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2. Diagnostic testing of COVID-19

2.1 Testing conducted on previous coronaviruses

2.1.1 Severe acute respiratory syndrome (SARS)

Severe acute respiratory syndrome, otherwise known as SARS, arose in South China in late 2002. Caused by the SARS caronavirus (SARS-CoV) and believed to have originated from horseshoe bats^[1], SARS eventually was contained in the summer of 2003. The last known infection was in April 2004, due to a laboratory accident.^[2] During that time, the following sample collection and test procedures evolved from the related outbreaks (note that this is only a summary; consult the cited literature directly for full details)^[3]^[4]^[5]^[6]^[7]:

Determine that the patient is indicating clinical and/or epidemiological evidence of SARS. As Knobler et al. put it: "SARS-CoV testing should be considered if no alternative diagnosis is identified 72 hours after initiation of the clinical evaluation and the patient is thought to be at high risk for SARS-CoV disease (e.g., is part of a cluster of unexplained pneumonia cases)."^[5]

Collect multiple specimen types at different time points of the patient's illness. Respiratory and plasma or serum specimens should be collected early into the first week of illness. Respiratory samples should be from nasopharyngeal aspirates and swabs in the upper respiratory tract, or in some cases fluids from the lower respiratory tract using bronchoalveolar lavage, tracheal aspiration, or a pleural tap. (Sputum can also be collected.) Whole blood (5 to 10 ml) is collected into either a serum separator tube for blood serum or EDTA tube for blood plasma. Stool samples are also of import early on for virus isolation or detection and are useful in at least the first and second weeks of the illness. Blood serum is usefull in weeks two and three for detecting a rising titre. Additionally, the literature also makes reference to methods of collecting specimens post-mortum.

Conduct testing. At the time, the two primary test types used were enzyme immunoassay (EIA; today more commonly known as ELISA^[8]) for detection of serum antibody and reverse transcription polymerase chain reaction (RT-PCR) for detection of the virus' RNA. The U.S. Centers for Disease Control and Preventions had this to say about these tests in May 2004^[4]:

Both the EIA and the RT-PCR tests are sensitive and highly specific for SARS-CoV. The ability to diagnose SARS-CoV infection in a patient is often limited, however, by either the low concentration of virus in most clinical specimens (RT-PCR assays) or the time it takes a person to mount a measurable antibody response to SARS-CoV (serologic assays). The likelihood of detecting infection is increased if multiple specimens (e.g., stool, serum, respiratory tract specimens) are collected at several times during the course of illness.

The literature also makes reference to an immunofluorescence assay (IFA) for detecting antibody, with the CDC calling its results "essentially identical to those for the EIA for SARS antibody."^[4] Tangentially, isolation of SARS-CoV in cell culture from a clinical specimen is also referenced, though such activity is reserved for Biosafety Level 3 (BSL-3) laboratories.

Confirm the results. Laboratory confirmation is based on one of 1. initial local lab detection and subsequent national reference lab confirmation of a validated serology-based test detection; 2. isolation of SARS-CoV in cell culture with subsequent confirmation from a validated test; or 3. initial local lab detection and subsequent national reference lab confirmation of SARS-CoV RNA from a validated RT-PCR test which used either two clinical specimens from different sources or two same-source clinical specimens from two different days.

Additionally, in the case of serology, one of the following must be true:

SARS-CoV serum antibodies are detected in a single serum specimen; or,
a "four-fold or greater increase in SARS-CoV antibody titer between acute- and convalescent-phase serum specimens tested in parallel"^[4] is detected; or,
a "negative SARS-CoV antibody test result on acute-phase serum and positive SARS-CoV antibody test result on convalescent-phase serum tested in parallel"^[4] is detected.

Of note is the WHO's January 2004 cautionary message about serological diagnostics in not only SARS-CoV but other types of coronaviruses. At that time, they showed a level of unsureness in regards to how coronaviruses elicited serological cross-reactions and generated antigenic recall. They also preached caution in interpreting serological results in non-epidemic periods and when no viral sequence data are available. Finally, they also mentioned the added difficulties of rate cases when coinfection with a related human coronavirus occurs, "although the use of expressed proteins in Western blots may help to sort this out."^[6] More than 15 years later, Loeffelholz and Tang put this concept into clearer terms, indicating that while "serological assays are not routinely used for diagnosis of [human coronavirus] infections due to the lack of commercial reagents," they still have important value "for understanding the epidemiology of emerging [human cornaviruses], including the burden and role of asymptomatic infections," as well as for antibody detection of novel and emerging coronaviruses.^[9]

Arrange for confirmatory testing to be performed by an appropriate test site in the case of a positive RT-PCR test.

Report to state or local health departments details of patients radiographically confirmed with pneumonia with at least one SARS-CoV risk factor for exposure, clusters of healthcare workers with unexplained pneumonia, and any positive SARS-CoV test results. Additional international reporting of SARS by WHO Member States in regards to probable and laboratory-confirmed cases is also requested.

