key: cord-0873988-7ulygkcg
authors: Fowler, V. L.; Armson, B.; Gonzales, J. L.; Wise, E. L.; Howson, E. L. A.; Vincent-Mistiaen, Z.; Fouch, S.; Maltby, C. J.; Grippon, S.; Munro, S.; Jones, L.; Holmes, T.; Tillyer, C.; Elwell, J.; Sowood, A.; Santos, H.; de Payer, O.; Dixon, S.; Hatcher, T.; Sivanesan, S.; Knight, H.; Laxman, S.; Walsh, C.; Andreou, M.; Morant, N.; Clark, D.; Houghton, R.; Moore, N.; Cortes, N.; Kidd, S. P.
title: 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
date: 2020-07-01
journal: nan
DOI: 10.1101/2020.06.30.20142935
sha: 34c2b7bac4b15a4514c32ea5ebcb60344a9ef090
doc_id: 873988
cord_uid: 7ulygkcg

The COVID-19 pandemic has illustrated the importance of rapid, accurate diagnostic testing for the effective triaging and cohorting of patients and timely tracking and tracing of cases. However, a surge in diagnostic testing quickly resulted in worldwide competition for the same sample preparation and real-time RT-PCR diagnostic reagents (rRT-PCR). Consequently, Hampshire Hospitals NHS Foundation Trust, UK sought to diversify their diagnostic portfolio by exploring alternative amplification chemistries including those that permit direct testing without RNA extraction. This study describes the validation of a SARS-CoV-2 RT-LAMP assay, which is an isothermal, autocycling, strand displacement nucleic acid amplification technique which can be performed on extracted RNA, (RNA RT-LAMP) or directly from swab (Direct RT-LAMP). Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1x101 and 1x102 copies when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT LAMP was 97% and 99% respectively, relative to the standard of care (SoC) rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly, evidence suggests there is a very low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct-RT LAMP was 67% and 97%, respectively. When setting CT cut-offs of <33 and <25, the DSe increased to 75% and 100%, respectively. Time from swab-to-result for a strong positive sample (CT < 25) was < 15 minutes. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increase in throughput, whereas Direct RT-LAMP could be used as a screening tool for triaging patients into appropriate hospitals wards, at GP surgeries and in care homes, or for population screening to identify highly contagious individuals (super shedders). Direct RT-LAMP could also be used during times of high prevalence to save critical extraction and rRT-PCR reagents by screening out those strong positives from diagnostic pipelines.

In December 2019, an unusual cluster of pneumonia cases were reported by the Chinese Centre for Disease Control (China CDC) in the city of Wuhan, Hubei province 1 It was quickly established by sequencing of airway epithelial cells that these patients were infected with a novel betacoronavirus 2 which was named by the International Committee on Taxonomy of Viruses as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to the close genetic relatedness to SARS-CoV 3 .

Since its first discovery, SARS-CoV-2 has spread around the globe reaching pandemic status, and by June 2020 has infected 9 million people and caused more than 460,000 deaths according to The World Health Organisation situation report (accessed 24 th June 2020).

Genomic regions suitable for targeting with molecular tests such as real-time reverse-transcription polymerase chain reaction (rRT-PCR) were published by Corman et al 4 early in the outbreak and comprised the RdRp, E and N genes. Diagnostic tests developed targeting these regions have since been utilised for routine use in many reference and hospital laboratories around the world. However, with the huge surge in diagnostic testing, laboratories began competing for the same test components and certain reagents such as RNA extraction kits became difficult to source.

Consequently, to ensure a robust, resilient diagnostic service with an increased capacity, Hampshire Hospitals NHS Foundation Trust (HHFT) sought to diversify the portfolio of testing strategies by exploring alternative chemistries which have separate reagent supplier pathways to those of rRT-PCR, and which also permit direct testing without the need for RNA extraction.

Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) satisfied these requirements by combining reverse-transcription and autocycling, isothermal, strand displacement DNA amplification to produce a highly sensitive, versatile and robust test 5-7 . LAMP chemistry is more resistant to inhibitors than rRT-PCR, enabling simplification and even removal of extraction procedures 8 . LAMP has been applied for the detection of a wide range of pathogens, including positive-sense RNA viruses and has been used extensively in the veterinary and plant industry [9] [10] [11] 4 and more recently in human diagnostics [12] [13] [14] [15] [16] . Herein we describe the validation of a novel SARS-CoV-2 RT-LAMP assay which can be performed on extracted RNA, or directly from viral transport medium (VTM) taken from combined oropharyngeal and nasopharyngeal swabs (ONSwab).

. CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06. 30.20142935 doi: medRxiv preprint 2. Methods

Diagnostic sensitivity (DSe) and specificity (DSp) were determined using ONSwabs submitted to HHFT, previously confirmed as either SARS-CoV-2 positive or negative by rRT-PCR. All ONSwabs were collected in Sigma Virocult® medium (Sigma-Aldrich Inc.).

Analytical sensitivity (ASe) of RNA-RT-LAMP was determined using a ten-fold dilution series of SARS-CoV-2 RNA purified from virus infected tissue culture fluid (BetaCoV/England/02/2020) obtained from Public Health England (Lot 07.02.2020) and a titration of a synthetic DNA fragment containing the SARS-CoV-2 RT-LAMP target in nuclease free water (NFW) (Integrated DNA Technologies).

ASe of Direct RT-LAMP was determined using a two-fold dilution series (1:8 to 1:2048) of VTM taken from a SARS-CoV-2 positive ONswab sample. A standard curve (Qnostics, Scotland, UK) was run on the rRT-PCR, allowing quantification of RNA in digital copies (Log 10 dC/ml). Analytical specificity (ASp) was determined using the NATtrol™ Respiratory Verification Panel (ZeptoMetrix Corporation, New York, United States) containing pathogens causing indistinguishable clinical signs to and a pool of meningitis encephalitis causative agents (n=7) (Table 1) .

Repeatability, inter-operator and inter-platform reproducibility were determined using combined ONSwabs submitted to HHFT, previously confirmed as SARS-CoV-2 positive, and a SARS-CoV-2 Medium Q Control 01 positive control (Qnostics, Scotland, UK) (diluted 1 in 10 and 1 in 100).

Preliminary evaluation of Direct RT-LAMP for detection of SARS-CoV-2 in other clinical samples was performed using fourteen saliva samples collected from hospital in-patients confirmed from paired ONSwabs as positive and negative for SARS-CoV-2. Collection of saliva involved the patient providing . CC-BY-ND 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)

The copyright holder for this preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint a fresh saliva sample into a 10 ml universal container. Prior to analysis the saliva was diluted 1:5, 1:10 and 1:20 in NFW.

RNA was extracted using the Maxwell® RSC Viral Total Nucleic Acid Purification Kit (Promega UK Ltd., Southampton, UK) according to manufacturer's instructions. Briefly, 200 µl of sample was added to 223 µl of prepared lysis solution (including 5 µl per reaction of Genesig® Easy RNA Internal extraction control, Primerdesign Ltd, Chandler's Ford, UK). Samples were then inactivated for 10 minutes at room temperature within the safety cabinet and 10 minutes at 56 o C on a heat block before automated RNA extraction using a Maxwell® RSC 48 Instrument (Promega UK Ltd., Southampton, UK). RNA was eluted in 50 µl of NFW. In the case of saliva, RNA was extracted from 200 µl of saliva diluted 1:20, as saliva volume was insufficient unless a dilution was performed.

rRT-PCR assays were performed in single replicates using 5 µl of RNA template with the COVID-19 genesig® Real-Time PCR assay (Primerdesign Ltd, Chandler's Ford, UK) according to the manufacturer's guidelines, on a MIC qPCR Cycler (Bio Molecular Systems, London, UK). Single replicates were performed to ensure an adequate supply of reagents. The cycling conditions were adjusted to the following: a reverse-transcription (RT) step of 10 minutes at 55 o C, a hot-start step of 2 minutes at 95 o C, and then 45 cycles of 95 o C for 10 seconds and 60 o C for 30 seconds. The Genesig® COVID-19 positive control included in the kit, a negative extraction control, and a no template control were also included on each rRT-PCR run.

