LII:COVID-19 Testing, Reporting, and Information Management in the Laboratory/Diagnostic testing of COVID-19 and other coronaviruses
- 1 2. Diagnostic testing of COVID-19 and other coronaviruses
- 1.1 2.1 Testing terminology
- 1.2 2.2 Testing conducted on previous coronaviruses
- 1.3 2.3 Organizational and agency guidance on COVID-19 testing
- 1.4 2.4 Current test methods and their differences
- 2 References
- 3 Citation information for this chapter
2.1 Testing terminology
As you read through this chapter, you may discover terms like "polymerase chain reaction" (PCR) and "lateral flow assay" (LFA). If you're a laboratorian or have a clinical background, you may already be familiar with these terms. However, it seems prudent to at least briefly discuss a few of them before delving into coronavirus testing itself.
Living organisms store information in their genetic material, using DNA or RNA as the information carrier. That information, or genetic code, essentially provides instructions for organism development, function, growth, and reproduction. In the late twentieth century, researchers were laying the groundwork for molecular diagnostics, the concept of examining an organism's genetic code and its associated biological markers to diagnose and treat disease on a more personalized basis. This requires an assay, an investigative procedure for assessing the presence of, or measuring the amount or functional activity of, a target analyte. In the case of molecular diagnostics, and more broadly molecular biology, the target is biological in nature, and thus biological assays are used. These biological assays are designed to accurately detect the presence of or enable counts of biological molecules, including DNA, RNA, proteins, cells, bacteria, and virus particles (e.g., viral plaque assays).
2.1.2 Polymerase chain reaction (PCR)
Polymerase chain reaction or PCR is a molecular biology method that takes small amounts of DNA sequences and makes copies of (amplifies) them to the point of having enough material to sufficiently study or work with. The base technique can yield results in several hours and has a high level of sensitivity, with its ability to amplify the DNA to counts of millions or billions. PCR has been used in molecular diagnostics for testing prospective parents for being genetic carriers of particular diseases (i.e., expanded carrier screening), tissue typing to ensure more effective organ transplants, and analyzing mutations in oncogenes to customize cancer treatments. However, the method has also been applied to forensic science and epidemiology.
PCR and its variations have been used to characterize and detect infectious disease organisms such as human immunodeficiency virus (HIV), pathogenic tuberculosis bacteria, and Bordetella pertussis, which causes whooping cough. Additionally, a selection of viruses can have their RNA detected using PCR, though the primers (short single-strand DNA fragments) used in the process must by sympathetic to the virus' genetic structure to ensure that only target virus material is amplified. As it turns out, coronaviruses are RNA viruses, having some of the longest genomes of any RNA virus, and, detrimentally, the highest known frequency of recombination (the exchange of genetic material with another organism); this broadly means high rates of virus mutation, which interferes with maintaining consistent diagnostic detection and therapy.
PCR comes in several variant methods. For example, while PCR monitors the amplification portion at the end of the overall process, real-time or "quantitative" PCR (qPCR) allows for the generation rate of the amplified product to be monitored at a particular point during each PCR cycle. Reverse transcription PCR (RT-PCR) is a combinatory process, applying reverse transcription (creating complementary double-stranded DNA [cDNA] from an RNA template) with PCR. If RT-PCR incorporates qPCR, you end up with "real-time RT-PCR" (rRT-PCR), sometimes referred to as "quantitative RT-PCR" (qRT-PCR). In the case of using PCR for detecting coronaviruses, more often than not we see some variation of RT-PCR, with or without real-time amplification monitoring. (It's important to not assume all RT-PCR processes incorporate real-time methods.)
How does PCR work in practice? The simplified version (see this JAMA Patient Page for a useful graphical explanation, using COVID-19 as an example) has a clinician obtaining a biological specimen from the appropriate location or source material. Then, special techniques are used to isolate viral (or in some cases, bacterial) genetic material from the specimen. (If RT-PCR is performed, the next step of reverse transcription of the isolated viral RNA into cDNA is also performed.) Once the viral genetic material is isolated, suitable primers that are sympathetic to the structure of the isolated genetic material are introduced. Those primers bind to the virus' genetic material and begin making copies of it. Fluorescent or other biomarkers that were attached to the copies during the PCR process eventually release from the copies, and an attempt is made to detect the presence of those biomarkers. The presence or absence of these markers drives the determination of a positive or negative detection for the sought-after virus.
2.1.3 Lateral flow assay (LFA)
The lateral flow assay or LFA is another molecular biology method for detecting the presence of a target analyte in specimen material. In comparison to PCR, LFA has the advantages of being more rapid, low-cost, easy-to-use, and applicable at the point of care, though the average LFA is at best semi-quantitative in its results and has slightly lower sensitivity. The method involves a cellulose-based strip with an ordered collection of specific "pads" and reagents that are reactive to a target analyte in a liquid, which is placed on the strip and moves across the various reagents using capillary and electrostatic interactions. LFA has been used in molecular diagnostics for testing urine, saliva, sweat, serum, plasma, and other biological fluids for the presence of specific antigens and antibodies, as well as signs of gene amplification. The method has also been effectively applied to other industries outside healthcare, including the food and beverage, chemical, and environmental industries.
