HPV nucleic acid detection made into POCT, only 45min
A team of Rice University researchers has developed a point-of-care assay for human papillomavirus that combines isothermal amplification and lateral flow technology.
The team describes the development and validation of the assay in a paper published this week in the journal Science Translational Medicine. Kathryn Kundrod, a former postdoctoral researcher in Rice's Department of Bioengineering and one of the authors, said the test uses recombinase polymerase amplification to amplify DNA from two HPV genotypes, combined with lateral flow technology to generate A visual positive or negative result. on the paper.
Running the test requires treating a swab sample (which can be collected by a doctor or patient) with a lysing enzyme and then centrifuging the sample after lysis to limit interference from cellular debris. The DNA is then diluted and added to the chamber containing the RPA reagent mix, where it is amplified. The tubes are then placed in a lateral flow box and results are returned 15 minutes later. The entire process, including the centrifugation and sample preparation steps, takes about 45 minutes, Kundrod said.
The integration of amplification tubes and lateral flow cassettes also avoids potential environmental cross-contamination, which could affect the accuracy of the test and lead to false-positive results, she noted.
Once the test was developed, the researchers validated it in resource-rich settings (a lab in Houston) and resource-poor settings (a hospital lab in Mozambique). For samples in the Houston group with at least 1,000 copies of HPV 16 DNA per reaction, the sensitivity was 100%, the positive predictive value was 86%, and the negative predictive value was 56%. The test was 93% sensitive for samples containing at least 500 copies of HPV 16 DNA per reaction. Because of the small number of HPV 18 samples in this group, the team only determined the specificity of the test, which is 100 percent, Kundrod noted. The overall percent agreement between the Rice test and the control test (Roche Cobas HPV test) was 85%.
The discordant samples missed by the Rice test in this group all had very low copy numbers, and the team "wasn't particularly concerned about misses," she added, because high-copy number samples are the most important to catch.
The Mozambique validation set had slightly different results, with some discordant samples having high cellular content in addition to low copy number. Kundrode attributes this in part to the fact that all the samples in the cohort were self-collected. The discrepancy between the clinician-collected samples and self-collected samples suggested "the need to include a cell control in the test" to catch any false negatives due to high cellular content, an issue the team is actively investigating.
In this cohort, testing showed a sensitivity of 80% for HPV 16 samples with a cycle threshold (Ct) value of less than 30, indicating a high DNA copy number. In HPV 16 samples with Ct values less than 35, where the DNA concentration was slightly lower, the detection was 83% sensitive. The specificity was 89%, the positive predictive value was 73%, and the negative predictive value was 85%. In HPV 18 samples, the specificity was 98% and the negative predictive value was 85%.
A major use case for the test is HPV screening and treatment planning in low-resource settings, Kundrod said. Although centrifugation is relatively common even in these settings, researchers are developing instrument-free sample preparation methods to completely eliminate the need for centrifugation and further reduce costs.
In addition to the new sample preparation method and cell controls, another major next step in making the test clinically useful is adding additional HPV genotypes beyond HPV 16 and HPV 18. The two genotypes included in the test account for about 70 percent of cervical cancer cases, Kundrod said, but the team hopes to add three to five more genotypes to make the test more robust. To that end, the team has been working on a way to divide the amplification tube into chambers containing reagents for different genotypes.
One of the key complexities of adding more genotypes is ensuring that the amplification reagents for each genotype do not interfere with each other, so physically separating the reagents will alleviate this problem. It would be "a very similar overall technique with some additional, comparable parallel reactions occurring in the same test," she said.
The researchers hope to validate the test in a larger, more diverse clinical trial to ensure that it works across different populations and across multiple sample types. Kundrod said they are also working with an external company to optimize the platform and ensure that it can be produced on a larger scale when commercialization arrives.
In her view, the main advantages of the test are that it is less complicated, less polluting and less expensive than commercial tests. In addition to the kit itself, the Rice test requires only a heating block and centrifuge, both of which cost about $500 each, she said. Cartridges and reagents are estimated to cost about $5 in total.
The comparative tests used in the validation study, Roche's Cobas HPV test in Houston and Cepheid's point-of-care Xpert HPV test in Mozambique, require a Cobas instrument and a GeneXpert instrument, respectively. Many other HPV tests, such as Hologic's Aptima HPV test, Abbott's RealTime High Risk HPV test, and Becton Dickinson's Onclarity HPV test, run on these companies' own platforms, although these tests detect Other HPV genotypes. Qiagen also offers the CareHPV test for limited resource settings, which uses the company's Hybrid Capture 2 technology to detect 14 high-risk HPV genotypes.
The researchers aim to use the test in settings where actionable results can be obtained within the time frame of a single appointment, such as in a community health clinic or hospital where the test could be administered and treatment delivered based on the results of a single appointment. "Swoop," said Kundrod.
“What we really envision is providing the entire continuum of care in one appointment closer to where people live, their own communities, to really increase access to services,” she said.
