key: cord-318204-t024w7h6
authors: Fang, Ferric C; Naccache, Samia N; Greninger, Alexander L
title: The Laboratory Diagnosis of COVID-19-- Frequently-Asked Questions
date: 2020-06-08
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa742
sha: 
doc_id: 318204
cord_uid: t024w7h6

Diagnostic testing has played and will continue to play a major role in the COVID-19 pandemic. The ability to detect the SARS-CoV-2 coronavirus in respiratory secretions is essential to determine when an individual is infected and potentially infectious to others. Viral detection is used for the identification, management and isolation of individual patients. Viral detection is also used to determine when the virus has entered a community and how rapidly it is spreading. As communities attempt to re-open following periods of shutdown, the detection of both SARS-CoV-2 and specific antibodies recognizing the virus will become increasingly important as a means to assess infection and immunity in individuals and communities. Here we discuss questions commonly asked by clinicians about COVID-19 diagnostic testing.

Limitations in the availability of PPE and testing supplies, in addition to the operational difficulty of scaling NP swab collections for asymptomatic screening, have led to the evaluation of alternative samples including nasal swabs, mid-turbinate swabs, oropharyngeal swabs, and saliva. Saliva is particularly attractive as it requires neither swabs nor transport media, although collection and processing of saliva presents other challenges (20) . The comparability of these specimen types for a qualitative test depends on the viral load present at the time of infection. Oropharyngeal swabs have shown less sensitivity compared to nasopharyngeal and nasal swabs (21) . Nasal swabs and nasopharyngeal swabs may be comparable in sensitivity, but more studies are needed to compare these specimens across different patient populations. New specimen types for an EUA test must receive a specific amendment for that specimen type before they can be reported. Some studies have indicated that self-collected samples are comparable in sensitivity to those collected by health care providers, which can obviate the need to use PPE for testing (22) (23) (24) (25) . Although sputum and bronchoalveolar lavage samples may have higher viral loads and can therefore provide greater test sensitivity than upper respiratory samples (26, 27) , particularly at later stages of illness, they entail a higher risk of aerosol generation or require an invasive procedure, so these sample types are obtained more selectively.

A c c e p t e d M a n u s c r i p t (28, 29) . This may be a consequence of the variable distribution of the ACE2 viral receptor protein in the respiratory tract (30) . Interestingly, viral tropism for the lower respiratory tract was also seen during the H1N1 influenza epidemic (31) .

Is it worth repeating a test after a patient has tested negative? In view of the less than ideal sensitivity of an NP swab to detect SARS-CoV-2 infection, it may be useful to repeat testing in a patient in whom the clinical suspicion is high (32) . In our experience, the yield of repeat testing from the same source is low (33) . However, the yield from repeat testing may be substantial in higher prevalence settings (34) .

shedding culturable virus after about one week from symptom onset, but can continue to have detectable viral RNA in their respiratory tracts for longer periods of time (35, 36) . More severely ill patients will remain PCR-positive for longer, sometimes extending for weeks-to-months (37) . SARS-CoV-2 RNA can also be found in stool samples and typically continues to be detectable in the feces for weeks (38) . Evidence indicates that human intestinal epithelial cells can support replication of A c c e p t e d M a n u s c r i p t 7 the virus (39) , and SARS-CoV-2 has been cultured from stool samples (40) . Fecal-oral transmission of SARS-CoV-2 has not been demonstrated, but there is concern that fecal shedding might contribute to the spread of infection.

Does a positive specimen mean that a patient is infectious? Not necessarily. Although viral nucleic acids can be detected during convalescence (35) , culturable virus is believed to represent a better correlate of infectivity (41) . However, SARS-CoV-2 culture requires a BSL-3 facility and there are no authorized clinical assays utilizing SARS-CoV-2 viral culture at this time, so clinical monitoring is dependent on RNA detection.

negative? Two negative PCR assays at least 24 hours apart are commonly used as a criterion to discontinue isolation (42) . However, a number of patients will revert to PCR-positivity after two negative samples (43) . This may reflect fluctuations in the quantity of viral RNA shedding during recovery. When monitoring for viral load as inferred by real-time PCR cycle threshold (Ct), cases that have reverted to positivity consistently exhibit high Ct values indicative of a low viral load (44) . There is presently little evidence of true virological and clinical relapse, and the prognosis for these patients appears to be good.

A c c e p t e d M a n u s c r i p t 8 Is quantitative PCR (viral load) useful? There is some correlation between illness severity and viral load on presentation, as inferred by the real-time PCR cycle threshold (Ct) (37, 45) . However, an isolated viral load estimate is of limited prognostic value, and even asymptomatic individuals may have high viral loads (46) . Viral load trends may have greater value and might help to inform decisions to initiate a trial of antiviral therapy, but viral loads typically decline regardless of the clinical course (3, 6, 47) .