In the realm of infectious disease, the LFA has played an important role in disease diagnosis and control, particularly in resource-constrained settings where resources are limited and point-of-care testing is critical to filling the gap. Testing for the presence of an infectious agent in body fluids using LFA can be performed in two ways: lateral flow immunoassay (LFI) or lateral flow nucleic acid (LFNA). The immunoassay variant looks for antibodies created as a result of the presence of an infectious agent, whereas the nucleic acid variant is built to detect an amplified nucleic acid sequence specific to a target infectious agent. Speaking specifically to the SARS-CoV-2 coronavirus, the antibody-based immunoassay method of lateral flow is predominantly used, targeting one of either a monoclonal antibody directed at a viral antigen, or a viral antigen that is recognizable by a patient's developed antibodies.
Testing using LFI is generally as follows. A specialized adsorbent sample pad at one end of the LFI strip receives the specimen material. That material then migrates to the next conjugate release pad, where the specimen material is exposed to "antibodies that are specific to the target analyte and are conjugated to coloured or fluorescent particles." The material then progresses to a detection zone containing antibodies or antigens that are fixed in the zone and intended to react to a specific analyte. If the analyte is present, a test line produces a visual, qualitative response, and a control line ensures proper liquid flow across the strip. A wicking pad at the other end properly maintains the flow of liquids across the strip.
2.2.1 Severe acute respiratory syndrome (SARS)
Severe acute respiratory syndrome, otherwise known as SARS, arose in South China in late 2002. Caused by the SARS coronavirus (SARS-CoV) and believed to have originated from horseshoe bats, SARS eventually was contained in the summer of 2003. The last known infection was in April 2004, due to a laboratory accident. 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):
- Determine that the patient is indicating clinical and/or epidemiological evidence of SARS (meets case definitions). 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)."
- 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 of 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 useful in weeks two and three for detecting a rising titre. Additionally, the literature also makes reference to methods of collecting specimens postmortem.
- Conduct testing. At the time, the two primary test types used were enzyme immunoassay (EIA; today more commonly known as ELISA) 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:
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 Centers for Disease Control and Prevention (CDC) calling its results "essentially identical to those for the EIA for SARS antibody." 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" 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" is detected.
- Of note is the World Health Organization's (WHO) 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 unsurety 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." 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.
- 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.
2.2.2 Middle East respiratory syndrome (MERS) 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, though it's not entirely clear how the rest of known cases have been caused (Alshukairi et al. suggest asymptomatic or mildly symptomatic camel workers may serve as a possible transmission source). 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):
- Determine that the patient is indicating clinical and/or epidemiological evidence of MERS (meets case definitions). "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."
- Collect at a minimum both lower respiratory and upper respiratory tract samples. 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 serum specimens, slight differences in guidance appear between WHO guidance and CDC guidance. The WHO appears to differentiate between symptomatic and asymptomatic patient testing, whereas 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). Serological testing of serum specimens should be used for symptomatic patients "only if NAAT is not available." If this is the case, the WHO recommends paired 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." Whether or not to collect a serum specimen in MERS diagnostics may depend on the assay used, however. For example, the CDC, in its Version 2.1 guidance, indicates that testing using the CDC MERS rRT-PCR assay requires collection of serum in addition to upper and lower respiratory tract specimens. For that specific assay, the CDC differentiates between patients who've had symptom onset less than 14 days prior and those who've had it 14 days or later: if prior, serology is for the rRT-PCR test, and if later, serology is for antibody testing. In either case, 200 µL of serum is required.
- Conduct testing. NAAT methods like real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays have been the most common tool for diagnosing MERS-CoV infection due to their high sensitivity. According to late 2018 research by Kelly-Cirino et al., at least 11 commercial single assay and five commercial multiplex assay kits are available (see Table S1, a PDF file, from their highly relevant paper), perhaps more as of April 2020. Serological antibody detection is performed using ELISA, indirect immunofluorescence (IIF), and microneutralization.
- Confirm the results. Laboratory confirmation of MERS-CoV infection is the same for both the WHO and the CDC: one of either a validated NAAT test providing a positive result for at least two different genomic targets, or a validated NAAT test providing a positive result for a specific genomic target along with sequencing confirmation of a separate genomic target. Persons under investigation who receive one negative NAAT result on a recommended specimen are considered to be negative for active MERS-CoV infection. The laboratory should consider testing additional specimens after the first negative. The CDC considers known MERS patients to be negative for active MERS-CoV infection after two consecutive negative NAAT tests on all specimens. The WHO adds: "A patient with a positive NAAT result for a single specific target without further testing but with a history of potential exposure and consistent clinical signs is considered a probable case." The WHO also has additional guidance on using serology for confirming MERS-CoV infection for purposes of reporting under the International Health Regulations.
- Report using national reporting requirements. More broadly, state or local health departments should receive details about received specimens to be tested for MERS-CoV, even before testing begins. Regardless of result, the final positive or negative laboratory confirmation should also be reported to national authorities. If the infection becomes widespread, updates for each new confirmed case or suspected positive should also be made.
2.2.3 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. 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, and laboratory testing isn't necessarily indicated for those immunocompetent individuals capable of self-limiting. 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, laboratory methods of respiratory virus diagnosis—particularly for those who are immunocompromised—have value.
RT-PCR, a molecular method, has been used for well over a decade for detecting coronaviruses. 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. 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. The cost associated with these sorts of tests, compared to their benefits, likely limits ubiquitous use at the first sign of a cold, 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.
