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It is clear that the coronavirus disease 2019 (COVID-19) pandemic, due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be with us for the foreseeable future, possibly entrenched as an endemic infection. This poses a grave threat for clinical dental practice as asymptomatic (viral) carriers unknowing of their status, may attend for treatment with the possibility of resultant disease spread to non-immune individuals in the clinic. Even if one is immune through vaccination, the temporal waning of protective antibodies may lead to mild–moderate variant-induced infection, as shown in a number of recent studies. Hence, it is likely that rapid and accurate identification of COVID-19 patients at the point-of-care (POC), that is in the clinic or during the pre-attendance period, will be a critical imperative in the foreseeable future, as a secondary safeguard, in addition to successful vaccination. There are now an array of POC diagnostic tests to detect SARS-CoV-2, and here we summarize their salient features, and the potential utility as an infection control measure in dentistry.
CPD/Clinical Relevance: To describe the currently available POC diagnostic tests for COVID-19, and their utility as a critical infection control measure during dental care delivery in the immediate post-pandemic period.
Article
The COVID-19 pandemic, due to SARS-CoV-2, which began in late 2019, has virtually engulfed the whole world while incessantly spreading in some regions, and gradually receding in others. The morbidity and the mortality of the disease is breathtaking, and makes for grim reading with 179 million infections and 3.88 million deaths worldwide at the time of writing (July 2021).1 The miraculous production of effective and efficacious COVID-19 vaccines within the relatively short span of 1 year, and their prompt administration have curbed the viral spread in most regions. For instance, in the People's Republic of China, where up to a billion people have been vaccinated thus far, the disease has been virtually conquered, with sporadic pockets of resurgent infection. In other parts of the world such as the UK, Europe and USA the infection is slowly but surely receding.
Unfortunately, although there are many regions, particularly Asia, where COVID-19 is rampantly spreading with few signs of disease abeyance in the near future, particularly due to vaccine insufficiency, as well as the waves of infection and re-infection caused by periodically emerging viral variants. At the time of writing, four major SARS-CoV-2 variants, alpha (UK), beta (African), gamma (Brazilian) and delta (Indian) have been identified, and more appear to be on the way, as long as the pandemic persists in any region of the world.2 While most of the currently available COVID-19 vaccines are highly efficacious in preventing the infections due to the original strain of SARS-CoV-2, they are significantly less effective in preventing variant-induced disease.2 Such statistics and data are a clear indication of the chronic, and dogged ability of the virus to survive and circulate in the community for the next few years, at least.
Given this scenario, healthcare workers, including dental care workers, have a stark choice in front of them for infection control during routine patient care. One major way to allay the constant worry and anxiety of either a symptomatic and/or asymptomatic, COVID-19 patient attending the clinic, and creating a cluster of infection, is to implement the so called, point-of-care (POC) diagnostic tests for the infection, prior to beginning any dental treatment. This will be in addition to taking the routine temperature measurements, medical and travel history eliciting potential patient exposure to COVID-19, as well as the patients' vaccination history, and related serological tests.
Additionally, a rapid diagnosis of an infected individual will help in his/her quick isolation and diminution of the community spread of the disease. In practical terms, a patient should be immediately tested on arrival at the clinic, possibly in a separate room, prior to entering the patient waiting area, and further steps taken in the event of a positive outcome. In such an event, it is likely that a confirmatory test will be required, with immediate patient referral to the local centre for COVID-19 assessment and care.
Point-of-care (POC) diagnostic tests for COVID-19
An array of new POC diagnostic tests are currently available. According to the World Health Organization (WHO) and the United States Food and Drug Administration (FDA) over 450 such tests have been developed thus far for rapid identification of COVID-19 patients in clinical settings.3 Although these are not widely used in routine dentistry, as yet, possibly due to the novelty and the initial expenditure outlay, it is hoped that some of these tests will be mass produced and available in the near future as inexpensive test kits to facilitate easy administration by any member of the dental team. Indeed the stated goal of some of the manufacturers is to produce simple, inexpensive kits akin to ‘home pregnancy tests’ so that they are widely available and easily accessible to the public.