Older patients tend to have higher viral loads (6), just as they are at greater risk for more severe or critical illness (48, 49) .

imply that only one of two PCR targets was detected, and confirmation with a different assay that detects alternative targets is recommended. Given the high specificity of the PCR assays, most inconclusive results will ultimately be confirmed as positive. Inconclusive or Indeterminate results could also indicate that the internal controls failed and may indicate a technical issue, such as the presence of a PCR-inhibitory substance in the sample. Different labs and tests may use different terminology, so it is worth contacting a given clinical laboratory to determine how they report low positive results. Diagnostic platforms used for the detection of specific antibodies to SARS-CoV-2 proteins include rapid diagnostic tests (RDT) such as lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), neutralization assays and chemiluminescent immunoassays (50) . Only neutralization assays can provide information regarding the ability of antibodies to inhibit viral growth.

The performance of various serologic tests is more variable than the RT-PCR assays for SARS-CoV-2 (8, (51) (52) (53) . This is particularly important because the positive or negative predictive value of a test is dependent not only on the intrinsic test accuracy but also on the prevalence of disease in the population. An insensitive test will have a poor ability to exclude the presence of disease when prevalence is high, but more germane to COVID-19, a test with low specificity will have a poor ability to indicate the presence of disease when the prevalence is low.

When only a few percent of the population have immunity to SARS-CoV-2, as is presently the case in most regions, a positive result from a serologic test with low specificity will be more likely to represent a false-positive.

There are four kinds of human coronavirus that cause mild-to-moderate seasonal respiratory tract infections: 229E, NL63, OC43 and HKU1. Cross-reactivity with antibodies to seasonal coronaviruses is a theoretical concern for a SARS-CoV-2 serologic test, but for most of the commercial assays evaluated thus far, this does not seem to be the case (54, 55) . To rule-out cross reactivity, a new assay should be tested against a panel of serum samples that pre-date the emergence of SARS-CoV-2, ideally more than 500, to A c c e p t e d M a n u s c r i p t 10 accurately ascertain specificity. Cross-reactivity between SARS-CoV-1 or MERS-CoV and SARS-CoV-2 is more likely (56) , but should be a limited concern.

Are serologic tests useful to diagnose acute COVID-19? Although the primary use of serologic tests is to determine prior exposure to SARS-CoV-2, the detection of specific antibodies may support the diagnosis of COVID-19 in a patient with a high clinical suspicion but negative PCR tests (57-59).

IgM and IgG directed against SARS-CoV-2 may appear as early as 3-6 days after the onset of symptoms. By three weeks, nearly all patients have seroconverted, and the antibodies persist for at least two months, with IgG showing greater persistence (4-8, 59).

The duration of antibody responses to SARS-CoV2 is unknown. Antibody responses to the common respiratory coronaviruses decay after a few years (56) , and it is suspected that immunity to SARS-CoV-2 will be similar.

Antibody responses have been observed in nearly all patients with COVID-19, although it is possible that some very mild or asymptomatic infections, or infections in immunocompromised patients, may not result in seroconversion (5, 60) .

A c c e p t e d M a n u s c r i p t 11 Does a positive antibody test mean that a patient is immune? It is likely that the detection of specific antibodies in a patient with a history of COVID-19-like illness will be indicative of at least some degree of immunity (61) . Experimental animals re-challenged with pathogenic coronaviruses exhibit resistance to re-infection (62) . However, a quantitative cutoff of antibody titer that correlates with protective immunity is undefined. As with other viruses, it is possible that a low titer of antibodies is not protective. Also, it is not presently known whether neutralizing antibodies are the primary mechanism of immune protection. In fact, higher antibody titers are observed in patients with more severe illness (5, 6, 51) . As patients with mild COVID-19 may recover despite low antibody titers, and patients with severe COVID-19 have persistent illness despite the development of high antibody titers, one may question whether neutralizing antibodies are in fact protective. The reported therapeutic benefits of convalescent plasma might be due to constituents other than neutralizing antibodies (63) . Moreover, the development of neutralizing antibodies is accompanied by T cell responses (64, 65) , which may contribute to protection.

Are quantitative serologies helpful? Because SARS-CoV-2 is a new human pathogen, pre-existing adaptive immunity is non-existent, so acute and convalescent titers are not required to establish the diagnosis of COVID-19. Until specific cutoffs are identified as a correlate of protective immunity, qualitative serologic results are sufficient to provide clinical guidance. However, reporting of quantitative serological read-outs could offer clinicians more information about the potential for false positives and false negatives for values near the positivity threshold.

A c c e p t e d M a n u s c r i p t 12 Is there a correlation between age and antibody titer? Older age correlates with a higher likelihood of severe illness from COVID-19 and with the development of higher antibody titers (59), perhaps due to a higher antigen load.

