Introduction
As the world recognized that this new virus was rapidly spreading, the need for rapid identification aired, and COVID-19 testing rapidly began. Unfortunately, the logistical issues with massive testing also evolved. To start massive testing efforts, a coordinated scientific and logistic approach must occur. An essential aspect of a containment strategy is employment of extensive testing, which allows health organizations to identify the individuals currently or previously infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate diagnostic testing is crucial to identify symptomatic and asymptomatic individuals to ensure adequate treatment and isolation measures, thus preventing further disease transmission.
This chapter describes the importance of testing, the refinement of different diagnostic tests, the differences between them, and the challenges faced while undergoing massive diagnostic testing.
Background
COVID-19 was first publicly announced in December 2019 to the world, following the outbreak in Wuhan, China. The virus, now recognized as SARS-CoV-2, was identified and sequenced in early January 2020.1–3 It consisted of a positive-sense single-stranded RNA β-family coronavirus that shares significant genetic homology with the SARS coronavirus and bat SARS-like coronaviruses, with a genomic size of 29.9 kb. Each intact virus consists of four structural proteins. The N protein holds the RNA genome of the virus, and S, E, and M proteins create the virus envelope together.4–6
Isolation and identification of the virus paved the way to the prompt development of diagnostic testing based on real-time reverse transcription polymerase chain reaction (RT-PCR) technology. Currently, this is one of the most extensively employed diagnostic tests for COVID-19.7,8
As testing evolved, the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) stressed massive widespread testing, tracking infected people, and tracing those exposed, as an effective strategy to decrease viral transmission.9
Emergency Development of COVID-19 Testing
During the beginning of the pandemic in Wuhan, China, where the first cases were reported, the primary scientific task was to identify why this atypical pneumonia was causing so many critical care admissions due to acute hypoxemic respiratory failure. The first step was an attempt to culture the pathogenic agent that was causing the disease. After identifying the single-stranded virus, the second step was to start the task to decode its genome, which was accomplished in a very short time thanks to the efforts of the scientific community.10 After that was achieved, the new goal was to develop accurate tests to help in the early diagnosis and identification of patients and begin containing measures. Immediately, the scientific community started the task of developing those tests, and soon three principal types of diagnostic tests for COVID-19 were developed: nucleic acid amplification tests (NAATs), antibody tests, and antigen tests.11,12 NAATs identify viral genetic material. Antibody tests examine the presence of antibodies generated from the human immune response to the viral infection. Antigen tests identify the presence of viral antigens.13
Early in the pandemic, the CDC developed real-time RT-PCR diagnostic panels for viral detection.14 The CDC’s Real-Time RT-PCR Diagnostic Panel is a test formulated to qualitatively detect two different regions of the N gene, N1 and N2, and the RNase P (RP) gene from specimens obtained from the upper and lower respiratory tract.14 The RP gene test performs as an internal control to confirm that the RT-PCR was performed properly.15 Because the virus is found in the upper and lower respiratory tract, specimens from the nasopharynx will represent the presence of viral RNA in the upper and lower respiratory tract.
The CDC’s Real-Time RT-PCR Diagnostic Panel was the first to obtain approval on February 4, 2020, and employs the N1 and N2 genomic segments.16 The CDC developed the primers and probes and made the materials obtainable for other diagnostic labs that wanted to employ the same test.16
Most of the antigen tests focus on the four viral structural proteins. The spike (S) and nucleocapsid (N) proteins are the main immunogens.17 The S1 subunit shares less homology with other coronaviruses and is very specific to SARS-CoV-2.18 Furthermore, the receptor-binding domain (RBD) of the S protein demonstrates a reduced amount of cross-reactivity between SARS-CoV-2 and other coronavirus strains.19
The serological test came into play to try to detect rapidly and inexpensively the immune response of a suspected patient for COVID-19, mainly focusing on the presence of immunoglobulin M (IgM) in the blood to diagnose an acute infection and the presence of immunoglobulin G (IgG) to address acquired immunity, as well as diagnose people who had the infection without noticing.
Although all of these tests were getting developed as fast as possible, limitations in sample collection, transportation, and kit performance during the beginning of the pandemic led regulatory agencies to decide to grant emergency and special approval to try to face the challenge of massive testing (Table 3.1).
