NEW YORK – Sample preparation is a major bottleneck in any diagnostics workflow, restricting most rapid point-of-care tests to simple sample and target types. Researchers at Vanderbilt University have now created an extraction-free method that, when paired with a previously developed PCR technology, can be used to detect nucleic acids of malaria parasites in whole blood with enough accuracy to enable profiling of drug resistance genes.
In a study recently published in the Journal of Molecular Diagnostics, the Vanderbilt team found that the method could discriminate between wild-type Plasmodium falciparum and a chloroquine-resistant mutant in the presence of whole blood, a typical PCR inhibitor.
Drug resistance is a growing problem for malaria treatment and control in some parts of the world, particularly in Southeast Asia. A PCR test is generally the most sensitive assay to detect any infectious disease, but it is not the easiest to execute, said Mindy Leelawong, lead author on the study.
In remote settings — with few labs, and limited instrumentation — using dried blood spots for malaria testing is typically the only option, Leelawong said in an interview. But once the DBS gets to the lab, the DNA extraction step still takes about one hour per sample, as does the PCR.
To make PCR-based testing faster, the technique described in the JDM article uses a sample prep technique that Leelawong developed in the lab of Frederick Haselton, a bioengineer at Vanderbilt. It also employs a PCR technology developed in the lab that relies on mirror image DNA, or L-DNA, called Adaptive PCR.
Leelawong surmised that the presence of PCR inhibitors and autofluorescence are two of the major problems limiting the use of whole blood in point of care settings. The method she developed first uses an OmniTaq 3, an inhibitor-tolerant polymerase developed by DNA Polymerase Technology, to address the inhibition issues.
And, because the optical interference from whole blood seems to occur in the lower end of the wavelength spectrum, the method also opts for higher-wavelength dyes that are not typically used in diagnostics. Specifically, it uses the reporter dyes Cy5 and TEX615, both on the red end of the wavelength spectrum.
The technique also uses LNA probes to enable SNP genotyping of P. falciparum directly from patient blood without DNA extraction.
The sample prep method Leelawong developed could also be adapted to be used with other PCR-based systems, she said, but the Adaptive PCR has advantages in speed and sensitivity. "The strengths play off each other," according to Leelawong, in particular because both technologies are designed for low-resource settings.
The Adaptive PCR-based instrument that the Vanderbilt researchers built has also been replicated in other sites and run successfully, and it is portable and easy-to-use, she said. To use the sample prep technique most effectively on other instruments, they would need to be adapted for continuous reading of the fluorescence, rather than the typical once-per-PCR cycle, she added.
In an interview, Haselton said that future directions for further development include perfecting the instrument and assays. "We'd now like to take what we've learned about instrument design and make it even smaller, and more portable," he said, adding that tests with no requirement for DNA extraction, like the malaria test, complement the point-of-care capabilities of the Adaptive PCR platform.
While others working on genetic resistance tests that can potentially be used at the point of care — such as Aldatu Biosciences, MolBio, and Genedrive plc — have tended to focus on HIV or tuberculosis, the Vanderbilt group chose to develop a resistance test for malaria, as opposed to other organisms, in part because Leelawong had prior experience with the pathogen. Drug-resistant malaria is also a growing problem globally, she said.
The previous generation of malaria treatment, chloroquine, was a potent drug until resistance developed in Southeast Asia. The resistance has spread to Africa, and now the drug can't be used there, Leelawong said.
Meanwhile, artemisinin, a potent antimalarial therapy, is currently being used frequently, but resistance to the drug is already being seen in some patients in Southeast Asia, she said. "What we want to do is avoid having another generation of a very powerful antimalarial rendered less useful," she said.
Other researchers have also used inhibitor-tolerant polymerases to expedite sample prep in whole-blood testing. For example, Stephanie Yanow at the University of Alberta School of Public Health has used polymerases from DNA Polymerase Technology for a whole-blood-based malaria PCR test. The method was later incorporated into a lab-on-a-chip diagnostic test on a platform called Accutas and is being commercialized by startup Aquila Diagnostic Systems.
The findings in the Vanderbilt group's JMD study "provide a new approach" to genotype the SNPs associated with drug-resistant malaria directly from blood samples, Yanow said in an email.
"The idea of using probes labeled with fluorophores that have a distinct emission spectra from blood overcomes a major obstacle in using probe-based detection methods with clinical samples in the absence of DNA extraction," Yanow said.
And, the Adaptive PCR instrument seems to have the potential to be developed for use in low-resource settings, where there is an urgent need to conduct molecular surveillance to track and contain drug-resistant parasites, she said.
"We need to expand our toolbox to support these efforts, and the methods presented in this paper offer an alternative strategy to PCR and DNA sequencing," Yanow added.
Yanow and her team are also developing assays for direct detection of SNPs associated with drug-resistant malaria, particularly targeting the SNPs in the kelch 13 gene that are associated with artemisinin resistance. Her group is currently preparing a manuscript that describes a SYBR Green-based assay to detect these SNPs directly from blood using similar reagents to those described in the JMD paper, she said. The technology has also been adapted to detect dengue virus directly from blood, and tested on a portable instrument. Yanow and her team have recently completed a field trial of the malaria diagnostic in a rural setting in India in partnership with Aquila Diagnostics.
Haselton noted that the Vanderbilt group has filed for patents on the Adaptive PCR method and is always looking for collaborators. The L-DNA method is also being commercialized in collaboration with a startup company called Mirror Molecular. The group is also now working on publishing a study applying the Adaptive PCR method to virus detection.
The Vanderbilt researchers are funded by the National Institutes of Health Small Business Technology Transfer (STTR) program in collaboration with a Murfreesboro, Tennessee-based company called BioVentures. Together, the collaborators were granted approximately $280,000 in Phase I funding and $2.2 million in Phase II funding in 2017 to develop PCR reaction additives and simplified instrumentation that enable single-tube diagnostics at the point of care.