key: cord-0875174-s9jr70y5 authors: Tolan, Nicole V.; Horowitz, Gary L. title: Clinical Diagnostic Point-of-Care Molecular Assays for SARS-CoV-2 date: 2022-03-04 journal: Clin Lab Med DOI: 10.1016/j.cll.2022.03.002 sha: 24b37a2f1be1cbe4b4bc28f1906c65a91c26ebbe doc_id: 875174 cord_uid: s9jr70y5 Laboratories faced many challenges throughout the COVID-19 pandemic including the demand for unprecedented volumes of molecular testing. Point-of-care (POC) SARS-CoV-2 nucleic acid amplification tests (NAATs) provided a key solution to the need for rapid turnaround time (TAT) in select patient populations and were implemented at the POC but also within laboratories to supplement traditional molecular assays. Clinical Laboratory Improvement Amendments (CLIA)-waived rapid POC SARS-CoV-2 NAATs offer the benefit of reduced educational requirements for operators and can be performed by non-laboratory trained individuals. However, it is essential that these methods are validated to ensure the manufacturer’s performance specifications are met and that they are found to be fit-for-purpose in the clinical workflows they are implemented. Laboratories faced many challenges throughout the COVID-19 pandemic including the demand for unprecedented volumes of molecular testing. Point-of-care (POC) SARS-CoV-2 nucleic acid amplification tests (NAATs) provided a key solution to the need for rapid turnaround time (TAT) in select patient populations and were implemented at the POC but also within laboratories to supplement traditional molecular assays. Clinical Laboratory Improvement Amendments (CLIA)waived rapid POC SARS-CoV-2 NAATs offer the benefit of reduced educational requirements for operators and can be performed by non-laboratory trained individuals. However, it is essential that these methods are validated to ensure the manufacturer's performance specifications are met and that they are found to be fit-for-purpose in the clinical workflows they are implemented. • Although certain states do not require formal validation of CLIA-waived POC SARS-CoV-2 NAATs, it is highly recommended that laboratories evaluate the method performance. • Even when reporting results qualitatively, it is particularly important to determine the limit of detection/analytical sensitivity using specimens with known cycle threshold (Ct) values that span the clinical range. • Additional considerations exist if Ct values are made available to treating clinicians, including the need to: • Determine assay precision (variability within sample) and • Perform method comparison (variability across methods) • Establish the clinical acceptability of the assay for differentiating very low viral copies (residual RNA) from high copies (acute infection, increasing over time) • POCT methods offer a number of advantages but implementation and regulatory compliance can be challenging. • CLIA-waived methods have reduced educational requirements for operators and can be run by non-laboratory trained staff. • While not well-suited for high throughput demands, POC SARS-CoV-2 NAATs can offer rapid TAT at the POC but also when implemented within-laboratory. • It can be particularly challenging to maintain compliance with assay and regulatory requirements at the POC, particularly with regards to: • Staying within the clinical indications of use (e.g. including testing only symptomatic individuals, and/or within a certain number of days since symptom onset) The coronavirus disease 2019 (COVID- 19) pandemic presented clinical laboratories with significant challenges in meeting the demands for molecular testing. These included: (1) the inability to implement laboratory developed nucleic acid amplification tests (NAATs) during the state of emergency, (2) managing perpetual delays in the availability of FDA emergency use authorization (EUA)-cleared assays due to supply-chain issues/reagent allocations, (3) the requirement to perform unprecedented volumes of molecular tests, and (4) Prior to the SARS-CoV-2 pandemic, most clinical laboratories were performing NAATs with conventional polymerase chain reaction (PCR) and reverse-transcriptase PCR (RT-PCR) assays. Examples include HIV and HCV viral loads, and HPV qualitative testing. These assays are FDAcleared as Clinical Laboratory Improvement Amendments (CLIA) moderately/highly complex assays or laboratory developed tests (LDTs) running on expensive laboratory-based instrumentation. They all have in common a TAT of several hours but are capable of testing large numbers of samples in that time frame. In the early stages of the pandemic, the laboratory-based SARS-CoV-2 NAATs developed by manufacturers were designed in similar fashion to these traditional molecular assays. A College of American Pathologists (CAP) SARS-CoV-2 proficiency testing (PT) survey from 2020 revealed that 93% (13/14) of assays in use at the time had analytic times that did not meet the requirements for rapid molecular testing. POC NAATs for other respiratory viruses such as influenza A/B (Flu) and respiratory syncytial virus (RSV) have been commercially available for a number of years. These