Research and analysis

Rapid evaluation of OptiGene RT-LAMP assay (direct and RNA formats)

Published 1 December 2020

Applies to England

Executive summary

The OptiGene RT-LAMP assay was evaluated according to the published Technical Validation Group protocol.

A collaboration across 9 NHS trusts and university partners who evaluated LAMP based molecular testing technologies, including an NHS asymptomatic staff pilot study

The OptiGene RT-LAMP assay was assessed on saliva and swab samples, both directly (native samples) and after RNA extraction.

The OptiGene RT-LAMP assay on oropharyngeal and nasopharyngeal swabs, with RNA extraction, had a sensitivity of 95% (CI 0.91-0.97) and specificity of 99% (CI 0.99-1.00) across all samples tested (CT <45 by comparator RT-qPCR).

The OptiGene RT-LAMP assay on saliva, without RNA extraction, had a sensitivity of 79% (CI 0.73-0.84) and specificity of 100% (CI 0.99-1.00) across all samples tested (CT <45 by RT-qPCR), increasing to a sensitivity of 94% (CI 0.87-0.98) for those samples with a higher viral load (CT <25 by RT-qPCR).

Nucleic acid amplification tests using LAMP technology on saliva samples demonstrates viral detection with sufficient sensitivity and specificity for an effective regular interval-based testing system, as indicated by the hospital and community programmes.

Verification of performance at scale is provided from a DHSC-funded population-based pilot study in Southampton, in which no false positives have been recorded in 51,022 saliva Direct RT-LAMP tests. As of 5 November 2020, all 64 positives were confirmed by RT-qPCR, with CT values ranging from 17.4 to 34.0.

1. Background

1.1. The importance of testing, isolation and contact tracing for control of SARS-CoV-2 transmission has been highlighted by the World Health Organisation (WHO) as a critical intervention to prevent the spread of infection and ensuing morbidity and mortality from COVID-19. International efforts have largely focused on the detection of infection in symptomatic individuals as well as evolving clinical testing systems to detect those with asymptomatic or pre-symptomatic (subsequently referred to collectively as asymptomatic) infection that may still be infectious to others.

1.2. The technology of choice for this provision to date has been reverse transcription quantitative polymerase chain reaction testing (RT-qPCR), typically provided in central laboratory facilities, with a sample receipt to result time of >24hours and reliance on international supply chains for a singular testing system. Considering these challenges, alternative technology approaches are being evaluated, including other forms of nucleic acid amplification (ie loop-mediated isothermal amplification (LAMP)) and protein based methods (ie lateral flow devices (LFD)) for viral antigen detection, potentially providing diversification to the overall supply chain and options for an integrated testing strategy. These additional testing assets have also been considered in the context of other components of the wider end to end testing system, including the source and ease of sample collection and frequency of provision (saliva v swab), RNA extraction requirements, and efficiency of returning results from a testing system into a clinical system for full maximal clinical utility, public health reporting and intervention requirements.

1.3. This report outlines the technical validation undertaken in conjunction with the NHS Test and Trace Technical Validation Group (TVG) and, given the need to prevent nosocomial spread and maintain NHS capacity and capability, it was conducted in collaboration with NHS trusts and university partners as a real-world evidence study. Additional verification data in a real-world mass population setting is provided by a DHSC-funded study in Southampton, in which 51,022 saliva Direct RT-LAMP tests were undertaken in school and higher education settings.

2. Technical validation

2.1. The Technical and Validation Group was established under NHS Test and Trace, inclusive of NHS and PHE experts and working closely with the Medicines and Healthcare products Regulatory Agency (MHRA) and research bodies. The Technical and Validation function considers manufacturers of SARS-CoV-2 (COVID-19) tests for viral detection (including LAMP technologies) and registers their interest in the national procurement process if their test meets, or are intended to meet the requirements of the relevant MHRA Target Product Profiles. The Technical and Validation function reviews product information; undertakes technical and clinical validation; and establishes and/or work with service evaluation projects and pilots.

Assay description and intended purpose

2.2 Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of DNA and a low-cost, rapid alternative to RT-qPCR. Reverse Transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with a reverse transcription step to allow the detection of RNA. Target sequence is amplified at a constant temperature. Typically, 4 different primers are used to amplify 6 distinct regions on the target gene, which increases specificity. Additional pairs of “loop primers” can further accelerate the reaction. The amount of amplified product produced in LAMP is considerably higher than PCR-based amplification.

2.3 LAMP is an isothermal nucleic acid amplification technique, in contrast to the polymerase chain reaction (qPCR) technology, in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler.