2.3 Organizational and agency guidance on COVID-19 testing
NOTE: Information shown here may rapidly become outdated given how quickly response to pandemic testing can change. A full attempt to keep the content relevant will be made. With any novel virus, clinicians and public health experts are dealing with unknown factors. However, public health organizations and agencies have had a base to work from when creating laboratory testing guidance for a novel coronavirus, with more than 40 years of experience with coronavirus biology, pathogenesis, and diagnosis. And while there are fundamental differences between SARS-CoV-2 and its predecessor SARS-CoV, they still share approximately 70 to 80 percent of their genetic code. In fact, the WHO had draft guidance for laboratory testing out as early as January 10, 2020, before gene sequencing was even completed. This guidance and similar draft guidance from national public health organizations and agencies have received steady revisions since as understanding of the virus has grown.
Similar to its predecessors SARS-CoV and MERS-CoV, RT-PCR is being recommended in guidance for detecting SARS-CoV-2's RNA in specimens and thus laboratory confirmation of COVID-19 cases. Serology has its place in testing as well, though with similar lessons from SARS and MERS that it's best used to test for past infection (typically after 14 days of suspected contact with a carrier, or mild symptoms) and thus potential short-term immunity due to the presence of antibodies in blood. In its March 19 guidance, the WHO said: "In cases where NAAT assays are negative and there is a strong epidemiological link to COVID-19 infection, paired serum samples (in the acute and convalescent phase) could support diagnosis once validated serology tests are available." On April 3, the U.S. Food and Drug Administration (FDA) approved the countries first COVID-19 serology test created by Cellex, though Mayo Clinic was also on the verge of rolling out its own in-house serology test as well As of July 7, the U.S. FDA has granted emergency use authorizations (EUA) for 26 serology/antibody tests. (Note: Johns Hopkins appears to be maintaining a page tracking approved serology tests around the world.)
The following sample collection and test procedures have evolved from the COVID-19 pandemic (note that this is only a summary; consult the cited literature directly for full details):
- Determine that the patient is indicating clinical and/or epidemiological evidence of COVID-19 (meets case definitions). Early on in the pandemic, case definitions and testing criteria were initially strict due to lack of test kits, but test kit availability has ramped up since, allowing for testing a wider group of symptomatic patients, as well as asymptomatic patients (though, the CDC still provides a recommended testing hierarchy for areas where testing is still limited). However, clinicians are still encouraged to consider other causes for respiratory illness.
- Collect at a minimum upper respiratory tract specimens and, whenever possible, lower respiratory tract specimens. Although too early to say with certainty, it appears lower respiratory tract specimens such as sputum and bronchoalveolar lavage fluid are typically the most reliable specimen type for RT-PCR applications, as they have been shown to contain the highest viral load, in comparison to upper respiratory tract specimens. As Wang et al. point out, "testing of specimens from multiple sites may improve the sensitivity and reduce false-negative test results," which is largely reflected in WHO, CDC, Public Health England (PHE), and Public Health Laboratory Network (PHLN; Australia) testing guidance.
- Slight differences in upper respiratory tract specimen collection procedures can be found between the WHO/CDC and PHE/PHLN. Both the WHO and CDC offer nasopharyngeal and oropharyngeal swabs as options. The WHO doesn't appear to give a preference, whereas the CDC has a preference for nasopharyngeal swabs but maintains oropharyngeal as still remaining "an acceptable specimen type." In comparison, the latest PHE and PHLN guidance prefer the approach of collecting from both pharynx locations—even with the same swab—"to optimize the chances of virus detection." Nasopharyngeal aspiration is also an acceptable sample collection method for the upper respiratory tract according to all mentioned entities, though the PHLN specifies that it is a substitution for only the nasopharyngeal (they now refer to it as "deep nasal") specimen.
- Regarding serum specimens, statements differ slightly. The WHO notes serology to be useful for retrospective case definition, using paired specimens from the acute and convalescent phases of the disease. The CDC doesn't make reference to serum or serology in their clinical specimen guidance. The PHE suggests hospital patients have "a sample for acute serology" taken but say little else. The PHLN initially provided similar advice as the WHO, but in late April they expanded their guidance to discuss the value of serology, as well as the questions that still persist in using serology for determining immunity to SARS-CoV-2. They have also added collection recommendations for serology, within seven days of symptom onset. The PHLN adds that it "should be stored and tested in parallel with convalescent sera collected two or more weeks after the onset of illness."
- Finally, and more recently, potential evidence of saliva having diagnostic value for detecting SARS-CoV-2 has arisen. As Xu et al. note in their mid-April paper, the "diagnostic value of saliva specimens for ... nucleic acid examination remains limited but promising." Another paper awaiting peer review by researchers at Yale School of Public Health provides similiar thoughts, though is generally more optimistic than the paper published by Xu et al., suggesting saliva from the opening of the mouth (in contrast to Xu et al. and their finding of better results from salive in the throat) may be viable specimen. In fact, an April EUA by the FDA has been made for the first saliva-based COVID-19 test, produced by Vault Health, Inc. As these and similar studies get peer reviewed and methods validated, preferred sample types (and the guidance recommending them) may change. However, it should be noted that as of July 7, none of the prior mentioned official guidance has been updated with any recommendations of using saliva as a substrate.
