National COVID-19 Convalescent Plasma Project

Virus Detection

3/27/21 Update

Testing Procedures

Rapid vs. Non-rapid Testing

Molecular Test Antigen Test
Detects Viral genetic material, through multiple amplification cycles in PCR testing Protein(s) from a virus particle
Sample type Nasal swab, nasopharyngeal swab, mid-turbinate swab, respiratory aspirate/lavage, or saliva sample, depending on the test Nasal swab or nasopharyngeal swab, depending on the test
Laboratory, point-of-care (POC), or at-home Most tests are authorized for use in traditional laboratories. A few tests are authorized for use at the point-of-care. Some tests are authorized for at-home sample collection and the patient mails the sample to a traditional laboratory for analysis. Some tests are authorized to be used completely at home with results provided in minutes. Most tests are authorized for use at the point-of-care
Turnaround time Several hours to days for traditional laboratory tests; less than an hour for point-of-care tests; minutes for at-home tests Less than an hour
Sensitivity and Specificity Generally, highly sensitive (especially traditional laboratory PCR tests) and highly specific Generally, highly specific, but less sensitive than molecular tests

Reverse transcriptase PCR (RT-PCR) is a laboratory testing technique that is used worldwide to make the diagnosis of COVID-19 by confirming the presence of virus in the patient’s nasopharynx or other tissues. The RT-PCR test detects the genetic material of the virus, which in the case of SARS-CoV-2 is RNA. The Real Time RT-PCR procedure approved for use by the CDC detects the presence of RNA sequences from the SARS-CoV-2 gene encoding the nucleocapsid of the virus. Since SARS-CoV-2 is an RNA virus, its genetic material is first reverse transcribed to yield a complementary DNA (cDNA) transcript from the targeted probe. In most living organisms it is DNA that is transcribed to produce RNA, and thus when we deal with an RNA virus which transcribes RNA to DNA, the process is referred to as “reverse transcription”. The cDNA produced by reverse transcription can then be amplified by the process called polymerase chain reaction (PCR) to increase the amount of cDNA making it easier to measure. As the viral RNA is transcribed, a fluorescent probe is cleaved, producing a signal that increases in intensity with each cycle of RT-PCR. The intensity of the fluorescence provides information about the quantity of viral RNA present in the sample.

Many laboratories have produced RT-PCR testing kits, and the current list of FDA-approved tests (approved under Emergency Use Authorization provisions) and their manufacturers can be found on the FDA's page for Emergency Use Authorizations.

The FDA separately tabulates tests approved for commercial use by any CLIA-certified laboratory and tests developed by hospitals and universities for use only in their own laboratories. The commercially approved tests number 49 as of this writing, of which 41 are RT-PCR tests, and 8 are antibody tests. The 21 listed single-laboratory tests are all RT-PCR tests. Thus, at this moment 62 RT-PCR tests have received FDA approval for use in the US.

Limitations to testing

Is the virus live or not?

It is important to note that although RT-PCR has been the most widely used diagnostic tool for detection of SARS-CoV-2, a positive test result only indicates presence of viral RNA and does not conclusively prove that the virus is alive and transmissible.i The gold standard for detection of live virus is viral culture. A study of SARS patients, published in 2004, indicated that while viral RNA was present in some patients for 30 days or more after the onset of symptoms, none of these individuals had a positive viral culture from 22 days after the onset of illness.ii However, these are findings in patients with SARS-CoV, the name given to the coronavirus that caused the severe respiratory epidemic in 2003-4, and not SARS-CoV-2, the related virus causing the pandemic we are experiencing now. We do not yet have publications on viral cultures of SARS-CoV-2.

With these limitations in mind, RT-PCR remains the preferred method of diagnostic testing, because viral culture is labor-intensive and time-consuming and requires use of a Biosafety level 2 laboratory (BSL2/3) to safely culture the virus.

