key: cord-0966897-55s8nnk0
authors: Ptasinska, A.; Whalley, C.; Bosworth, A.; Poxon, C.; Bryer, C.; Grippon, S.; Wise, E.; Armson, B.; Goring, A.; Cortes, N. J.; Howson, E.; Snell, G.; Forster, J.; Mattocks, C.; Frampton, S.; Anderson, R.; Cleary, D.; Parker, J.; Boukas, K.; Graham, N.; Cellura, D.; Garratt, E.; Skilton, R.; Sheldon, H.; Collins, A.; Ahmad, N.; Friar, S.; Godfrey, K.; Williams, T.; Deans, S.; Douglas, A.; Hill, S. L.; Kidd, M.; Porter, D.; Kidd, S. P.; Fowler, V.; Richter, A. G.; Beggs, A. D.
title: Diagnostic accuracy of Loop mediated isothermal amplification coupled to Nanopore sequencing for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations
date: 2020-12-16
journal: nan
DOI: 10.1101/2020.12.15.20247031
sha: dfccf7a6dc66066d2223763bae1b542c1239bda5
doc_id: 966897
cord_uid: 55s8nnk0

Introduction: Rapid, high throughput diagnostics are a valuable tool, allowing the detection of SARS-CoV-2 in populations, in order to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as reverse transcriptase quantitative PCR (RTqPCR), particularly throughout the first months of the pandemic. We investigated the use of LamPORE, where loop mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS CoV 2 in symptomatic and asymptomatic populations. Methods: In an asymptomatic prospective cohort; health care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self swabbed with nasopharyngeal swabs weekly for three weeks and supplied a saliva specimen daily. These samples were tested for SARS CoV 2 RNA using the Oxford Nanopore LamPORE system and a reference RTqPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza like illness from March 2020 to June 2020, were similarly tested from nasopharyngeal swabs. Results: In the asymptomatic cohort a total of 1200 participants supplied 23,427 samples (3,966 swab, 19,461 saliva) over a three-week period. The incidence of SARS CoV 2 was 0.95% using LamPORE. Diagnostic sensitivity and specificity was > 99.5% in both swab and saliva asymptomatic samples as compared to the reference RTqPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. Conclusions: LamPORE is a highly accurate methodology for the detection of SARS CoV 2 in both the symptomatic and asymptomatic population settings and can be used as an alternative to RTqPCR.

3 Birmingham (C17422/A25154) and the Birmingham Experimental Cancer Medicine Centre (C11497/A25127).

Role of funders: The Department of Health and Social Care gave input into the study design and coordination but had no influence on the analysis and conclusions of the paper. This report presents independent research and the views expressed in this publication are those of the authors and not necessarily those of the NHS, or the Department of Health and Social Care.

Competing interests: ADB -has received travel funding to the Oxford Nanopore Community Meeting 2019 from Oxford Nanopore. The rest of the authors declare no competing interests.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Methods: In an asymptomatic prospective cohort, for three weeks in September 2020 health care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self-swabbed with nasopharyngeal swabs weekly and supplied a saliva specimen daily.

These samples were tested for SARS-CoV-2 RNA using the Oxford Nanopore LamPORE system and a reference RT-qPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza like illness from March 2020 -June 2020, were similarly tested from nasopharyngeal swabs.

Results: In the asymptomatic cohort a total of 1200 participants supplied 23,427 samples (3, 966 swab, 19,461 saliva) over a three-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% in both swab and saliva asymptomatic samples when compared to the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%.

Conclusions: LamPORE is a highly accurate methodology for the detection of SARS-CoV-2 in both symptomatic and asymptomatic population settings and can be used as an alternative to RT-qPCR.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 16, 2020.

COVID-19, caused by an emergent novel betacoronavirus known as SARS-CoV-2;

represents a public health emergency. First identified in Wuhan, China(1) in December 2019 it has spread rapidly across the world, causing, at the time of writing, over 700,000 deaths with 19.4 million cases detected.

