As the epidemic outbreaks of novel respiratory tract infectious diseases SARS, MERS, and the ongoing pandemic of covid-19 have shown, the development of accurate diagnostic tests play an important role in outbreak management.^1,2

While serological tests are inexpensive, practical to use, and provide rapid point-of-care tools for screening and diagnosis, they require further confirmatory tests. In addition to this, there is typically a lag period of 5 to 14 days for virus-specific IgM antibodies to appear in the body after infection which is a consideration for this form of testing. 

By comparison, molecular assays targeting the viral genome are specific and can be quickly developed. This makes them ideal for the rapid detection of new pathogens. SARS-CoV-2 is an RNA virus and therefore most molecular assays apply reverse transcription quantitative PCR (RT-qPCR), although alternative amplification chemistries are also being used. The unprecedented pace with which the scientific community has responded to and collaborated together on covid-19³ has resulted in the rapid development and application of molecular assays developed in-house and, subsequently, commercial diagnostic solutions.^4,5 Without these in-house developed molecular assays it is difficult to envisage how the world could have responded so quickly to detect and manage patients, and also begin to identify the clinical and epidemiological characteristics of the covid-19 pandemic. 

These rapidly developed assays developed in-house play a fundamental role in the identification of new diseases which are not catered for by the available commercial diagnostic platforms that currently underpin health service laboratories. They also serve as a reference for confirmation of results from other assays and as a backup when the supply of commercial tests is disrupted, for example, the current reagent shortages reported for the platforms that enable SARS-CoV-2 detection. Furthermore, they are also important for when pricing structures for commercial platforms may render them unaffordable in low resource settings.

PCR provides the ability to design, validate and roll out assays targeting any genetic sequence of interest at very short notice. For these reasons, PCR tests are currently playing a crucial role in identifying SARS-CoV-2 infected patients. However, some key questions remain on the accuracy of these diagnostic tests for identifying and confirming new cases, guiding clinical management, infection control, disease surveillance, patient discharge, and follow up. In-house developed molecular assays differ from commercially available tests because they are often applied using ‘research use only’ reagents without the quality assurance required for the mass production and use of commercial assays. Thus, optimal diagnostic performance and traceability, to account for discrepant results between laboratories, is more challenging for in-house developed molecular assays.

A range of covid-19 molecular assays are currently being used globally, by multiple laboratories, of varying quality and expertise. Fundamental to RT-qPCR is the selection of primers and molecular probes, the combination of which are referred to as panels, and these make the major contribution to the assay characteristics. The WHO highlights seven different assay panels⁶ and selection of which to use is complicated by the availability of an increasing number (currently over 500) commercial tests which are being marketed.⁷

While the pace of test adoption reflects the urgent global response to the covid-19 pandemic, it is crucial that accurate assessment of the analytical, diagnostic, and operational performance of each assay is monitored. The importance of quality systems for diagnostics cannot be overstated; a fact that is not helped in the current pandemic since the systems required (such as reference materials) to ensure test quality are challenging to implement on a timeline comparable to the assays they need to support.

Currently recommended molecular assays^4-6 detect different regions of the SARS-CoV-2 viral genome. While this can provide resilience by accounting for sequence variation between populations, it can also lead to diagnostic discrepancies associated with genomic variability or analytical performance. Often overlooked are the pre-analytical and processing steps of the recommended protocols, these include specimen sampling tools and techniques, storage and transport, extraction required prior to performing the molecular assay. Guidelines for standardisation of molecular assays developed in response to emerging infectious diseases are required. The potential impact of ignoring this is well known. When using non-standardised molecular assays for viral analysis, differences in excess of a hundred-fold are not uncommon⁸  and an artificially low signal (e.g. due to poor sample processing) can manifest as a false negative result.

While the world approaches a million daily tests for detecting SARS-CoV-2, it becomes important that national and global public health actors and regulatory bodies take forward solutions to assure quality control for covid-19 testing as well as any future pandemic. First, the quality systems which assist the rapid development and confident application of diagnostic molecular assays should be an integral part of a global emergency response planning, including access to reference materials and biobanked clinical samples for test validation. Second, standardised generic protocols and standards to assist with test quality, which could then be adapted to specific scenarios, would better prepare the world for pandemics and the diagnostic testing component of the response. The molecular standards community are already working on solutions; systems more common to clinical chemistry, which combine high accuracy measurements and well characterised reference materials, are increasingly available to support harmonisation of molecular diagnostic procedures.^9,10

Third, molecular diagnostic methods such as digital PCR and advanced sequencing technologies offer increasingly accurate and robust diagnostic approaches.^11,12 These simplify the routes to ensure traceability, evaluate test performance and increase confidence in the result providing reliable detection. Fourth, wide collaborative efforts to facilitate ring trials and proficiency testing of testing laboratories are needed, including those performing commercial assays. Finally, comparative studies, assessing the performance of various assays while taking into account high quality clinical and epidemiological data and also other reference testing modalities, such as virus culture and quantitation, should be encouraged to facilitate assay harmonisation.