platforms are capable of providing rapid TAT results albeit with low throughput, and many are also CLIA-waived, which allows them to be performed in clinical settings by non-laboratory trained individuals such as nurses and medical assistants. As the pandemic proceeded, these platforms were expanded to diagnose SARS-CoV-2 alone or as a multiplex panel, thus offering a pathway for using rapid molecular results in select patient populations. Accordingly, a significant increase was observed in the number of laboratories using rapid NAAT platforms as their primary method for SARS-CoV-2 diagnostics, from 680 in the 2020 CAP survey to 2739 labs in 2021. This is likely an underestimate given that CLIA-waived devices do not formally require participation in a proficiency testing program. There is a clear use case for many laboratories to supplement their large laboratory equipment with these single-use, cartridge-based methods within the laboratory itself. Manufacturers developed SARS-CoV-2 NAATs for systems that were already on the market, with the major examples being the Abbott ID NOW COVID-19, 1 the Cepheid GeneXpert Xpress SARS-CoV-2, 2 and the Roche LIAT. 3 These methods leveraged existing technologies, and either substituted the primer probe sets with those specific to SARS-CoV-2 or added them to the influenza/RSV sets to offer a respiratory virus panel. As detailed in Table 1 An interesting approach to providing rapid and inexpensive NAATs for SARS-CoV-2 detection at the POC has been the adaptation of reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology 4, 5 In contrast to conventional RT-PCR, which requires multiple cycles of heating and cooling (thermocycling) to amplify target RNA ( Figure 1A ), LAMP assays are isothermal. RT-LAMP significantly reduces the amplification time typically required for thermocycling of the primers that extend the DNA template ( Figure 1B ). Typical RT-LAMP assays can be completed in under an hour, and in some cases as quickly as 15 minutes. One commercial assay, the Lucira CHECK-IT COVID-19 Test Kit, 6 received FDA EUA for home use with a physician's order and then later, for over-the-counter (OTC) and direct-to-consumer (DTC) use. This method can provide positive results in as little as 11 minutes, however it has yet to be widely adopted for clinical diagnostic use due to concerns over sensitivity compared to traditional RT-PCR. 7 interpreting Ct values of SARS-CoV-2 molecular assays. 12 However, aside from the Abbott m2000, the remaining seven methods analyzed in the letter had median Ct results with relatively good agreement using the same batch of proficiency testing material, across multiple manufacturers' methods and various gene targets. Therefore it would seem reasonable to be able to use this data to reliably differentiate high vs. low Ct values in the clinical context of a given patient's presentation. However, the Ct ranges observed for patients without symptomatic disease or with low levels of virus require additional studies beyond those currently available; 12-14 the details of which are beyond the scope of this manuscript. Regardless, if a laboratory were to choose to report Ct values, the comparability across clinically reported methods must be evaluated and the results accompanied by sufficient interpretation, by way of automatic result comments or other means of appropriately interpretive comments. Beyond these components of the validation, even for qualitatively reported assays, it is particularly important to verify the manufacturer's claims of performance in terms of the analytical sensitivity and limit of detection (LOD). The traditional method of assessing analytic sensitivity of NAATs is to prepare a series of reference standard dilutions, which are then run in replicate. The conventional definition of the LOD represents the lowest concentration that can be detected at least 95% of the time (e.g., 20 of 21 replicates). Most laboratories do not have the ability to run such extensive numbers of replicates, but they can make a reasonable assessment of the LOD with fewer replicates. As shown in Figure 3A , multiple dilutions of a J o u r n a l P r e -p r o o f SARS-CoV-2 standard (5336 copies/mL, AccupPlex SARS-CoV-2 Reference Material, SeraCare, Milford, MA) confirmed that the Cepheid assay detected concentrations down to roughly 60 copies/mL. An alternative approach to determining analytic sensitivity, which is perhaps more informative, is to compare clinical specimens against a previously validated, highly-sensitive comparison method. As shown in the top panel of Figure 3B , 47 patient samples with estimated copy numbers (log copies/mL) determined using the Abbott m2000 were evaluated by the Cepheid Xpert Xpress, which in this case is the 'test' method. The LOD of the Abbott m2000 method had previously been formally confirmed using a reference standard, as described above. With this set of clinical specimens, the lowest positive concentration that the Cepheid could determine (i.e., with no false negative results) was approximately 80 copies/mL, which was roughly equivalent to the previous study using the externally purchased standard material. Examples of similar comparisons of two other SARS-CoV-2 NAATs is reflected for an LDT method ( Figure 3C ) and a traditional NAAT ( Figure 3D ). Neither of these could reliably detect concentrations as low as the Cepheid, with their cut-off limits being roughly 180 and 8500 copies/mL, respectively. In the absence of a standard curve relating Ct values to viral RNA copy numbers, many studies were reported comparing positive and negative percent agreements (PPA, NPA) with highly sensitive NAATs, the method required by the FDA EUA authorization template for molecular tests. 