Abbreviations: NAAT, nucleic acid amplification test; RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
FDA Emergency Approval
In the United States, transmission of the virus outpaced the ability to test for it, with more patients developing the symptoms and no available test to obtain a certain diagnostic test. It was one of the main reasons why the Food and Drug Administration (FDA) eventually used its Emergency Use Authorizations (EUAs) on February 4, 2020, to permit for prompt and widespread development and deployment of in vitro testing.14
Any laboratory or commercial company developing molecular tests to detect SARS-CoV-2 that wanted to obtain this approval had to submit an application to the FDA and obtain EUA status to offer the test for diagnostic purposes to release it to the market.20 However, all these tests had to contain the legend “Emergency Use Authorization,” either in the boxes of the testing supplies or in the reports given to the patients.21
Several more tests were added in early March 12, 2020, under a different permission by a Presidential directive, enabling laboratories with Clinical Laboratory Improvement Amendment accreditation to add tests without regulatory clearance.22 This produced an unprecedented scenario in which the medical profession and the general public were unaware about the new COVID-19 tests that were being given to patients and health care facilities. However, the objective of all of these measures was to provide as many feasible testing resources available for all levels of care.
Screening for Testing
The determination to test ought to be predicated on clinical, epidemiological circumstances and linked to appraising the probability of infection.23 At the beginning of the pandemic, testing of asymptomatic or presymptomatic individuals who have had contact with COVID-19-positive patients was contemplated and screening protocols were then adapted to different locals and laws.24
The clinical manifestations of infection are often nonspecific, including pyrexia, fatigue, cough, anosmia, and dyspnea. Also, a large proportion of individuals demonstrate no symptoms throughout their disease course, and this poses difficulty in determining who should be tested.25
Only a few studies have matched the timing of detection of the viral RNA, and the timing of the detection of antibodies, with the course of the disease beginning with the incubation phase ending with recovery.26–28 However, the available information at this point suggests that a patient can be carrying the virus for 2 to 14 days and can be a “negative” patient in a first test but become “positive” in a test done in 6 to 8 days after the first day.29
Both the WHO and CDC advise testing individuals exhibiting symptoms and close contacts of individuals with identified infections.9 Furthermore, both advise testing for a wide range of respiratory pathogens on patients’ samples suspected for COVID-19 to reduce the risk of untreated coinfection.30 The same guidelines advise testing asymptomatic or presymptomatic patients only if they have had contact with a COVID-19-positive patient.9,30
In the United States and most countries, the decision to test a person had to be precise, as there was a lack of resources and materials to test everyone. This brought to importance the clinical diagnosis of COVID-19, which was solely grounded on epidemiological history and clinical manifestations.23 However, this process had several flaws, such as patients not recalling a complete history of present illness or lying to be tested.
Due to the uncertainty and different presentations of COVID-19, it comes only as an intelligent decision to treat every patient as a possible COVID-19 until a PCR negative is obtained.30 Moreover, even though a negative result comes back if the patient is still displaying symptoms or the clinical presentation changes, it is a good clinical practice to retest the patient.30
Mutations
The mutation of this virus is of great concern for the scientific community, primarily because of the velocity that these mutations are taking place.31 In the context of COVID-19, this poses an unprecedented threat to the efforts to contain the virus because most of these variants are also more transmissible or the infection the patients develop is worst.32
An unexpectedly increased quantity of mutations may occur involving all locations of the viral genome and will have substantial effects on diagnostic kits, vaccines, and the development of therapeutic agents.32 That is why every test that has been developed in COVID-19 will have to be rechecked in a short period of time. For example, the nucleocapsid gene may not be an optimum target for diagnostic kits, and the existing test kits targeting the nucleocapsid gene should be appropriately modified for testing accuracy.
Types of Tests
The three main types of tests available to detect SARS-CoV-2 and accurately diagnose COVID-19 include NAATs, serological tests, and antigen tests (Table 3.2).
Abbreviations: NAATs, nucleic acid amplification tests; FDA, Food and Drug Administration; IgG, immunoglobulin G; IgM, immunoglobulin M; NAATs, nucleic acid amplification tests.