- Conduct testing. NAAT methods like rRT-PCR have been the primary tools for diagnosing SARS-CoV-2 infection due to their high sensitivity. The PHLN provides the most background about PCR in their guidance, noting that laboratories in its network are confirming positive infections "either with RT-PCR assays detecting a different target gene, or broadly reactive PCR tests with sequencing of amplicons." The latter option is less common due to long turnaround time. They also note that other zoonotic viruses such as SARS-CoV are capable of being detected from PCR assays, though endemic coronaviruses like -229E won't be. The WHO, CDC, and PHLN underscore the idea that viral cultures for routine diagnoses are not practical and, if attempted, should only be performed in Biosafety Level 3 (BSL-3) laboratories. As of July 7, only the PHLN has made any specific recommendations for how serological testing should be conducted for testing past cases of COVID-19, emphasizing it " be performed using a validated assay meeting acceptable and documented performance standards." The current set of approved serology tests from around the world appear to use lateral flow immunoassay, ELISA, or neutralization methods.
- Confirm the results. The strongest public guidance for considering a potential case as being laboratory-confirmed for SARS-CoV-2 infection comes from the WHO. In their guidance, they differentiate between cases by NAAT "in areas with no known COVID-19 virus circulation" and "in areas with established COVID-19 virus circulation." In the first case, one of these conditions must apply: either a validated NAAT test providing a positive result for at least two different genomic targets, or a validated NAAT test providing a positive result for the presences of betacoronavirus along with sequencing confirmation of a separate genomic target, "as long as the sequence target is larger or different from the amplicon probed in the NAAT assay used." In the latter case of established virus circulation, the WHO notes that "a simpler algorithm might be adopted in which, for example, screening by rRT-PCR of a single discriminatory target is considered sufficient." However, if testing produces one or more negative results, that doesn't necessarily rule out SARS-CoV-2 infection. If suspicion of infection remains high, particularly if only upper respiratory tract specimens were collected, additional specimens from the lower respiratory tract should be collected and analyzed. They also emphasize that both external and internal controls should be applied to NAAT runs.
- Report using state and, if applicable, national reporting requirements. (See the next chapter for more on reporting.) Regardless of result, the final positive or negative laboratory confirmation should also be reported to state and national authorities. In the U.S., for example, this means reporting to the local or state health department using the CDC's PUI and Case Report Form. In Canada, reports are sent to the Public Health Agency of Canada (PHAC) via their Coronavirus Diseases (COVID-19) Case Report Form.
Mitigating risk associated with false negatives
Before moving on, words of caution should be issued in regard to any COVID-19 testing conducted: false-negative results can be problematic. One of the primary reasons they are problematic is that it may leave an otherwise asymptomatic individual to continue to unknowingly spread the virus further. Those individuals may relax physical distancing measures and become lax with their mask wearing, affecting others outside the clinical setting. Inside a clinical setting, a patient with a false negative "may be sent to the frontlines of care and inadvertently transmit the virus to patients and colleagues, further straining the already precarious ability of the health care system to respond to the pandemic."
In a perspective piece published in Mayo Clinic Proceedings, West et al. of the Mayo Clinic offer four critical recommendations for society as we attempt to mitigate the risk associated with false negatives when performing clinical testing for COVID-19. Those recommendations are:
- 1. Continue protective and preventative measures inside and outside the testing facility. This includes efforts such as physical distancing, regular hand-washing, regular disinfection of surfaces, and adequate personal protective equipment (PPE) for clinical staff (as well as the encouragement of proper mask wearing by others).
- 2. Develop and improve PCR and serological assays to be more sensitive and specific. The development and improvement process must include methodologically rigorous studies designed to limit the risk of biased results, as well as clearly reported test performance characteristics.
- 3. Assess patients carefully for their potential risk level for being infected. Confidence in negative test results may need to be lowered for health care workers and individuals in other high-risk groups. In general, given the uncertainty around viral load, asymptomatic transmission, and other disease characteristics, caution should be used with negative results in general.
- 4. Establish risk-based protocols for managing negative COVID-19 results. Truly low-risk individuals may not be a major concern when results come back negative. However, individuals in higher-risk categories may require more judicious protocols, e.g., delaying a return to a workplace (for self-isolation) despite receiving a negative and having no symptoms. (This may require a more sensitive follow-up test or at least a second negative in a repeat test, particularly among clinical workers.)
2.3.1 Regulatory considerations: HIPAA and CLIA
The Health Insurance Portability and Accountability Act (HIPAA) is a set of U.S. federal regulatory requirements that attempts to modernize the flow of healthcare information, stipulate how personally identifiable information (PII) (often referred to as "protected health information" or "PHI") maintained by healthcare providers and insurers should be protected from fraud and theft, and address limitations on healthcare insurance coverage in the U.S. HIPAA spans five sections or "titles," mandating health care information access, portability, privacy, and security, as well as stipulations on medical savings accounts, group health insurance requirements, and other tax- and legal-status-related issues.
