One of healthcare’s largest and most rapidly expanding market segments, point-of-care (POC) and at-home rapid diagnostic testing, has been a hot topic in the medical world since COVID-19 was first classed a pandemic. Spurred by sudden dire need and fed by the cooperation of regulatory agencies worldwide, diagnostic technology has advanced in a few short years what it may not have in decades otherwise, leaving us now with innovative new platforms and future applications with the potential to revolutionize healthcare overall. Here, we explore the most popular and emerging diagnostic testing methods and discuss their value as applied to POC and at-home settings.
Antigen tests are immunoassays that indicate current infection status by detecting the presence of specific viral antigens. Collected simply with a nasal or throat swab, specimens are placed into the assay’s extraction buffer, then exposed to a reagent that reveals proteins identified as part of the target virus. Familiar to most in the form of over-the-counter COVID-19 test kits, antigen tests have accounted for the vast majority of rapid diagnostic tests until recent history, used most commonly in diagnosing respiratory pathogens such as COVID, influenza, and respiratory syncytial virus or RSV. Because of this, they are best suited to identifying individuals at or near peak infection.
Requiring no expensive or complex equipment or training to complete, antigen testing is generally faster and less costly than other detection methods, with accessibility that has made it an obvious choice for at-home diagnostics. Yet, the technology contains a fatal flaw that has pushed the development of alternative methods: its relatively low accuracy compared to other methods. Because of the considerable lag between infection and detectability, antigen tests performed too far from the peak of infection can and often will return negative results, even as individuals may be showing symptoms and are actively contagious. Meanwhile, false positives can occur when other viruses are present, improper collection techniques are used, or other bodily substances produced during infection interfere with test results.
Though antigen testing is undoubtedly an invaluable diagnostic tool, there remains the need for methods that allow for earlier and more reliable detection both in and outside medical facilities.
Molecular Diagnostics: NAATs
A type of test with varying methods, Nucleic Acid Amplification Tests, or NAATs, are viral diagnostic tests that detect the genetic material of viruses within patient samples by amplifying nucleic acids. More specifically, NAATs identify the RNA sequence comprising a particular virus’s genetic material and force replication of that material until it can be detected, making them able to identify even minimal viral loads. With various amplification methods available, NAATs are reliable, highly sensitive diagnostic tests unlikely to return false negative results.
Since the onset of COVID-19, the FDA has authorized the emergency use of many NAAT methods and technologies. The most common methods of amplification used in NAATs include:
- Reverse Transcription Polymerase Chain Reaction (RT-PCR)
- Isothermal amplification, including:
- Nicking Endonuclease Amplification Reaction (NEAR)
- Transcription Mediated Amplification (TMA)
- Loop-mediated Isothermal Amplification (LAMP)
- Helicase-Dependent Amplification (HDA)
- Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Diagnostic
- Strand Displacement Amplification (SDA)
Authorized for use in a wide range of settings from laboratories to non-healthcare locations, NAATs vary significantly in sensitivity and timeliness, with some performed at or near the site of sample collection and providing results within minutes, the same as other rapid testing methods. Still, laboratory-based NAATs will generally be more sensitive than those performed at home or a point of care.
We will focus on molecular diagnostic applications utilizing RT-PCR, LAMP, and CRISPR.
RT-PCR and LAMP
Each test that allows for the detection of viruses in both symptomatic and asymptomatic patients, PCR and LAMP utilize nasal swabs much the same as antigen tests.
One of the most widely used methods in molecular biology for almost thirty years, PCR testing was the first established method of COVID-19 detection and the standard by which subsequent methods have since been judged. Within this method, RNA from a patient sample is isolated to remove proteins, fats, and other molecules before being converted to DNA and amplified via a process known as reverse transcription, or RT. This requires enzymes to undergo thermocycling, a process in which they are repeatedly cycled through specific temperatures. During this, fluorescent markers added to the amplified DNA will produce light to be read by a detector to determine test results, with positive results being recorded once that light’s intensity has passed a particular threshold. Many cycles may be required to reach the appropriate fluorescence threshold, the exact number of which is indicative of a patient sample’s viral load.
A relatively newer testing method, LAMP is widely considered to be more efficient and cost-effective than PCR testing. While both target the viral RNA of a sample to extract and amplify, LAMP amplification is performed isothermally, at around 60-65°C, rather than necessitating multiple temperature cycles. This eliminates the need for expensive thermal cycling equipment and reduces the time and complexity of the process required to return a result.
Of the two similar but distinct methods, RT-PCR is the gold standard in diagnostic testing.