2.4 OptiGene’s COVID-19 RT-LAMP assay is a test for the detection of SARS-CoV-2 in clinical specimens (nasopharyngeal swabs/oropharyngeal swabs and saliva). The RT-LAMP assay targets the positive sense viral genomic RNA within the ORF1ab region. The assay has two formats, an RNA version for use on extracted RNA and a Direct version for use on crude clinical samples. The assay is intended for use by professionals trained in laboratory settings in the detection of SARS-CoV-2 viral RNA in association with the Genie® platforms.

2.5 The OptiGene RT-LAMP assay was performed using the OptiGene Genie instruments consisting of the Genie® HT (High Throughput) and Genie® II. All of the results from the NHS asymptomatic pilot were performed on the Genie® HT.

2.6 The validation was performed on nasopharyngeal swabs/oropharyngeal swabs and saliva. Swabs were collected in viral transport medium (Virocult). Saliva was collected in universal plastic tubes. The samples were all collected in clinical settings.

2.7 The specific equipment required to perform testing on the OptiGene RT-LAMP assay was supplied by the manufacturer, whilst other laboratory consumables, RNA extraction kits, saliva collection containers and nasopharyngeal swabs/oropharyngeal swabs were separately sourced.

Performance characteristics

2.8 Analytical Sensitivity of SARS COV-2 targets

The analytical sensitivity (ASe) for the RNA and Direct RT-LAMP assays were evaluated using a blinded panel of NIBSC inactivated virus ranging from 107/ml to 102/ml, noting that the process of viral inactivation may result in a degree of RNA degradation, which may impact on limit of detection studies to an extent (table 1 and table 2). These viral samples were run in both the Direct (bypassing the heat and lysis step) and RNA RT-LAMP master mixes. The genesig® SARS-CoV-2 Winterplex assay (PrimerdesignTM Ltd, Camberley, Surrey, UK) includes a SARS-CoV-2 gene target, which has proximity to the LAMP assay target, was used as a comparator after the RT-LAMP reactions had been performed and the panel unblinded by NIBSC. Both RT-LAMP assay formats detected to 103 copies/ml (1000 copies/ml).

Table 1: performance of RT-LAMP assay on NIBSC inactivated virus (direct RT-LAMP)

NIBSC Control	Concentration	RT-LAMP Tp	Winterplex ORF1ab (RT-qPCR CT)
Inactivated virus	106/ml	00:08:12	25.24
Inactivated virus	105/ml	00:09:35	28.57
Inactivated virus	104/ml	00:10:14	32.09
Inactivated virus	103/ml	00:11:07	34.55
Negative Control	Negative control	NEG	NEG

Table 2: performance of RT-LAMP assay on NIBSC inactivated virus (RNA RT-LAMP)

NIBSC Control	Concentration	RT-LAMP Tp	Winterplex ORF1ab (RT-qPCR CT)
Inactivated virus	106/ml	00:08:01	25.24
Inactivated virus	105/ml	00:09:15	28.57
Inactivated virus	104/ml	00:10:56	32.09
Inactivated virus	103/ml	00:11:30	34.55
Negative Control	Negative control	NEG	NEG

2.9 Precision and robustness

Repeatability and inter-operator reproducibility were measured by running eight replicates of four clinical samples with three different operators (table 3). Each operator independently mixed the sample 1:1 in OptiGene sample buffer (RapidLyze) ensuring separate processing workstreams for each of the replicates. Inter-platform reproducibility was measured by running eight replicates of a clinical sample across two platforms. The same 1:1 sample to buffer ratio was used across the two platforms.

Table 3: inter-operator precision

Mean time to positivity in minutes (% coefficient of variation)

Mean anneal temperature [number of replicates positive]

RT-qPCR CT	Operator 1	Operator 2	Operator 3	Reproducibility between operators
19.43	06:45 (0.67) 84.19°C [8/8]	06:34 (0.94) 84.04°C [8/8]	07:03 (0.95) 84.11°C [8/8]	06:48 (3.59)
22.59	10:30 (4.95) 84.72°C [8/8]	10:16 (3.64) 84.36°C [8/8]	12:01 (3.39) 84.27°C [8/8]	10:55 (8.68)
20.15	06:55 (1.86) 84.19°C [8/8]	06:43 (1.14) 84.08° [8/8]	08:03 (2.36) 84.06°C [8/8]	07:13 (9.90)
23.13	14:20 (10.14) 84.47°C [8/8]	14:03 (16.79) 84.24°C [6/8]	13:52 (14.62) 84.26°C [7/8]	14:05 (1.64)

Table 3 criteria for acceptance: (i) mean time to positivity does not vary more than 20% and (ii) the mean anneal temperatures are within +/- 1°C. TP: time to positivity (minutes: seconds). Samples were collected on 24/09/2020; tests were performed on 18/10/2020 [samples stored short term at 4°C and long term at -20°C). VTM: viral transport medium.