15,16 These reports used a wide range of comparator methods and patient populations, but they still provide useful information. Table 2 shows a compilation of the data from a metaanalysis of the ID NOW, 17 indicating that it has excellent NPA but a suboptimal PPA of approximately 70% (with 30% false negative results). An interesting aspect of the Lucira package insert is that it provided clinical performance data as a function of viral RNA concentration. 6 In two studies in community settings, the Lucira NAAT showed an overall PPA of 92% on 404 specimens from symptomatic and asymptomatic patients, including 10 specimens with very low viral RNA concentrations. Excluding these specimens, whose Ct values were >37.5, the assay was shown to be 98% accurate in comparison to high sensitivity laboratory assays. The ability to maintain a single sample type was challenging throughout the pandemic without reliable sources of collection swabs and transport media. Further, experts were concerned about the loss of sensitivity as samples deviated from the preferred NP swabs. In addition to traditional NP swabs, three major POC methods used in clinical laboratories did receive EUA for some combination of anterior nares (nasal) swabs, nasal mid-turbinate (NMT), oropharyngeal (OP), and in some cases, nasal wash/aspirate collection types. A few studies have also suggested reliable results from alternate specimen types including oral fluid, 18 stool and ocular secretions, 19,20 but outside of saliva, these are not in widespread use. In order to ensure comparability in method performance across sample types, it is essential for laboratories to separately validate alternate collection materials/conditions, with particular focus on specimens with low viral RNA copy numbers. The actual implementation of assays categorized by the FDA as waived and designed to be performed at the POC requires additional considerations beyond assay performance. Assays that are CLIA-waived have a major advantage in that they have reduced educational requirements for the testing personnel and can be performed by laboratory accessioning staff, medical assistants, nurses, and other non-technical staff. This offers the opportunity for implementing these assays within the clinical laboratory itself for triaging specimens with rapid TAT requests, but without requiring the same technologists who meet the educational requirements of moderate-and high-complexity testing. Further, while federal CLIA and state requirements apply whether performing the test in the lab or truly at the POC, the laboratory setting is much more conducive to the rigorous laboratory quality essentials as compared to the clinical settings like the emergency department, which are staffed by personnel focused on patient care. When one considers the workflows affecting the TAT of NAATs used in the laboratory setting, one needs to take into account many factors beyond the analytic time of the assay. These include the time required for: • preparing the specimen for transport to the lab • transporting the specimen, often in a batched process • receiving and/or accessioning the specimen into the laboratory information system • pre-analytical processing, which depends upon the NAAT used • resulting and transmitting the result to the ordering provider and clinical team If the analytic time is measured in hours, these other factors may represent a relatively small fraction of the overall TAT. However, when they are all combined, they lead to significant additional delays compared to implementing these rapid methods at the POC (Figure 4 ). Although POC NAATs cannot compete with traditional NAATs within the lab, in terms of providing high-throughput testing volumes on a daily basis, they can outperform these tests in providing reliable, actionable results within minutes to an hour of specimen collection (depending on the workflow). In the best case scenario, sample transport and receipt into the laboratory would be limited to one hour, there would be no batching delays and specimens would be tested as they were received, analytic times would be no longer than two hours and the instrument would be interfaced with results reporting into the electronic record without significant delay in the provider reviewing the results. With these parameters, the laboratory testing workflow could be as short as 3 hours from the time of collection ( Figure 4 ). However, this certainly is not the case for all