Normally, HIPAA regulations would put strict requirements on how and when PII can be managed, used, shared, and stored. However, the COVID-19 pandemic has seen a relaxation of some of those requirements by the U.S. Department of Health and Human Services' (HHS) Office for Civil Rights (OCR). The HHS is currently maintaining a list of announcements, notifications, guidance documents, bulletins, and other resources as they relate to HIPAA and the public health emergency. Important notes pulled from that information reveal:
- Family, friends, and others identified by the patient as being involved in their care may receive PHI from a covered health care provider, particularly when they deem that sharing in the patient's best interest. Additionally, "[a] covered entity also may share information about a patient as necessary to identify, locate, and notify family members, guardians, or anyone else responsible for the patient’s care, of the patient’s location, general condition, or death."
- "Covered health care providers will not be subject to penalties for violations of the HIPAA Privacy, Security, and Breach Notification Rules that occur in the good faith provision of telehealth during the COVID-19 nationwide public health emergency. This Notification does not affect the application of the HIPAA Rules to other areas of health care outside of telehealth during the emergency." This exercising of enforcement discretion will last until OCR provides a notice otherwise. Telehealth should optimally be performed in a private setting, whenever possible, using non-public facing communication technologies. Providers should opt to use the most secure services possible, "but will not be penalized for using less secure products in their effort to provide the most timely and accessible care possible to patients during the public health emergency."
- The OCR extended its enforcement discretion to include business associates of covered health care providers, allowing them to share PHI data "without risk of a HIPAA penalty." The OCR adds that "[s]ome HIPAA business associates have been unable to timely participate in these efforts [to ensure the health and safety of the public] because their BAAs do not expressly permit them to make such uses and disclosures of PHI." Like the enforcement discretion of telehealth provision by covered health care providers, the business associate must still make a good-faith effort in the "use or disclosure of the covered entity’s PHI" for public health and health oversight activities. Similarly, this exercising of enforcement discretion will last until OCR provides a notice otherwise.
- The OCR also extended its enforcement discretion to include covered health care providers and business associates operating community-based COVID-19 testing sites (CBTS)—"which includes mobile, drive-through, or walk-up sites that only provide COVID-19 specimen collection or testing services to the public"—during the public health emergency. This enforcement discretion, however, "does not apply to covered health care providers or their business associates when such entities are performing non-CBTS related activities, including the handling of PHI outside of the operation of a CBTS."
- "[T]he HIPAA Privacy Rule permits a covered entity to disclose the protected health information (PHI) of an individual who has been infected with, or exposed to, COVID-19, with law enforcement, paramedics, other first responders, and public health authorities1 without the individual’s HIPAA authorization, in certain circumstances." OCR provides numerous examples, including: "when the disclosure is needed to provide treatment," "when such notification is required by law," when a public health authority must be notified "to prevent or control spread of disease," "when first responders may be at risk of infection," when preventing or lessening "a serious and imminent threat to the health and safety of a person or the public," and when responding to a law enforcement inquiring concerning a lawfully detained inmate or other individual.
- "The HIPAA Privacy Rule permits HIPAA covered entities (or their business associates on the covered entities’ behalf) to use or disclose PHI for treatment, payment, and health care operations, among other purposes, without an individual’s authorization." ICR provides more details in its guidance, noting however that reasonable efforts must still be made to protect PHI, and that this does not apply to activities that constitute marketing.
- The public health emergency does not change restrictions on disclosing PHI to the media. This includes the prohibition of media crews in, for example, emergency departments where COVID-19 patients are being treated, as PHI is found everywhere in that setting. Only when every patient who is or will be in a potentially filmed area has signed a HIPAA authorization form can this be done.
The Clinical Laboratory Improvement Amendments (CLIA) are a set of U.S. federal regulatory requirements applied to all non-research-based clinical laboratory testing performed on humans. These requirements are intended to further ensure a higher standard of quality in clinical laboratory testing, focusing in on improving the accuracy, reliability and timeliness of tests. (The implications of these requirements on U.S. clinical labs and their ability to test for COVID-19 are discussed later, in the next section.)
CLIA uses seven different criteria to gauge and assign one of three complexity levels to laboratory devices and assays: high, moderate, and waived. Additionally, CLIA mandates clinical laboratories handling specimens originating from the U.S. and its territories to apply for a CLIA certificate that is appropriate for the type of testing it performs. Labs using complex devices and assays would have to apply for a high complexity certificate, and so on. Waived tests are recognized as simple to perform with a low risk of erroneous results and include among others urinalysis for pregnancy and drugs of abuse, blood glucose and cholesterol tests, and fertility analysis.
Anything but waived testing requires meeting "the CLIA quality system standards, such as those for proficiency testing, quality control and assessment, and personnel requirements. The standards for moderate and high complexity testing differ only in the personnel requirements." As the Centers for Medicare and Medicaid Services (CMS) points out in a frequently asked questions (FAQ) document—and as can be verified on the FDA's EUA page—a huge majority of COVID-19 tests are only authorized for moderate or high complexity testing, and thus labs certified to do that sort of testing. As of July 7, only four of 101 FDA EUAed molecular diagnostic tests are approved to be performed in a CLIA-waived laboratory setting.
- CLIA regulations remain firmly in effect during the U.S.-declared public health emergency; a Section 1135 waiver, under the Social Security Act, that modifies or suspends CLIA requirements is not within the authorizing jurisdiction of the CLIA program. Additionally, CMS in general does not have the authority "to grant waivers or exceptions that are not established in statute or regulation."
- Laboratories choosing to use temporary testing sites for remotely (from home or another temporary location) viewing and reporting on cytology slides and images may do so if certain defined conditions are met. (Consult the memo for those defined conditions.)
