Harvard University and Akonni Biosystems have teamed up to develop and test an amplification array-based diagnostic for multi-drug-resistant and extreme-drug-resistant tuberculosis.
The work is one of several projects that will be supported by a five-year, approximately $29 million Centers of Excellence for Translational Research awarded to Harvard by the National Institute of Allergy and Infectious Diseases to establish a translational research center to develop molecular diagnostic technologies for TB.
As reported in Genome Web Daily News, NIAID also awarded similar funding to a project out of Columbia University.
The CETR award intends to stimulate academic and industry partnerships, and calls for the development of a commercial product at the end of the grant.
The Harvard team, led by Megan Murray at the School of Public Health, will lead a TB bioinformatics and sequencing project, as well as proof-of-principle experiments on resistance mutations uncovered.
Akonni will integrate these new targets into an entirely closed consumable, based on its amplification microarray technology. The groups will also collaborate to optimize sample preparation, particularly cell-free DNA from urine, to improve diagnosis of TB in children.
In an interview with PCR Insider, Murray explained that this work will build upon results her group recently published in Nature Genetics. In that study, they looked at the evolution of MDR and XDR TB, using "an evolutionary analysis method to try to detect selection pressure in the parts of the phylogenetic tree in which drug resistance had occurred," she said.
"We found that when you do targeted sequencing of drug-resistant strains you can't really explain all the resistance," Murray said. Thus they will focus, in part, on strains that have resistance profiles but no known drug resistance mutations. The project will also include whole-genome sequencing to more broadly understand the portions of the TB genome most likely to lead to resistance.
The center grant also allocates funds for a basic science lab to do mutagenesis experiments through a process of allelic exchange and recombineering, Murray said. Here, mutations will be inserted into TB strains, queried, and quantified for drug resistance and fitness. This will be an extension of a proof-of-principle experiment in their Nature Genetics paper, in which they "took one mutation out of the many hits ... put it into a wild-type strain, and tested it and showed that it actually did have an increase in its mean inhibitory concentration to rifampicin," one of the first-line TB drugs, Murray said.
In parallel with this scientific work, Murray said that the industry partner, Akonni, will develop the diagnostic. She said that according to the National Institutes of Health's call for submissions, "there is a more distal piece to [the funding], where it's supposed to go to [a] usable product," adding, "we don't really do products."
Murray said she had been in discussions with Akonni for many years. "Ever since I started working in this area about a decade ago, we were looking for a tool that's going to be able to pick up all the mutations that we think are relevant to choosing the best drugs to treat people as soon as they're seen. It did seem, even back then, that a microarray was going to offer advantages in the diversity of targets." She said the conversations began when she called Akonni "out of the blue" upon learning that they were developing array technologies for TB diagnostics.
Akonni CSO Darrell Chandler agreed. "There's an inherent synergy between the nature of drug resistance in TB and the capability of microarrays to address the problem," he said. The Harvard team's approach to detecting resistance mutations is unique, he said, and he believes it will have some staying power. He added that Murray is "outstanding in the field, and has an excellent grasp on the nature of the problem and where a technology like Akonni's microarrays can fit in and add value, so there is synergy at that level as well."
Akonni has previously received funding to apply its amplification microarray technology to an MDR-TB diagnostic. The new project differs in format, Chandler said, with previous work being "a high-complexity manual workflow and set of consumables" and the new project "a medium-complexity closed amplicon consumable."
Both Murray and Chandler pointed to Cepheid's GeneXpert as a model for this kind of sample-to-answer diagnostic development. Subsidies from non-governmental organizations and endorsement from the World Health Organization might enable commercialization of the diagnostic, although Chandler asserted that they are simplifying workflow for the device and moving to manufacturing with plastics such that "the cost of the consumable approaches the price point that the TB community expects in the absence of any subsidies."
However, while the Cepheid device is useful for triaging patients, sending them for further drug sensitivity testing, or quarantining them, "it doesn't actually tell you what drugs to treat that person with today," Murray said. "You have to start another process of repeating the drug resistance testing, and that can take weeks to months. We think, even with the advances of the last few years, there's still not a tool that's doing what we need it to do, which is to help us make decisions about how to treat patients when we see them."
The proposed diagnostic will include Akonni's TruTip sample preparation, which has recently been shown to enrich cell-free fetal DNA. The project also includes optimizing nucleic acid enrichment for pediatric TB diagnosis.
According to Murray, TB in children can have a more subtle presentation than in adults. Also, for unclear reasons "kids tend not to be able to cough up sputum," she said. The project will assay other biological fluids from infected children to determine whether these can be used for TB diagnosis.
Both Murray and Chandler are particularly interested in determining whether fragments of TB cfDNA can be amplified from urine in pediatric cases. Chandler said that there is precedent in the literature for transrenal recovery of DNA. "If it is possible for us to adapt TruTip to extract TB DNA out of urine and detect markers of drug resistance from that DNA, that would be a significant breakthrough in and of itself," he said.
Another advantage to this partnership will be the international pilot testing. Chandler pointed out that the Harvard team had been "working at international sites and has collaborators at international locations prepared and set up to receive prototype instruments and assays from Akonni, and test them in a real clinical use environment, before we at Akonni have to go through the expense of a full-on clinical trial," he said.
This aspect of the project will include clinical work in Peru, which Murray said has been an ongoing collaboration for the past seven years. "In the context of that we have recruited and followed 4,000 patients, about 11 or 12 percent of [whom] have multi-drug resistant TB. We have all these strains archived, and the drug resistance phenotypes and the patient outcomes are very well described," she said.
The amplification microarray will be based on one described in the journal Microarrays in 2012, Chandler said. A PCR master mix containing purified DNA, primers, buffer, and dNTPs is loaded over the top of a gel element array. Each gel element, or spot, on the array contains a unique probe for one or more genes, and one or more resistance-conferring mutations, and each array typically has about 200 spots.
The microarray is subjected to thermal cycling, and amplified targets hybridize to the microarray. Chandler said that "for the Harvard project, the microarray will contain an integrated waste chamber, so the entire process is closed." After wash, the arrays are imaged through the coverslip, and automated software analyzes the image and reports the results. This will be combined with automated TruTip sample prep into a single sample-to-answer system. "The footprint will be slightly larger than a laptop computer, [and] the power requirements are such that it could be run off a battery, if necessary," he said.
He also added that the amplification reagents and chemistry will be in a dry pellet form within the array, so that they can be stored at room temperature. This will solve the cold chain storage problem common in low resource settings, and make the consumable more useful in places with limited access to electricity.
Chandler said the company will work toward obtaining an ISO13485 certificate with this project, as well as CE marking prior to further clinical trials.
There is no exclusivity arrangement in terms of the Harvard research, and Murray said she hopes the basic science "will inform not only Akonni's tool, but any other tool that wants to diagnose TB using drug resistance mutations, whether that's going directly to whole-genome sequencing or targeted sequencing, or any one of a thousand possible tools that could be used this way," she said. "We will make this data public and hope that as many different companies use it as possible, because we'd love to see competitive tools out there so people have choices."
The ultimate goal, she said, is to both "improve clinical outcomes for patients by treating them earlier and to have an impact on the epidemic of TB by interrupting transmission."