At-Home Molecular Diagnostics: Just around the Corner or Years Away?

Expansion of rapid testing for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), especially as the Omicron variant of concern began circulating in late 2021, was a key public health effort for COVID-19 (1). This strategy involved rapidly expanding the use of over the counter (OTC) or home testing for SARS-CoV-2, predominantly using lateral flow immunoassay rapid antigen tests. Dozens of rapid antigen tests have now received Emergency Use Authorization (EUA) by the US FDA; and elements of various executive orders as part of the public health emergency facilitated their use by requiring insurance companies to pay for them. In addition, the federal government provided free tests distributed by the United States Postal Service. In contrast to the dozens of lateral flow rapid antigen tests receiving EUA approval, only 2 OTC molecular tests (Cue Health and Lucira home testing kits) for SARS-CoV-2 have received FDA EUA as of August 1, 2023. One of these tests is no longer available after the company producing it declared bankruptcy. So that leaves us with the question, what is the future for OTC molecular diagnostic testing for infectious disease? Rapid antigen testing, primarily using lateral flow immunoassays, has been used for decades and is well understood. The primary limitation to rapid antigen testing using lateral flow immunoassay is limited analytical and clinical sensitivity. Independent studies of rapid antigen tests for SARS-CoV-2 demonstrated 35%–96% sensitivity depending on the population tested, comparator or reference test, and other factors (2–4). Molecular tests, specifically nucleic acid amplification tests (NAAT), are considered the gold standard for viral detection. Reverse transcription quantitative PCR (RT–qPCR) is the most common NAAT method used for detection of viral pathogens including SARS-CoV-2, and in general has a lower limit of detection (increased analytical sensitivity) compared to other NAAT methods. However, RT–qPCR is a difficult technology to incorporate into OTC methods due to the need to cycle between room temperature and high temperature to amplify nucleic acids. Isothermal amplification is more amenable to OTC use, and both EUA approved OTC molecular SARS-CoV-2 tests used this method. However, current isothermal amplification methods have a higher limit of detection (decreased analytical sensitivity) compared to RT–qPCR. Whether this translates into a decreased clinical sensitivity when used on either asymptomatic or symptomatic patient populations is not clear. There are several factors that favor the widespread adoption of OTC (at-home or self-testing) NAAT testing for infectious disease. First and foremost is the increased desire and acceptance of at-home testing from patients. A 2021 survey of members of the public on the East and West coasts of the United States found that between 2020 and 2021 attitudes toward at-home testing shifted. More people surveyed felt that point-of-care testing could improve their health and provide faster treatment compared to the same questions asked in 2020. There were overall favorable attitudes toward 13 elements or survey questions regarding the use and value of point-of-care tests. The only significant concern was related to insurance not covering the cost of testing (5). The most important test characteristic identified by end users in both years was test accuracy (5). Health systems are also looking for ways to implement population and rural health initiatives that allow disease diagnosis and management without clinic or hospital visits. During the pandemic, both at-home testing and remote or virtual care became more common and accepted. Well before the COVID-19 pandemic health systems were developing population health approaches to manage chronic and acute disease while minimizing patient and healthcare provider time and resources. Technologies for remote or virtual physical examination, while currently available only within healthcare environments, may logically extend to the home. This would allow history, physical examination, and diagnosis to be conducted without a visit to a clinic or hospital. This would be a great benefit to patients in rural areas that must travel great distances to receive care, and also facilitate population health efforts to prevent hospital readmission. Time to treatment could be significantly reduced by OTC especially for patients in rural or remote areas. Today, this would likely be accomplished by use of antigen tests relying on lateral flow immunoassay technology. However, the vast amount of information disseminated during COVID-19 to raise awareness of the limitations of antigen testing and reinforce NAAT testing as superior in various settings has primed the pump among both members of the public and healthcare providers to be open to at-home NAAT testing. The knowledge that diagnosis or screening may be accomplished by a single NAAT test in comparison to serial antigen testing may create a desire to promote at-home NAAT testing over less costly antigen tests. The same concepts