During the early phase of the coronavirus disease 2019 (COVID-19) pandemic, design, development, validation, verification and implementation of diagnostic tests were actively addressed by a large number of diagnostic test manufacturers. Hundreds of molecular tests and immunoassays were rapidly developed, albeit many still await clinical validation and formal approval. In this Review, we summarize the crucial role of diagnostic tests during the first global wave of COVID-19. We explore the technical and implementation problems encountered during this early phase in the pandemic, and try to define future directions for the progressive and better use of (syndromic) diagnostics during a possible resurgence of COVID-19 in future global waves or regional outbreaks. Continuous global improvement in diagnostic test preparedness is essential for more rapid detection of patients, possibly at the point of care, and for optimized prevention and treatment, in both industrialized countries and low-resource settings.
You are watching: What are the three factors influencing laboratory diagnostics?
During a pandemic there are multiple concurrent clinical priorities, including the need to understand the pathophysiology of the disease, optimized patient care and prevention of future infections1. The detection and characterization of the etiological agent or its immunological consequences in the host are the necessary starting points2. Being able to define the pathogen, biologically and genetically, and whether it is inducing (protective) immunity are key in the development of protective and curative protocols against future persisting disease. The current diagnostic procedures are twofold. First there is the direct detection of (parts of) the virus. This can be done by culture of the virus, detection of one or more of its proteins and, the method used most frequently during the present pandemic, direct detection of nucleic acids or detection via amplification of nucleic acids. The latter are what are currently called ‘molecular tests’. Immunological tests detect the consequences of infection by the virus in the host. This is most frequently focused on the detection of virus-specific antibodies, whereas some specialized laboratories may also be capable of defining the cellular immune response. Here we will mostly focus on the nucleic amplification tests, with illustrations of how immune tests may complement molecular tests in several cases.
Diagnostics can be used in various manners, the so-called use cases. These include triage of symptomatic individuals in an epidemic or endemic setting, triage of at-risk presymptomatic and symptomatic individuals in endemic settings, confirmatory testing, diagnosis of symptomatic individuals in endemic or epidemic settings, differential diagnosis in endemic or epidemic settings, testing of patients with previous exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; the cause of the coronavirus disease 2019 (COVID-19) pandemic), surveillance at sites of previous or potential outbreaks and environmental monitoring (Foundation for Innovative New Diagnostics (FIND)). The use case determines the way in which diagnostic tests are used optimally3.
The ongoing COVID-19 pandemic has underpinned the central position of diagnostic testing in outbreak control4. Ending the pandemic involves the accurate application of diagnostic testing in high volumes and the rapid use of the results to help implement the appropriate therapy and prevent further spread. The value of integrated diagnostics in the management of the current COVID-19 wave and possible future COVID-19 waves is high, especially for the molecular detection of the virus, and for the qualification and quantification of the immunological host response5. The rapid implementation of COVID-19 tests requires critical assessment and adequate ‘jumping’ of the initial hurdles during the developmental and regulatory process. Test design, validation and verification, emergency use approval and the manufacturing of test kits in (very) high numbers are just a few examples of such obstacles. From the perspective of a routine-diagnostic microbiology laboratory, the setting up of high-throughput diagnostic pipelines, the logistics involved and the optimization of pragmatic use of test results were encountered as important problems during the first wave of the ongoing COVID-19 pandemic. Ultimately, optimized diagnostic tools will provide guidance in the development of therapeutics and vaccines (Fig. 1). Diagnostic lessons learnt during the first wave of the COVID-19 pandemic should be used to help prepare for the next wave, which is anticipated by many.
A schematic overview of the key innovation drivers, technologies, institutes and partners needed for the development of new diagnostic tests, drugs and/or vaccines. The boxes on the left identify some of the important medical, scientific and industrial deliverables where an interconnected approach covering each steps from fundamental research to commercialization is needed. Translational medicine relies on the interconnection of multidisciplinary teams of life scientists able to translate basic scientific discoveries into changes in clinical practice supported by expertise from engineering, law and financial sciences. The most important facilitators are indicated in each box. The column on the right lists the innovation drivers. Current innovation drivers combine the ability to process large amounts of data and to facilitate access to biological material through easy access to a biobank. The intense collaboration between academia and industry, with detailed sharing of research goals and directions during the coronavirus disease 2019 (COVID-19) pandemic, through the identification of an optimal collaborative approach capitalizing on the strengths of both made possible the rapid development of new diagnostic tests, drugs and possible vaccines against COVID-19.