Is point-of-care testing available? Affordable point-of-care (POC) diagnostics for SARS-CoV-2 could facilitate the widespread testing and contact tracing strategies proposed for post-pandemic wave containment (66) . However, performance characteristics and usability are critical parameters for these tests, as they are typically deployed without the quality assurance apparatus of a high complexity laboratory. A POC antigen detection assay was recently authorized by the FDA, but is known to be less sensitive than PCR, so its clinical role has yet to be defined. Negative results using this assay should be confirmed by a more sensitive method in most instances. Other assay technologies, including a CRISPR-based nucleic acid detection system (67), may be utilized in POC formats in the future if appropriate sensitivity can be achieved.

A variety of biomarkers, including lymphocyte count, neutrophil-tolymphocyte ratio, CRP, troponin T, D-dimer, LDH, procalcitonin, IL-6 and ferritin are predictive of disease progression and mortality in COVID-19 (table 1) (68, 69) . These laboratory tests play a vital role in identifying patients at risk for complications and to guide treatment interventions.

Early transmission dynamics in Wuhan, China, of novel coronavirusinfected pneumonia

The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application

Temporal dynamics in viral shedding and transmissibility of COVID-19

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Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

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A COVID-19 Transmission within a family cluster by presymptomatic infectors in China

Contact tracing assessment of COVID-19 transmission dynamics in taiwan and risk at different exposure periods before and after symptom onset

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Diagnostic testing for severe acute respiratory syndrome-related coronavirus-2: A narrative review

Food and Drug Administration. Emergency Use Authorizations

Comparison of four molecular in vitro diagnostic assays for the detection of SARS-CoV-2 in nasopharyngeal specimens

Comparison of commercially available and laboratory developed assays for in vitro detection of SARS-CoV-2 in clinical laboratories

Performance of Abbott ID NOW COVID-19 rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a New York City academic institution

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Comparison of nasopharyngeal and oropharyngeal swabs for SARS-CoV-2 detection in 353 patients received tests with both specimens simultaneously

Consistent detection of 2019 novel coronavirus in saliva

Effect of throat washings on detection of 2019 novel coronavirus

Saliva as a non-invasive specimen for detection of SARS-CoV-2

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Detection of SARS-CoV-2 in different types of clinical specimens

SARS-CoV-2 Viral Load in Clinical Samples of Critically Ill Patients

Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19) in China: A Report of 1014 Cases

Diagnosis of the coronavirus disease (COVID-19): rRT-PCR or CT

SARS-CoV-2 reverse genetics reveals a variable infection gradient in the respiratory tract

Diagnosis of influenza from lower respiratory tract sampling after negative upper respiratory tract sampling

Infectious Diseases Society of America guidelines on the diagnosis of COVID-19

Occurrence and timing of subsequent SARS-CoV-2 RT-PCR positivity among initially negative patients

Clinical performance of SARS-CoV-2 molecular Testing

Virological assessment of hospitalized patients with COVID-2019

Predicting infectious SARS-CoV-2 from diagnostic samples

Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China

Prolonged presence of SARS-CoV-2 viral RNA in faecal samples

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Infectious SARS-CoV_2 in feces of patient with severe COVID-19

SARS-CoV-2 shedding and infectivity

Discontinuation of transmission-based precautions and disposition of patients with COVID-19 in healthcare settings

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Findings from Investigation and Analysis of Re-Positive Cases

Viral dynamics in mild and severe cases of COVID-19

Presymptomatic SARS-CoV-2 infections and transmission in a skilled nursing facility

Clinical and virological data of the first cases of COVID-19 in Europe: a case series

Estimating clinical severity of COVID-19 from the transmission dynamics in Wuhan, China

OpenSAFELY: factors associated with COVID-19-related hospital death in the linked electronic health records of 17 million adult NHS patients

The role of antibody testing for SARS-CoV-2: Is there one?

Severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease

Test performance evaluation of SARS-CoV-2 serological assays

Evaluation of nine commercial SARS-CoV-2 immunoassays

Patients with common cold coronavirusaes tested negative for IgG antibody to SARS-CoV-2

Validation and performance comparison of three SARS-CoV-2 antibody assays

A systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of of disease

Profiling early humoral response to diagnose novel coronavirus disease (COVID-19)

Early detection of SARS-CoV-2 antibodies in COVID-19 patients as a serologic marker of infection

Longitudinal change of SARS-Cov2 antibodies in patients with COVID-19

Different longitudinal patterns of nucleic acid and serology testing results based on disease severity of COVID-19 patients

COVID-19 and postinfection immunity: Limited Evidence, Many Remaining Questions

SARS-CoV-2 infection protects against rechallenge in Rhesus Macaques

Convalescent plasma in Covid-19: Possible mechanisms of action

Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals

Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals

Laboratory diagnosis of emerging human coronavirus infections -the state of the art

Point-of-care testing for COVID-19 using SHERLOCK diagnostics

Hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (COVID-19): a meta-analysis

Elevated levels of IL-6 and CRP predict the need for mechanical ventilation in COVID-19

Interpreting diagnostic tests for SARS-CoV-2

A c c e p t e d M a n u s c r i p t 15 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t