- Proficiency testing (PT) is still required if a CLIA-certified lab is still performing testing and issuing patient results. However, should a PT provider need to postpone, suspend, or cancel a proficiency testing event, "[l]aboratories will not be penalized for lack of PT results ... so long as the cancelation is documented (including the notification from the PT program), and PT is conducted in a timely manner after the public health emergency ends. However, labs should consider performing their own self-assessment to ensure reliable testing."
- Alternate specimen collection devices (e.g., viral transport media, flocked nasopharyngeal swabs) used outside the manufacturer's instructions still require the establishment of performance specifications and assay validation prior to patient use. The FDA provides additional guidance on this topic.
- Laboratories performing laboratory developed tests (LDTs) are still required to be CLIA-certified and meet the requirements for high complexity testing. However, if the state government of such a laboratory has opted to take responsibility for authorizing an LDT (in order to expedite COVID-19 testing), then engagement with the FDA is not required.
- As of May 2020, a CLIA specialty or subspecialty has not yet been assigned to COVID-19 testing.
2.4 Current test methods and their differences
NOTE: Information shown here may rapidly become outdated given how quickly response to pandemic testing can change. A full attempt to keep the content relevant will be made.
2.4.1 Background on the laboratory testing environment
Before continuing, it should be noted that many elements of the prior-mentioned COVID-19 testing guidance have governmental public health laboratories in mind. However, as the scale of the epidemic has grown, the need for commercial laboratories and assay developers to get involved with efforts towards increasing analytical testing throughput—through a more rigorous public-private partnership—has become abundantly clear. Even so, at least in the United States, turnaround times have been slow due to a variety of factors, from lack of in-house laboratory resources to handle high test volumes and a slower-than-expected ramping up of test kit production, to actually getting diagnostic assays that are more rapid (yet still accurate) in their diagnosis, simpler to use, and useable at the point of care. The good news is examples of these rapid point-of-care molecular test kits are now becoming available around the globe, including the United States as part of the U.S. Food and Drug Administration's EUA process.
As the demand for expanded diagnostic testing grows in the face of a pandemic, it's important to compare the U.S. laboratory testing environments of public health and large commercial testing labs with those of small, in-office clinical labs. In the U.S., all but research-based laboratory testing of human specimens is regulated under CLIA, including public health laboratories. Of the more than 256,000 non-exempt CLIA-registered labs in the U.S., only 33,212 or 13 percent of them are certified to perform moderate- and high-complexity testing. Your public health labs and commercial diagnostic labs fall into this category, with investments in the personnel, training, certifications, and equipment to conduct those sorts of tests. Contrast this with the small yet numerous physician office laboratories (POLs) and how they operate. As of October 2019, nearly 46 percent of CLIA-certified laboratories in the United States are POLs. Located in an ambulatory or outpatient care setting, these labs test specimens from human patients to assist with the diagnosis, treatment, or monitoring of a patient condition. Testing in the clinical lab generally depends on three common methodologies to meet those goals: comparing the current value of a tested substance to a reference value, examining a specimen with microscopy, and detecting the presence of infection-causing pathogens.
These POL's operate in a different environment than your average public health laboratory or reference lab that receives, processes, and reports on specimens en masse. The POL is typically a smaller operation, performing simple laboratory testing that can produce useful diagnostic data cheaply and rapidly. Rather than performing advanced pathology and molecular diagnostic procedures that require specific equipment and expertise, the POL typically focuses on blood chemistry, urinalysis, and other testing domains that don't require significant resources and provide rapid results. This can be seen in Centers for Medicare and Medicaid Services statistics reported in October 2019 that show nearly 67 percent of POLs in the U.S. are certified to provide CLIA-waived tests, "simple tests with a low risk for an incorrect result."
As of early July, with 1. all but a handful of the current EUAed molecular in vitro diagnostic COVID-19 test kits being limited to moderate- and high-complexity CLIA labs (the FDA claims that EUAed SARS-CoV-2 tests authorized for "use at the point of care" are considered CLIA-waived tests), and 2. local government entities such as New York City's Department of Health and Mental Hygiene sending reminders to local laboratories that serology testing (as of April 7) is also still considered high-complexity in nature (though the FDA has EUAs for serology tests that are approved for both moderate and complex CLIA labs), a significant majority of clinical laboratories are shut out from assisting with the effort to test the U.S. population for SARS-CoV-2 infection. Given the rapid rate of change at multiple levels of government and society, and wildly varying levels of reliable information being given to the public, it's important to remember these fundamental differences in laboratories when trying to explain to someone why they are, as of yet, unable to go to their primary care physician and get tested for SARS-CoV-2 in the doctor's office. Should researchers develop and the FDA provide EUAs for more CLIA-waived point-of-care assays, these differences may become less noticeable, and more people will be able to be tested.
As of July 7, the U.S. Food and Drug Administration (FDA) has 101 EAUs for molecular in vitro diagnostic test kits. A huge majority use some form of RT-PCR methods, with most using real-time versions of RT-PCR (rRT-PCR). Eighty-four of those 101 are only authorized to be used in CLIA-certified high-complexity laboratories, with 15 being rated for both moderate- and high-complexity. Two RT-PCR kits—Mesa Biotech's Accula SARS-Cov-2 Test and Cepheid's Xpert Xpress SARS-CoV-2 Test—have an additional authorization for point-of-care (POC) use (and thus CLIA-waived use) when used with their authorized POC devices.