It is critical that clinicians understand the difference between all the available tests, the correct diagnostic test, and, more importantly, interpret the results (Fig. 3.1). One should always remember that all of these tests are aiding the diagnosis.
As a rule, all tests have main and dependent variables, accuracy, and cost. When we select to lower the price, the accuracy of the test reduces. Therefore, it is crucial to understand that even though a test might seem accessible, this does not guarantee that it will be accurate.
Nucleic Acid Amplification Test
Because viremia is frequently detected early in the course of an illness, NAATs are the most sensitive assays and widely recommended diagnostics for detecting early viral infections,9 For a quick and reliable diagnosis of COVID-19, many NAATs have been developed. Most assays have a detection limit of between 3.4 and 4.5 log10 copies/mL of virus.9
All methods based on nucleic acid are complex (and expensive), involving sophisticated equipment and testing reagents and requiring highly trained technicians. As a consequence, it is not feasible to employ this type of testing as point-of-care diagnostic or bedside tests in resource-limited settings. Furthermore, the tests take 4 to 6 hours to complete on average, but the necessity to transport clinical samples takes time, thus delaying reporting of results.
These types of tests have three main processes that can influence their results. These are the collection of the specimen, handling of the specimen, and processing of the specimen.33 Proper technique and well-trained medical staff reduce the chances of erroneous results from a deficient collection of a sample. When it comes to handling the specimens, the manufacturer of each collection kit specifies different temperatures at which the samples must be stored; the last part is processing the sample.34
Real-Time Reverse Transcription Polymerase Chain Reaction Test
The RT-PCR test was one of the first tests to be developed and the first one that the CDC approved for the diagnosis of COVID-19.9 To this day, it retains its position as the gold standard at diagnosing suspected cases of COVID-19.35 RT-PCR has been deemed the “gold standard” for diagnosis as it has been demonstrated to be highly sensitive for accurately identifying viral genomes present, down to just one molecule of RNA.36
RT-PCR is an example of molecular testing. RT-PCR is a NAAT that detects unique sequences of SARS-CoV-2 (Fig. 3.2).
The first RT-PCR assays were intended to identify viral RNA from upper or lower respiratory tract samples.9 However, later development and curiosity from researchers allowed obtaining specimens to test from vaginal and anal swabs and even urine samples.37
Apart from nasopharyngeal (NP) and oropharyngeal sample, sputum samples can be collected to diagnose infection by expectorating deep cough into a sterile container.37 In contrast to oropharyngeal samples, increased positive rates are observed with NP swabs performed by trained medical personnel.38
When it comes to the results of this test, clinicians must be cautious; different panels available can vary drastically from one laboratory to another. For example, some tests detect two or more viral genes.37 Test results are interpreted differently in various assays. Some assays require that both viral genes be identified for the test result to be interpreted as positive, while others require detecting one of two viral genes for a positive interpretation.37 Furthermore, it is important to recall that due to the small amount of RNA that can be identified, these test results can be positive during the incubation period, which occurs several days prior to the development of disease symptoms, and remain positive for the duration of symptoms.9 That is why it has been an ideal test to detect patients who report themselves as asymptomatic. Indeed, in our practice, we have seen patients remain “positive” for weeks after they had no more symptoms of the illness. This has been specially observed to be true in specimens obtained from anal swabs.39
In general, a positive result in an RT-PCR test indicates the presence of viral nucleic acid in the specimen obtained. However, testing in conjunction with the clinical evaluation, clinical history, doctors’ perception, and further diagnostic techniques should be used to establish the patient’s infection status.9,23,29,37,40 Negative results should not mislead to rule out SARS-CoV-2 infection and must be used in combination with other clinical features and testing to determine patient management.9,23,29,37,40
The results of the RT-PCR tests might take anywhere from a few hours to a few days, depending on the PCR version and the panel.37 This test can be done in a one-step or two-step process. The one-step method, which combines RT and DNA polymerase to carry out their reactions in the same tube, is the recommended method for identifying the virus.41 The two-step approach entails reverse transcription of RNA in one tube and ensuing DNA polymerization in a different reaction tube.42
False-positive results as a result of the cross-reactivity with other coronaviruses, the human genome, and microflora can be reduced with sequence fidelity.9 A reliable RT-PCR test contains a control, which means that the test is also looking for human epithelial cells as a safety measure of quality control to confirm that the specimen was correctly obtained.9,23,29,37,40
The CDC recommends the following:
For initial diagnostic testing for SARS-CoV-2, collecting and testing NP or oropharyngeal samples, sterile swabs must be employed to collect samples. Swabs must be transferred directly into a sterile transport containing 2 to 3 mL of either viral transport medium, Amies transport medium, phosphate-buffered saline, or sterile saline. For personnel obtaining samples or working within 6 feet of individuals suspected to be infected, it is essential to maintain appropriate infection control and use the recommended personal protective equipment (PPE) while obtaining samples.43
Providers handling sampling but not involved in the collection (e.g., self-collection) and not working within 6 feet of the patient should follow standard precautions. Providers are recommended to wear a face mask at all times while in the facility.43
Samples should be stored at 2 to 8°C for up to 72 hours after collection. If a delay is anticipated, store samples at −70°C or below.
