NEW YORK – A team of researchers from Rice University have developed a point-of-care test for human papillomavirus that combines isothermal amplification and lateral flow technology.
The team described the development and validation of the assay in a paper published this week in Science Translational Medicine. The test uses recombinase polymerase amplification to amplify the presence of DNA from two HPV genotypes, combined with lateral flow technology to produce a visual positive or negative result, said Kathryn Kundrod, a former postdoctoral researcher in the bioengineering department at Rice and one of the authors on the paper.
Running the test requires treating the swab sample, which can be collected either by a doctor or by the patient, with a lysis enzyme and then centrifuging the sample after lysis to limit the interference of cellular debris. The DNA is then diluted and added to a chamber containing a mixture of RPA reagents where it is amplified. Afterward, the tube is put into the lateral flow cartridge, and results are returned in 15 minutes. Including the centrifugation and sample preparation steps, the entire process takes about 45 minutes, Kundrod said.
The integration of the amplification tube and the lateral flow cartridge also avoids potential environmental cross-contamination that could impact the test's accuracy and lead to false positive results, she noted.
Once the test was developed, the researchers validated it in a high-resource setting, a laboratory in Houston, and a low-resource setting, a hospital laboratory in Mozambique. For samples with at least 1,000 copies of HPV 16 DNA per reaction in the Houston group, sensitivity was 100 percent, positive predictive value was 86 percent, and negative predictive value was 56 percent. For samples with at least 500 copies of HPV 16 DNA per reaction, the test's sensitivity was 93 percent. Kundrod noted that because there were few HPV 18 samples in the group, the team only determined the test's specificity, which was 100 percent. The overall percent agreement between the Rice test and the comparator test, Roche's Cobas HPV assay, was 85 percent.
She added that the discordant samples missed by the Rice test in this group all had very low copy numbers, which the team is "not particularly worried about missing" since the high copy number samples are the most important to catch.
The Mozambique validation group had slightly different results, with some of the discordant samples having high cellular content in addition to low copy numbers. Kundrod partially attributed this to the fact that all of the samples in that cohort were self-collected. The variations between clinician-collected and self-collected samples show a "need to incorporate a cellular control" into the test to catch any false negatives due to high cellular content, which the team is actively working on.
In that cohort, the test showed 80 percent sensitivity in HPV 16 samples with cycle threshold (Ct) values less than 30, indicating high DNA copy numbers. In HPV 16 samples with Ct values less than 35, which includes slightly lower concentrations of DNA, the test's sensitivity was 83 percent. Specificity was 89 percent, positive predictive value was 73 percent, and negative predictive value was 85 percent. In HPV 18 samples, specificity was 98 percent and negative predictive value was 85 percent.
One primary use case for this test is for HPV screen-and-treat programs in low-resource settings, Kundrod said. Although centrifuging is relatively common even in those settings, the researchers are developing instrument-free sample preparation methods to remove the need for centrifuging altogether and further cut down the cost.
In addition to new sample preparation methods and cellular controls, the other major next step to make the test clinically useful is adding other HPV genotypes beyond HPV 16 and HPV 18. The two included in this test account for approximately 70 percent of cervical cancer cases, Kundrod said, but the team would like to add three to five more genotypes to make the assay more robust. To do that, the team has been working on an approach that would separate the amplification tube into multiple chambers that would contain reagents for different genotypes.
One of the key complexities in adding further genotypes is ensuring the amplification reagents for each genotype don't interfere with each other, so physically separating the reagents would alleviate that issue. It would be "a really similar overall technology, just with a couple of additional, comparable parallel reactions happening within the same test," she said.
The researchers hope to validate the test in larger, more diverse clinical trials to ensure the test works in diverse populations and with multiple sample types. They have also worked with an outside company to optimize the platform and ensure it is manufacturable on a larger scale when the time for commercialization comes, Kundrod said.
In her view, the test's major benefits are the lack of complexity, the contamination-free format, and the lower cost compared to commercially available tests. Outside of the cartridge itself, the Rice test requires only a heat block and centrifuge – both of which are available for about $500 each, she said. The cartridge and reagents are estimated to cost about $5 total.
The comparator tests used in the validation studies, 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 assay, Abbott's RealTime High Risk HPV assay, and Becton Dickinson's Onclarity HPV assay, run on those companies' own platforms, although those tests detect additional HPV genotypes beyond HPV 16 and HPV 18. Qiagen also offers its CareHPV test for limited resource settings, which uses the firm's Hybrid Capture 2 technology to detect 14 high-risk HPV genotypes.
The researchers' goal is for the test to be used in settings that would allow for actionable results within the time frame of one appointment, such as in a community health clinic or hospital that can run the test and provide treatment based on the result in one fell swoop, Kundrod said.
"What we envision really is the whole continuum of care happening in the context of one appointment closer to where people live, in their own communities, to really increase access," she said.