key: cord-0956701-vukh2ka5
authors: Davies, Emma; Farooq, Hamzah Z.; Brown, Benjamin; Tilston, Peter; McEwan, Ashley; Birtles, Andrew; O’Hara, Robert; Ahmad, Shazaad; Machin, Nicholas; Hesketh, Louise; Guiver, Malcolm
title: An Overview of SARS-CoV-2 Molecular Diagnostics in Europe
date: 2022-03-08
journal: Clin Lab Med
DOI: 10.1016/j.cll.2022.02.005
sha: dfc092f10dc0c2ffa143534eb67b8af128b87db9
doc_id: 956701
cord_uid: vukh2ka5

The COVID-19 pandemic has led to rapid development of a plethora of molecular diagnostic assays with RT-PCR at the forefront. In this review, we will discuss the history and utility of SARS-CoV-2 molecular diagnostics and the associated current and future regulatory process in Europe. We will assess the performance characteristics of a range of the most common SARS-CoV-2 molecular tests currently utilized in Europe with focus on as rapid molecular platforms, stand-alone RT-PCR kits, the role of low-throughput and high-throughput end-to-end testing platforms and the rapidly evolving field of SARS-CoV-2 variant of concern identification.

• The COVID-19 pandemic has led not only to an influx of new molecular diagnostics but also a drive 22

to modify existing technologies to allow testing of thousands of patients daily over a variety of 23 settings. 24

• The need for rapid turn-around times for SARS-CoV-2 testing for public health actions and patient 25 care has led to the necessity for synchronously utilizing multiple assays and platforms. 26

• Testing solutions exist for any scale of SARS-CoV-2 testing strategy. 27

• Overall SARS-CoV-2 molecular diagnostics appear to perform well however market saturation has 28 left peer-reviewed real-world data lacking. 29

• With these new developments, diagnostic testing regulations for SARS-CoV-2 are paramount to 30 aid manufacturers in achieving assay performance and for laboratories to use as a tool alongside 31 local verification to determine the suitability of an assays and platforms for use in future 32 epidemics. 33 34 Synopsis 35

The COVID-19 pandemic has led to rapid development of a plethora of molecular diagnostic assays 36 with RT-PCR at the forefront. In this review, we will discuss the history and utility of SARS-CoV-2 37 molecular diagnostics and the associated current and future regulatory process in Europe. We will 38 assess the performance characteristics of a range of the most common SARS-CoV-2 molecular tests 39 currently utilized in Europe with focus on as rapid molecular platforms, stand-alone RT-PCR kits, the 40

An emerging viral pneumonia of unknown aetiology was detected in patients from several healthcare 45 facilities in the city of Wuhan in China on the 30th of December 2019. 1 

identified initially termed "2019-nCoV" and designated as Severe Acute Respiratory Syndrome 47 antigen tests being freely available and actively encouraged in the asymptomatic general population 142 and in laboratory NAAT being used for more sensitive screening of all hospital admissions including 143 day case and those with symptoms consistent with COVID- 19. 35,36 The ECDC recommends the use of 144 NAAT for all symptomatic cases but also acknowledges the role for rapid antigen tests in population 145 screening. 33, 34 The use of sensitive molecular diagnostic assays are important to the control of 146 transmission. If SARS-CoV-2 infection is allowed to spread unchecked the emergence of novel variants 147 is likely to be enhanced as mutations in key genes continue to accumulate as part of the natural error 148 prone replication of RNA viruses. As mutations accumulate it is not only possible that they can lead to 149 increased pathogenicity or vaccine escape but that they may also lead to detection failures in well-150 established diagnostic assays. It is now recommended that the presence of SARS-CoV-2 in clinical 151 samples is determined through the detection of at least 2 distinct targets to mitigate for this risk. The 152 observation of the ThermoFisher S gene PCR assay failure in the UK for the B.1.1. 7 Alpha variant, which 153 would have led to significant numbers of false negative tests being reported if this was being utilized 154 as a single target assay, highlights the importance of a multi-target approach. 37 155

In order to achieve testing on such an immense scale testing a diverse approach has been required 156 with laboratories often utilizing multiple assays and platforms in unison. The following is by no means laboratories. They are simple to use, provide accurate results within 1-2hrs, have minimal hands-on-165 time and permit on-demand testing of urgent specimens. 166

An overview of the main sample-to-answer platforms is presented in Table 4 . These single-use tests 167 often automate nucleic acid extraction, purification, amplification, detection and interpretation of 168 results. All the platforms presented are internally controlled yet only three utilise an endogenous 169 sample control, which monitors for an adequately taken sample and sample degradation. 170

Independent studies evaluating performance of rapid RT-PCR tests have varied with few head-to-head 171 comparisons although evaluations of these platforms are more extensively published due to their 172 widespread use in non-specialist laboratories. 173