Reverse Transcription Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplification (LAMP) is a qualitative PCR-based nucleic acid amplification, which can precisely amplify the target sequence very efficiently, rapidly under isothermal conditions (usually 60–65°C).44 The method employs multiple primers, which recognize specific areas of the target gene.45
Although RT-LAMP-based molecular technologies are available, they have not met with widespread use as a result cross-reactivity and poor sensitivity of the assays. RT-LAMP does not necessitate skilled providers or high technological equipment. In contrast to PCR, this technique can be executed in a limited-resource setting by heating the specimens and reagents in a single reaction tube.46
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Test
CRISPR technology has been applied as a diagnostic tool by COVID-19 iinvestigators.47
Recently, assays combining isothermal amplification and CRISPR technology have been developed as diagnostic tools for the fast identification of viral RNA. A distinct set of Cas nucleases were observed to demonstrate DNA and RNA cleavage activity.48–50
A technique termed SHERLOCK has been harnessed for sensitive DNA or RNA viral identification. Employing this technology requires very little laboratory resources and facilitates amplification of target RNA, and this technique is able to detect viral RNA in under 1 hour.48–51
There are many adaptations of CRISPR platform technology and amplification techniques in development that will be valuable in assisting the diagnosis of COVID-19; however, they are beyond the scope of this chapter.47
Antigen Tests
Rapid antigen tests are rapid diagnostic methods (point-of-care tests) due to the practicality of testing the patient at the bedside and with fast turnaround time of results.52 Most of these tests are based on lateral immunochromatography; they are already in use for other respiratory viruses (e.g., influenza virus, syncytial virus) and do not require specific and expensive equipment or supplies, which makes them very accessible for clinics or places where a high complexity laboratory is not in the near vicinity.53
Most of these tests do rapid qualitative viral identification. They detect viral antigen, which is usually a protein, by the immobilized coated viral antibody on the apparatus. They usually come in immunofluorescence-based lateral flow technology.5 These tests do not employ amplification technology and are more likely to produce a false-negative results than NAATs. Their main advantage is that they are fast to deliver results, with the fastest turnaround times for all tests.
It is necessary that a negative antigen test be confirmed by further molecular testing before infection is ruled out in a COVID-19-suspected individual.54
Trusting just an antigen result would be a big mistake by the clinician. In the authors’ experience, a false-negative rate of 30% is commonly encountered with some of these tests.
Three different main types of antigens are commonly used: (1) the recombinant nucleocapsid protein, from SARS-CoV-2, which is highly conserved among all seven members of coronaviruses and led to poor specificity in tests in the general population; (2) the Spike1 protein from SARS-CoV, which has very different antigenicity from its counterpart in SARS-CoV-2; and (3) the RBD of SARS-CoV-2 Spike1. Among these antigens, the nucleocapsid shares significant homology with other coronaviruses, leading to low specificity of the test.55
Most of the antigen tests’ specimens have to be collected from an NP or nasal sample. This means the existence of same limitations as noted in the PCR tests about requiring well-trained health care workers.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
