremely convenient to have a neutralization assay that does not involve live virus, with its attendant requirement for biosafety level 3 containment. One of the studies is based upon inhibition of binding of the receptor binding domain (RBD) of the S protein to immobilized ACE2 protein (15). The results are read colorimetrically and are near 100% sensitive and specific. An advantage of this test is that it would measure neutralization by IgA, IgM, and IgG all at the same time, increase sensitivity. This type of test will obviously be highly useful as vaccines begin to be widely deployed.

Regarding the sampling procedures, we are still using taking nasal swabs; this is a sensitive method but is prone to be false negatives due to poor sampling. Saliva is clearly far simpler to collect than nasal swabs or blood. How do tests of saliva compare with those of nasal swabs and blood?  For RNA detection by RT-PCR, tests of saliva appear to have at least comparable sensitivity to those of nasal swabs(2-4), representing a considerable simplification of testing. Potentially better is RNA detection in saliva using loop-mediated isothermal amplification (LAMP), which avoids the necessity for thermal cycler instrumentation and for RNA purification. Results appear to be equally sensitive and specific to those from RT-PCR(5). Another isothermal method that is fully automated with less sensitivity, delivers results on a dipstick in 90 minutes(6). What about antibodies in saliva? Saliva is much easier to collect. It appears that antibodies are as detectable in saliva as in serum. It’s been shown that viral antibodies persist in saliva for months(16). Another study showed persistent IgG activity against both N protein and the RBD of the S protein(17). The anti-N antibody showed 100% of sensitivity and the RBD antibody 100% specificity. Thus, a multiplexed assay should be 100% accurate.

We are heading into a period of perhaps explosive increases in rates of infection. Widespread and frequent testing with contact tracing can be a practical way to actively prevent transmission of virus, particularly, in big groups of people (i.e., school settings). The tests described above are playing a role in curbing the pandemic.

 

1. J. Bullard et al., Predicting infectious SARS-CoV-2 from diagnostic samples. Clin Infect Dis, (2020).
2. L. Azzi et al., Saliva is a reliable tool to detect SARS-CoV-2. J Infect 81, e45-e50 (2020).
3. M. Baghizadeh Fini, Oral saliva and COVID-19. Oral Oncol 108, 104821 (2020).
4. K. K. To et al., Consistent detection of 2019 novel coronavirus in saliva. Clin Infect Dis, (2020).
5. S. Wei et al., Field-deployable, rapid diagnostic testing of saliva samples for SARS-CoV-2. medRxiv, (2020).
6. D. A. Collier et al., Point of Care Nucleic Acid Testing for SARS-CoV-2 in Hospitalized Patients: A Clinical Validation Trial and Implementation Study. Cell Rep Med 1, 100062 (2020).
7. Z. Ali et al., iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res 288, 198129 (2020).
8. J. Arizti-Sanz et al., Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. bioRxiv, (2020).
9. X. Ding et al., Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun 11, 4711 (2020).
10. T. Hou et al., Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19. PLoS Pathog 16, e1008705 (2020).
11. C. M. Ackerman et al., Massively multiplexed nucleic acid detection with Cas13. Nature 582, 277-282 (2020).
12. W. N. Chia et al., Serological differentiation between COVID-19 and SARS infections. Emerg Microbes Infect 9, 1497-1505 (2020).
13. K. W. Ng et al., Preexisting and de novo humoral immunity to SARS-CoV-2 in humans. Science, (2020).
14. K. Bond et al., Evaluation of Serological Tests for SARS-CoV-2: Implications for Serology Testing in a Low-Prevalence Setting. J Infect Dis 222, 1280-1288 (2020).
15. C. W. Tan et al., A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Nat Biotechnol 38, 1073-1078 (2020).
16. B. Isho et al., Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients. Sci Immunol 5, (2020).
17. N. Pisanic et al., COVID-19 serology at population scale: SARS-CoV-2-specific antibody responses in saliva. J Clin Microbiol, (2020).