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What approaches could accelerate large-scale screening?

Text updated on 2020-11-01

Tracking cases requires accelerated and widespread testing. Simple and rapid tests are already available as alternatives to reference tests (nasopharyngeal swab and measurement by "RT-qPCR"): 1) to make sampling simple, autonomous, without risk of contamination and minimally invasive: sampling can be based on saliva which contains a lot of virus (see "Which sample to test for the presence of coronavirus: nasopharyngeal or oral? "); 2) in order to reduce the length (e.g., wait time) of the test, rapid detection methods allow direct detection of virus antigens (so-called "antigenic" tests). In order not to miss cases with a low viral load, another test with a rapid amplification step exists: the "RT-LAMP" method; 3) in order to reduce the number of tests to be done to detect cases on a large scale, the samples can be pooled with a provision for combining the samples into groups, a method known as "pooling". See "Combining tests ("pooling", "pooling"): why and for what purpose? ».

NOTE: A distinction must be made between the virus detection tests that tell you if you are currently infected with the virus (see " What is the reference test that finds out if I am infected with SARS-CoV-2 ?" ) and serological tests that tell you that you have been infected with the virus by detecting your immune response (see " What are the tests to find out if I've ever had COVID-19 ?").

Tests for the detection of the virus in our body secretions can either be highly sensitive using a virus amplification process (PCR or RT-LAMP methods) or have low-sensitivity if the virus is detected directly without virus amplification (so-called "antigenic" tests).

Saliva: a simple, self-contained collection method that is less prone to contamination. Recently, tests based on saliva analysis have proven to be sensitive due to high viral load. Because saliva collection is easy, individuals can even collect saliva on their own.

Surprisingly, the virus is very stable in saliva: it resists more than a week at room temperature, more than 2 weeks at 4°C and months in the freezer at -80°C. Thus, the system for collecting saliva samples can be simple and inexpensive.

For all these reasons, saliva tests are very promising for rapid and large-scale testing. See the question " Which sample to test for coronavirus: nasopharyngeal or oral? ".

Fewer tests to identify positive cases and lower testing costs through sample pooling. Combining samples from several people in the same household into a single pool could optimize the detection of infected outbreaks in the population. See the questions "Pooling tests: why and why not? "and " What are the risks of false negatives in pooled testing? ". The technique of sample pooling reduces the number of standard PCR tests that need to be performed and thus saves time and efficiency in case detection, as well as reducing costs. It is possible to pool samples, to test pools, and in case of positivity to re-test individuals separately.

Detection of the virus by rapid amplification of the viral sequence for individual and collective screening: the RT-LAMP method and the hope of the LAMP-seq approach. 

Tests based on Polymerase Chain Reaction ("PCR") that are faster than RT-qPCR exist such as RT-LAMP which is a loop-mediated isothermal amplification technique that can be performed in half an hour. This method can be used for individual screening and is marketed in France for saliva samples by the company SkillCell ("EasyCov" test).

The RT-LAMP method has been adapted in a recent proof of concept for large-scale screening: the LAMPseq approach. By adding "bar codes" in the sample of each individual, it is possible to combine samples from multiple individuals (up to tens of thousands) and identify the result of each in a single test! The barcode is a small sequence of DNA stuck to primers that are used to amplify the virus genome. So, if one of the samples contains the coronavirus, the viral sequence will be amplified at the same time as the individual's identification bar code. Through massive sequencing, it is possible to trace the virus to the person carrying the virus within the pool. The "LAMPseq" necessarily requires more time than the half-hour of RT-LAMP in order to process the tens of thousands of samples, to sequence, and interpret the sequences. On the other hand, it is not necessary with this approach to re-test the same samples since the identity of each individual in the pool of samples is known thanks to the bar code. Note that the positivity rate of cases COVID-19 in the test population impose constraints on the number of samples to be pooled with this method.

Finding highly contaminating positive cases quickly by detecting molecules present on the surface of the virus: low-sensitivity but rapid antigenic tests. While PCR tests detect the genetic material of SARS-CoV-2 (RNA), antigenic tests detect the molecules present on the surface of the virus, usually the nucleocapsid protein. These tests are rapid (less than 30 minutes) and can, therefore, be useful for detecting people with a high viral load who would be expected to infect effectively during a large event. These tests are highly specific for the virus, but they are not as sensitive as PCR-based measures that detect the virus sequence after an amplification process. Therefore, there is a higher risk of making false-negative errors with these antigenic tests. Due to the low sensitivity of these tests, symptomatic individuals or contact cases with a negative antigenic test are encouraged to confirm their results with a more sensitive PCR test.