Point of care diagnostic technologies: definition and ideal properties
POC diagnostic tests can be defined as ‘a diagnostic test that is performed near the patient or treatment facility, has a fast turnaround time, and may lead to a change in patient management’.4
The following are considered the ideal attributes for POC diagnostic technology.4
Simple to administer and user-friendly: the major desirable property of a POC diagnostic test is that it should be simple so that the user/s can easily administer, with minimal training, without the help of a highly trained medical/dental professional.
Inexpensive: the POC test must be inexpensive and affordable so that it could be administered in large numbers either in routine clinical practice or in mass screening programmes particularly, in resource-poor regions.
High sensitivity: ideally, the sensitivity of a POC test should be 100%, if possible, with minimal, if any, false-negative test results.
High specificity: similarly, the specificity should reach 100%, ideally with no false-positive test results, as far as possible.
Rapid functionality: this is required to perform all steps, from sample collection to test result readout, within minutes, if not seconds, is important so that the test could be quickly interpreted. This attribute is critical, especially in a busy clinic/hospital to facilitate rapid patient turnover.
Robust and equipment-free: ideally a POC test should be self-contained in a kit with a reasonably long expiry period, with the possibility of storage at ambient temperature, and not requiring low temperature fridges or freezers.
Classification of current POC diagnostic tests
The Food and Drugs Administration (FDA) USA classification of the POC tests for COVID-19 fall into three main categories.5 In each category there are over 100 ‘kits’ produced by various commercial manufacturers, and this list is dynamic with new products added virtually on a daily basis. Owing to the severity of the pandemic, the FDA usually gives emergency user authorization (EUA) for these tests, which is recalled in the event of any adverse events/reports. The tests are basically divided into three categories:
Molecular tests: to detect molecular RNA of the pathogen (Figure 1);
Rapid antigen tests: to detect protein markers on the outside of the virus (Figure 1); and
Serology and adaptive immune response tests: these antibody tests are not used in POC tests for SARS-CoV-2 as they look for the serological response from a previous infection or vaccination, and hence, not further discussed here.
Figure 1. SARS-CoV-2 viral components used in POC testing. Components of the nucleic acid amplification tests (NAATS) are shown on the left, and the rapid antigen tests are on the right.
Molecular tests and rapid antigen tests
Molecular tests
These are also called viral nucleic acid amplification tests (NAATS) because they detect and amplify the viral genomic material specific to SARS-CoV-2 (Figure 1). The SARS-CoV-2 viral genomic material evaluated is its ribonucleic acid (RNA), which is present in the body only when the virus is still replicating.6 These tests can detect even very low levels of the virus. NAATs include the polymerase chain reaction (PCR) tests, loop-mediated isothermal amplification tests (LAMP), and next generation sequencing (NGS) assays (Table 1).
EUA: Emergency user authorization; FDA: Food and Drug Administration, USA; MHRA: Medicines and Healthcare products Regulatory, Agency, UK; rtPCR: real-time polymerase chain reaction; LAMP: loop mediated isothermal amplification. Data from the quoted sources. Note: There are over 220 EUA approved tests in the FDA directory, at the time of writing and fewer than 20 are currently approved as POC tests, other tests need to be performed in a hospital setting. EUA status is also temporary, so it is desirable for the EUA tests to eventually become FDA cleared under normal regulatory pathways to enable full-fledged long-term usage.
Figure 2. Some examples of currently available devices used in point-of-care diagnostic tests for COVID-19. (a) Fisher Scientific (Mesa Biotech) ACULA Biotech test kit. (b) ABBOTT Laboratories test kit. (c) Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV for use on GeneXpert Xpress system. (d) Abra and TT Electronics Virolens system.
The patient samples for these tests could either be nasopharyngeal swabs, deep throat saliva or sputum. The nasopharyngeal swabs, however, are the most reliable, as they essentially sample the primary colonization site of SARS-CoV-2 once it infects an individual. Hence, these swabs are likely to yield adequate quantities of viral particles from very early, asymptomatic disease. Indeed, these are the patients who need to be detected as early as possible, not only to prevent aerosol generated cross-infection in the dental clinic, but also ‘silent’ viral transmission in the community – for they are still asymptomatic. The sensitivity and the specificity of the newly developed NAATS are so good that saliva samples are thought to be adequate for a number of test kits currently in circulation.