Rapid detection of infected cases in order to limit transmission, remains challenging as most validated methods utilise reverse transcription quantitative polymerase chain reaction (RT-qPCR) (2) targeting one or more gene targets for SARS-CoV-2. Although considered the gold standard for diagnosis, RT-qPCR is laborious can be difficult to scale up for masstesting, and competition for reagents/equipment from many laboratories may lead to widespread reagent shortages. Initially in the outbreak, labs throughout the United Kingdom utilised primers that were designed to target sequences within the RNA dependent polymerase gene (RdRp) (3), however these lacked sensitivity (4) . RT-qPCR Primer sets were introduced that targeted the envelope (E), nucleocapsid (N) and ORF1ab genes, which provided necessary increased sensitivity (5) . As all these assays depend on RT-qPCR technology, if viral gene targets are amplified singly for a sample within a series of wells, this reduces the overall capacity of RT-qPCR. Multiplexing of gene targets (6) using several fluorescent dyes within the same reaction volume increases capacity, but the majority of machines in common use can only utilise a maximum of 4-5 dyes (limiting to 4-5 gene targets) and also process between 96-384 samples simultaneously. A typical RT-qPCR reaction requires approximately 60-90 minutes to process, meaning that these are rate limiting steps, impeding the ability to expand capacity to allow rapid and widespread detection of cases.

Loop mediated isothermal amplification (LAMP) offers an alternative to RT-qPCR (8) . It utilises a strand displacement, replicating DNA polymerase utilising multiple primer sets (and specific "Loop primers" to increase specificity) to rapidly amplify DNA using a continuous temperature. This reaction typically takes 20-30 minutes which is considerably quicker than . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20247031 doi: medRxiv preprint 6 PCR. The LAMP reaction can also be coupled to a reverse transcriptase, allowing detection of RNA (RT-LAMP) (9) Nanopore sequencing allows rapid sequencing using protein nanopores embedded in a lipid membrane. Electrical current passes through the nanopores and changes in electrical conductivity occur as molecules of nucleic acid translocate through the pores. Each base produces a characteristic change in current which is detected and converted into a readable trace (10). This allows the sequencing data to be analysed in real time, and the process can be halted once sufficient data is recorded. Nanopore sequencing technology allows all the advantages of conventional next generation sequencing technology, especially the capacity to perform very high (limited only by the number of barcodes available) sample multiplexing within the same sequencing run.

LamPORE is a combination of LAMP and Nanopore sequencing, developed by Oxford Nanopore (11) that utilises barcoded LAMP reactions to generate amplicons from SARS-CoV-2 viral RNA. These can then be multiplexed via sample barcoding and pooled onto a flow cell for sequencing. This technology has a theoretical maximum capacity of 15,000 samples per GridIon Mk 1 machine (Oxford, Nanopore) per 24 hours, allowing scalability and high throughput.

Use of alternative sampling strategies, such as saliva, could theoretically increase capacity over swabbing (because of less pressure on the supply chain) and increase compliance because of the less invasive nature of sampling of saliva. There is also evidence that viral loads in saliva are high prior to symptoms and early in infection (7) .

This study aimed to assess the assay performance characteristics of the LamPORE SARS-CoV-2 Detection Assay against the gold standard RT-qPCR for SARS-CoV-2 detection in both symptomatic and asymptomatic populations from multiple independent centres. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20247031 doi: medRxiv preprint 7

The study consisted of a retrospective and prospective diagnostic accuracy study comparing the performance of LamPORE sequencing of the ORF1ab, N2 and E gene targets of SARS-CoV-2 against RT-qPCR of the ORF1ab and N1 gene targets of SARS-CoV-2 (12) .

For the prospective study 1200 health care workers at high risk of asymptomatic transmission were recruited as part of a consented National Health Service England and NHS Improvement (NHSE/I) service evaluation in September 2020. Prospective participants performed naso-pharyngeal self-swabs at day 0, 7, 14 and 21 as well as daily saliva sampling for 21 days ( Figure 1 ). Any symptoms were reported in a symptom diary. 

Sampling: For swab-based tests, participants underwent self-directed nasopharyngeal swabbing using flocked swabs containing viral transport medium. For saliva-based tests participants were instructed to dribble at least 1ml of saliva into a universal specimen container without any additive. Samples were tested immediately if returned on the day of testing or if received on Friday then stored for a maximum of 4 days at 4 o C then tested.