clinical laboratories and may not even be consistent throughout the day/week ( Figure 5) . Therefore, from a purely TAT standpoint, implementing POC rapid NAATs in the clinic at the point of specimen collection, rather than the lab, reduces the TAT to less than 60 min and is mostly reliant on the analytical time of the method. POC tests can be performed by operators with limited laboratory skills as compared with the highly trained and qualified laboratory personnel required for most traditional NAATs. Indeed, the same person collecting the specimen can run the test, while wearing the same personal protective equipment. To protect staff, laboratories that perform infectious disease testing, whether it be culture or NAAT, often use biologic hoods for specimen preparation. However, for SARS-CoV-2 testing , the use of biologic hoods is not required by the EUAs covering POC NAATs. Arguably, the greatest risk of acquiring SARS-CoV-2 for staff at the POC relates more to their direct interactions with patients, and specifically specimen acquisition, as opposed to specimen processing on the assay. Thus, the personal protective equipment requirements for specimen J o u r n a l P r e -p r o o f collection are sufficient for testing personnel to guard against any potential aerosolizing steps (e.g. vortexing) of the testing procedure. We took advantage of a number of these aspects at our respective institutions. At BWH, LIATs were deployed in the microbiology laboratory, as well as in the laboratory of an affiliate urgent care clinic, separate from the main hospital. While not truly implemented at the POC, the laboratories found great value in offering this rapid, cartridge-based method to meet the TAT needs of hospital-based urgent testing (e.g. asymptomatic patients presenting to the ED, preprocedural, admit/discharge) and reduce the time for results from the off-site urgent care clinic testing symptomatic patients, that would otherwise incur long-delays in batched transport back to the main campus laboratory. This workflow allowed the laboratory to provide a TAT of approximately one hour from sample collection by reducing the analytical time to approximately 20 min on the Roche LIAT (Table 1) . At Tufts, LIATs and GeneXpert methods were deployed in the central laboratory to achieve the same goal, and the use of these methods was not restricted to just patients in the emergency room, but was expanded, as reagent supplies allowed, to any patient for whom the infectious diseases consulting physicians needed rapid results. In addition, LIATs were deployed as genuine POC devices at four individual affiliated urgent care sites, where results were reported in real-time. The total test volumes peaked at roughly 600 per week and often exceeded the volumes of rapid NAATs performed by the laboratory (approximately 250 per week), indicating that they were providing an extremely valuable service in the communities they served. As previously discussed, attention to specimen types and specimen acquisition is important for all SARS-CoV-2 NAATs. However, this deserves special emphasis for POC testing, where non- Once collected, specimen handling issues become paramount. Whether testing is performed at the POC or in a traditional laboratory, appropriate labeling of specimens is critical, but the temptation to be more lax with this requirement is far greater at the POC. In most cases, specimen swabs have to be placed in viral transport media, at which point specimen stability for testing is assured for suitable lengths of time. In some cases however, specimens need to be tested directly within an hour of collection (e.g. Abbott IDNOW). In all cases, the testing process must follow the manufacturer instructions and laboratory validated protocols to remain unmodified and CLIA-waived. Once testing is complete, it is critical not only to inform the patient's caregivers but also to enter the results into the medical record and to report them to local public health authorities. One of the advantages of POC testing is that informing the patient's caregivers is usually straightforward, especially if the testing is completed while the patient and caregivers are still present. But it is critically important that the information be accurately captured in the medical record, particularly for patient precautions, but to also ensure reporting to public health J o u r n a l P r e -p r o o f authorities. Many of the POC devices are capable of being interfaced with laboratory information systems, a feature whose importance should not be understated. The effort required to build and maintain this interface must also not be underestimated. Manual transcription of results is not as reliable and requires error-checking for accuracy, something that can be challenging to enforce at the POC, particularly when testing volumes are high. As compared to other POC methods (e.g. rapid Streptococcal antigen tests), relying on a physician's note to capture SARS-CoV-2 NAAT results severely limits the ability to interface with state reporting mechanisms, challenges the ability for local institution