In this Review we address early COVID-19 test design and the design-, development-, production- and distribution-associated hurdles. We discuss the importance of quality control and options for mass production as well as the practical issues around broad and rapid implementation of entirely new tests that have not undergone classic evaluation and validation. We also estimate the effect of new-generation COVID-19 tests on laboratory medicine practice, the need for new approaches towards biobanking and the economic consequences of the pandemic. Of note, we focus on molecular assays, with limited presentation and explanation of serological tests.
SARS-CoV-2 is an RNA virus, and thus all available RNA detection formats can potentially be applied to detect the virus6. For adaption towards the more frequently used diagnostic DNA detection formats, the viral genome needs to be transcribed into a DNA complement by reverse transcriptase. Currently, the preferred SARS-CoV-2 test is DNA amplification by PCR, and the real-time versions of such tests were among the earliest available. Such tests were previously developed during the emergence of SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), and therefore a PCR-based testing approach for SARS-CoV-2 was an obvious route to take7. Moreover, monitoring the host response is important in identifying individuals who have already been infected with SARS-CoV-2 as well as for assessing future vaccine efficacy. For that purpose, again similarly to tests previously developed for SARS-CoV and MERS-CoV, a broad variety of tests detecting specific SARS-CoV-2 antigens and antibodies were developed. Over the past months, all currently available technologies have been exploited to rapidly develop highly sensitive and highly specific detection and characterization assays for SARS-CoV-2. In this section we briefly discuss these test formats, but we will not consider functional tests that assess virus inactivation or the therapeutic effect of cellular immune responses1. Such assays are mostly limited to highly specialized laboratories and do not yet have a major impact on current global health-care practice.
Diagnostic tests developed and their application
Direct diagnostic testing to detect active SARS-CoV-2 infections mostly involves reverse transcriptase real-time PCR (rtPCR), although different molecular technologies, such as CRISPR-mediated detection or loop-mediated isothermal amplification, have also been applied8,9,10,11,12. Operation and application of these molecular tests is in keeping with those for previously developed tests that detect infectious agents13.
Moreover, rapid antigen detection tests have also been developed to detect active infection, although a limited number of such tests are available14,15. However, in comparison with rtPCR, rapid antigen detection tests lack sensitivity, and owing to the increased risk of false-negative results, they are considered as an adjunct to rtPCR tests16,17. Olfactory tests using electronic ‘noses’ or even dogs have also been presented, but these tests are not yet directly applied in patient care18,19.
Antibody testing can have a mostly complementary role to rtPCR tests in the diagnosis of COVID-19, at approximately 10 days or more after the onset of symptoms, in assessing past infections and defining the dynamics of the individual humoral responses in individual patients or in patient cohorts undergoing certain forms of treatment20,21. Immune-based assays, such as lateral flow assays, are usually designed for detecting human IgA, IgM and/or IgG antibodies or virus antigens22,23. Targets for the tests have been identified by comparative screening for genomic regions that have a low mutation frequency to avoid primer and antibody mismatches, and enhance test quality and stability24. Hundreds of such diagnostic tests have now been developed (Supplementary Table 1), and technical reviews of their comparative performance assessment have been published recently25,26,27,28,29,30,31,32. Very recently, the Journal of Clinical Microbiology dedicated nearly an entire issue to COVID-19 testing33.
Data are available on more than 240 Emergency Use Authorization (EUA)-level COVID-19 diagnostic tests (as of 5 September 2020), and the number of commercially manufactured COVID-19 molecular tests and the number of commercially manufactured immunoassays are approximately equivalent. FIND is or has been assessing more than 800 diagnostic assays, more than 250 of which are so-called rapid tests taking less than 30 minutes to generate a result. The use of immunoassays at the point of care (POC) remains to be universally accepted as part of the postrestriction COVID-19 control strategy34. It is important to note that all novel tests urgently need useful clinical cut-off values to help enhance their medical value35. At present, negative results in either of these test types do not completely rule out current or past infections owing to possible false-negative results36,37. Whether COVID-19 tests need to be quantitative or qualitative is subject to continued debate38. Quantitative test results may be a prerequisite for the choice of COVID-19 treatment strategy, for treatment follow-up or for the support of vaccine trials.
