Chapter 23: Serology and Molecular Detection of Viral Infections

COVID-19 ADDENDUM
By Deborah Josko, PhD, MLT(ASCP)M, SM

LEARNING OUTCOMES

After finishing this addendum, you should be able to:

1. Describe characteristics of coronaviruses.

2. Discuss viral properties associated with SARS-CoV-2.
3. Define the acronym COVID-19.
4. List signs and symptoms of COVID-19.
5. Discuss the significance of the ACE2 receptor in COVID-19 cases.
6. Explain how COVID-19 is transmitted.
7. Compare and contrast various SARS-CoV-2 laboratory assays, including specimen
collection and methodology.

8. Describe the Food and Drug Administration (FDA) approved SARS-CoV-2 vaccines.

On December 31, 2019, the World Health Organization (WHO) learned of a cluster of

viral pneumonia cases originating in Wuhan, People’s Republic of China.1 The causative agent

responsible for this viral outbreak was identified as a novel coronavirus called Severe Acute

Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which quickly spread throughout China,

Europe, and the globe, becoming one of the deadliest pandemics the world has encountered. This

new SARS-CoV-2 is the causative agent of coronavirus disease 2019 referred to as COVID-19.1

Coronaviruses (CoVs) are not new viruses. Severe Acute Respiratory Syndrome (SARS-

CoV) and Middle East Respiratory Syndrome (MERS), both coronaviruses, emerged in 2002 in

Guangdong Province, China, and in 2012 in Saudi Arabia, respectfully.2

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 The Coronavirinae, a subfamily of the Coronaviridae, contains four genera:

Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. SARS-CoV

and MERS belong to the Betacoronavirus lineage A and C, respectfully. Betacoronaviruses are

also called “bat-coronaviruses” because evidence suggests bats may be the natural reservoir for

coronaviruses, including SARS-CoV-2. Recent RNA sequencing analysis of SARS-CoV-2

shows greater than 90% similarity to a bat coronavirus RaTG13.3

Coronaviruses are termed as such because of their surface projections resembling a

“crown,” suggestive of the “solar corona” seen on electron microscopy. CoVs contain large,

linear, positive single-stranded RNA genomes ranging from approximately 25 to 32 kb in size. 3

The viral structure is enveloped and consists of two major proteins, the spike (S) glycoproteins

radiating from the surface of the virus and the transmembrane (M) proteins. The M protein plays

a role in viral assembly and formation of the viral envelope, whereas the S glycoprotein is an

antigen that binds to the human angiotensin-converting enzyme 2 (ACE2) receptor and is

responsible for membrane fusion.4,5 The ACE2 receptor is the major receptor that provides the

portal of entry for viral invasion into the host. ACE2 receptors are found in virtually all organs;

therefore, once the virus gains access to the host, it can infect a wide variety of cells and organs

such as the oral and nasal mucosa, nasopharynx, lungs, stomach, small intestine, colon, skin,

lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain cells.6

SARS-CoV-2 is a highly communicable and infectious virus transmitted human to human

by close contact and inhalation of infected respiratory droplets. The virus can also gain access

via oral, nasal, and eye mucosa contact.7 In addition, transmission can occur indirectly because of

contact with contaminated objects or surfaces.8 The presence of SARS-CoV-2 in intestinal

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infections has been reported; however, there is not enough evidence to date to indicate that

SARS-CoV-2 is transmitted via the fecal-oral route.4

Although SARS-CoV-2 can infect a wide range of cells and organs, the lungs are the

primary target for infection. CT scans have shown the appearance of “bilateral ground-glass

opacity lesions” in the lungs.9 Biopsy samples of the lung, liver, and heart from patients who

died from COVID-19 complications revealed that the lung is the organ mainly affected, resulting

in pathological changes including alveolar damage.10

SARS-CoV-2 and influenza are both contagious respiratory illnesses; however, SARS-

CoV-2 appears to spread easier than influenza and can cause more severe symptoms in certain

individuals. Signs and symptoms appear 2 to 14 days after exposure and range from

asymptomatic to mild to severe depending on the individual’s age and immune status. Younger

children and adolescents tend to be asymptomatic or exhibit mild symptoms whereas older

individuals and individuals with underlying medical conditions tend to develop more severe

symptoms.

Some of the common symptoms include fever, chills, cough, shortness of breath or

difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste and/or smell, sore

throat, congestion, nausea or vomiting, and diarrhea.11 In addition, severe symptoms include

pneumonia, coughing up blood, and kidney failure.4 The Centers for Disease Control and

Prevention (CDC) recommend that individuals experiencing trouble breathing, persistent pain or

pressure in the chest, new confusion, inability to wake or stay awake, and bluish lips or face seek

emergency medical care immediately.11

According to the CDC, if an individual is exposed to a person who tested positive for

SARS-CoV-2, they should self-quarantine for 14 days because this will help prevent further

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spread of the virus. Individuals should monitor their temperature (100.4° F) and watch for signs

such as shortness of breath, cough, and other COVID-19 symptoms.12 If an individual becomes

symptomatic, SARS-CoV-2 testing should be performed, which is available at walk up clinics,

urgent care centers, pharmacies, hospitals, and so on.