Table 4: inter-platform reproducibility

Mean time to positivity in minutes (% coefficient of variation)

[Number of replicates positive]

Sample	RT-qPCR CT	Genie® HT	Genie® III	Reproducibility between platforms
Clinical patient sample 1 (Swab VTM)	19.43	06:45 (0.67) 84.19°C [8/8]	06:58 (2.64) 83.90°C [8/8]	06:51 (2.26)
Clinical patient sample 2 (Saliva)	22.59	10:30 (4.95) 84.72°C [8/8]	10:30 (5.31) 84.20°C [8/8]	10:30 (0.04)
Clinical patient sample 3 (Swab VTM)	20.15	06:55 (1.86) 84.19°C [8/8]	07:06 (1.53) 83.88°C [8/8]	07:01 (1.91)
Clinical patient sample 4 (Saliva)	23.13	14:20 (10.14) 84.47°C [8/8]	14:11 (12.17) 83.97°C [7/8]	14:15 (0.76)

Table 4 criteria for acceptance: (i) mean time to positivity does not vary more than 20% and (ii) the mean anneal temperatures are within +/- 1°C. TP: time to positivity (minutes: seconds). Samples were collected on 24/09/2020; tests were performed on 18 October 2020 [samples stored short term at 4°C and long term at -20°C]. VTM: Viral Transport Medium.

2.10 Analytical specificity (Interferences and cross-reactions)

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). No cross reactivity was observed in either assay (Direct RT-LAMP and RNA RT-LAMP) (table 5).

Table 5: specificity assessment for direct and RT-LAMP

Panel member	Strain	Panel member	Strain
Influenza A H1N1	A/New Caledonia/20/99	Coronavirus NL63	N/A
Influenza A H3	A/Brisbane/10/07	Coronavirus 229E	N/A
Influenza A 2009 H1N1pdm	A/NY/02/09	Coronavirus OC43	N/A
Influenza B	B/Florida/02/06	Coronavirus HKU-1	N/A
Metapneumovirus 8	Peru6-2003	M. pneumoniae	M129
Respiratory Syncytial virus A	N/A	C. pneumoniae	CWL-029
Rhinovirus 1A	N/A	B. Pertussis	A639
Parainfluenza virus Type 1	N/A	Adenovirus Type 31	N/A
Parainfluenza virus Type 2	N/A	Adenovirus Type 1	N/A
Parainfluenza virus Type 3	N/A	B. parapertussis	A747
Parainfluenza virus Type 4	N/A	Negative	N/A
Adenovirus Type 3	N/A

3. Diagnostic sensitivity and specificity

Clinical validation with confirmed positives and negatives

3.1 Samples selected for the validation were representative of the range of low and high viral loads to avoid increasing or lowering diagnostic sensitivity and specificity. Table 6 illustrates the sample sets used for the validation included spiked saliva, lighthouse laboratory (Milton Keynes) and clinical laboratory contributions from each of the study sites. The number and range of viral loads of positive samples found within each of the evaluations is shown in table 7. See section 6 for data sources for both tables.

Table 6: provenance of samples used for validation

	Spiked samples	Clinical samples: lighthouse laboratory	Clinical samples: study sites
RNA swab	n/a	72	12,505
RNA saliva	n/a	105	12,372
Direct swab	n/a	72	487
Direct saliva	66	67	7,790

Table 7: range of viral loads for validation samples

CT Range RNA swab LAMP	Sample number (n)
CT <25	107
CT <33	180
CT <45	212

CT Range RNA saliva LAMP	Sample number (n)
CT <25	56
CT <33	106
CT <45	111

CT Range direct swab LAMP	Sample number (n)
CT <25	113
CT <33	182
CT <45	199

CT Range direct saliva LAMP	Sample number (n)
CT <25	88
CT <33	166
CT <45	226 (59 were spiked saliva samples)

3.2 Diagnostic sensitivity: confirmed clinical samples from patients (positive RT-qPCR result) were compared across platforms. The CT values or equivalent for both the assessed and comparator assays were included in the submitted validation data.

