key: cord-0968766-xz7rmapf
authors: Fowler, Veronica L.; Armson, Bryony; Gonzales, Jose L.; Wise, Emma L.; Howson, Emma L.A.; Vincent-Mistiaen, Zoe; Fouch, Sarah; Maltby, Connor J.; Grippon, Seden; Munro, Simon; Jones, Lisa; Holmes, Tom; Tillyer, Claire; Elwell, Joanne; Sowood, Amy; de Peyer, Oliver; Dixon, Sophie; Hatcher, Thomas; Patrick, Helen; Laxman, Shailen; Walsh, Charlotte; Andreou, Michael; Morant, Nick; Clark, Duncan; Moore, Nathan; Houghton, Rebecca; Cortes, Nicholas; Kidd, Stephen P.
title: A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection
date: 2020-11-30
journal: J Infect
DOI: 10.1016/j.jinf.2020.10.039
sha: 077a387ef9e190f28bc439263f7fc70f16d05252
doc_id: 968766
cord_uid: xz7rmapf

The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 10(1) and 1 × 10(2) copies per reaction 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 rRT-PCR. When a C(T) cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) was 67% and 97%, respectively. When setting C(T) cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively, time from swab-to-result, C(T) < 25, was < 15 minutes. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and a care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.

increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) was 67% and 97%, respectively. When setting C T cut-offs of <33 and <25, the DSe increased to 75% and 100%, respectively, time from swab-to-result, C T < 25, was < 15 minutes.

We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and a care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.

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 30.6 million people and caused more than 950 000 deaths according to The World Health Organisation situation report (accessed 24 th September 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] [6] [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] 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).

Diagnostic sensitivity (DSe) and specificity (DSp) were determined using adult inpatient ONSwabs submitted to the Microbiology department at HHFT during March and April 2020, which had previously been confirmed either SARS-CoV-2 RNA positive or negative by SoC rRT-PCR. All ONSwabs were collected in Sigma Virocult® medium (Medical Wire & Equipment, Corsham, UK).

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, Coralville, United States).

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 2 (ZeptoMetrix Corporation, New York, United States) containing pathogens causing indistinguishable clinical signs to COVID-19 (n=22) 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 HHFT in-patients during May 2020 confirmed from paired ONSwabs as positive and negative for SARS-CoV-2. Collection of saliva involved the patient providing 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. 

rRT-PCR assays were performed in single replicates using 5 µl of RNA template with the COVID-19 

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 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 patient's probability of being infected under different clinical scenarios where Direct RT-LAMP could be applied. Final diagnosis in these scenarios is given by linking the patient's pre-test probability of infection (P pre ) with the Direct RT-LAMP's LRs to estimate the post-test probability of infection (P post ).

To estimate these pre-and post-test probabilities of infection a scenario-tree model was used 19 which allowed estimation of risk-based probability estimates for scenarios where patients are: 1) 

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/reaction) ( 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 22.7 (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 -100) ( 

To perform RT-LAMP directly from the swab VTM a series of dilutions were evaluated comprising 1:5, 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 24.2 and 24.8 were not detected by Direct RT-LAMP. This contrasts with 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 sample and this likely reflects the difference observed. Using a standard curve to measure genome copies was not 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, including four seasonal coronaviruses Direct-RT-LAMP. No cross reactivity was observed.

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 ). The ONSwab samples ranged in C T value from 

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 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 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 others [23] [24] [25] [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 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 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 rapid track and trace methods deployed -essential in surveillance 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 care home settings. However, it must be highlighted that a test with this demonstrated sensitivity will require negative verification from a suitability sensitive diagnostic test in line with the WHO diagnostic test Target Product Profile (TPP).

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 (~40% positivity of submitted samples) and on a limited number of samples available at the time from largely symptomatic adult 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. Consequently, further evaluation using a larger sample set and from different scenarios is recommended, for example during periods of low prevalence or in asymptomatic individuals. Indeed, during the period of evaluation of the RT-LAMP assays, the laboratory was quickly evolving based on testing and staffing requirements, and therefore contamination of equipment or reagents occurred infrequently, which may have been the reason for the less than perfect diagnostic specificity of the RT-LAMP assays. Further evaluation within established laboratories may therefore result in an increase in diagnostic specificity, where contamination may be less likely.

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 further research needs to be undertaken to explore saliva as a matrix for detection of SARS-CoV-2 both by rRT-PCR and Direct RT-LAMP. The drop in performance that we observed when compared to ONSwabs could be due to a number of factors causing either 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 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.

The standard of care assay used as the reference standard for both RT-LAMP assays targeted the RdRp gene. There were two reasons for choosing the assay; one it was the assay currently validated, available and in clinical use and secondly the RdRp gene sits within the Orf1b region that the Optigene Ltd. assay targets making it appropriate at the time. There has been some discussion about the sensitivity of the RdRp gene for SARS-CoV-2 due to the poor performance of some of the early assays containing this target 35 . However, Primer Design have demonstrated in silico versus the submitted SARS-CoV-2 sequences throughout the pandemic that the region within RdRp gene that is targeted by the genesig assay is very robust as a diagnostic 36 . However, the authors would recommend that further benchmarking should be carried out versus other more sensitive Orf1ab/RdRp assays with additional gene targets 37 on RT-LAMP naïve clinical sites to generate more performance data and help determine if this level of sensitivity is clinically relevant.

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 evaluation study.

Initial reagents were supplied free of charge by Optigene Ltd. (Horsham, UK), the remainder of the service evaluation was self-funded by Hampshire Hospitals NHS Foundation Trust. Optigene Ltd.

representatives played no part in study design or data analysis.

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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