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One of the first studies indicating the reliability of saliva tests.

Azzi, L., Carcano, G., Gianfagna, F., Grossi, P., Dalla Gasperina, D., Genoni, A., ... & Maurino, V. (2020). Saliva is a reliable tool to detect SARS-CoV-2. Journal of Infection.

One of the first studies indicating the reliability of saliva tests.

Williams, E., Bond, K., Zhang, B., Putland, M., Williamson, D.A. (2020) Saliva as a non-invasive specimen for detection of SARS-CoV-2. J Clin Microbiol. pii: JCM.00776-20.

Researchers at Yale University show that saliva tests are more sensitive than those performed on nasopharyngeal samples (n = 70 patients COVID):

Wyllie, A. L., Fournier, J., Casanovas-Massana, A., Campbell, M., Tokuyama, M., Vijayakumar, P., ... & Petrone, M. E. (2020). Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs. New England Journal of Exp. Medicine, Sept 24 2020, 383: 1283-1286.

The virus is stable in saliva for more than a week at room temperature, more than 2 weeks at 4°C, and months in the freezer at -80°C. These observations suggest that a simple and inexpensive collection system is possible:

Ott, IM, Strine, MS, Watkins, AE, Boot, M, Kalinich, CC, Harden, CA, Vogels, CBF, Casanovas-Massana, A, Moore, AJ, Muenker, MC, Nakahata, M, Tokuyama, M, Nelson, A, Fournier, J, Bermejo, S, Campbell, M, Datta, R, Dela Cruz, CS, Farhadian, SF, Ko, AI, Iwasaki, A, Grubaugh, ND, Wilen, CB, Wyllie, AL. Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices.

The French COVISAL study conducted in the Amazonian forest in French Guyana shows the interest and ease of saliva collection. The field study reveals variations between groups sampled under laboratory conditions (Yale University, Hokkaido University) and in the field. This study, based on 25 asymptomatic cases, led the Haute Autorité de Santé to carry out salivary tests only on people with symptoms (https://www.has-sante.fr/jcms/p_3202317/fr/).covid-19-the-salivary-test-can-complete-nasopharyngeal-test-symptomatic-persons)

Nacher, M, Mergeay-Fabre, M, Blanchet, D, Benois, O, Pozl, T, Mesphoule, P, Sainte-Rose, V, Vialette, V, Toulet, B, Moua, A, Simon, S, Guidarelli, M, Galindo, M, Biche, B, Faurous, W, Abad, F, Fahrasmane, A, Rochemont, D, Vignier, N, Vabret, A, Demar, M. COVISAL Guyane, Nacher, M, Demar, M. Prospective comparison of saliva and nasopharyngeal swab sampling for mass screening for COVID-19. COVISAL Guyane, Nacher, M, Demar, M. Prospective comparison of saliva and nasopharyngeal swab sampling for mass screening for COVID-19.

Pooling the samples within a household could optimize the detection of infected outbreaks in the population:

Hogan, C. A., Sahoo, M. K., & Pinsky, B. A. (2020). Sample Pooling as a Strategy to Detect Community Transmission of SARS-CoV-2. JAMA.

A recent Japanese study of 55 asymptomatic cases of COVID shows that saliva is a very efficient collection method for detecting SARS-CoV-2 even in asymptomatic patients: 92% sensitivity for saliva collection and 86% sensitivity for nasopharyngeal sampling, with a 99.8% probability of agreement. The tests have been performed on more than 1,950 contact persons or on arrival at airports:

Yokota, I, Shane, P, Okada, K, Unoki, Y, Yang Y, Tasuku, I, Sakamaki, K, Iwasaki, S, Hayasaka, K, Sugita, J, Nishida, M, Fujisawa, S, Teshima, T. (2020). Mass screening of asymptomatic persons for SARS-CoV-2 using saliva. 10.1101/2020.08.13.20174078.