Of course, there are many more test kits than those approved in the United States. The Foundation for Innovative New Diagnostics (FIND) is currently "collating an overview of all SARS-CoV-2 tests commercially available or in development for the diagnosis of COVID-19." As of July 7, their site shows more than 220 commercialized manual NAAT tests around the world (most being RT-PCR), with nearly 30 in development. The AdVeritasDx test and controls database is also useful.
2.4.3 LFA- and LAMP-based
LFAs are currently rare, but due to their advantages of being quick and useable at the point of care, some have suggested that as a format for antigen and antibody (serology) testing, they could positively change the testing landscape. As of July 7, seven of 26 serology tests that have received EUAs by the FDA for moderate- and high-complexity CLIA-certified labs are LFAs. An article by Sheridan in Nature Biotechnology highlights a handful of others developed around the world (see their Table 1). FIND shows more than 200 commercialized rapid diagnostic immunoassay tests around the world, though it's not clear how many of them actually LFAs (from their list, only six are explicitly stated as being LFA). At this point, it's safe to say that LFA are still being developed, and it may be a while before we start seeing more of them, at least in the United States.
Also of note are the loop-mediated isothermal amplification (LAMP) and reverse transcription LAMP (RT-LAMP) methods. Abbott's ID NOW and Cue Health's Cue COVID-19 tests are described by the FDA as using "isothermal nucleic acid amplification technology for the qualitative detection of SARS-CoV-2 viral nucleic acids." These are presumably loop-mediated isothermal amplification or LAMP tests, the only two so far approved by the FDA. Among FIND's list of more than 220 commercialized manual NAAT tests around the world, four of them are explicitly shown to be some form of LAMP test. Multiple preprints on medRxiv and bioRxiv, as well as published papers, suggest that RT-LAMP could provide rapid results for SARS-CoV-2 testing, and Abbott is stating its EUAed ID NOW COVID-19 test can be completed within five minutes. However, mid-May findings by New York University put the test's accuracy into question. On July 1, an FDA spokesperson allegedly indicated receipt of 126 reports of "adverse events" concerning the test. The FDA is still investigating the data and working with Abbott to have additional studies performed on the test's accuracy.
2.4.4 Blood serum As discussed prior, LFAs are intended to be rapid point-of-care tools for qualitatively testing body fluids for patient antibodies or viral antigen. The ELISA is, in contrast, a more lab-bound method which produces results that are qualitative or quantitative. In the context of COVID-19 testing, ELISA tests for the presence of patient antibodies in a given specimen based upon whether or not an interaction is observed with the viral proteins present on the test plate. However, even if antibodies are present, ELISA isn't able to tell a clinician if those antibodies are able to protect against future infection. Neutralization assays are the lengthiest to complete, taking from three upwards to five days. This is largely due to the fact that the assay depends on culturing cells that encourage growth of the target virus. Afterwards, introduced patient antibodies, if present, will fight to prevent viral infection of cells. This process is performed in decreasing concentrations, giving the clinician an opportunity to "visualize and quantify how many antibodies in the patient serum are able to block virus replication." In contrast to ELISA, a neutralization assay is able to determine if a patient's antibodies are actively fighting against the target virus, even after recovering from the infection.
Johns Hopkins' Center for Health Security appears to be tracking serology-based COVID-19 tests that are in development or have been approved in various parts of the globe. However, for the most up-to-date list of serology tests that have received EUAs in the United States, the FDA's EUA list appears to be the best source. As of July 7, the FDA shows 26 serology assays approved for diagnostic use in the U.S. Of those 26, five are explicitly listed as being ELISA-based.
A review of Johns Hopkins' tracking list showed more LFA-based tests among those approved in other parts of the world. ScanWell Health's kit appears to be proprietary, but given its claims of rapid results, it's probably LFA. A Singapore-based operation has two tests, one that is advertised as rapid and another as a neutralization test. Of the 29 assays approved for research or surveillance, seven appear to be based on ELISA, two are unknown, and the rest are other types of assays. Among those still in development, an LFA stands out for integrating CRISPR detection. CRISPR (clustered regularly interspaced short palindromic repeats) represents bacterial and archaeal DNA sequences derived from DNA fragments of previous infection. This genetic material can then be used as an activator of biomarkers when attached RNA "guides" find a match with target viral RNA in patient specimen.
2.4.5 Testing alternatives and challenges
We’ve never faced this before, where clinical labs needed to very quickly be able to ramp up a test so fast.
- Jennifer Doudna, Executive Director of the Innovative Genomics Institute, University of California, Berkeley
Though the dismantling and fund-cutting (proposed and real) of government programs designed to protect the populace from pandemics—as well as shortfalls in funding overall—have likely hobbled local, national, and global response to COVID-19, it should be recognized that this pandemic may arguably represent a once-in-a-century type of event. That said, even the most well-prepared governments would still face challenges in quickly learning about, controlling, and developing therapies for a novel disease agent. Shortages in supplies, workers, funding, and other resources are inevitably caused with a pandemic as people across all types of infrastructure fall ill. This requires the additional human elements of adaptability, drive, and shared knowledge to find new and alternative solutions to fighting the challenges inherent to fighting against a novel disease.