Another important aspect is surveillance: the rapid and continuous detection efforts aimed at early recognition, isolation and treatment of those infected with the virus29. When an infection has been diagnosed, usually on the basis of a combination of clinical parameters (for example, fever, sore throat or loss of smell and taste) and a direct COVID-19 test, search and control policies will be initiated for the detection of those people who were in recent direct contact with the patient and who will then be subjected to confinement and/or COVID-19 testing. For adequate surveillance and tracing, both regionally and globally epidemiological virus typing is important. Next-generation nucleotide sequencing is used to define polymorphisms and to define interrelatedness between virus strains39,40. Such approaches have been instrumental in defining the global spread of the virus and may also help to define virus variants with different biological capacities (for example, ease of spread, pathogenicity and tissue tropism). Metagenomic next-generation nucleotide sequencing can also be used diagnostically for virus detection in patients41 or in environmental samples (such as wastewater)42.
Considerations for the development, production and distribution of diagnostic COVID-19 tests
The superficial sketch of test design provided in the previous subsection represents only the first steps in test development. Initial design, experimental small-scale laboratory validation and, if at all possible, clinical evaluation using high-quality and patient specimens are followed by industrial scale-up. The test format needs to be compatible with large-scale production, which in the case of COVID-19 was possible for tests that were supported on pre-existing platforms43. Any test that was developed rapidly but was not applicable on an existing instrument had a substantial disadvantage to reach the market44. Possible exceptions are tests that are presented in a platform-agnostic layout and that can be combined with any type of instrument already available to laboratory-based diagnosticians45.
Moreover, instruments and tests need to be abundantly available at a local and global scale to ensure scale-up of clinical testing. The preavailability of a platform also enables the broad geographical spread of the test. If an installed base of instruments already exists, then new tests in the already existing format can be rapidly and reliably added to the testing repertoire of a laboratory. In such cases, assay transport and storage are two remaining hurdles, and test distribution in itself may be an important obstacle. The shelf life of a test, the temperature tolerance of the test components and simple characteristics such as the size and weight of the package are all important parameters in the perceived ease of distribution. Once the instrument and assays are available to users, instrument availability and human expertise may still be limiting factors in high-throughput test application46. Finally, there needs to be a balance between the laboratory test capacity and the number of requests for tests, and the fluctuation in the number of tests requested and changes in priority test recommendations pose additional problems. It is clear that the entire global population cannot be tested (repeatedly) at the same time, and choices need to be made to prioritize patient groups or groups at increased risk of being infected (for example, health-care workers)47. When these groups have been identified, sampling processes (and their logistics) need to be designed and implemented. Simplicity of sampling and homogeneity of the sample itself are important parameters to consider, and other sources, such as saliva, have been considered as alternative specimens for COVID-19 testing48. Testing of sample pools has been suggested as a solution to minimize test costs while maintaining test sensitivity and specificity, specifically in settings where the incidence of infection is low49. Pooling of samples may generally induce practical pretesting burden and may lower traceability, and thus sample pooling should perhaps be restricted to times of reagent shortage. The jury is still out on whether pooling is diagnostically robust and cost-effective, with conflicting reports having been published50,51,52. In addition, the current consensus is that individual laboratories should perform validation studies before embarking on large-scale pooling strategies52.
Many ‘diagnostic streets’ or drive-through test facilities were established as soon as COVID-19 tests became available, and many laboratories opted for externalization of testing (using tents, dedicated buildings and separation between sample taking and actual testing)53. Finally, there is a continuous need for means of rapid and reliable result dissemination, an issue that is covered in privacy loopholes but also the need to use test results beyond the privacy of an individual patient.
See more: Café Con Aroma De Mujer: Conoce Por Qué Carmen Villalobos Y Su Hijo Mayor
Test results are key in surveillance and outbreak management and should be used to inform infection prevention measures. Diagnostic tests need careful consideration and validation before being launched. This is often underestimated and underappreciated by scientists and the community, and involves processes that are costly and time-consuming.