Validation of COVID19 diagnostic testing by LCMSMS [COVID-19]
Research type
Research Study
Full title
Validation of COVID19 diagnostic testing by liquid chromatography mass spectrometry
IRAS ID
296024
Contact name
Holly Gettings
Contact email
Sponsor organisation
Guy's and St Thomas' NHS Foundation Trust
Duration of Study in the UK
0 years, 2 months, 10 days
Research summary
Research Summary
COVID testing by LC-MS/MS is part of the DHSC ‘Operation Moonshot’ programme which is a collaboration between industry, academia and the NHS. A novel method to detect COVID-19 has been developed using liquid chromatography mass spectrometry (LC-MS/MS). The initial validation indicates that this method compares well to existing PCR based methods. The next stage is validation of the method in a clinical laboratory and UKAS accreditation. The LCMSMS test offers a number of advantages; it relies upon a different supply chain to the existing COVID tests; it will be provided by a different group of scientists (biochemists) thus relieving pressure on Virology staff; it has high throughout with a single instrument being able to analyse approx. 1000 samples each day.
To translate the LC-MS/MS test from the academic laboratory and into a NHS clinical laboratory, we urgently need access to additional samples to validate the new method and gain UKAS accreditation.
This application is a request to collect an additional combined nose and throat swab and a saliva sample from patients presenting to the Emergency Department who are already having a routine nose and throat swab collected for SARSCoV-2 PCR testing. The research swab and saliva samples will be collected into an ethanol buffer which deactivates the virus at the point of collection, thus simplifying the logistics of sample handling and processing. The samples will be pseudoanonymised and there will be no impact on the clinical care of the patient, nor will the research results be returned to the participant. There will be no impact on standard of care for the participants as a result of this study.Summary of Results
Executive summary.
Methodology
The detection of SARS-CoV-2 by liquid chromatography tandem mass spectrometry (LC-MS/MS) is an antigen test based upon the identification of three target peptide sequences derived from the nucleocapsid (NCAP) protein. A respiratory sample is collected on a combined nose and throat swab and placed in viral transport medium (VTM). The protein present in the VTM is denatured prior to digestion with trypsin which cleaves the NCAP into its characteristic peptide fragments, three of which, AYNVTQAFGR (AYN), ADETQALPQR (ADE) and NPANNAAIVLQLPQGTTLPK (NPA) are then isolated via Stable Isotope Standard Capture AntiPeptide Antibody (SISCAPA), prior to separation and detection by LC-MS/MS. The Waters RUO SARS-CoV-2 reagent kit was used to prepare all samples.
The test is essentially qualitative. Although the target peptides are quantified in each patient sample against synthetic peptide calibration curves, this is simply a measurement of the amount of each target peptide injected ‘on column’. It is not possible to relate the amount detected ‘on column’ to an in vivo concentration because the amount of biological material collected from the respiratory swab is uncontrolled and depends upon the efficacy of the sample collection process. From the UKAS perspective, the test is considered semi-quantitative because in addition to a visual assessment, numerical results are used to assist with the classification of test results as SARS-CoV-2 detected/not detected.Method comparison
Method comparison was performed against samples (n=315) analysed in Virology, Viapath on a two-step PCR assay, the AusDiagnostics SARS-CoV-2, Influenza and RSC 8-well panel using the High-Plex 24 System (REF 91501). This assay utilises a multiplex-tandem polymerase chain reaction (MT-PCR) for the enrichment of targets and then amplification of targeted RNA. It should be noted that the LC-MS/MS and MT-PCR methods are not directly comparable. One measures viral RNA, the other measures peptides derived from nucleocapsid protein. Furthermore, PCR is very sensitive and detects the presence of viral RNA (with PCR the virus is detected by targeting one or more gene fragments). The gene fragment might be detected and the virus ‘positively’ found but whether that RNA represents infectious virus is unclear. It is possible that because of the long-duration of this ‘RNA-positive tail’ some of the infected people in the validation may have been identified after the infectious period had passed i.e. PCR testing has poor specificity when used during this phase.
Concordance testing showed near total concordance with the AusDiagnostics MT-PCR assay (Cohen’s Kappa = 0.8315 across all samples and 0.9572 across samples with take-off value < 20) and passed acceptance criteria set at a minimum of 0.8 (above a level of good to excellent).
The LC-MS/MS method has a diagnostic sensitivity of 75.0% (95% CI 62.3 – 84.5) and a diagnostic specificity of 100% (95% CI 98.5 – 100.0). For samples with a take-off value < 20, the method has 92.9% (95% CI 81.0 – 97.5 sensitivity and 100% (95% CI 98.5 – 100.0) specificity.Analytical Performance Characteristics
Analytical performance of the three target peptides was satisfactory.
The formal limit of quantitation is 2.5 attomol/µL for ADE (AYE), 5.0 attomol/uL for AYN and 10.0 attomol/uL NPA.
Repeatability was measured by replicate analysis (n=5) of three levels of IQC material (negative, low positive, high positive) across five different days.
Qualitatively, the negative IQC material was consistently classified as SARS-CoV-2 not detected (25/25). AYN, AYE and ADE were negative on 25/25, was NPA negative on 24/25.
Qualitatively, both the low positive and high positive IQC material was consistently classified as SARS-CoV-2 detected (25/25).
For completeness the analytical sensitivity was also determined quantitatively on these materials. Replicate analysis (n=5) of the three levels of IQC material (negative, low positive, high positive) across five different days gave co-efficient of variation of 9.6%, 8.0% and 11.2% in the high IQC material for AYN, AYE and NPA respectively and 18.9%, 26.4% and 19.1% in the low IQC material for AYN, AYE and NPA respectively.
The assay demonstrates analytical specificity for other viral respiratory pathogens with no cross reactivity from Influenza A, Influenza B or Rhinovirus.REC name
Wales REC 3
REC reference
21/WA/0101
Date of REC Opinion
18 Mar 2021
REC opinion
Favourable Opinion