will apply for at-home tests for additional respiratory pathogens and sexually transmitted infections. The lack of insurance coverage for at-home testing and increased cost of NAAT (compared to antigen) methods are the obvious barriers to widespread adoption of at-home NAAT testing for infectious diseases. While an antigen test may cost $10–20, NAAT are likely to be in the $40–60 range. While the COVID-19 pandemic increased both awareness and acceptance of at-home testing, executive orders signed as part of the COVID-19 public health emergency resulted in most people having access to antigen tests without out-of-pocket cost. The move back to accepting out-of-pocket costs for testing that would otherwise be covered in part or in full by insurance benefits may be hard for many to accept. OTC can also impede the gathering of public health information such as disease transmission rates and prevent sequencing of samples to track the spread of new variants. Beyond the obvious limitations of cost and insurance coverage, performance specifications of current NAAT technology for at-home testing also present a barrier to widespread adoption. While increased analytical sensitivity and avoiding the need for serial testing are potential upsides to this technology, false positives and invalid test results remain a significant concern. A study of one of the 2 isothermal amplification tests approved for OTC use (the Cue Health device), conducted at a drive-through swabbing site using point-of-care testing personnel, found that the negative percentage agreement (specificity) of the test compared to central laboratory RT–qPCR was 98.4% (n = 243 RT–qPCR negative patients). The same study found that 8.6% of tests that performed point of care resulted in initial invalid results (6). Our experience (unpublished observations) with the same test in a rapid response laboratory environment demonstrated a 0.7% rate of presumed false positives for the first 6455 tests performed. The invalid rate varied monthly over 15 months of use between 5% and 15%. A single published study of the other isothermal amplification test approved for OTC use (Lucira) noted 100% negative agreement (specificity) with a central laboratory RT–qPCR test, but used only 100 negative samples in the evaluation (7). While no EUA SARS-CoV-2 OTC methods using RT–qPCR are currently available, there is one single use, disposable device using RT–qPCR methodology with EUA for point-of-care healthcare use (Visby Medical). Both studies of this device found that the limit of detection for the RT–qPCR device was lower than that reported for isothermal amplification methods (8, 9). One study found no false positives but among only 17 RT–qPCR negative samples (8), with 1 of 78 results invalid on initial testing. The other study of this disposable RT–qPCR device found 98.6% (69 of 70) negative percentage agreement in samples testing RT–qPCR negative by a central laboratory test, with 2 out of 100 initial invalid results (9). Both studies were retrospective benchtop studies using residual samples rather than prospective studies performed at the point of care. While a 0.5%–2% false-positive rate may seem like overall good test performance, the utility of OTC antigen testing during COVID-19 was driven in large part by the extremely low rate of false-positive results with these methods. In a large study of twice weekly screening of asymptomatic employees (with RT–qPCR confirmation of any positives), the rate of false-positive screening tests (among 903 408 tests) was only 0.05%. Despite this extremely low rate of false positives, only 42% of positive antigen tests were confirmed by positive RT–qPCR (10). This demonstrates the math problem behind population screening, and highlights the need for extremely low false-positive rates for any technology used outside traditional healthcare settings where pretest probability and reasons for testing cannot be controlled. While antigen tests can malfunction and not return results, when used according to instructions invalid results are very rare. By contrast, the invalid rate for OTC (and NAAT technologies that might be adopted to OTC use) is much higher and could frustrate individuals paying out of pocket for testing on the basis of convenience and speed of results. While the promise of at-home molecular testing for infectious disease is much more promising today than it was just a couple of years ago, significant technical barriers remain before it can become commonplace. Per-test costs must come down to better approximate the cost of antigen tests, and the false-positive and invalid rates for these technologies must be improved to avoid frustration on the part of individuals choosing at-home testing for its convenience and speed. The good news is that these are technical limitations that can be overcome if the demand for OTC infectious disease testing remains strong. Nonstandard Abbreviations: SARS-CoV-2, severe acute respiratory syndrome-coronavirus-2; OTC, over the counter; EUA, Emergency Use Authorization; NAAT, nucleic acid amplification tests; RT–qPCR, reverse transcription quantitative PCR.