Rapid antigen tests
Rapid antigen tests detect specific viral proteins (not the RNA) of SARS-CoV-2, such as the S or spike protein, M or membrane proteins and E or envelope protein (Figure 1). These surface antigens of the virus are copious in either symptomatic or asymptomatic patient samples, such as a nasal or nasopharyngeal swabs or saliva, during viral replication. Most of the current rapid antigen tests detect basic levels of the antigen/s providing only a qualitative ‘yes’ or ‘no’ response, similar to a pregnancy test, and do not quantify the viral load in the sample. Currently there are over 100 of these tests, but only a handful are useful for POC testing in a dental clinic.
Another noteworthy point for both the molecular tests and the antigen tests described above is that a positive test is indicative only, and needs to be confirmed with a more robust laboratory test.
POC test technology
Although PCR technology has been the backbone of diagnostic tests so far, newer methods such as lateral flow testing and LAMP technology, which do not require temperature changes for the process, are gaining popularity in POC tests. Additionally, cutting edge technologies such as holography and artificial intelligence5 are likely to be new genres that will be used in future for POC testing.
The technology underpinning the current POC diagnostics and their pros and cons are discussed below.
Rapid antigen tests
Rapid antigen tests detect easy-to-find surface markers on the outside of the virus and avoid extraction and amplification steps. Researchers or clinicians collect samples from easy-to-reach areas (such as the nasal passage) where the virus tends to replicate the most.
Advantages: can detect active production of viral proteins, fairly rapid tests (minutes to results). RNA extraction and amplification steps are not needed as in PCR tests.
Disadvantages: the test may yield false negatives if viral protein production is low, or if there is not enough virus replication in the sampled area. Hence confirmatory tests are required.
Molecular tests or nucleic acid amplification tests (NAATs)
rRT-qPCR identifies and quantifies the presence of SARS-CoV-2 nucleic acids in a sample through the process of detection, amplification, and output measurement.
Advantages: this test has extremely high specificity and could be quickly modified to detect new viral variants to fit its new iterations. Many (virtually hundreds) of samples can be run at once, and it detects low copy numbers of viral RNA, hence it is extremely sensitive (low limit of detection).
Disadvantages: requires trained personnel and special equipment, and the test may take 1–3 hours. Hence, not ideal for the clinic situation where quick sample throughput is necessary, not only for identification of viral carriers, but also for efficient functionality of busy clinics.
Here the rapid amplification of viral RNA is coupled with a colour- or light-based readout. The foremost advantage of this method, as opposed to the standard PCR-based technology, is that the test can be performed at a single (isothermal) temperature.
Advantages: extremely rapid with results in seconds to minutes with the test performed at a single temperature 60–65°C. Extremely high sensitivity to defined SARS-CoV-2 sequences. Ideal for POC diagnostics.
Disadvantages: Provides only a qualitative ‘yes’ or ‘no’ response and viral quantification is not possible.
Recombinase polymerase amplification (RPA)
Basically, these tests detect RNA sequences through exact matches of an enzyme called recombinase and then amplify the specific viral genes.
Advantages: requires a single temperature only, as in LAMP tests. Extremely rapid with result in seconds/minutes. Has high sensitivity and specificity for defined SARS-CoV-2 sequences.
Disadvantages: Provides only a qualitative ‘yes’ or ‘no’ response, and viral quantification is not possible.
CRISPR-based diagnostics
As per the principles of clustered regularly interspaced short palindromic repeats (CRISPR), this method uses highly specific targeting and cleaving action of CRISPR-Cas systems to locate and cut a specific part of the SARS-CoV-2 RNA genome sequence. The cleaving action results in a visual signal that indicates the presence of the virus.
Advantages: extremely rapid with result in seconds/minutes. Has high sensitivity and specificity for defined SARS-CoV-sequences
Disadvantages: depending on the specific kit system, the samples may need to undergo RNA extraction before the test can be run, adding another 1–2 hours, depending on lab capacity. The technology is still very complex for clinics.