The modern genome-editing technology at the center of much progress and debate, CRISPR, known in full as Clustered Regularly Interspaced Short Palindromic Repeats, is based upon a process occurring naturally in nature by which bacteria protect themselves from viruses. Within this process, a cutting protein called Cas is guided by an RNA molecule to its matching DNA sequence originating from the invading virus. Once at its target, the Cas protein becomes more or less a pair of molecular scissors which penetrate the bacterium and cut apart the invading virus, effectively killing it. In 2012, researchers successfully extracted the CRISPR protein from bacteria. They reprogrammed the guide RNA to target any RNA sequence possible, creating, in essence, a tool for editing the genome of any species.
CRISPR’s application in Molecular Diagnostics, however, has little to do with genome editing; rather, scientists have recently discovered methods of leveraging the technology to perform nucleic acid testing rapidly, accurately, and inexpensively with single base-pair specificity– all without the need for complex laboratory equipment.
Diagnostic methods based on CRISPR rely on identifying a particular nucleic acid sequence associated with a pathogen or disease, then cleaving it to produce a readable signal either in the form of a fluorescent reporter or electric signal that occurs upon target binding. Several CRISPR-based diagnostic platforms exist, each utilizing a different Cas protein. These include:
- Cas9 platforms: Including the FLASH-NGS platform and the CRISPR-CHIP system, these target and cleave DNA via the Cas9 protein.
- FLASH-NGS combines Cas and next-generation sequencing, or NGS, technologies for precise identification of a pathogen, with the Cas9 system cleaving target nucleic acid sequences into fragments for NGS. Applications include identifying antibacterial-resistant strains such as MRSA and VRE.
- The CRISPR-Chip system combines dCas9 with a graphene transistor film to create an electrical signal upon target binding, producing a digital readout within fifteen minutes or less. This platform has been used to detect the deletion of two exons associated with muscular dystrophy.
- Cas12a platforms: Within the DETECTR and AIOD-CRISPR (all-in-one dual) platforms, when Cas12a detects and cleaves the target DNA sequence, it also cleaves ssDNA linked to a molecule producing a fluorescent signal. The DETECTR (DNA Endonuclease Targeted CRISPR Tans Reporter) system has been employed in the detection of human papillomavirus as well as SARS-CoV-2.
- Cas13a: The SHERLOCK system uses Cas13a to detect RNA molecules associated with the target virus. Once bound to the target ssRNA, Cas13a releases a fluorescent signal through collateral cleavage of an ssRNA fluorescent reporter. SHERLOCK has been deployed to detect Zika, Dengue, West Nile, and yellow fever.
Though each has its competitive strengths and weaknesses, the AIOD-CRISPR and DETECTR methods are generally considered better diagnostic methods than others when comparing the time taken and cost associated with each test and their proficiency in detecting SARS-CoV-2 in clinical samples. As they continue to advance and evolve, these CRISPR-based methods will likely facilitate new point-of-care applications in the following generation of novel diagnostics.
CRISPR vs. RT-PCR
Even compared to gold-standard RT-PCR testing, CRISPR-based systems have several significant advantages, including their ability to target the nucleic acid sequences of different pathogens with single base specificity. For example, where Cas9 and Cas12 proteins target DNA, Cas13 targets RNA. This is in contrast to PCR’s generated primers, which amplify target sequences but leave potential for off-target effects and non-specific amplification.
Diagnostic applications link target sequence binding with a readout, such as color changes in lateral flow assays or fluorescence. In the latter’s case, researchers have leveraged the indiscriminate cleaving properties of Cas12 and Cas13 to produce a fluorescent signal enabling detection. By including nucleic acid reporters that fluoresce when cleaved collaterally upon target binding by the Cas protein, detection methods can directly link cleavage of the target sequences to a fluorescent signal.
Further, CRISPR-based molecular diagnostics are well-suited to POC testing in low-resource settings; unlike PCR methods, they require no complex lab or clinic setup and no thermocycling, instead using only simple reagents.
Yet, these detection methods have their weaknesses, the most notable being that they are slightly less sensitive than the more widely used RT-PCR tests. To spur clinical adoption, significant advancements must be made toward improving test sensitivity and simplifying the workflow involved in these methods. Still, CRISPR-based detection systems are poised to become the next generation of POC diagnostic testing platforms, marrying the sensitivity and specificity of RT-PCR testing with the ease and convenience of rapid test kits while remaining accessible and cost-effective.
Today, the advancement of both at-home and POC diagnostic solutions has allowed for a higher standard of patient care with improved safety and more satisfying outcomes while reducing healthcare spending and easing the burden of resource usage. Patients are now more aware of their power and responsibility in monitoring their health. They also take a much greater interest in them, with consumers now having more options to track and monitor health status and symptoms than ever. Moreover, early detection and diagnosis are now recognized as key contributors to positive patient outcomes across conditions, while accessibility and equitability are more emphasized in healthcare than ever.
With the rapid innovation of POC and at-home diagnostic applications, we are now at the precipice of a radical shift in how healthcare is viewed, sought, and received– one in which next-generation diagnostics will be at the forefront of providing access to effective, affordable medical care for a wide variety of common conditions.
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