The uncertainty of test reliability in the covid-19 pandemic has highlighted the imperative of standardisation in diagnostic test development, it must be part and parcel of the global response, not only for the current pandemic but also setting a precedent for novel emerging pathogens.

Jim Huggett, University of Surrey, UK.

Kathryn Harris, Great Ormond Street Hospital NHS Foundation Trust, UK, National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust , UK, and University College London, UK.

Timothy D McHugh, Professor of Medical Microbiology & Director UCL Centre for Clinical Microbiology, University College London, UK.

Jacob Moran-Gilad, Ben-Gurion University of the Negev, Israel.

Alimuddin Zumla, University College London, UK, and London Hospitals NHS Foundation Trust, UK.

Acknowledgements: Dr Jim Huggett is a principal scientist at the National Measurement Laboratory which is supported the UK National Measurement System and the European Metrology Programme for Innovation and Research (EMPIR), co-financed by the Participating States and the European Union’s Horizon 2020 research and innovation programme. Prof Jacob Moran-Gilad and Dr Kathryn Harris are a co-PIs in the EMPIR Project. Prof Sir Alimuddin Zumla and Prof Timothy D McHugh are members of the Pan-African Network on Emerging and Re-emerging Infections (PANDORA-ID-NET), funded by the European & Developing Countries Clinical Trials Partnership, supported under Horizon 2020. Prof Zumla is in receipt of a National Institutes of Health Research senior investigator award. 

Competing interests: None declared

References:

1. Memish ZA, Perlman S, Van Kerkhove MD, et al. Middle East respiratory syndrome. Lancet 2020 doi: 10.1016/S0140-6736(19)33221-0 [published Online First: 2020/03/08]
2. Zumla A, Gant V, Bates M, et al. Rapid diagnostics urgently needed for killer infections. Lancet Respir Med 2013;1(4):284-5. doi: 10.1016/S2213-2600(13)70099-7 [published Online First: 2014/01/17]
3. Ghebreyesus TA, Swaminathan S. Scientists are sprinting to outpace the novel coronavirus. Lancet 2020;395(10226):762-64. doi: 10.1016/S0140-6736(20)30420-7 [published Online First: 2020/02/28]
4. WHO. Coronavirus disease (COVID-19) technical guidance: Laboratory testing for 2019-nCoV in humans 2020 [Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance.
5. WHO. Laboratory testing for coronavirus disease (COVID-19) in suspected human cases. WHO/COVID-19/laboratory/2020.5. https://www.who.int/publications-detail/laboratory-testing-for-2019-novel-coronavirus-in-suspected-human-cases-20200117. 2020
6. WHO. Molecular assays to diagnose COVID-19. In-house developed molecular assays 2020 [Available from: https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf?sfvrsn=de3a76aa_2.
7. FIND-Diagnostics. SARS-CoV-2 diagnostic pipeline 2020 [Available from: https://www.finddx.org/covid-19/pipeline/.
8. Fryer JF, Baylis SA, Gottlieb AL, et al. Development of working reference materials for clinical virology. J Clin Virol 2008;43(4):367-71. doi: S1386-6532(08)00298-9 [pii]
9. 10.1016/j.jcv.2008.08.011 [published Online First: 2008/10/01]
10. Whale AS, Jones GM, Pavsic J, et al. Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine. Clinical chemistry 2018;64(9):1296-307. doi: 10.1373/clinchem.2017.285478 [published Online First: 2018/06/16]
11. Zook JM, Catoe D, McDaniel J, et al. Extensive sequencing of seven human genomes to characterize benchmark reference materials. Sci Data 2016;3:160025. doi: 10.1038/sdata.2016.25 [published Online First: 2016/06/09]
12. Dong L, Zhou J, Niu C, et al. Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR. medRxiv 2020
13. Hoenen T, Groseth A, Rosenke K, et al. Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool. Emerg Infect Dis 2016;22(2):331-4. doi: 10.3201/eid2202.151796 [published Online First: 2016/01/27]