Cutting edge technologies evaluated for POC diagnostics
Apart from molecular technology, such as PCR and LAMP-based tests, newer, more cutting-edge technologies are being evaluated for rapid testing of COVID-19. One example of this is the use of holography and artificial intelligence (AI)-based methods that are in the developmental stages. With these tests, the results of a saliva sample for instance, could be delivered within 60 seconds. A UK-invented POC test is currently being trialled for mass use in airports and public spaces for rapid detection of infection in individuals (Table 1 and Figure 2).
Other technologies under development include the use of biological sensors, or biosensors, such as electrochemical sensors, field-effect transistor (FET)-based biosensors, magnetic biosensors, immunosensors, enzyme-based sensors and DNA biosensors.7 These analytical systems comprise a transducer and an immobilized biological component. The biological component recognizes a target biomarker in the sample and the transducer converts the corresponding biological signal into an electrical signal.8 Biosensors have been evaluated for detection of infectious disease such as MERS-CoV9 and influenza.10
Examples of currently used POC diagnostic tests for COVID-19 (Table 1)
Five examples of brand-name POC tests for COVID-19 currently in use in different jurisdictions are given with some of their specifications in Table 1.
This test relies on isothermal nucleic acid amplification, targeting a unique region of the RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2. As mentioned above, a constant temperature, between 60 and 65°C, using two to three sets of primers and a polymerase with high strand displacement activity, is used, avoiding the need for thermal cycling. To achieve good specificity, four different primers are used to amplify six distinct regions of the target gene.
This test is a combination of RT-PCR and lateral flow immunoassay.13 It targets the N gene of SARS-CoV-2 from nasal and throat samples. As in a pregnancy test, the results are simple to read, with colour lines indicating positivity/negativity. The reported analytical sensitivity is 200 viral copies/mL.
This is a rapid, portable assay that delivers results to a mobile phone in less than 25 minutes. Similar to Abbott's test, Cue's test also uses isothermal amplification on nasal swabs, but it detects the SARS-CoV-2 N gene. Additionally, Cue's disposable POC test cartridge forms a connected diagnostic platform with a mobile phone that enables a patient to have convenient access to their health information.
Virolens artificial intelligence test system15
The Virolens system uses a portable machine that creates a microscopic holographic image to detect the virus in saliva samples in 20 seconds. Developed in UK, the system uses a digital camera attached to a microscope, which then runs data through a computer that can identify the virus. The device has been trialled at Heathrow Airport, with satisfactory preliminary results.
Future perspectives
It is generally recognized that the COVID-19 pandemic will be with us at least for the next few years, and is most likely to end up as an endemic disease, as in the case of the Ebola infection in some parts of Africa that raises its ugly head sporadically. Although the latter disease is endemic only in Africa, only time will tell whether COVID-19 will also be endemic, and if so, in which regions of the world. A likely future scenario is that COVID-19 could be virtually eradicated in the West and some Asian countries, such as China, due to the development of herd immunity through mass vaccination programmes. Nevertheless, pockets of COVID-19 infection are likely to smoulder in less-developed countries for many reasons, including vaccine deficiency, and above all, the emergence of new viral variants and the so-called ‘escape mutants’ that emerge during viral replication in unvaccinated cohorts. These variants, in turn, raise the spectre of new waves of infection throughout the world.
Such a grim scenario is bound to impact the clinical dental practice, particularly in the UK with its cosmopolitan population mix. Hence, it will be critical that practitioners ensure that the patients they are handling on a daily basis do not have ‘viral reservoirs’ that may lead to new bouts of infection. One way to obviate such an outcome is to perform POC diagnostic tests for the SARS-CoV-2 antigens for all clinic attendees using simple, inexpensive and reliable tests that do not require training and are easily deployable in clinics and outpatient settings.
Almost all of the current POC systems yield qualitative, yes/no data, but not quantitative data for the viral load in the processed samples. Yet, in the longer term, it is also important to develop systems that can provide quantitative diagnoses, in order to understand disease progression after symptom onset. Newer POC diagnosis systems are also currently in development for both qualitative and quantitative data yields within seconds. These will certainly allay the anxieties of clinicians and patients alike, and hopefully assist in delivery of safe and wholesome clinical dental care in a post-COVID-19 world.
