NEW YORK – A clinical evaluation study has called Oxford Nanopore Technologies' LamPore sequencing assay a "promising method" for high-throughput SARS-CoV-2 diagnostic testing as the company is awaiting regulatory approval for the test, but the authors recommended improvements to the workflow.
The test, first announced in May, runs on Oxford Nanopore's GridIon and MinIon nanopore sequencing platforms, and the company said it expects to receive the CE mark for LamPore in the near future. Obtaining regulatory approval would allow the firm to sell the assay for clinical use in Europe and other countries that accept the CE designation.
Last month, a consortium of researchers from the University of Oxford's Nuffield Department of Medicine, Public Health England Porton Down, and the University of Sheffield published a preprint of a retrospective clinical validation study of the LamPore assay in MedRxiv, demonstrating its potential for high-throughput diagnostic testing.
But the test may need to undergo further development before it can be implemented in such a setting, the authors cautioned. "Obviously for this to be used on a large scale it needs to have a streamlined workflow, ideally including automation, and as with all tests would be more appealing if we could also use it on saliva and without RNA extraction," said Leon Peto, corresponding author of the study and a researcher at Oxford University Hospitals NHS Foundation Trust and the Nuffield Department of Medicine at the University of Oxford, in an email.
"Their sensitivity and specificity look very promising, and since it uses extracted RNA, a logical next step might be to avoid the RNA purification altogether," said Chris Mason, associate professor in the department of physiology and biophysics at Weill Cornell Medicine, who was not involved in the study. His lab has developed a colorimetric LAMP SARS-CoV-2 assay, which they published as a preprint this past spring. He pointed to Yale's SalivaDirect test as a successful example of an RNA-extraction-free molecular SARS-CoV-2 test.
Peto added that RNA extraction is currently a bottleneck for SARS-CoV-2 testing at Oxford University Hospital's own laboratory. He said his team is working on some of these improvements for LamPore, as are other laboratories. "As soon as we have any more data, we'll make it public," he wrote.
Oxford Nanopore declined to provide additional comment on the test, its commercialization, and expected improvements at this time. In August, the company said that under an agreement with the UK's Department of Health and Social Care, it will make an initial 450,000 LamPore tests available to several UK National Health Service testing laboratories. It also said it is working on a version of LamPore that tests for additional pathogens, including influenza A and B and respiratory syncytial virus.
LamPore combines loop-mediated isothermal amplification (LAMP) with nanopore sequencing and has the potential to analyze thousands of samples a day on a single Oxford Nanopore sequencing device – up to 2,000 on the MinIon Mk1C and 15,000 on the GridIon, according to the company. It joins a variety of other molecular SARS-CoV-2 assays that have replaced PCR with LAMP, employing a variety of readout methods, such as pH-sensitive dyes.
For their validation study, Peto and his colleagues compared the LamPore assay side by side with RT-PCR using the Altona Diagnostics SARS-CoV-2 RT-PCR assay and an in-house RT-PCR assay running on an Applied Biosystems 7500 RT-PCR system.
To be able to analyze multiple patient samples on a single sequencing run, LamPore introduces DNA barcodes into the LAMP products during sample preparation. At the moment, 96 samples can be combined on a single flow cell, they wrote. The workflow includes 40 minutes of amplification, a library preparation step of unspecified duration, and a 60-minute sequencing run, which they wrote is comparable to an RT-PCR workflow that starts with extracted RNA.
The study analyzed viral RNA from two sets of samples: a saliva sample set that was spiked with SARS-CoV-2 RNA and a clinical sample set from Oxford University Hospitals and from Sheffield Teaching Hospitals consisting of previously tested nose or throat swabs.
Based on the spiked saliva samples, the researchers found the limit of detection for LamPore to be seven to 10 genome copies per microliter RNA, more than three times higher than that of the RT-PCR assays, which could detect two copies per microliter. However, they wrote, "this did not correspond to a significant loss of diagnostic sensitivity in the clinical samples."
Mason said that the detection limit shown for LamPore in this study places it near the middle of the pack of current SARS-CoV-2 tests that have received Emergency Use Authorization from the US Food and Drug Administration, including most RT-PCR tests.
The UK team also found LamPore to be 99.1 percent sensitive, detecting 226 of 228 samples that tested positive with RT-PCR, and 99.6 percent specific, providing negative results for 278 of 279 RT-PCR-negative samples, including 153 that had other respiratory pathogens detected by PCR. A small fraction of samples, 1.4 percent, yielded indeterminate results on first testing with LamPore.
The test was highly reproducible, giving identical results in 478 of 494 samples, or 96.8 percent, that were tested twice by LamPore.
The clinical samples came from a time period earlier this year when testing was limited to symptomatic individuals, who likely had a high viral load. People carrying less virus, where testing with RT-PCR had a high cycle threshold, or Ct value, might not test positive with the LamPore assay, according to the authors. "Our data suggest that LamPore is most likely to miss weak positive samples with Ct values above 35, so could have had lower diagnostic sensitivity if tested in such groups," they wrote.
"However, this may not be a significant practical disadvantage, as although weak positives have some value for contact tracing, they are likely to come from individuals with low infectious potential."
Going forward, establishing an RNA-extraction-free protocol for LamPore would "greatly streamline the workflow, but further evaluation is required," they wrote.
"We also did not evaluate how the throughput and turnaround time of LamPore would compare to RT-PCR during routine use in a clinical laboratory or centralized testing center," they wrote, adding that for testing tens or hundreds of thousands of samples per day, a streamlined workflow that includes automated sample handling and integration with a laboratory information management system would be required.
"LAMP has been used successfully for COVID-19 diagnostics since February, and I am glad to see another application of it here," Mason said.