• 212 positive swabs analysed by RNA RT-LAMP
• 111 positive saliva samples analysed by RNA RT-LAMP
• 199 positive swabs analysed by Direct RT-LAMP
• 226 positive saliva samples analysed by Direct RT-LAMP

3.3 Diagnostic specificity: confirmed clinical samples from patients (negative RT-qPCR result) were used. The CT values or equivalent for both the assessed and comparator assays were included in the submitted validation data.

• 12,365 negative swabs analysed by RNA RT-LAMP
• 12,366 negative saliva samples analysed by RNA RT-LAMP
• 360 negative swabs analysed by Direct RT-LAMP
• 7,697 negative saliva samples analysed by Direct RT-LAMP

3.4 The diagnostic sensitivity (DSe) and specificity (DSp) derived from section 3.2 and 3.3 are as follows:

3.4.1 DSe and DSp (95% Confidence Intervals (CI)) for all samples (including clinical and spiked samples) with a CT <45 are summarised below:

• RNA RT-LAMP on swabs: DSe 95% (CI 0.91-0.97); DSp 99% (CI 0.99-1.00)
• RNA RT-LAMP on saliva: DSe 80% (CI 0.71-0.97); DSp 100% (CI 0.99-1.00)
• Direct RT-LAMP on swabs: DSe 70% (CI 0.63-0.76); DSp 100% (CI 0.98-1.00)
• Direct RT-LAMP on saliva: DSe 79% (CI 0.73-0.84); DSp 100% (CI 0.99-1.00)

3.4.2 To inform potential use cases for Direct RT-LAMP in applications for identification of medium to high viral loads in upper respiratory tract / salivary samples, it is also informative to illustrate the DSe and DSp with a RT-qPCR cut off of <25 and <33:

• <25 Direct RT-LAMP on swabs: DSe 100% (CI 0.96-1.00); DSp 100% (CI 0.98-1.00)
• <25 Direct RT-LAMP on saliva: DSe 94% (CI 0.87-0.98); DSp 100% (CI 0.99-1.00)
• <33 Direct RT-LAMP on swabs: DSe 77% (CI 0.70-0.82); DSp 100% (CI 0.98-1.00)
• <33 Direct RT-LAMP on saliva: DSe 83% (CI 0.75-0.89); DSp 100% (CI 0.99-1.00)

4. Verification of assay performance in real world asymptomatic mass testing studies

Hospitals programme

4.1 In response to nosocomial COVID-19 clusters, the Direct RT-LAMP assay was used for whole hospital testing of all staff at Royal Hampshire County Hospital, Winchester. Testing was undertaken between 16th – 23rd October 2020. Over the course of 7 days, 3563 asymptomatic staff were identified and approached for testing. Of those approached 3314 samples were processed at the NHS laboratory in the Basingstoke and North Hampshire Hospitals, part of the same trust. Across the 3314 samples analysed over the 7 days, 2 positives were identified. These positive samples were confirmed by RT-qPCR. The information from the testing exercise was then integrated into the ‘Test and Trace’ programme and infection control system of the trust.

Community programme

4.2 In parallel to the NHS asymptomatic pilot programme, a DHSC supported Direct RT-LAMP asymptomatic study in the Southampton community has been in operation. This pilot project has utilised the Animal and Plant Health Agency (APHA) laboratory, testing samples collected from asymptomatic participants in hospital, university and school settings. As of the 5th November 51,022 saliva samples had been tested by Direct RT-LAMP, with 64 positives. All the positive Direct RT-LAMP samples were confirmed by RT-qPCR, with all 64 confirmed, representing CT values ranging from 17.4 to 34.

5. Conclusions

5.1 The technical performance of the OptiGene RNA RT-LAMP assay on swabs demonstrated a sensitivity of 95% (CI 0.91-0.97) and specificity of 99% (CI 0.99-1.00) in comparison to current standard of care RT-qPCR testing after RNA extraction, performed on viral oropharyngeal and nasopharyngeal swabs taken from symptomatic individuals (CT <45). These data support a potential application for this alternative nucleic acid amplification technology after RNA extraction, in diagnostic testing.

5.2 When the OptiGene Direct RT-LAMP assay is evaluated using saliva samples, without RNA extraction (Direct RT-LAMP) the sensitivity is 79% (CI 0.73-0.84) and specificity 100% (CI 0.99-1.00) across all samples tested (CT <45). The sensitivity increases with viral load and when the CT <33 the sensitivity is 83% (CI 0.75-0.89) and specificity 100% (CI 0.99-1.00) and when the CT <25 the sensitivity is 94% (CI 0.87-0.98) and specificity 100% (CI 0.99-1.00).

