Diagnosis of COVID-19

Clinical Presentation

The most common presenting signs and symptoms of COVID-19 are fever, dry cough, and fatigue. Other common presenting symptoms are detailed below (WHO-China Joint Commission Report, 2020):

Of note, though a majority of patients have fever at some point in their disease course, a study of 1,099 patients (both hospitalized and outpatient) with laboratory-diagnosed COVID-19 from 522 hospitals in 30 provinces of China found that only 44% of patients were febrile at the time of diagnosis, which highlights some of the diagnostic challenges associated with variable clinical presentations (Guan et al., NEJM 2020).

Thought questions:

• Diane wakes up feeling “off,” and she calls her doctor to ask about the possibility of having coronavirus. If you were her doctor, what questions might you ask? What is on your differential alongside COVID-19?
• Does the lack of a fever mean a patient is not infected with SARS-CoV-2? Why not? What about the presence of a sore throat or nasal congestion?

• So if Brian is correct and the majority of people will have “just a bad cold” with fever, dry cough, and fatigue, why are we taking such drastic measures to contain this virus?

Another clinical feature of COVID-19 that has received considerable media attention is olfactory and taste disorders (OTDs), including anosmia (NPR, 4.1.20). Frequency of reported anosmia ranges from 22%-65% in the literature. Frequency of taste disorders depends on the definition (dysgeusia - 33% vs ageusia - 20%) (Larco, et al., Wellcome Open Res 2020).

In summary, no ONE symptom or set of symptoms can reliably diagnose or exclude COVID-19 infection but it is clear that cough, fever, and fatigue are most commonly seen. As discussed in the basic virology section, it is also important to remember that a person begins shedding the virus (i.e. can transmit the virus to others) prior to symptom onset and some people will never develop symptoms and remain asymptomatic carriers (Li et al., Science 2020; Pan et al., Lancet 2020; Roth et al., NEJM 2020).

Presenting symptoms in children are similar to those in adults (fever, dry cough, and fatigue) although often less severe and asymptomatic cases have also been reported (Hoang et al., EClinicalMedicine 2020).

Diagnostics

Thought question:

• Given that treatment for COVID-19 is primarily supportive, what are the benefits of testing for SARS-CoV-2?

• Based on what you have learned about the basic virology of SARS-CoV-2, how would you design a test to look for infection? From where would you collect samples?

Molecular assays

Most tests for SARS-CoV-2 utilize RT-PCR against the RNA-dependent RNA polymerase (RdRp), E (envelope), N (nucleocapsid), S (spike protein), and/or ORF1b transcripts (video review of RT-PCR) (review: Sheridan, Nature 2020). These PCR tests use respiratory specimens, primarily from nasopharyngeal, and sometimes oropharyngeal, swabs. RT-PCR is highly specific, and is therefore considered the gold standard diagnostic for confirming COVID-19 infection. Sensitivities across kits vary dramatically, however, and can be as low as 70% compared against clinical suspicion with positive CT findings, especially early in the disease course (Fang et al., Rad 2020; Ai et al., Rad 2020). Given this higher likelihood for false negatives, if clinical suspicion remains for COVID-19 despite a negative initial test, the WHO recommends resampling and retesting from multiple sites. Though there has been new data in preprint suggesting saliva swabs may be more sensitive than nasopharyngeal swabs at least in inpatients, current clinical guidelines recommend nasopharyngeal swabbing only (Wyllie et al., medRxiv 2020). Additional PCR testing can be done on stool samples, though whether this represents active infection or continued viral shedding is debated (see Pathogenesis).

Multiple companies have also developed isothermal nucleic acid amplification tests (NAAT)-- the same technology used for rapid influenza and strep tests-- against SARS-CoV-2 (Cepheid, Abbott). By avoiding thermal cycling for denaturing and annealing, isothermal NAAT can amplify user-specified nucleic acid sequences at a much faster rate than conventional PCR. Notably, however, unlike other modalities, most isothermal strategies would only be able to run one test at a time. They may be better suited for outpatient clinic settings or point-of-care testing sites, rather than high-throughput clinical laboratories. Clinical sensitivity and specificity data for these tests have yet to be released.

Given the risk of inducing coughing and consequently aerosolizing droplets, the CDC recommends healthcare workers wear N95 respirators, eye protection, gloves, and a gown for all lower respiratory tract specimen collection (e.g.: sputum induction, bronchoalveolar lavage), and, if resources allow, for nasopharyngeal swabbing as well.