Because SARS-CoV-2 is a novel virus, no laboratory assays were available to detect the

presence of either nucleic acids, antigen, or antibody once the virus was identified. Many

medical companies quickly developed molecular and antigen assays in addition to antibody tests;

however, these assays did not undergo rigorous method validation, resulting in some assays with

low sensitivity and specificity. Because of the urgent need for diagnostic testing, the Food and

Drug Administration (FDA) approved many in vitro diagnostic tests for Emergency Use

Authorization (EUA). A list of all approved in vitro diagnostic EUAs can be found on the FDA

website.13

If an individual is exposed to a person with COVID-19, laboratory testing should be

performed, especially if the individual becomes symptomatic. Molecular and antigen tests are

available to detect the presence of SARS-CoV-2, and antibody tests are available to determine if

the patient had a past infection and has acquired immunity to the virus. On average, individuals

become symptomatic 5 to 6 days after exposure, so SARS-CoV-2 testing should be performed

around day 5. Testing too early may result in a false-negative result.

Specimen types utilized in the various molecular and antigen tests include

nasopharyngeal (NP) swab, wash/aspirate or oropharyngeal (OP) specimens collected by a

trained health-care provider, a nasal mid-turbinate specimen or anterior nares specimen collected

by either a trained health-care provider or self-collected at home, and a saliva specimen collected

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by the person being tested.14 The specimen type depends on the test kit used. Serum is used to

detect the presence of antibodies.

The method of choice and “gold standard” for detecting SARS-CoV-2 is by molecular

assays utilizing the reverse-transcription–polymerase chain reaction (RT-PCR). These assays

target two to three SARS-CoV-2 genes: nucleocapsid (N) genes–N1 and N2 and RNase P (RP)

gene, with most assays yielding high sensitivity results (greater than 90%).15 RT-PCR test results

can take anywhere from a few hours to several days to be reported.16

Rapid methods are also available and include rapid molecular and rapid antigen assays.

Rapid molecular assays are more sensitive (greater than 90%) because genetic/nuclear material

specific to the SARS-CoV-2 virus is amplified through multiple PCR cycles.17 Rapid antigen

tests detect one or more specific proteins of the virus; however, cross contamination can occur

resulting in false-positive results.18 Rapid antigen tests tend to be highly specific but have a low

sensitivity (~50%) and are not recommended in asymptomatic individuals because false-negative

results can occur.17 Rapid molecular and rapid antigen test results are usually available in under 1

hour.16 Antibody testing is available to determine if an individual who tested positive for SARS-

CoV-2 produced antibodies; however, long-term immunity is yet to be determined.

SARS-CoV-2, also known as COVID-19, is responsible for over 27 million cases and

over 472,000 deaths in the United States alone.19 To date, there are over 108 million confirmed

cases including over 2.4 million deaths worldwide.20 In order to prevent the spread of this highly

contagious, communicable disease, the CDC recommends wearing masks, maintaining a distance

of 6 feet apart, washing hands for 20 seconds or using hand sanitizer with at least 60% alcohol,

and avoiding large gatherings and crowds.21 Despite these guidelines, the virus has rapidly spread

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throughout the United States and the world. Therefore, development of a vaccine is imperative.

Researchers all over the world began working on a vaccine based on some of the information

from studies on the SARS-CoV-1 virus. Although many pharmaceutical companies and scientists

worldwide are working on a vaccine, the Pfizer-BioNTech vaccine was approved first by the

FDA for EUA in December 2020. The first dose of the vaccine was administered in the United

States to a critical care nurse in Queens, New York, on December 14, 2020. The Pfizer-

BioNTech vaccine requires two doses spaced 3 weeks apart and has shown to be 95% effective

with minimal side effects.22 On December 18, 2020, the FDA approved the second EUA vaccine

manufactured by Moderna, Inc. The Moderna vaccine also requires 2 doses administered 1

month apart.23 Both vaccines utilize an mRNA-based methodology.

The vaccines work by introducing mRNA, the genetic material used to make proteins,

into the host. The mRNA in the vaccines contains a special coating that protects it from

proteolytic enzymes in the body and allows it to enter macrophages and other dendritic cells.24

The mRNA does not enter the nucleus, nor does it interact with the host’s DNA. It also does not

contain a live or attenuated virus, thereby reducing the risk of causing disease in recipients.24

The COVID-19 mRNA vaccine provides the instructions for our cells to make the “spike

protein” that is found on the surface of the virus. Once the protein piece is made, our immune

system recognizes the protein as foreign and begins making antibodies against it while activating

T cells to fight off the infection, similar to what happens in a natural, active infection.22, 24

In a recent study published in the New England Journal of Medicine by Polack et al.