RT-LAMP reactions were performed using OptiGene Ltd. (Camberley, UK) COVID-19_RT-LAMP kits which target the ORF1ab region of the SARS-CoV-2 genome: (i) COVID-19_RNA RT-LAMP KIT-500 kit . CC-BY-ND 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)

The copyright holder for this preprint this version posted July 1, 2020. To confirm the specificity of the amplification reaction, an anneal curve was performed: RT-LAMP products were heated to 98°C for 1 min, then cooled to 80°C decreasing the temperature by 0.05°C/s. Genie® embedded software (OptiGene Ltd., UK) was utilised to analyse RT-LAMP results and define thresholds for result calling. All RT-LAMP reactions were performed at least in duplicate, and a sample was considered positive when a Tp was observed in at least one replicate with amplification above 5000 fluorescence points and had an anneal temperature of between 81.50 o C and 84.05 o C with a derivative above 2500 F/ o C.

For RNA RT-LAMP 5 μl of extracted RNA was added to the reaction and for Direct RT-LAMP 5 μl of VTM from the swab diluted 1:20 in NFW, or saliva diluted 1:5, 1:10 and 1:20 in NFW was added to the reaction.

Repeatability and inter-operator reproducibility for the RNA RT-LAMP and Direct RT-LAMP were measured by running eight replicates of samples with three different operators. Inter-platform . CC-BY-ND 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)

The copyright holder for this preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint reproducibility was measured by running eight replicates of the samples across two Genie® platforms. For RNA RT-LAMP, operators used the same RNA extraction for each sample; for Direct RT-LAMP operators used the same 1 in 20 dilution of a combined swab sample in NFW.

DSe, DSp, positive and negative likelihood ratios (LR) including 95% confidence intervals (CI), and the Cohen's Kappa statistic (κ) 17 were determined using contingency tables in R 3.6.1 18 . Assessment of the diagnostic performance was made under three scenarios: 1) "No C T cut off" (low-to-high viral load), 2) "C T cut off <33" (moderate-to-high viral load) and 3) C T cut off <25 (high viral load and significant risk of shedding).

To further explore the practical application of the RT-LAMP assay in clinical practice, we estimated a . CC-BY-ND 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)

The copyright holder for this preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint 3. Results

Using a synthetic DNA template titrated in NFW, the RNA-RT-LAMP and Direct-RT-LAMP assays were able to detect 1x10 1 copies each, in one of two duplicates (detection limit between 1x10 1 and 1x10 2 copies) ( Table 2) . To compare the ASe of the RNA RT-LAMP with the rRT-PCR assay a 10-fold decimal dilution series of SARS-CoV-2 RNA extracted from a virus infected tissue culture media was used. The RT-LAMP detected to a dilution of 10 -3 , equivalent to a rRT-PCR C T value of 36.0 (Table 1 ). In the case of RNA RT-LAMP the dilution with a corresponding rRT-PCR C T <30 was detected in duplicate and C T >30 and <39 were detected in one of the duplicates (Table 3) .

To compare the analytical sensitivity of the Direct RT-LAMP to the rRT-PCR assay a 2-fold decimal dilution series of SARS-CoV-2 positive VTM from a combined swab was used. The Direct RT-LAMP detected dilutions spanning 1:8 to 1:512, equivalent to a rRT-PCR C T value of 22.65 (Table 3) . This would equate to between 5 -6 log 10 digital copies (dC)/ml. The rRT-PCR detected dilutions spanning 1:8 to 1:2048 (Table 4 ).

The performance of the RT-LAMP on extracted RNA was determined using 196 individual clinical samples tested in duplicate and compared to the results of the rRT-PCR (tested in single) ( Figure 1 ).