CRISPR

CRISPR-based technologies have also emerged as novel diagnostic strategies for COVID-19 (video review of CRISPR). The Broad Institute is in the process of validating their CRISPR-based SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing) technique against the SARS-CoV-2 S and ORF1ab genes (Zhang, Abudayyah, and Gootenberg, 2020, not peer-reviewed), while Mammoth Biosciences out of the University of California San Francisco simultaneously validated their DETECTR protocol, against a panel of N, E, and RdRP genes (Broughton et al., Nature Biotechnology 2020). These tests are similar in design: after nucleic acid extraction from respiratory samples, both SHERLOCK and DETECTR make use of simultaneous reverse transcription and isothermal amplification. Guide RNAs paired with Cas enzymes first cleave these specific sequences, and then cut reporter substrates to generate a visual read-out. Using lateral flow (a technique used in commercial pregnancy tests, for a review: Koczula & Gallota, Essays Biochem 2016), results can then be read on a paper dipstick. Both would be rapid diagnostic tests that could turn around results in under an hour.

Thought questions:

• What are the limitations of testing via nasopharyngeal swab?

• What might be the benefits of testing for previous infection with SARS-CoV-2, rather than active infection?

Immunoassays

Blood samples of patients with COVID-19 mount the expected dynamic pattern of IgM followed by sustained IgG antibody levels against SARS-CoV-2 within two weeks time (Zhao et al., Clin Inf Dis 2020) (video review of ELISA, the primary method of antibody/antigen detection). IgM/IgG testing could reveal not only those with active infection but also those with a missed previous infection. It is currently unknown whether the presence of these antibodies confers immunity, and if so, for how long. A rapid diagnostic antibody test against the SARS-CoV-2 spike protein (both the full length protein, and its smaller receptor binding domain) has recently been designed and validated against 3 samples from COVID-19+ patients (Amanat et al., Nat Med 2020). Rapid point-of-care lateral flow immunoassay testing for an IgM/IgG panel was also validated in 397 PCR-confirmed positive patients and 128 negative patients, with 89% sensitivity and 91% specificity within 15 minutes (Li et al., J Med Virol 2020). Testing from venous blood and fingerstick were consistent across samples. Obtaining blood rather than respiratory samples, especially if blood is collected by fingerstick onto a paper strip, might also decrease risk of transmission to healthcare workers handling the specimens.

United States Testing Capacity

Testing capacity in the United States initially lagged behind that of other countries due to regulatory requirements by the FDA, faulty tests provided by the CDC, and limitations in healthcare infrastructure such as not enough laboratory personnel, supplies, and/or testing facilities such as tents and drive-through centers. These policies resulted in “rationing” of tests, with eligibility based not only on viral pathophysiology and clinical judgment, but also on epidemiology and public safety. The limitations of testing in the U.S. prevented early contact tracing and individual isolation, as has been done in South Korea (discussed in Module 2).

Commercial lab tests and hospital-specific protocols were quickly developed based on CDC protocol, underwent FDA Emergency Use Authorization (EUA), and began to address the need for PCR gold standard diagnostics in the United States (Roche EUA 3/16/20, Thermo Fisher EUA 3/16/20, Broad Institute for Massachusetts). Point-of-care tests using isothermal NAAT also dramatically increased outpatient testing capacity (Cepheid Xpert Xpress SARS-CoV-2 EUA 3/21/20, Abbott ID NOW COVID-19 EUA 3/27/20). Rapid antibody testing has also been approved (Cellex EUA 4/1/20). Though antibody testing is gaining attention, it is currently only used to support a diagnosis of SARS-CoV-2 infection rather than to guide practices such as use of PPE and return to work given that it is unknown if and for how long these antibodies confer immunity. As this is a rapidly changing area of diagnostics, the CDC has published interim guidelines for serologic testing.

Given rapid development of EUA SARS-CoV-2 virology tests, with over 150 tests having undergone EUA to date, there has emerged a need to compare test performance (e.g., limit of detection, sensitivity, specificity) and scalability across the myriad assay and sample types in a standardized fashion. Doing so could better enable the selection of the most economically viable, scalable, and best-performing tests for the much needed global, ubiquitous temporal monitoring, involving diverse clinical settings and sample types. Recent efforts to perform such a standardized comparison of virology tests include the Resilience Health online tool, a continually updated comparative dataset of tests that have undergone EUA (Mackay et al., Nat Biotechnol 2020).