Unlike other applications the rapid testing platforms exhibit significant variation in the technologies 174 employed. Cepheid Xpert Xpress, QiaStatDx and VitaPCR SARS-CoV-2 rely on classic multiplex RT-PCR 175

Novodiag COVID-19 38 is unique in its utilisation of qPCR and microarray technology for the detection 176 of SARS-CoV-2. GenomEra SARS-CoV-2 39 and GenomEra SARS-CoV-2 with Flu A/B + RSV 40 Evaluations often used small sample sets, due to limited availability of reagents and utilised various 201 SARS-CoV-2 reference controls, making LOD comparisons difficult. Reported LODs varied from 100 202 copies/ml for Xpert Xpress SARS-CoV-2 to 3000 genome copy equivalents for the Aries SARS-CoV-2 53 203

Several platforms fail to achieve the MHRA TPP "acceptable" LOD criteria of 1000 copies/ml; 204 GenomEra SARS-CoV-2, Flu A/B + RSV at 2,857 copies/mL, 40 Novodiag COVID-19 38 at 1,815 copies/mL 205 when using collection devices other than the provided mNAT medium 54 and both the GenMark ePlex 206 SARS-CoV-2 42 and the QIAstat-Dx Respiratory SARS-CoV-2 Panel 46 at 1000 copies/ml. 207

The main limitations of the rapid sample-to-answer platforms include their high cost per test and low 208 sample throughput. In addition, despite their low complexity, rapid platforms are not infallible, they 209 are sensitive molecular tests that can be compromised without meticulous sample processing and 210 good laboratory practice. Notably BioFire and ePlex platforms do not output Ct values meaning there 211 is no indication of SARS-CoV-2 viral burden which can be of interest to the clinician as higher viral loads 212 have been associated with increased SARS-CoV-2 mortality 55 . 213 214 J o u r n a l P r e -p r o o f

One of the biggest barriers to implementation of SARS-CoV-2 testing in non-specialist laboratories 216 early in the pandemic was the availability of the correct equipment to enable rapid introduction of 217 testing. The solution to this problem for many manufacturers was the rapid introduction to market of 218 stand-alone assays encompassing kits which include the reagents necessary for reverse-transcription 219 PCR, including controls, but which are not tied to a specific extraction or PCR platform. They offer 220 flexibility over more "closed" systems as they can potentially be run on existing instrumentation, 221 precluding the requirement for purchasing new and often expensive equipment. Use of such reagents 222 requires more extensive validation than end-to-end systems and the onus on providing this validation, 223 including sample preparation and the compatibility of any instrumentation with a particular kit, will 224 fall on the individual laboratory. Some suppliers provide details of compatible platforms, but many do 225 not and it is this lack of data which has allowed many substandard kits to enter the market. Over two 226 hundred CE-marked manual RT-PCR kits are listed on the COVID-19 In Vitro Diagnostic Medical 227 Devices database 28 , a selection of which are shown in Table 5 along with some of their main 228 attributes. 18,56-73 229 Kit formats are broadly similar and include minimal necessary reagents (primer/probe mixes, 230 controls). Reagents may be provided either lyophilised or "wet", most commonly in tubes, but also as 231 8-well strips. Although earlier kits relied on a single viral gene target, these have now been largely 232 superseded by dual or triple target assays which focus on some combination of the E, N, S and Orf1a 233 genes. While this has made the assays more robust in dealing with the emergence of novel SARS-CoV-234 2 variants, it has also complicated the interpretation of results when some gene targets fail to amplify. 235

In addition, most kits supply an internal control (IC) which may be either endogenous (eg RNase 236 P) 18 J o u r n a l P r e -p r o o f

The number of tests per kit ranges from 48-4800 allowing for a wide range of throughputs although 240 this will also depend on the number of wells required per sample and whether they are being tested 241 in 96 or 384 well format. Many assays exploiting real-time PCR can typically use up to 4 different 242 fluorescent reporter dyes, including the internal control, but others are not so comprehensively 243 multiplexed and require two or even three wells for each sample. At least one kit (Menarini) 74 uses 244 melt curve analysis in preference to hydrolysis probes, negating the requirement for multiple 245 fluorescent reporter dyes. Although not shown in Table 5 , many SARS-CoV-2 kits are also formulated 246 as multiplexes with other respiratory viruses, most commonly influenza and respiratory syncytial virus 247 eg Altona, 75 Viasure, 76 ThermoFisher 77 . This will usually require the addition of an extra well for each 248 sample and/or the use of a single dye for multiple gene targets of the same virus. The actual 249 throughput for these assays will depend heavily on the extraction and PCR equipment chosen for use 250 and the level of automation. Use of an automated end-to-end system like the Roche FLOW could 251 produce in excess of 1000 results in a 24-hour period from experience in our local laboratory. 252

