key: cord-0999460-98helrto authors: Peto, L.; Rodger, G.; Carter, D. P.; Osman, K. L.; Yavuz, M.; Johnson, K.; Raza, M.; Parker, M. D.; Wyles, M. D.; Andersson, M.; Justice, A.; Vaughan, A.; Hoosdally, S.; Stoesser, N.; Matthews, P. C.; Eyre, D. W.; Peto, T. E.; Carroll, M. W.; de Silva, T. I.; Crook, D. W.; Evans, C. M.; Pullan, S. T. title: Diagnosis of SARS-CoV-2 infection with LamPORE, a high-throughput platform combining loop-mediated isothermal amplification and nanopore sequencing date: 2020-09-25 journal: nan DOI: 10.1101/2020.09.18.20195370 sha: 75255088a52f30e5f06d2380f8b9272073f21780 doc_id: 999460 cord_uid: 98helrto LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA that combines loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and from stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 7-10 genome copies/microlitre of extracted RNA. This is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9-99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0-100.0%]). Overall, 1.4% (7/514 [0.5-2.9]) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494 [94.8-98.1]). This indicates that LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing. LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA that combines loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and from stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 7-10 genome copies/µl of extracted RNA. This is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09. 18.20195370 doi: medRxiv preprint Kingdom testing program more than seven months after the SARS-CoV-2 pandemic was declared a public health emergency of international concern by the WHO. Further expansion of testing to include screening of asymptomatic individuals, which may be needed to prevent SARS-CoV-2 circulation, would require a significant further increase in testing capacity 1,2 . In the UK, clinical laboratories have struggled to expand conventional RT-PCR workflows to meet the demand for SARS-CoV-2 testing, and many have explored alternative methods that would be more scalable or allow near-patient use 3, 4 . At the Oxford University Hospitals NHS Foundation Trust (OUH) and Sheffield Teaching Hospitals NHS Foundation Trust (STH), we evaluated LamPORE, a novel diagnostic platform for SARS-CoV-2 that combines loopmediated isothermal amplification (LAMP) with nanopore sequencing 5 . During sample preparation a unique combination of DNA barcodes is incorporated into the LAMP products from each specimen so that these can be pooled into a single sequencing run. The protocol currently allows up to 96 samples to be analysed on one flow cell, potentially allowing thousands of samples to be analysed per day on a single instrument. The workflow involves a 40-minute amplification, followed by a library preparation and a 60-minute sequencing run, generating results in a comparable time to RT-PCR when starting with extracted RNA. As well as molecular barcoding, using sequencing to detect the outcome of the LAMP reaction offers other advantages compared with simpler LAMP assays that detect the presence of DNA synthesis by measurement of pH, turbidity, or fluorescent dyes. Sequenced reads from a specific target will contain sequences not present in the primers, avoiding false positives caused by non-specific amplification 6 . Conversely, reads confidently assigned to . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint SARS-CoV-2 target can indicate a true positive even if present at relatively low levels, potentially improving the low sensitivity seen in several LAMP assays compared to RT-PCR 7 . LamPORE uses standard Oxford Nanopore Technologies (ONT) flow cells compatible with several sequencing instruments, including the portable MinION device, and high-throughput GridION and PromethION platforms, so could potentially be used both for mobile and centralised testing. In this evaluation we aim to compare the performance of LamPORE, which is awaiting regulatory approval, with RT-PCR on extracted RNA from respiratory specimens. Initially, we use spiked samples to determine the analytical limit of detection of the assay. We then use stored clinical samples to determine the assay's diagnostic sensitivity, specificity and reproducibility. The evaluation was conducted across three sites: OUH, STH and the Public Health England National Infection Service at Porton Down (PHE Porton Down). LamPORE is described in detail in James, et al. 5 , and was performed identically at each site using a GridION instrument with operators blinded to sample identity. The assay takes 20µl RNA input into a single multiplex reaction targeting three regions of the SARS-CoV-2 genome; ORF1a, envelope and nucleocapsid genes, plus human β-actin mRNA as a control of sampling adequacy and assay performance. LamPORE sample preparation uses a 96-well . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint plate format, with each sample having one of eight LAMP Forward Inner Primer (FIP) barcodes and one of 12 transposase (rapid) barcodes added before pooling. In these experiments a single LAMP barcode (FIP7) was not used, as it had previously been associated with lower β-actin read counts and was awaiting replacement (unpublished data). As a result, plates contained up to 80 samples, plus two no-template controls and two positive controls consisting of synthetic SARS-CoV-2 RNA (Twist Bioscience). We used the LamPORE protocol dated 1 st July 2020 (version 1, revision 4), the full text of which is available in the supplementary material. Briefly, this consists of adding sample RNA to LAMP master mix and primers, then incubating at 65-80˚C in a thermocycler for 40 minutes, during which time amplification occurs and the LAMP primer barcodes are incorporated into concatemers containing the target sequence. Following this, a second set of barcodes are incorporated using a rapid transposase-based method and samples are pooled into a single sequencing library. The pooled library has a bead-based cleanup, then is added to a MinION flow