All samples with a C T <30 were detected within 16 minutes. The overall DSe was calculated as 97% (95% CI: 90 -99) and the overall DSp was 99% (95 -1.00) ( CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint likelihood ratio: 0.00 [0.00 -0.03]), indicating almost perfect agreement between the two assays (Table 5B) . By employing a rRT-PCR cut-off of <C T 

To perform RT-LAMP directly from the swab VTM a series of dilutions were evaluated comprising 1:5, 1:10, 1:20 and 1:40 in NFW. The optimal dilution whereby inhibition was limited, but Tp was agreement using a C T cut off <33 and almost perfect agreement using a C T cut off <25. When the ASe was determined independently from the DSe using a dilution series of SARS-CoV-2 patient swab VTM, it was noted that a C T value of 24.15 and 24.80 were not detected by Direct RT-LAMP. This is in contrast to the results from the DSe evaluation when these range of C T were detected. A C T of 24 directly from VTM is not necessarily comparable to a C T of 24 derived from a serially diluted swab . CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint sample and this likely reflects the difference observed. Using a standard curve to measure genome copies was performed for DSe, but it was used for ASe.

The incorporation of subsequent confirmatory rRT-PCR testing to verify a negative Direct RT-LAMP result increased the overall DSe of this pipeline to 99%, with a DSp of 98.4%. ASp was determined using a panel of respiratory pathogens for Direct-RT-LAMP. No cross reactivity was observed, including against four seasonal coronaviruses.

A selection of paired ONSwab and saliva samples were compared to evaluate saliva as a potential diagnostic matrix for SARS-CoV-2 detection ( Table 7) 

When it comes to repeatability and inter-operator reproducibility, 100% of the replicates were detected for each sample by the three operators. The percentage coefficient of variation (%CV) was below 10 both when comparing within and between operators (Table 8) . When comparing between platforms, 100% of the replicates were detected on both the Genie® HT and Genie® III, with the %CV below 10 (Table 9 ).

The practical application of using Direct RT-LAMP during the growing phase of an epidemic where the prevalence of infection is around 0.14 (14%) (Supplementary Information 1) was modelled. In . CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint practice a clinical team will assess patients who have clinical signs (symptomatic) or not (asymptomatic) and those that have either had contact or not with sick or infected individuals (risk contact). These patients all have different risks and therefore different pre-test probabilities of being infected ( Figure 3 ). Pre-and post-test probabilities of infection are presented for different risk categories of patients and different risk categories of viral shedding levels (no C T cut off, C T <33, C T <25) (Figure 3 ). For example, consider a symptomatic patient who had no risk contact. As shown in Figure 3 , the pre-test probability that they are infected is on average 0.19 (19%), after testing positive in the Direct RT-LAMP test, the (post-test) probability of this patient being infected increased to 0.81 (81%). On the other hand, if the Direct RT-LAMP result was negative the probability of the patient being infected decreases to 0.07 (7%). Assuming this probability is considered too high, the clinical team would recommend isolation until confirmatory diagnosis is obtained.

Consider now an asymptomatic patient with a confirmed contact awaiting a test result. The pre-test probability of this patient is 0.12 (12%), after a negative Direct RT-LAMP result the post-test probability of this patient being infected is 0.05 (5%). The clinical team, before sending the sample for confirmatory testing, may look at the post-test probability of this patient shedding moderate to high levels of virus if they were infected (C T < 33, C T <25). These probabilities are lower than 0.05 (5%) ( Figure 3 ) so the clinical team may consider these probabilities low and infer that the patient does not represent a risk for spreading infection, and diagnose the patient as "not infected". These kinds of decisions may be necessary when there are limited diagnostic resources available.

This study describes the development and validation of a rapid, accurate and versatile SARS-CoV-2 RT-LAMP assay. This assay demonstrates excellent concordance with rRT-PCR when performed on extracted RNA and when used directly on diluted VTM can detect samples with a high viral load . CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint which would be considered significant for viral transmission 21 . No cross reactivity was observed against common respiratory pathogens including seasonal coronaviruses.

The overall DSe of the RNA RT-LAMP assay was calculated as 97% and the overall DSp was 99% with all samples of C T <30 detected within 16 minutes. We therefore recommend that when using RNA RT-LAMP, the length of the assay should be a maximum of 16 minutes to avoid detection of degraded nucleic acid which may be derived from the clinical sample or the environment 22 .

