key: cord-0917838-s5vpg0bg
authors: Ptasinska, Anetta; Whalley, Celina; Bosworth, Andrew; Poxon, Charlotte; Bryer, Claire; Machin, Nicholas; Grippon, Seden; Wise, Emma L.; Armson, Bryony; Howson, Emma L.A.; Goring, Alice; Snell, Gemma; Forster, Jade; Mattocks, Chris; Frampton, Sarah; Anderson, Rebecca; Cleary, David; Parker, Joe; Boukas, Konstantinos; Graham, Nichola; Cellura, Doriana; Garratt, Emma; Skilton, Rachel; Sheldon, Hana; Collins, Alla; Ahmad, Nusreen; Friar, Simon; Burns, Daniel; Williams, Tim; Godfrey, Keith M.; Deans, Zandra; Douglas, Angela; Hill, Sue; Kidd, Michael; Porter, Deborah; Kidd, Stephen P.; Cortes, Nicholas J.; Fowler, Veronica; Williams, Tony; Richter, Alex; Beggs, Andrew D.
title: Diagnostic accuracy of loop mediated isothermal amplification coupled to nanopore sequencing (LamPORE) for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations
date: 2021-04-24
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2021.04.008
sha: 9b048d9434bb8837d3523e524de72e9d2cca4911
doc_id: 917838
cord_uid: s5vpg0bg

OBJECTIVES: 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 (RT-qPCR), particularly throughout the first months of the COVID-19 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, 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% (reducing to ∼ 98% when clustered estimation was used) 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.

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

represents a public health emergency. [1] .

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] . Although considered the gold standard for diagnosis, RT-qPCR is laborious can be difficult to scale up for mass-testing, 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] . [6] Loop mediated isothermal amplification (LAMP) offers an alternative to RT-qPCR [7] . This reaction typically takes 20-30 minutes which is considerably quicker than PCR. [8] Nanopore sequencing allows rapid sequencing using protein nanopores embedded in a lipid membrane. [9] . 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 [10] 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. [11] .

J o u r n a l P r e -p r o o f

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 To assess the limit of detection and precision, 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 used and tested in triplicate. . A panel of respiratory viruses (Zeptometrix Respiratory Panel R2, Buffalo NY 14202) was used to assess specificity of the LamPORE assay. QuantStudio 5 or BioMolecular Systems MIC instruments, using 5µL of extracted RNA per reaction. [2, 12] Comparator test (LamPORE): For each sample, 20µL of RNA sample underwent amplification and sequencing using the LamPORE technique as per the manufacturers protocol .A proprietary Guppy/VSEARCH/SnakeMake pipeline (algorithmic details as per James et al [13] ) that aligned reads to the viral target genes and a human beta-actin (ACTB) internal control and reported results in absolute reads per sample per gene.

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 3.6.3 [14] ) from the methodology of Stark et al. [15] 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 J o u r n a l P r e -p r o o f comparison was made and the sample was removed from the analysis. Standard analyses of variability in diagnostic precision were made, and modification to the analysis was made to carry out clustered analyses of diagnostic precision using clustered logistic regression with a sandwich estimator (Stata 16.1, StataCorp, TX), on the basis that each separate study represented a cluster within the whole and may bias estimates of sensitivity and specificity [16] .

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 In order to understand the context in which LamPORE operates, a comparative experiment was carried out examining the sensitivity of our reference RT-qPCR assay vs. flourimetric LAMP (Optigene LAMP kit) vs. LamPORE (supplementary table 4) . This showed that LamPORE detected three additional positive samples detected by RT-qPCR that were not detected by RT-LAMP.

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 LamPORE (comparator assay) and RT-qPCR (reference assays) were performed on all 23,427 samples (Table 2) 

When modelled at 1% population prevalence the PPV dropped to 66.24% and at 0.1% population prevalence the PPV was 16.3%. NPV remained at > 99.99% in all population scenarios.

When a clustered regression analysis was performed to calculate clustered sensitivity and specificity on all participants (when the cohorts were used as the cluster definition), sensitivity reduced to 98.52% (95% CI 94.75%-99.82%) and specificity was 97.39% (95% CI 97.14-97.62%. Positive predictive value droped to 22.62% (95% CI 21.03-24.29%) and negative predictive value to 99.99% (95% CI 99.95-100%).

If sensitivity and specificity was calculated as regards to the cohort (n=1200, Supplementary J o u r n a l P r e -p r o o f

We carried out a very large asymptomatic cohort study of health care workers using a novel technology, LamPORE, comparing it to a reference RT-qPCR assay. 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.

LamPORE has the advantage that it is scalable [10] to allow testing of very large population levels because of the use of sample barcoding allowing pooling of up to 3,500 samples on a single GridIon instrument. Due to the increased sensitivity of LAMP as part of the LamPORE system, it gives very high sensitivity for SARS-CoV-2. With combinatorial barcoding as has been adopted in other population level assays [17] 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 [18] 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 [19] . Given the advantages of LAMP in terms of speed of amplification [8] 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. However, LAMP plus sequencing introduces several steps into the workflow which means that LamPORE becomes inherently "nonlinear" i.e. the relationships between the genes amplified and sample viral load is not linear and thus LamPORE may only be used to infer positivity rather than any measure of viral load.

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.

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 [20] 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 [21] , 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. . We found two false negative samples within the saliva cohort. These had high Ct values (ORF1ab Ct 36.5 and 37.1) in the PCR, and we hypothesise that this may have occurred because of low viral loads as the extracted RNA went through a freeze thaw before it was run on LamPORE.

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. 07 08 09 10 11 12 13 14 15 16 17 18 19 20 J o u r n a l P r e -p r o o f

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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.