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The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20247031 doi: medRxiv preprint 8 Clinical material used for analytical performance:

To assess the limit of detection, a tenfold? dilution series (from 20,000 copies/ml to 0.2 copies/ml) of droplet digital polymerase chain reaction (ddPCR) quantified SARS-CoV-2 was constructed from the lysate of the Public Health England SARS-CoV-2 reference strain grown on Vero E6 cells and diluted in nuclease free water. This was tested by RT-qPCR and LamPORE in triplicate. For intra-and inter-assay precision, and reproducibility, 20,000 copies/ml of the same extracted SARS-CoV-2 RNA from the prepared dilution series was used. A panel of respiratory viruses (Zeptometrix Respiratory Panel R2, Buffalo NY 14202) was used to assess specificity of the LamPORE assay. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Sample size was determined pragmatically, based on the incidence seen in the United Kingdom at the time of the study (1%). Sample size was calculated using R code (using R . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20247031 doi: medRxiv preprint 3.6.3 (13)) from the methodology of Stark et al (14) for binary diagnostic test outcomes (a=0.05,b=0.90) setting a base sensitivity and specificity of RT-qPCR of 95% and 99% respectively. We aimed to be able to detect a change of sensitivity & specificity of 10% in LamPORE respectively giving a sample size of greater than 9,600 in the prospective cohort.

The readers of RT-qPCR and LamPORE tests were blinded to any clinical information relating to study participants. For the RT-qPCR reference assay, SARS-CoV-2 was said to be detected if the following conditions were met; amplification of the kit internal control, amplification of either the ORF1ab or N gene with a cycle threshold of Cycle threshold (C T ) < 38, detection of the positive control on the sample plate, detection of the RNA extraction control on the sample plate, and no SARS-CoV-2 specific amplification in the negative control. C T values were calculated automatically using instrument software with automatic baseline setting calculated. All curves were manually inspected by two investigators in order to check for quality and inhibition of reaction.

LamPORE: Aligned read counts were generated via the LamPORE pipeline against ORF1ab (labelled AS1), E1 and N2 genes, as well as a human ACTB gene internal control. Any unaligned reads were marked as "Undetermined". Samples were called positive if any of the SARS-CoV-2 target genes had > 50 reads/sample, indeterminate if between 20-50 reads and negative if less than 20 reads. ACTB gene counts were not used as part of the calling algorithm but were used to infer sufficient sampling.

Test results were compared using a 2x2 table and standard measures of sensitivity, specificity, positive predictive value and negative predictive value were calculated using R. If results from either the RT-qPCR or LamPORE test were missing or indeterminate then no comparison was made and the sample was removed from the analysis. Standard analyses of variability in diagnostic precision were made.

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For the prospective asymptomatic study, a total of 1200 participants who were at work and reported to be well were recruited across the four sites (Birmingham n=600, Southampton n=200, Basingstoke/Winchester n=200, Manchester n=200). There were no adverse events.

Sample flow is shown in Figure 2 .

LamPORE reliably detected SARS-CoV-2 to 20 copies/ml of sample. SARS-CoV-2 reads were detected in the 0.2 copies/ml sample but this was below the threshold for calling as positive sample in LamPORE but were not detected via RT-qPCR (Table 1, Figure 3 ).

Intra-and inter-assay precision was calculated against the ORF1ab gene. For intra-assay precision on a single day, the standard deviation of ORF1ab was 50 reads with a coefficient of variation (CV) of +/-2.3% (supplementary table 1 ). For inter-assay precision across multiple days, the standard deviation of ORF1ab was 178 reads with a CV of +/-7.8% (Supplementary table 2) .

For reproducibility for 24 replicates the standard deviation for ORF1ab gene was 128 reads with a CV of +/-3.9% (Supplementary table 3) .

For analytical specificity of the LamPORE assay, SARS-Cov-2 was not detected in any of the samples within the respiratory virus panel.