A shortage in the supply of RNA extraction reagents was a critical rate-limiting step affecting COVID-19 diagnostic capacity, thus the ability to bypass this step and test directly from swab has significant advantages. Various simple sample preparation methods have been reported which can circumvent RNA extraction, including the use of syringe filtration, Chelex TM 100, dilution in NFW, or a heat step, among other 23-26 . In this study the best performance for Direct RT-LAMP was achieved using a 1:20 dilution of VTM in NFW. This sample preparation method is simple and quick to perform (<5 mins) and does not require any additional equipment, therefore it is well-suited for near-patient testing.

Recent publications have demonstrated that there is a strong correlation between rRT-PCR C T values and the ability to recover live virus, and therefore it is unlikely that patients providing samples with high C T values pose a high risk of transmission 21 . One previous study demonstrated that live virus could only be recovered reliably from samples with a C T between 13 to 17, when using a rRT-PCR targeting the E gene 21 . Additionally, the ability to recover live virus then dropped progressively with virus unrecoverable from samples with a C T above 33 21 . Bullard and colleagues 27 found no virus was recoverable from clinical samples taken from symptomatic patients with rRT-PCR (targeting the Egene) C T values of >24. In the same study 27 each unit increase in C T value corresponded to a 32% decrease in the odds of recoverable live virus. Consequently, as the risk of SARS-CoV-2 transmission is still not fully understood, a range of C T cut-off values were set in our study, to understand in particular the performance of the Direct-RT-LAMP assay at different viral loads. The overall DSe of . CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint Direct RT-LAMP was 67%, however, when setting C T cut-offs of <33 (low-medium viral load) and <25

(high viral load and significant risk of shedding) the Direct RT-LAMP DSe increased to 75% and 100%, respectively. DSp was unchanged and remained at 97%. As no samples were detected beyond 14 minutes, we recommend that when using Direct RT-LAMP the length of the assay should be a maximum of 14 minutes to avoid detection of degraded nucleic acid which may be derived from the clinical sample or environment 22 .

The ability to detect patients with high viral load (C T <25) directly from diluted swabs, demonstrates significant potential for the use of Direct RT-LAMP for the rapid diagnosis of symptomatic patients and also for rapid screening of asymptomatic individuals. This is largely supported by studies reporting similar viral loads in asymptomatic and symptomatic patient groups 28 . CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint Rapid testing of symptomatic SARS-CoV-2 positive patients within healthcare facilities allows their rapid isolation or cohorting, significantly reducing onward transmission and improving bed management and patient flow. Additionally, screening of asymptomatic patient groups or at the community level may enable the rapid identification of those with high viral loads who may pose a high risk of onward transmission. This would allow for swift public health intervention with instruction to self-isolate/ quarantine and the rapid tracking and tracing of their contacts -essential in screening programmes aiming to reduce the reproductive number (R 0 ) and spread of the disease in a community.

Direct RT-LAMP offers speed, robustness and portability making it attractive as an option for nearpatient testing outside the conventional clinical laboratory, subject to the necessary risk-assessments to ensure safety of the operator 34 . Within HHFT we are exploring its application in settings such as: a multi-disciplinary non-specialist laboratory; the emergency department; primary care and nursing/ care home settings.

In this study, clinical validation of the RT-LAMP assay took place in March, April and May 2020, largely during a period of high local COVID-19 prevalence (around 40% positivity of samples submitted) and on samples from largely symptomatic patients and hospital staff. It is possible that RT-LAMP assay performance on samples from asymptomatic subjects may vary dependent on the level of detectable RNA (as a surrogate of live viral shedding) in this different patient group.

Additionally, the RT-LAMP assay was validated using ONSwabs in VTM. Assay performance on a limited number of salivary samples was also explored. This preliminary analysis suggests that saliva may not be as suitable when compared to ONSwabs as the detection of SARS-CoV-2 both by rRT-PCR and Direct RT-LAMP was poorer using saliva samples. More work is required to understand if this is due to degradation of the RNA within the sample (e.g. salivary enzymes), or inhibition due to the complex nature of this matrix. Assay performance was not evaluated on lower respiratory tract . CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint samples or non-respiratory tract samples, and therefore future research may aim to determine the performance of both the RNA-and Direct-RT-LAMP assays using these various sample types.