cell and sequenced for 60 minutes, after which a report is generated automatically within seconds for each barcode set. Unlike RT-qPCR, LamPORE is not designed to be a quantitative assay, as measurement only occurs after amplification is complete. The number of reads assigned to each target is used to generate a report as follows: Invalid: <50 classified reads in total detected from SARS-CoV-2 and β-actin targets is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint Spiked samples were prepared and analysed at PHE Porton Down to establish the limits of detection of LamPORE. Aliquots of pooled volunteer saliva were used for spiking experiments, which were confirmed SARS-CoV-2 negative by RT-PCR. These were spiked with cultured SARS-CoV-2 (Victoria/01/202026 passaged twice in Vero/hSLAM cells) and serially diluted with the remaining material to create a dilution series of positive samples. From each spiked sample a 140µl aliquot was inactivated by addition to 560µl buffer AVL (Qiagen), incubated at ambient temperature for 10 minutes, then added to 560µl 100% molecular grade ethanol. The entire inactivated volume was then extracted manually using the QiaAMP Viral RNA mini kit (Qiagen), with RNA elution into 50µl of nuclease-free water. For the quantitation of SARS-CoV-2 by RT-PCR, 5µl of RNA extract was used in each of duplicate reactions using the CDC NS1 assay 8 Testing of stored clinical samples was performed at OUH and STH. All samples were nose and/or throat swabs collected into viral transport media during routine clinical care and stored at -80˚C. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint Sample selection SARS-CoV-2 positive samples: At OUH sequentially available positive samples were chosen without reference to RT-PCR cycle threshold (Ct) value. These were collected from March-April 2020, during which time the PHE RdRp RT-PCR assay was in use 9 , and testing was mostly restricted to hospitalised patients and symptomatic staff. At STH a stratified random sample of specimens collected from April-May 2020 were selected based on their initial SARS-CoV-2 E gene Ct value, with 50% <30 and 50% ≥30. During this collection period, testing at STH was also largely restricted to hospitalised patients and symptomatic staff, using an in-house assay based on the Corman et al. protocol 10, 11 . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. For samples originating from STH, an in-house RT-PCR assay based on Corman et al. 10, 11 provided the comparator, run on an Applied Biosystem 7500 Real Time PCR system. The is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint assay takes 6µl RNA input into a multiplex reaction targeting SARS-CoV-2 E and RdRp genes, plus human RNAse P as an internal control 13 To assess the reproducibility of the assay, LamPORE replicates were performed on aliquots of the same RNA extract, thawed just prior to analysis. Results from the first replicate were used to report overall diagnostic sensitivity and specificity. To ensure that RT-PCR and LamPORE results were comparable between OUH and STH, a subset of SARS-CoV-2 positive and negative samples were exchanged between sites, with both LamPORE and comparator RT-PCR repeated. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint Results were analysed using R version 3.5.0, with exact binomial confidence intervals calculated for proportions. The initial LamPORE replicates were used to derive estimates of sensitivity and specificity, with second replicates used to estimate LamPORE reproducibility. The full dataset is available in the supplementary material. Using spiked samples spiked with cultured virus, LamPORE had a limit of detection of 7-10 SARS-CoV-2 genome copies/µl of extracted RNA or 140-200 copies per 20µl reaction, detecting 24/25 (96%) samples in this range (table 1). With the RNA extraction protocol used, this would correspond to a concentration of 2,500-3,600 SARS-CoV-2 genome copies/ml of sample. Although LamPORE did not consistently detect spiked samples at concentrations below this, it was positive in 15/21 (71%) samples at the highest dilution tested, 3-4 genome copies/µl of extracted RNA or 50-70 copies per 20µl reaction, equivalent to 900-1,250 copies/ml of sample. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint Diagnostic performance of LamPORE was assessed using 514 stored nose and throat swabs, 400 from OUH and 114 from STH (details in table S1). Sixty cross-site replicates demonstrated good correlation between RT-PCR Ct values for E gene targets at both sites despite different assays being used at OUH and STH, so this was used as the reference Ct Negative Inconclusive Invalid Total Positive 226 2 0 1 229 Negative 1 278 3 3 285 Total 227 280 3 4 514 Table 2 Clinical diagnostic performance of LamPORE versus RT-PCR . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint targets ( figure 1) . This showed that the pre-specified cut-off of ≥50 for a positive result was optimal, with any cut-off in the range of 25-182 producing a maximal Youden index (sensitivity+specificity-1) of 0.988. As the rate at which reads are detected becomes roughly constant within a few minutes of sequencing, the effect of a sequencing run longer or shorter than 60 minutes can be inferred. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. Inconclusive Invalid Total Positive 222 3 2 0 227 Negative 1 255 2 3 261 Inconclusive 0 2 0 0 2 Invalid 1 2 0 1 4 Total 224 262 4 4 494 Table 4 Reproducibility of LamPORE on aliquots of the same RNA extract. In this evaluation we found that LamPORE had a high diagnostic sensitivity (99.1%) compared to reference RT-PCR in our clinical sample set, consistent with initial development work 5 . The limit of detection of LamPORE, at 7-10 genome copies/µl of extracted RNA, was somewhat higher than the 2 copies/µl achievable with high-performance RT-PCR 14 , but this did not correspond to a significant loss of diagnostic sensitivity in the clinical samples. Our spiking experiments used an extraction in which RNA from 140µl transport medium was eluted in 50µl, which is typical of commonly used protocols. However, commercially . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint available extraction methods can use input volumes of 1ml or more of transport medium, potentially meaning that LamPORE with an input volume of 1ml could have the same limit of detection as RT-PCR with an input volume of 140-200µl. This assumes minimal increase in assay inhibition at higher input volumes, which is currently being evaluated. LamPORE also showed high diagnostic specificity (99.6%) and reproducibility (96.8%), both within and across sites, supporting its practical use for high-throughput testing in a low-prevalence population. Although no clinical metadata are available for the samples used in this evaluation, they will mainly have been derived from patients with acute symptomatic infection, often requiring admission to hospital, as testing was mainly limited to this group during the first wave of infection. The distribution of Ct values may be higher in a population with more mild or asymptomatic infection, and would be markedly higher among those who remain RT-PCR positive weeks after recovering from acute infection 15, 16 . Our data suggest that LamPORE is most likely to miss weak positive samples with Ct values above 35, so could have had lower diagnostic sensitivity if tested in such groups. However, this may not be a significant practical disadvantage, as although weak positives have some value for contact tracing they are likely to come from individuals with low infectious potential 17 . Our evaluation has several limitations. It was conducted after the first wave of COVID-19 in the UK, when there were few incident cases, so we were unable to prospectively collect samples and instead relied on frozen transport media, which could differ from fresh material. Positives were defined by a positive RT-PCR at the time of initial sample collection and by repeat positive RT-PCR simultaneously with LamPORE, but although RT-PCR is used . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint as a reference test for SARS-CoV-2, there are many reports of its suboptimal sensitivity in clinical infection 18 . This early evaluation of LamPORE compared its performance against RT-PCR using extracted RNA, as this is the standard material used for detection of SARS-CoV-2. However, the requirement for viral inactivation and RNA extraction in a clinical laboratory produces bottlenecks that mitigate the potential benefit of LamPORE for high-throughput or mobile testing. LAMP reactions are reported to be more robust than RT-PCR to inhibitors present in clinical samples so may have superior performance with extraction-free protocols 19, 20 . This could greatly streamline the workflow, but further evaluation is required. We also did not evaluate how the throughput and turnaround time of LamPORE would compare to RT-PCR during routine use in a clinical laboratory or centralised testing centre. Using LamPORE for high-throughput testing of tens or hundreds of thousands of samples per day would be dependent on an streamlined workflow, including automated sample handling and integration with laboratory information management systems. In conclusion, we show that LamPORE on extracted RNA offers a promising method of highthroughput SARS-CoV-2 testing, and could be of much broader use if shown to work with extraction-free methods of sample preparation and automated workflows. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint We are grateful to all the clinical microbiology/virology staff at OUH and STH who helped to process the specimens used in this evaluation, and to Dr Kevin Bewley, PHE Porton Down, for providing the cultured virus. The process for collection of the donated saliva was approved by the PHE Research Ethics and Governance Group. The protocol for the use of stored clinical samples at OUH and STH was reviewed by the Joint Research Office of OUH and the University of Oxford (our Institutional Review Board), and it was determined that the activity constituted service evaluation and service development. As such, it required neither sponsorship nor research ethics review. Materials for the evaluation were supplied by Oxford Nanopore Technologies, but all experiments and analyses were conducted independently by the investigators. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted September 25, 2020. . https://doi.org/10.1101/2020.09.18.20195370 doi: medRxiv preprint Comparison of molecular testing strategies for COVID-19 control: a mathematical modelling study Weekly COVID-19 testing with household quarantine and contact tracing is feasible and would probably end the epidemic A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 within nasopharyngeal and oropharyngeal swabs at Hampshire Hospitals NHS Foundation Trust A handheld point-of-care system for rapid detection of SARS-CoV-2 in under 20 minutes LamPORE: rapid, accurate and highly scalable molecular screening for SARS-CoV-2 infection, based on nanopore sequencing LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples CDC 2019 Novel Coronavirus (nCoV) Real-Time RT-PCR Diagnostic Panel -Instructions for Use. fda.gov Available at Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Improved sensitivity using a dual target, E and RdRp assay for the diagnosis of SARS-CoV-2 infection: Experience at a large NHS Foundation Trust in the UK Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay SARS-CoV-2 molecular assay evaluation: results -FIND. SARS-CoV-2 molecular assay evaluation: results -FIND Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19 A Systematic Review of the Clinical Utility of Cycle Threshold Values in the Context of COVID-19 Viral cultures for COVID-19 infectivity assessment. Systematic review False Negative Tests for SARS-CoV-2 Infection -Challenges and Implications Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances Technique for quantitative detection of specific DNA sequences using