In total 23,427 samples were obtained from all participants, of which 22,401 were from the asymptomatic study and 848 were from the retrospective symptomatic cohort. Both . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20247031 doi: medRxiv preprint LamPORE (comparator assay) and RT-qPCR (reference assays) were performed on all 23,427 samples (Table 2) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20247031 doi: medRxiv preprint 13 Symptomatic cohort There was complete agreement between the RT-qPCR and LamPORE assays for 116 positive samples and 752 negative samples, for this cohort resulting in a DSe of 100%, DSp of 100%, PPV of 100% and NPV of 100%. The incidence of SARS-CoV-2 was 13.4% over the study period.

In order to understand the utility of LamPORE across the time course of infection, a single participant who was identified as the beginning of their infection, early in the study with a long time course (5 days) of positivity was studied with daily saliva sampling as per protocol ( Figure 4) . Initially a high viral load, indicative of a C T value of 19.5 was observed which increased (indicating decreasing viral load) over the five days to 23.5 and then became undetectable at day 6. Only LamPORE N2 reads were detectable at day 1, but E1 reads became detectable at day 2 and ORF1ab reads at day 4.

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We carried out a very large asymptomatic cohort study of health care workers using a novel technology, LamPORE, comparing it to a reference CE-IVD marked RT-qPCR assay. In addition, we have also carried out a large symptomatic cohort study based on "real world" samples typically found in clinical practice. We found that LamPORE has high sensitivity and specificity (>99%) in both the asymptomatic and symptomatic populations, directly comparable to RT-qPCR and therefore has comparable predictive ability across a range of use cases in varying levels of population prevalence. We studied a population with a wide range of viral loads as determined by cycle threshold, with LamPORE demonstrating good detection across the range, suggesting it has applicability across the whole spectrum of use cases.

LamPORE has the advantage that it is scalable (11) With combinatorial barcoding as has been adopted in other population level assays (15) on a larger flow cells (e.g. a Promethion flow cell), even greater sample multiplexing may be achievable. Another potential inherent advantage is the ability to multiplex gene targets allowing the detection of multiple respiratory pathogens (16) such as SARS-CoV-2, influenza and respiratory syncytial virus (RSV). It is not known what the upper limit of multiplexing of LAMP primers is, and they are considerably more complex to design than PCR primers (17) .

Given the advantages of LAMP in terms of speed of amplification (9) and sensitivity of detection, an exploration of LAMP multiplexing is urgently required. Also, the assay chemistry uses different enzymes and methodologies to PCR, meaning a diversification of supplies and therefore potentially less reagent shortages in a pandemic setting.

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The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20247031 doi: medRxiv preprint 15 A potential disadvantage of LamPORE is the differing workflow needed to prepare samples, including the LAMP step and library preparation, barcoding then sequencing. This requires more sample preparation steps than a RT-qPCR workflow. RNA extraction remains the real rate limiting step, for both LamPORE and RT-qPCR and ourselves and other groups are currently working on direct alternatives removing the need for RNA extraction.

During the testing of the asymptomatic cohort, we observed a number of false positives using LamPORE when compared to the RT-qPCR assay. There are a number of possible explanations for this observation. Firstly, LAMP amplification is more sensitive than PCR amplification (18) and so contamination risk is high but as the laboratories refined the technique, contamination issues seemed to resolve. Secondly, it is feasible that some of the samples are in fact, true positive as demonstrated by the ability of LamPORE to detect spiked, killed virus beyond the limit of detection of RT-qPCR. This may have useful implications for sample pooling (19) , as greater sensitivity would allow more samples to be pooled and tested. Finally, the LamPORE protocol requires multiple manual liquid handling steps which can lead to error and increases the number of opportunities for contamination to occur. The use of automated liquid handling methods (as developed by the manufacturer) is likely to reduce or eliminate this phenomenon.

In conclusion, we have demonstrated the accuracy of LamPORE across a range of population use cases, maintaining a high specificity and sensitivity, reproducibility and limit of detection, as well as working well on saliva samples, making it suitable for the detection of symptomatic and asymptomatic patients with SARS-CoV-2.

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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted December 16, 2020. ; It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20247031 doi: medRxiv preprint 26 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20247031 doi: medRxiv preprint