Due to the pressure to manufacture diagnostic kits rapidly as the pandemic took hold, much of the 253 technical and clinical validation data employed minimal data sets. Unlike for the rapid platforms which 254 are in widespread use peer reviewed literature is sparse for many stand-alone kits and in some cases 255 completely absent. For those referenced assays in Table 5 N gene assay which has been shown to be highly sensitive. 16, 17, 80 The Amplidiag COVID-19 assay was 286 highly sensitive showing >98% agreement when compared directly to Cobas 6800 SARS-CoV-2. All 287 J o u r n a l P r e -p r o o f other assessed platforms as shown in Table 6 were also found to have acceptable sensitivity and 288 specificity of >96% based on manufacturers data only. 79,81-85 289

All assessed platforms were shown to have good analytical sensitivity as outlined in Table 6 with the 290 exception of AUS Diagnostics SARS-CoV-2, Influenza and RSV which has an LOD on 2150-4325 291 copies/ml. 83 Real world testing of the Amplidiag COVID-19 also highlighted a failure to detect an EQA 292 sample at 3300 copies/ml suggesting the manufacturer published LOD of 313 copies/ml may not be 293 reliable. 84 Local verification of the manufacturers claims is important prior to introduction of any test 294 into routine use to ensure discrepancies such as this are detected. 295

The expected 24hr throughput for these systems is modest and these systems are likely to be sited in 296 laboratories which do not undertake 24/7 working meaning their full potential cannot be met. Whilst 297 this may be the case these automated solutions can offer easy to use solutions for laboratories with 298 limited molecular experience. This has been important in providing the ability to decrease time to 299 result over sending samples to specialist reference laboratories for testing which in turn can reduce 300 transmission risk particularly in healthcare settings. 301 302

Several high throughput platforms have been introduced for the detection of SARS-CoV-2 RNA offering 304 end to end automated testing of samples from nucleic acid extraction through to amplification and 305 detection. The introduction of high throughput screening platforms into laboratories can improve 306 laboratory efficiency and turnaround times while reducing staff hands on time 86 , facilitating a 307 substantial increase in testing capacity. The main high throughput testing platforms and associated 308 assays are listed in Table 7 . All are RT-PCR based assays except the Hologic Aptima SARS-CoV-2 assay 309 which uses transcription-mediated amplification (TMA). All assays listed uses a minimum of 2 different 310 SARS-CoV-2 targets to reduce the risk of false negatives due to primer/probe mismatches caused by 311 J o u r n a l P r e -p r o o f sequence variability 87 . Multiple comparisons between the high throughput platforms and standard 312 RT-PCR demonstrate a high level of diagnostic performance. The Panther Fusion had an overall 313 agreement of 96.4% when compared to the Roche Cobas 6800 SARS-CoV-2 assay 88 with a similar 314 finding in a separate study 89 . An agreement of 98.3% was found when comparing the Cobas to the 315 Abbott Alinity M SARS-CoV-2 AMP 90 and in a three way comparison between these platforms and the 316 Panther Fusion the overall agreement was 99.7% 91 . When the TMA based Aptima assay was compared 317 to both the Panther Fusion and rapid low throughput BioFire Defense COVID-19 test it produced a 318 positive percent agreement of 98.7% compared to the consensus and a 100% agreement for negative 319 results 92 . 320

Comparing analytical sensitivity is difficult due to differences in methods between studies, but 321 generally all have high analytical sensitivities with LODs of 200 copies/ml or below, as collated from 322 several studies and listed in Table 1 . The TMA based Aptima assay was shown to have a lower LOD 323 when compared to standard RT-PCR 13 , though when compared directly against the Roche Cobas and 324

Abbott m2000 the Cobas test had the lowest LOD 85 , a similar finding when the Cobas was directly 325 compared to the Abbott m2000 and Panther Fusion 93 . 326

All systems offer a throughput of 1000 samples or more in a 24-hour period. The highest throughput 327 systems are the Roche Cobas 8800 system and the recently introduced Thermofisher Amplitude 328 running the Taqpath COVID-19 assay, which claims a very high throughput of 8000 samples from a 329 single platform over 24 hours. The Taqpath COVID-19 assay has been evaluated as a standard RT-PCR 62 330 assay but no published data exists for the diagnostic performance of the complete Amplitude system. 331

Assays for these high throughput platforms are being updated to include additional respiratory targets 332 to meet the predicted increases in respiratory syncytial virus (RSV) and seasonal influenza infections permit on-demand testing of urgent specimens which is pertinent for non-COVID patient care. 404

• High-throughput platforms improve laboratory efficiency and turnaround times whist 405 reducing staff hands-on time. This leads to an increase in testing capacity of diagnostic 406 laboratories to help meet the clinical demand throughout pandemics. 407

• The use of SNP genotyping assays for the detection of SARS-CoV-2 VOCs can be an effective 408 Table 7 : An overview of high-throughput molecular diagnostic platforms for SARS-CoV-2 J o u r n a l P r e -p r o o f

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