Many strategies are being implemented to limit transmission of SARS-CoV-2 from those awaiting testing to other patients in healthcare waiting rooms or healthcare workers themselves. Often, patients are screened remotely via virtual visit or telephone. For those who are determined to require testing by institution-specific protocols, many institutions have developed “drive-through” testing capabilities to limit exposure. On April 21, the FDA approved the first at-home RT-PCR nasal swab test kit for COVID-19 through LabCorp. On November 17th, the FDA approved the first home self-testing providing rapid at-home results, The Lucira COVID-19 All-In-ONe Test Kit (FDA News Release, 11/20).

This is an active area of innovation, and tests are being developed at a rapid pace to meet need. Here is a frequently updated list of what diagnostic tests for COVID-19 are being developed worldwide. For a complete list of FDA EUAs, please refer to the COVID-19 Emergency Use Authorizations for Medical Devices.

Thought questions:

• For whom would you recommend PCR testing in an ideal resource situation? With limited testing resources, who should be tested?

Massachusetts Case Study: Testing on a State Level

Massachusetts began to see COVID-19 cases in early March; case numbers and state and commercial testing capacity rapidly increased since then. Compare the following two testing algorithms, adapted from Massachusetts Department of Public Health (MA DPH) guidelines (version 1 published 3/13/20, version 2 published 4/2/20) (highest quality images here and here). Thought question:

• What changes were made to the testing guidelines, and why?

Note that several eligibility categories in MA DPH’s guidelines in both versions query public safety factors, such as the individual’s risk of disease transmission to others, rather than the probability of an individual having COVID-19. Also note how epidemiological risk factors were adjusted (removal of recent travel categories, no requirement for “close contact” with known cases, expansion of testing to essential workers besides healthcare workers). Other institution-specific testing guidelines may approach eligibility from a purely clinical view, and may have a specific set of signs, laboratory findings, and/or imaging findings needed for inpatients, or telemedicine triage protocols to assess symptoms and signs for outpatients. As testing capacity has increased, clinical sites are able to test asymptomatic individuals such as close contacts of known COVID-19 patients and people without symptoms of COVID-19 who are admitted to the hospital.

Thought question:

• What factors would be important to consider in implementing a testing protocol for asymptomatic individuals? How might you weigh test characteristics, clinical management strategies, risks of testing, and economic implications?

Ancillary Studies

Laboratory diagnostics

Thought question:

• Given what you know about laboratory values in viral infections in general, what would you predict the laboratory values to be in patients with COVID-19?

The hallmark laboratory findings in COVID-19 cases reported thus far is lymphocytopenia. In the Guan cohort of 1099 patients discussed above, these trends were seen: lymphocytopenia (83%), elevated CRP (61%), thrombocytopenia (36%), and leukopenia (33%). Less commonly, elevations were seen in ALT, AST, CK, and d-dimer (Guan et al., NEJM 2020). These laboratory trends are represented below in the typical fishbone format but REMEMBER - a patient need not have all or any of these laboratory values to be infected:

(Nick Mark, A Seattle intensivist’s one-pager on COVID-19)

A strong push has been made for identifying laboratory markers that can be used as clinical predictors of disease severity. Unsurprisingly, patients with more severe disease have been seen to have more prominent laboratory abnormalities across the board than those with nonsevere disease (Guan et al., NEJM 2020). Additionally, some inflammatory markers have been found to be significantly different between admitted patients that recover from COVID-19 compared to those who die. Specifically, those who died had higher levels of troponin, myoglobin, CRP, IL-6, ferritin, procalcitonin, LDH, creatine kinase, D-dimer, and lower lymphocyte counts, platelet counts and albumin (Ruan et al., Intensive Care Med 2020; Zhou et al., Lancet 2020). These findings are detailed in the graph below. In a study that longitudinally monitored immunologic data from 326 COVID-19 patients, IL-6 kinetics were highly correlated with disease severity (i.e. if IL-6 increased over the course of a patient's disease, so too did their disease severity). Additionally, this study found that lymphocyte count on admission was associated with disease severity, with increased lymphopenia being associated with a more severe disease course (Zhang et al, Nature 2020).