In our experience, during the diagnostic response to this current pandemic caused by a novel emergent pathogen (SARS-CoV-2), diversity in diagnostic platforms and routes to deliver a result based on the ability and agility to switch between methodologies has been key to allowing delivery of a resilient and sustainable diagnostic service. Factors such as: analyser availability; staff-skill mix;

dynamic changes in patient groups tested or disease prevalence; and particularly in the UK; consumable and reagent supply, have highlighted the need for diagnostic services to have adaptability and capability to explore novel and alternative techniques.

No ethical approval was required for this service improvement study.

We would like to thank the clinical teams and Helen Denman the Microbiology Laboratory manager at Hampshire Hospitals NHS Foundation Trust.

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The copyright holder for this preprint this version posted July 1, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 1, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint detection and characterization of foot-and-mouth disease virus in East Africa using a field-PHE. COVID-19: safe handling and processing for samples in laboratories. Available at https://www.gov.uk/government/publications/wuhan-novel-coronavirus-guidance-forclinical-diagnostic-laboratories/wuhan-novel-coronavirus-handling-and-processing-oflaboratory-specimens#risk-assessment. Accessed 19 June 2020, 2020. . CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint . CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint . CC-BY-ND 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 preprint this version posted July 1, 2020. . https://doi.org/10.1101/2020.06.30.20142935 doi: medRxiv preprint Table 5 . Overall diagnostic sensitivity (DSe) and specificity (DSp) of the RNA RT-LAMP with all rRT-PCR C T values considered (A), and with a C T value cut-off of <33 (B) and <25 (C). . CC-BY-ND 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 preprint this version posted July 1, 2020. CC-BY-ND 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 preprint this version posted July 1, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 1, 2020. . CC-BY-ND 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 preprint this version posted July 1, 2020. . CC-BY-ND 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 preprint this version posted July 1, 2020. . CC-BY-ND 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.

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Linking pre-and post-test probability of infection In clinical practice diagnosis is made using a combination of the patient pre-test probability of being infected and the test result. The combination of these two will lead to an estimation of the post-test probability of infection. It is this final estimate which would help the practitioner's decision making. In our study, the pre-and post-test probability of infection were estimated using an scenario-tree model, where different risks for infection in the estimation of the pre-test probabilities are taken into consideration 19 . Pre-test probability of infection In this model the pre-test probability of infection was given by: = * * Where is the prevalence of infection in the population, is the adjusted risk ratio for infection of a symptomatic or asymptomatic patient and is the risk ratio of a patient being infected who did have a risk contact compared with a patient who did not have a risk contact (Table S1 ). The ARR were calculated as follows: These LRT were calculated using the Direct RT-LAMP's DSe and DSp estimates for the different viral load scenarios considered (estimated from C T 's: 1) "No C T cut off" (high-low viral load), 2) "C T cut off <33" (high-moderate viral load) and 3) C T cut off <25. Finally, the post-test odds were transformed to post-test probabilities of infection = /(1 + ) The model was implemented in Microsoft® Excel® using the add-in software Poptools 20 . For estimation of pre and post probabilities (mean and 95% confidence intervals), stochastic simulations of 1000 iterations were performed. Table S1 summarises the parameter values used. It should be noted that these values are crude approximations which were made only as an example of and to help understand the use of Direct RT-LAMP in practice. We encourage the readers who would like to use this model to quantify pre-and post-test probabilities of infection to better estimate the parameter values according to the epidemiological situation of the country/region where the test would be applied. Alternatively, once the pre-test probabilities are estimated, post-test probabilities can be approximated using a Fagan nomogram 35 .

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The copyright holder for this preprint this version posted July 1, 2020. distribution (a,b,c) where a = the minimum, b = the most likely and c = the maximum values.

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