Diagnosing COVID-19: Testing is essential

COVID-19 (coronavirus disease 2019) is the disease resulting from infection with a newly emerged coronavirus named SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2).[1] Coronaviruses are a family of RNA viruses usually found in animals. A few coronaviruses have been identified as causing (typically) mild infection in humans.

The emergence of the first much more pathogenic SARS virus in 2003 and the MERS-CoV virus in 2012 shifted the coronavirus landscape, as many patients developed severe respiratory illness with significant mortality.[2,3] The sudden appearance, severity of COVID-19 in an estimated 20% of symptomatic infections, and its rapid worldwide spread, has produced the first truly global pandemic since the great influenza pandemic of 1918.[4-6]

On average, someone with the COVID-19 virus is thought to infect two or more other people.[7,8] This high infectivity rate and environmental stability of the SARS-CoV-2 pathogen necessitates testing to identify infected individuals.

Diagnosis cannot be made solely on signs or symptoms as these overlap with other respiratory illnesses including influenza, so confirmation of the presence of the virus is essential. Table 1 describes the range and percent of symptoms seen in confirmed COVID-19 infections in a Chinese patient cohort.[4]

Testing for the viral RNA (the genetic material of the virus) is useful for the diagnosis of both symptomatic patients and individuals with known or potential exposure. Confirming current infection is an important tool for triage and for limiting transmission. Individuals with confirmed infection but not requiring hospitalization can self-isolate to reduce further spread.

Early data from the United States suggests that in patients meeting the COVID-19 symptomatic testing criteria, about one in five tested were confirmed to have the virus, highlighting the significant overlap with other respiratory illness (though false-negatives can occur for a range of reasons related to both sample collection, storage, and testing limitations). These numbers are preliminary however, and the percent of infections that are COVID-19 might rise significantly if contagion control measures fail to mitigate spread, and as access to testing increases.

Recommended testing for diagnosis of existing infection relies on detection of the viral genetic material (RNA) in a patient’s sample.[9] While virus has been identified in some COVID-19 patients from blood and fecal samples, respiratory secretions are the recommended sample type for molecular testing as they are the most likely to contain the pathogen. The initially recommended technique (nasopharyngeal swab) requires trained personnel and an extended swab for collection far back in the throat. Collected swabs are commonly placed in a transport medium for delivery to the testing location.

Other sample collections such as mid- or anterior nares have also been used.[9,10] These may be preferable as they are less invasive, might potentially be self-collected, and don’t require the extended swab used by a medical professional for nasopharyngeal collections. The FDA recently changed guidance to allow alternate methods of collection.[11] One study indicates the methods may be comparable in their ability to collect virus.[12] Testing recommendations are fluid and evolving rapidly as data is collected.

As a truly emergent human pathogen, a specific test for presence of the virus did not exist with the onset of cases, necessitating rapid development. Most current testing for infection relies on detection of the viral RNA using a reverse-transcription polymerase chain reaction (rtPCR), though other techniques include isothermal amplification. With rtPCR, viral RNA is first converted to DNA, and then amplified using specialized equipment for the detection of the viral genetic material. Both quantitative (viral load or “how much”) and qualitative (viral RNA detected “yes or no”) methods have been utilized.

Initial testing in the U.S. was performed by the CDC, which was limited in its ability to deliver large-scale testing services. Subsequent Emergency Use Authorization (EUA) has been granted by the FDA to some companies and labs that have developed related methods of testing for the viral RNA.[13] As availability and supplies for molecular testing for COVID-19 increase, testing turn-around times should improve, but remain contingent on sample collection, proper preanalytical handling (RNA is highly degradable), time to test result, and access to testing (point-of-care vs. lab-based).

Testing utilizing a blood sample can identify antibody to the virus (IgG and or IgM or other).[14,15] These tests will likely prove extremely valuable for surveillance and could potentially provide assessment for immunity, as well as aiding identification of acute infection. Performance of antibody tests will be contingent on good sensitivity and specificity for test accuracy.

Evaluation of resolving/resolved infection through community-based antibody testing will likely reveal significant insight into the virus’s prevalence within populations, particularly in infections that were mild or asymptomatic. Antibody testing may also find utility in identifying immune individuals or potential donors for immunoglobulin therapy should that prove efficacious. Immunity with resolved infection has yet to be established; additional data is necessary to confirm or refute.

NextGen sequencing methods are proving useful for investigation into the genetic sequence, mutations, and distributions of circulating virus. A low rate of mutation in the virus harbors potential for vaccine efficacy if, unlike seasonal flu, the regions targeted for immunity remain conserved and the vaccine induces protective immunity.

It is currently uncertain how the various molecular techniques compare for both sensitivity and specificity. As with all tests, both false-negatives and false-positives have been observed for both molecular and serology. While available testing can detect the viral genetic material of SARS-CoV-2, methods, throughput, equipment required, and turn-around time can substantially differ. All approaches require specialized (and sometimes proprietary) equipment and processes, some fully automated and others involving manual steps or the need to first extract the RNA from the sample prior to testing. Sample extraction can be a rate-limiting step. The process and time from sample collection to delivery of a result can be a significant challenge.

Delivery of results and testing capacity have also been challenged with limited collection kits or lack of testing venues. Molecular testing relies on obtaining viral RNA in the sample, so false-negative results can occur if the collected sample fails to contain the virus or is improperly stored prior to testing. Molecular testing remains the earliest method to detect infection. Serology testing for antibodies may prove complimentary, or as an alternative method informing a diagnosis. As the rapidly evolving testing environment and recommendations progress, access to testing, types and performance of tests, and improvements in the delivery of test results are anticipated.

For further reading, the CDC is compiling articles related to COVID-19 and listing those on its Emerging Infectious Diseases web resource.[17] Updated information on available COVID-19 testing can be accessed online.[18]