False negative

Four studies from Wuhan, China raise the possibility of false negative RT-PCR tests in individuals thought to have COVID-19 disease. In one study, among 290 patients thought clinically to have the disease and ultimately testing positive, 14% were negative on the first in-hospital test, although later tests showed presence of the virus.iii The test kit used was manufactured by the Shanghai Bio-germ Medical Technology Co Ltd. Interestingly, patients who were negative on the first test, and who thus might have had lower viral loads, had milder disease. The early production of antibodies was found useful in a second Wuhan study to validate RT-PCR findings.iv

In a third study from Wuhan, performed in a hospital reserved for the treatment of COVID-19, among 610 patients with the clinical diagnosis and a typical CT scan appearance of COVID-19 pneumonia, fewer than 40% had positive RT-PCR tests. The identity of the test kit in this study was not reported.v The combination of typical chest CT findings and RT-PCR testing was used in the largest of the four Wuhan studies, with more than 1,000 patients, to confirm the clinical diagnosis of COVID-19 disease.vi

Implementation Difficulties

Logistical obstacles have prevented thousands of COVID-19 tests from being used in the United States. Many reasons are reported by these certified laboratories, including federal regulation, and lack of communication between hospital electronic health records and external laboratories. Universities who have developed molecular tests have scrambled to obtain CLIA certification to broaden the impact of their testing.

Given the shortage of testing materials, as of this writing, many states still restrict testing of individuals for SARS-CoV-2 to symptomatic individuals of a specified level of severity. Hopefully this will change, since it has often proven difficult for individuals who are recovering from COVID-19 to find the viral testing to show viral absence that is required by many donor centers before they can provide plasma.

Implications for Plasma Donation

Although not yet published, we hear from several convalescent plasma donation sites that it is not uncommon for people recovering from COVID-19 to have positive RT-PCR throat swabs after as many as 28 days of being asymptomatic. We know of some centers that are sending these late positive swabs to laboratories for viral cultures, but, as noted above, this requires access to a BSL-2 Laboratory. This is important as most donor centers require recovering patients who had a positive RT-PCR test when ill to have a negative test before coming to donate plasma.

Another concern is the possible presence of SARS-CoV-2 RNA in the plasma itself. Among Chinese blood donors, a very few (fewer than one per thousand) individuals were found to have SARS-CoV-2 RNA, often at very low levels of detections.vii There were indications that some of these people may have been in the early pre-symptomatic stages of the illness. Although concentrations of virus during the viremic phase of the disease are low, and usually transient, and while respiratory viruses are generally not thought to be transmitted by blood, monitoring the safety of blood and plasma donations is of the utmost importance.viii

References

  1. Joynt and Wu: Understanding COVID-19: what does viral RNA load really mean? Lancet Infectious Disease 2020; March 27; https://doi.org/10.1016/S1473-3099(20)30237-1.

  2. Chan KH, Poon LLLM, Cheng VCC et al: Detection of SARS Coronavirus in Patients with Suspected SARS. Emerg Infect Dis. 2004 Feb; 10(2): 294–299.

  3. Zhang JJ, Cao YY, Dong X et al : Distinct characteristics of COVID‐19 patients with initial rRT‐PCR positive and negative results for SARS‐CoV‐2. European Journal of Allergy and Clinical Immunology 2020;. First published:13 April 2020. https://doi.org/10.1111/all.14316.

  4. Guo L, Ren L, Yang S et al: Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19) Clinical Infectious Diseases 2020; Published: March 21, 2020. ciaa310, https://doi.org/10.1093/cid/ciaa310
  5. Li Y, Yao L, Li J et al: Stability issues of RT‐PCR testing of SARS‐CoV‐2 for hospitalized patients clinically diagnosed with COVID‐19. J Med Virol. First published:26 March 2020. https://doi.org/10.1002/jmv.25786.

  6. Ai T, Yang Z, Hou H et al: Correlation of Chest CT and RT-PCR Testing in Coronavirus Disease 2019 (COVID-19) in China: A Report of 1014 Cases. Radiology. Feb 26 2020, https://doi.org/10.1148/radiol.2020200642.

  7. Chang L, Zhao L, Gong H et al: Severe acute respiratory syndrome coronavirus 2 RNA detected in blood donations. Emerg Infect Dis. 2020 Jul. https://doi.org/10.3201/eid2607.200839.

  8. Chan L, Yin Y, Wang L: Coronavirus Disease 2019: Coronaviruses and Blood Safety. Transfusion Medicine Reviews. Available online 21 February 2020

Virology