Temporal changes in laboratory markers from illness onset in patients hospitalised with COVID-19 (Zhou et al., Lancet 2020).

Elevated procalcitonin has been shown to be associated with a higher risk of more severe SARS-CoV-2 infection. Whether this represents bacterial superinfection or an inherent feature of the immune system’s response to SARS-CoV-2 remains unclear (Lippi et al., Clin Chim Acta 2020).

Coagulation abnormalities in COVID-19 patients, notably elevated D-dimer and fibrinogen levels, increased Factor VIII activity, as well as the following abnormal thromboelastography (TEG) findings in a population of 24 selected intubated patients with COVID-19 pneumonia (Panigada et al., J Thromb Haemost 2020):

• Reaction time (R) shortened (50%)

• Clot formation time (K) shortened (83%)

• Maximum amplitude (MA) increased (83%)

• Clot lysis at 30 minutes (LY30) reduced (100%)

While COVID-19 coagulation abnormalities are reminiscent of disseminated intravascular coagulation (DIC), a major distinction can be seen in the prominence of thrombosis in COVID-19 versus bleeding in DIC.

MGH’s COVID-19 Management Guidelines (7.1.20) recommend daily CBC, CMP, CPK, and Ferritin/CRP as well as PT/PTT/fibrinogen and d-dimer every other day for all patients admitted with confirmed or suspected COVID-19. Additionally they recommend LDH, troponin, and baseline ECG for risk stratification.

Imaging

Thought question:

• What might be barriers to using imaging to routinely screen for COVID-19?

Studies have found that abnormal lung findings can be seen on chest CT for patients with COVID-19, even in asymptomatic cases (Shi et al., Lancet Inf Dis 2020). Despite this, concerns over resource allocation, infection control, and the limited diagnostic specificity of chest imaging for COVID-19 have resulted in recommendations against using chest radiographs or CT as a first-line form of diagnosis (American College of Radiology Position Statement, 3/11/20).

Chest CT may however be indicated for hospitalized patients with severe respiratory symptoms, and the most commonly seen findings for COVID-19 are described below.

Thought questions:

• What imaging findings would you expect to see in a viral pneumonia?

• Are the imaging findings for COVID-19 different from other viral pneumonias? How?

For a concise summary and multiple images of CT findings, please watch this video.

The majority of imaging findings for COVID-19 are consistent with a viral pneumonia, with diffuse, bilateral involvement of the lung. The most common patterns seen are ground-glass opacities (GGOs), air-space consolidations, crazy paving (pattern of GGOs with inter/intra-lobular septal thickening), vascular enlargement, and traction bronchiectasis. Of note, GGOs, vascular thickening, and the peripheral distribution of these findings have been the most helpful in allowing radiologists to distinguish COVID-19 pneumonia from other viral pneumonias, but specificity remained quite variable across radiologists (24-100%) (Bai et al., Rad 2020). Imaging findings evolve over time, with abnormalities peaking at 10 days post symptom onset (Pan et al., Rad 2020), and fibrous stripes appearing with resolution (Pan et al., Eur Rad 2020). Imaging abnormalities, perhaps unsurprisingly, also correspond to disease severity, with dramatic increase in lung involvement correlating to rapid decline in patient prognosis (Shi et al., Lancet Inf Dis 2020).

Transverse thin-section CT scans in patients with COVID-19 pneumonia. (A) 56-year-old man, day 3 after symptom onset: focal ground-glass opacity associated with smooth interlobular and intralobular septal thickening in the right lower lobes. (B) 74-year-old woman, day 10 after symptom onset: bilateral, peripheral ground-glass opacity associated with smooth interlobular and intralobular septal thickening (crazy-paving pattern). (C) 61-year-old woman, day 20 after symptom onset: bilateral and peripheral predominant consolidation pattern with a round cystic change internally (arrow). (D) 63-year-old woman, day 17 after symptom onset: bilateral, peripheral mixed pattern associated with air bronchograms in both lower and upper lobes, with a small amount of pleural effusion (arrows). (Shi et al., Lancet Inf Dis 2020)

Lung ultrasound has also been used to evaluate critically ill COVID-19 patients; lung consolidation, B lines, septal thickening, and A lines may be seen during recovery (letter, Peng et al., Intensive Care Med 2020).