tracking, and managing patient precautions for the safety of other healthcare providers. Beyond the qualitative result reporting, simply as "positive" or "negative" (or "detected" or This was a niche that could be filled extremely well by POC rapid NAATs. The analytic times, almost always less than an hour, were the most attractive aspect of this strategy. One downside of these assays is throughput, which can be made mitigated by limiting testing to those patients who need it most. In our experience, the analytic sensitivity of POC NAATs is often adequate and in some cases, even better than some conventional laboratory-based RT-PCR NAATs. However, it is critical that they be validated by the laboratory rather than rely solely on manufacturer data. Although the FDA template for EUA for these POC CLIA waived assays mandated comparison to highly sensitive methods, it did not prescribe that a range of viral concentrations be included in those assessments. Because of differences in the comparison method used, in the populations tested, and in the specimen types used, the NPA and PPA might be excellent even for an assay with less J o u r n a l P r e -p r o o f than optimal analytical sensitivity. As one example, the Abbott ID NOW assay was determined by many independent investigators to have lower sensitivity than other NAATs, with differences large enough to be potentially clinically significant. Abbott ultimately updated the ID NOW package insert, indicating that tests could only be done in patients whose symptoms were compatible with COVID- 19 With respect to implementation, several aspects of CLIA waived POC rapid NAATs should be noted. The first is the reduced educational requirements for testing personnel, allowing the tests to be performed by non-technical staff within the laboratory and at the POC. As a result, workflows can be optimized, allowing for TATs of less than an hour from specimen collection. Second, especially at sites outside the laboratory, vigilance is required in maintaining adherence to specimen types and specimen handling. The third is the importance of capturing results in the medical record and transmitting them to public health authorities, requiring sufficient support and resources to implement and maintain electronic interfacing. One of the more interesting aspects of POC SARS-CoV-2 NAATs is their costs. In general, the actual cost to produce results with a POC test is far in excess of the cost to do the same test in the central laboratory. This is often related to the reagent costs and inability to leverage economies of scale like in large central laboratories, as well as incremental labor and capital equipment costs. It was our experience that the costs associated with POC SARS-CoV-2 NAATs J o u r n a l P r e -p r o o f were often comparable to their lab-based counterparts, unless one pooled specimens or developed one's own assay, which the overwhelming majority of laboratories did not do. Regardless, even if POC NAATs clearly did cost more, their beneficial impact in terms of added safety to the patient, to staff, and to other patients in hospitals and residents in community living settings arguably more than offset those costs. It is important to point out that rapid POC NAATs existed pre-pandemic. A particularly good example is for influenza testing. At a time when rapid antigen tests were not sensitive enough to reliably rule out influenza, it was strongly suggested that all negative tests done at the POC be confirmed by NAAT. Since the majority of tests were negative, this translated into excessive delays to get definitive results on most specimens with additive costs for both assays. POC NAATs for flu provided an excellent solution to the problem and enhanced patient care, providing real-time patient physician interaction and ability to prescribe anti-viral medication when indicated. Looking to the future, one wonders whether we can provide POC NAATs for other diseases, like HIV or HCV, where many patients are lost to follow-up before they can be connected to care. 22 In summary, POC SARS-CoV-2 NAATs, properly validated and implemented, represent extremely valuable resources, for POC sites as well as traditional laboratories. They should not be seen, as many POC assays are, as inferior and more expensive counterparts to moderately and highly complex traditional laboratory assays. Central laboratories can, and should, assist with validating and implementing these assays, helping to ensure they are fit for purpose. In doing so, they may find that these assays can play a vital role in addressing their own needs for J o u r n a l P r e -p r o o f reliable, appropriately sensitive, rapid turnaround time assays. POC SARS-CoV-2 NAATs represent another example of the continuum of laboratory testing, and it is vital that traditional laboratories embrace them in helping us meet our role in delivering the highest level of quality patient care. 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PR Newswire: Cision 22 Dedicated laboratory professionals supporting patient testing throughout the COVID-19 pandemic.