Send off for an additional verification by an external member of the WHO's SARS Reference and Verification Laboratory Network before internationally announcing results as a laboratory-confirmed case.

2.1.2 Middle East respiratory syndrome (MERS)

Unlike SARS, Middle East respiratory syndrome, or MERS, continues to appear in the human population. Since its appearance in 2012, several thousand laboratory-confirmed cases of MERS have been reported to the WHO.^[10] The virus MERS-CoV is believed to have originated from bats, which at some unknown point spread to Dromedary camels. Approximately 55 percent of MERS-CoV infections have come from direct contact with such camels, thought it's not entirely clear how the rest of known cases have been caused^[11] (Alshukairi et al. suggest asymptomatic or mildly symptomatic camel workers may serve as a possible transmission source^[12]). The following sample collection and test procedures have evolved from working with the MERS-CoV virus (note that this is only a summary; consult the cited literature directly for full details)^[13]^[14]^[15]^[16]:

Determine that the patient is indicating clinical and/or epidemiological evidence of MERS. "Testing for other respiratory pathogens using routinely available laboratory procedures, as recommended in local management guidelines for community-acquired pneumonia, should also be performed but should not delay testing for MERS-CoV."^[15]

At a minumum, both lower respiratory and upper respiratory tract samples should be collected. Lower respiratory tract specimens are typically the most revealing, as they have been shown to contain the highest viral load (due to the expression of the virus's cellular receptor DPP4 in the lower respiratory system). Bronchoalveolar lavage, tracheal aspiration, or a pleural tap can be used to collect specimens from the lower respiratory tract. (Sputum can also be collected.) Upper respiratory tract specimens (in this case, both a nasopharyngeal and an oropharyngeal swab are recommended) are also valuable in diagnosis, though extra care should be taken to ensure nasopharyngeal swabs gather secretions from the nasopharynx and not just the nostril. Nasopharyngeal aspiration is also an acceptable sample collection method for the upper respiratory tract.

Regarding serology, slight differences in guidance appear between WHO guidance and CDC guidance. The WHO appears to differentiate between symptomatic and asymptomatic patient testing, wheras the U.S. CDC seems to only indirectly differentiate the two. The WHO suggests if testing symptomatic patients, stick with lower and upper respiratory tract specimens, which will be tested using nucleic acid amplification (molecular) testing (NAAT). Serology should be used for symptomatic patients "only if NAAT is not available."^[15] If this is the case, the WHO recommends paires samples, one collected within the first week of illness and the second about three to four weeks later. For asymptomatic patients in high-contact outbreak scenarios, the WHO recommends all three sample types (with respiratory samples taken preferably within 14 days of last documented contact).

The current CDC guidance differentiates between molecular testing for active infections and serology for previous infections. The CDC adds that "MERS-CoV serology tests are for surveillance or investigational purposes and not for diagnostic purposes."^[13]

2.1.3 The common cold

Approximately 10 to 15 percent of cases of what we call the "common cold" are associated with an endemic coronavirus, of which are two distinct groups: HCoV-229E and HCoV-OC43.^[17] Disease symptoms associated with these coronaviruses—typically in the form of respiratory infection and the symptoms that come with it—by themselves are typically mild^[9], and laboratory testing isn't necessarily indicated for those immunocompetent individuals capable of self-limiting.^[18] However, symptom overlap with pharyngitis and bronchitis, as well as the complication of pharyngitis and sinusitis also potentially having bacterial origin, can complicate clinical diagnosis. Additionally, as more antivirals that target a specific virus are created, and as concerns of unnecessarily using antibiotics to treat viral diseases grows^[19]^[20], laboratory methods of respiratory virus diagnosis—particularly for those who are immunocompromised—have value.^[17]^[18]

RT-PCR, a molecular method, have been used for well over a decade for detecting coronaviruses.^[17]^[21] However, as molecular methods of analysis have expanded over the years, more rapid solutions for testing have been developed. For example, the GenMark ePlex rapid multiplex molecular diagnostics instrument and the ePlex Respiratory Pathogen Panel were evaluated in a multicenter trial by Babady et al. in 2017.^[18] The panel is capable of testing for the presence of 15 viral types—including the -229E, -OC43, and two other coronaviruses—and two bacterial types in nasopharyngeal swab specimens, with results in typically less than two hours.^[18] The costs associated with these sorts of tests, compared to their benefits, likely limits ubiquitous use at the first sign of a cold^[18], but as molecular diagnostic technologies become more compact and easy-to-use, testing for infection by endemic human coronaviruses may become slightly more commonplace. However, as the authors point out, with no treatment for these endemic coronaviruses, any additional utility beyond diagnosing an illness as viral rather than bacterial would primarily be found in epidemiological studies of the associated genotyping data.^[18]