(December 2020), the Pfizer-BioNTech vaccine (BNT162b2) was evaluated for efficacy and

safety. After an “ongoing multinational, placebo-controlled, observer-blinded” clinical trial, the

authors concluded the vaccine is safe and provides 95% protection against COVID-19 in

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individuals over 16 years of age.25 It should be noted the use of mRNA as a vaccine is not new

and has been studied before for influenza, Zika, rabies, and cytomegalovirus (CMV).26

The fact that vaccines received FDA EUA approval in less than 1 year after SARS-CoV-

2 became a global public health threat is a tremendous and unprecedented feat. With other

vaccines on the horizon, it is hoped that the COVID-19 pandemic, which has taken and continues

to take millions of lives worldwide, will be under control and that the magnitude of infections

will be greatly reduced.

Copyright © 2021 F.A. Davis 7

References
1. World Health Organization. Coronavirus disease (COVID-19).
https://www.who.int/emergencies/diseases/novel-coronavirus-2019/question-and-answers-hub/q-
a-detail/coronavirus-disease-covid-19. Accessed December 2020.

2. National Institutes of Allergy and Infectious Diseases. COVID-19, MERS & SARS.
https://www.niaid.nih.gov/diseases-conditions/covid-19. Accessed December 2020.

3. Gadsby NJ, Templeton KE. Coronaviruses. In: Jorgensen JH, Pfaller MA, Carroll KC, et al.,
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4. Esakandari H, Nabi-Afiadi M, Fakkari-Afjadi J, et al. A comprehensive review of COVID-19

characteristics. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402395/. Accessed December
2020.

5. Ujike M, Taguchi F. Incorporation of spike and membrane glycoproteins into coronavirus

virions. Viruses. 2015;7(4):1700–1725. doi: 10.3390/v7041700.

6. Hamming I, Timens W, Bulthuis ML, et al. Tissue distribution of ACE2 protein, the functional
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(COVID-19) through postmortem core biopsies. doi: 10.1038/s41379-020-0536-x.
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https://www.cdc.gov/coronavirus/2019-ncov/if-you-are-sick/quarantine.html. Accessed
December 2020.

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13. Food and Drug Administration. In vitro diagnostics EUAs. https://www.fda.gov/medical-
devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/
vitro-diagnostics-euas#individual-antigen. Accessed December 2020.

14. Centers for Disease Control and Prevention. Interim guidelines for collecting, handling, and
testing clinical specimens for COVID-19.
https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html. Accessed
January 2021.

15. Centers for Disease Control and Prevention. Research use only 2019–novel coronavirus
(2019-nCoV) real-time RT-PCR primers and probes. https://www.cdc.gov/coronavirus/2019-
ncov/lab/rt-pcr-panel-primer-probes.html. Accessed December 2020.

16. Food and Drug Administration. A closer look at COVID-19 diagnostic testing.
https://www.fda.gov/health-professionals/closer-look-covid-19-diagnostic-testing. Accessed
January 2021.

17. Infectious Diseases Society of America. Rapid testing. https://www.idsociety.org/covid-19-

real-time-learning-network/diagnostics/rapid-testing/. Accessed December 2020.

18. Food and Drug Administration. Potential for false positive results with antigen tests for rapid
detection of SARS-CoV-2—letter to clinical laboratory staff and health care providers.
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19. Centers for Disease Control and Prevention. Covid data tracker. https://covid.cdc.gov/covid-
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22. Pfizer. Pfizer and BioNTech conclude phase 3 study of COVID-19 vaccine candidate,
Meeting all primary efficacy endpoints.
https://www.pfizer.com/news/press-release/press-release-detail/pfizer-and-biontech-conclude-
phase-3-study-covid-19-vaccine. Accessed December 2020.

23. Food and Drug Administration. FDA takes additional action in fight against COVID-19 by
issuing Emergency Use Authorization for second COVID-19 vaccine. https://www.fda.gov/news-
events/press-announcements/fda-takes-additional-action-fight-against-covid-19-issuing-
emergency-use-authorization-second-covid. Accessed January 2021.

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24. Centers for Disease Control and Prevention. Understanding and explaining mRNA COVID-
19 vaccines. https://www.cdc.gov/vaccines/covid-19/hcp/mrna-vaccine-basics.html. Accessed
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25. Polack FP, Thomas SJ, Kitchin N, et al. Safety and efficacy of the BNT162b2 mRNA
COVID-19 vaccine. N Engl J Med. 2020 Dec 31;383(27):2603–2615.
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26. Centers for Disease Control and Prevention. Understanding mRNA COVID-19 vaccines.
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