See for example a non-peer-reviewed paper published on bioRxiv in early April 2020, where Schmid-Burgk et al. point out that though RT-PCR methods are the most common for currently testing for SARS-CoV-2, "global capacity for testing using these approaches, however, has been limited by a combination of access and supply issues for reagents and instruments." They propose "a novel protocol that would allow for population-scale testing using massively parallel RT-LAMP by employing sample-specific barcodes." They claim that a single heating step, pooled processing, and parallel sequencing with computational analysis would allow for the testing and tacking of "tens of millions of samples." Though the protocol has not been validated with clinical samples, and concerns about sensitivity levels of RT-LAMP (an isothermal nucleic acid amplification technique that allows for RNA amplification) have been raised, the authors' work exemplifies the immediacy and ingenuity going into finding workable solutions to a once-a-century problem.
Another example of ingenuity in the face of difficult circumstances can be found at the University of California, Berkeley. Its Innovative Genomics Institute (IGI) has rapidly repurposed a 2,500-square-foot scientific lab into an automated diagnostic laboratory that can initially process more than 1,000 patient samples per day, with the ability to ramp up to 3,000 per day thanks to robotics and a streamlined workflow. Partnering with dozens of people from Thermo Fisher Scientific, Salesforce, Third Wave Analytics, and Hamilton Corp., the lab is focused on not only turnaround time but also accuracy of results through automation. Their continued success, of course, relies on a steady supply of reagents and related supplies from Thermo Fisher.
Others have also expressed concerns about the global supply of reagents necessary to test for SARS-CoV-2. Successful testing using RT-PCR requires two different enzymes: reverse transcriptase, for converting RNA to DNA, and polymerase, for amplifying the converted DNA. These enzymes and other reagent components may be instrument-specific, and at least one component has to be sympathetic to detection of the target virus' RNA. Little of this can be prepared without a proper sequence of the virus in question. Dr. Ronald Leonard, president and medical director of Cytocheck Laboratory and medical director of the Labette Health hospital, has expressed the difficulties associated with reagent manufacturing thusly:
With the instant demand for SARS-CoV-2 testing, the manufacturing process had to start from scratch for the SARS-CoV-2 specific components, and this did cause a lag time before reagents were available. The increased demand coupled with the decision to only allocate reagents to two national laboratories, some state health departments, and to "hot spots" has compounded the difficulty for laboratories like ours to obtain the necessary reagents to perform the testing.
Reports of reagent shortages from various sources have appeared every month since March 2020, though whether the shortages are a real supply issue or "a consequence of restrictive policies on where and how testing could be completed" has been previously argued by some. Others have taken matters into their own hands in regards to reagent component shortages. Noting Irish laboratories' difficulties sourcing lysis buffer (for isolating molecules of interest and keeping them stable), Cork Institute of Technology's Dr. Brigid Lucey worked with several other virologists and microbiologists, as well as pharmaceutical company Eli Lilly, to produce a custom-formulated yet high-quality lysis buffer for not only Irish laboratories but also other countries can take advantage of. "We are happy to share what we found with other countries and it’s important our scientists retain their skills to make this kind of formulation because we may need to do this again in the future if we get other pandemics," she said.
One other challenge lies in the accuracy of serology-based antibody tests, let alone how much they actually tell us about immunity. FierceBiotech's Conor Hale touches upon this in late April:
Compared to molecular tests—which sequence and match the RNA of the novel coronavirus to produce a result—the FDA has described antibody tests that gauge the body’s immune system response as a less-complicated endeavor that could proceed without review, dubbed “regulatory flexibility” by Commissioner Stephen Hahn. This policy shift has led to confusion, with some antibody test developers falsely claiming their tests are FDA-approved or could diagnose COVID-19 at home. Still others have sold outrightly fraudulent tests online.
At least in the U.S., these problems are compounded by company participation in test validation of EUAs being voluntary. As a late April memorandum from Congress puts it: "FDA is unable to validate the accuracy of antibody tests that are already on the market, and companies are ignoring requests from the Department of Health and Human Services (HHS) to voluntarily submit their tests for validation ... FDA has failed to police the coronavirus serological antibody test market, has taken no public enforcement action against any company, and has not conveyed any clear policy on serological tests..." The entire memorandum is revealing in the challenges of attempting to relax social distancing measures under the pretense of the effectiveness of antibody testing. Entities such as the University of California - San Francisco and the University of California - Berkeley have been emphasizing the importance of elements such as sensitivity, specificity, proper training, and the unknowns of the predictive ability of the test. With public confusion growing and expectations increasingly out of line with the science, it's more important than ever for leaders across government, healthcare, and the media to continue to not spread misinformation and not make decisions based on poor scientific evidence. It will take organized efforts from multiple stakeholders—such as that found with a June NIH workshop on expanding and improving COVID-19 antibody tests—to find responsible solutions to the challenges we still face with this pandemic.
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Citation information for this chapter
Chapter: 2. Diagnostic testing of COVID-19 and other coronaviruses
Edition: Edition 1.1
Title: COVID-19 Testing, Reporting, and Information Management in the Laboratory
Author for citation: Shawn E. Douglas
License for content: Creative Commons Attribution-ShareAlike 4.0 International
Publication date: July 2020