A Japanese study of 103 positive cases comparing different RT-qPCR systems and one RT-LAMP system concludes that the sensitivity of RT-qPCR is better. Note that in this study there was no pre-treatment of saliva prior to RT-LAMP, which could affect the results obtained.

Ikeda, M, Imai, K, & Tabata, S, Miyoshi, K, Mizuno, T, Murahara, N, Horiuchi, M, Kato, K, Imoto, Y, Iwata, M, Mimura, S, Ito, T & Tamura, K, Kato, Y. (2020). Clinical evaluation of self-collected saliva by RT-qPCR, direct RT-qPCR, RT-LAMP, and a rapid antigen test to diagnose COVID-19. 10.1101/2020.06.06.20124123.

The EasyCOV detection kit using RT-LAMP after pre-treatment of saliva allows the detection of SARS-Cov-2 in 40 min with a sensitivity of 87.5% according to their first study carried out on 220 people.

A total of 720 people should be integrated by the end of the study.

The LAMP-Seq technique was developed and validated on a group of 28 individuals, 12 of whom were RT-PCR positive. 12 out of 12 individuals were identified as positive with the LAMP-Seq approach. The rates of false-negatives and false-positives will depend on the precise conditions of implementation and imposes constraints on the maximum rate of positive cases in the target population:

Schmid-Burgk, JL, Schmithausen, RM, Li, D, Hollstein, R, Ben-Shmuel, A, Israeli, O, Weiss, S, Paran, N, Wilbring, G, Liebing, J, Feldman, D, Słabicki, M, Lippke, B, Sib, E, Borrajo, J, Strecker, J, Reinhardt, J, Hoffmann, P, Cleary, P, Hölzel, M, Nöthen, MM, Exner, M, Ludwig, KU, Regev, A, Zhang, F. LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding.

Tests grouped from 5 to 20 in saliva samples by the Yale University team also behind the SalivaDirect test:

Watkins, A. E., Fenichel, E. P., Weinberger, D. M., Vogels, C. B., Brackney, D. E., Casanovas-Massana, A., ... & Cruz, C. S. D. (2020). Pooling saliva to increase SARS-CoV-2 testing capacity. medRxiv.

Pooled tests of 5 to 30 in Germany, in the Saarland, for epidemic prevention purposes :

Lohse, S., Pfuhl, T., Berkó-Göttel, B., Rissland, J., Geißler, T., Gärtner, B., ... & Smola, S. (2020). Pooling of samples for testing for SARS-CoV-2 in asymptomatic people. The Lancet Infectious Diseases.

Study examines specificity and sensitivity of the rapid antigenic test, "Coris COVID-19 Ag Respi-Strip", on 106 samples: if the test is specific (100%), the detection rate of positive cases is very low (30%).

Scohy, A., Anantharajah, A., Bodéus, M., Kabamba-Mukadi, B., Verroken, A., & Rodriguez-Villalobos, H. (2020). Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis. Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology, 129, 104455.

A Japanese study examining the performance of the LUMILUSTRE antigen test on 313 samples reveals that these tests are very specific for SARS-CoV-2 (specificity of 99.6%) but not very sensitive (sensitivity of 55.2% compared to PCR tests for virus detection).

Hirotsu, Y, Maejima, M, Shibusawa, M, Nagakubo, Y, Hosaka, K, Amemiya, K, Sueki, H, Hayakawa, M, Mochizuki, H,Tsutsui, T, Kakizaki, Y, Miyashita, Y, Yagi, S, Kojima, S, Omata, M. Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients. Int J Infect Dis. 2020 Oct; 99: 397-402. doi: 10.1016/j.ijid.2020.08.029

Rapid antigen fluorescence immunoassays from SARS-CoV-2 can identify patients within the first 5 days of symptom onset when respiratory secretions carried high viral loads, suggesting that these tests could play an important role in future PCR-independent strategies to detect early or infectious cases:

Porte, L, Legarraga, P, Iruretagoyena, M, Vollrath, V, Pizarro, G, Munita, JM, Araos, R, Weitzel, T. Rapid SARS-CoV-2 antigen detection by immunofluorescence - a new tool to detect infectivity

Further reading

Grouping tests ("pooling", "pools"): why and for what purpose?

How long is the coronavirus infectious?

Is spitting on the floor dangerous to others?