5.3 Across a variety samples types including spiked saliva, saliva and swab samples from the lighthouse laboratory pillar 2 service for symptomatic infection, clinical samples from pillar 1 laboratories and daily saliva samples from the symptomatic pilot programme, the performance of RT-LAMP demonstrated excellent specificity and sufficient sensitivity to be used as part of the infection control strategy for SARS-CoV-2.

5.4 The aim of an asymptomatic healthcare worker testing service is to detect unsuspected COVID-19 and thereafter to reduce the risk of SARS-CoV-2 transmission to other workers, to patients and to workers’ families. The vulnerability of patients is a key issue here, and such testing offers the opportunity to support infection control measures that are already in place. An early detection system could also support staff workforce resilience and so maintain NHS capacity.

5.5 Within a use case scenario of regular, frequent, testing of asymptomatic individuals within a population (for example healthcare worker staff group) for SARS-CoV-2, the ability to rapidly and accurately identify individuals with high levels of infective virus to allow for their prompt isolation will be an important contribution to reducing transmission of infection. Direct RT-LAMP has been successfully applied in this use case to populations of healthcare workers with the aim of curtailing transmission between staff and to patients within healthcare settings. The NHS asymptomatic staff saliva testing pilot successfully facilitated daily on-site testing and subsequent broader, hospital and community, programmes reinforcing the utility of Direct RT-LAMP as an additional test to support SARS-CoV-2 detection.

5.6 With the addition of an RNA extraction step to the amplification testing technology it has comparable results on swabs to RT-qPCR, thereby adding additional diagnostic capacity. In the Direct mode, for asymptomatic NHS staff testing, it complements the existing mixed economy of Lateral Flow Devices and Point of Care testing solutions. Overall LAMP technology adds depth and resilience to the existing suite of tests within an integrated testing landscape.

5.7 The further development of the RT LAMP technology will need to consider any further challenges of scaling across differently sized organisations, the adoption of a new technology in clinical laboratories and systems that are unaccustomed to this nucleic acid amplification approach and address the information technology integration that is needed to support a quality assured clinical service.

5.8 The diversification and stratification of the SARS-CoV-2 testing technologies and their applied stratification by defined use case testing scenarios in either clinical diagnostic or public health population-based interventions promises to provide additional capacity and capability to the emerging use case scenarios of testing as a public health intervention measure, however the scaling and other operational challenges are well recognised.

6. Additional data

Local verification reports

6.1 The OptiGene RT-LAMP assay has been locally verified in 9 sites, with the activities of each site listed below.

Site	RT-LAMP evaluations
Hampshire Hospital Foundation Trust (Basingstoke and North Hampshire Hospitals site)	Optimisation and original validation of RNA and Direct RT-LAMP protocols Limit of detection and analytical sensitivity (ASe) and specificity (ASp). RNA RT-LAMP as routine screening tool Direct RT-LAMP in decentralised settings (Lab Van) Direct and RNA Saliva and Swab RT-LAMP Asymptomatic Staff Pilot. Direct and RNA Saliva and Swab RT-LAMP pair saliva/swab lighthouse lab evaluation.
University Hospital Southampton	RNA RT-LAMP Asymptomatic Staff Pilot. RNA RT-LAMP Lighthouse laboratory sample evaluation. Analytical specificity.
Animal and Plant Health Agency/ MRC Lifecourse Epidemiology Unit (University of Southampton)	Direct Saliva RT-LAMP Mass Population Screening.
Manchester University Foundation Trust	Direct and RNA Swab and Saliva RT-LAMP Asymptomatic Staff Pilot.
Leeds Teaching Hospital NHS Trust	Direct Swab RT-LAMP Evaluation under CONDOR.
Institute of Cancer & Genomic Science University of Birmingham	Direct and RNA Saliva and Swab RT-LAMP Asymptomatic Staff Pilot. Direct and RNA Saliva and Swab RT-LAMP pair saliva/swab lighthouse lab evaluation.
Division of Virology at Porton Down	Direct and RNA Swab RT-LAMP as a surrogate for infectious virus recovery.
Public Health University Laboratory, Gibraltar Health Authority	Direct and RNA Swab and Saliva RT-LAMP for population-based screening in a UK overseas territory as an alternative to RT-qPCR.
Hampshire Hospitals NHS Foundation Trust (Royal Hampshire County Hospital, Winchester site)	Direct and RNA Swab RT-LAMP in an acute hospital setting without an on-site microbiology laboratory. Limit of detection and analytical sensitivity and specificity.

Sample provenance by site

6.2 Data tables showing sample provenance by site and by methodology are available on request from paul.chambers@dhsc.gov.uk.