FDA Mulls Guidelines for NGS-Based Infectious Disease Diagnostics

SAN FRANCISCO (GenomeWeb) – Since releasing draft guidelines in 2016 on next-generation sequencing-based infectious disease diagnostics, a US Food and Drug Administration sponsored workgroup has been gathering stakeholder input and considering how to move forward to regulate such tests.

Traditionally, diagnosis of infectious diseases has relied on culturing the organism. Testing for antimicrobial resistance was a separate step involving trying to grow the pathogen in the presence of various compounds.

Now, however, "NGS has the capability to replace all these previous methods with a single approach and accomplish what might have required several steps in one step," Heike Sichtig, a principal investigator of microbiology and chair of the FDA working group, said.

Over the last several years, NGS has become more common as a tool used to study outbreaks. For instance, the New York State Department of Health now uses whole-genome sequencing for influenza surveillance, and researchers at the University of Birmingham in the UK used nanopore sequencing to track the West Africa Ebola outbreak in 2014.

And, in the UK, Public Health England has even begun using sequencing as its standard diagnostic for tuberculosis and plans to move other infectious disease testing to NGS platforms.

In the US, while many academic groups and companies are developing NGS-based infectious disease tests, none have been brought through FDA clearance. The laboratory-developed tests offered by companies such as Fry Labs, IDbyDNA, and Karius, and academic groups like the University of California, San Francisco, use a broad range of NGS-based methods, from targeted 16S approaches to shotgun metagenomic sequencing, as well as a test that analyzes circulating cell-free microbial DNA.

Sichtig said that the FDA decided to get involved because although such tests are proliferating and hold great potential to improve on existing infectious disease diagnostics, "there are no existing comprehensive standards that would be applicable to these tests. And, the FDA thinks these standards are needed to provide assurance of the safety and effectiveness of such tests."

The working group includes representatives from the FDA's Center for Devices and Radiological Health, the Center for Biologics Evaluation and Research, and the Center for Drug Evaluation and Research. In addition, Sichtig said, the group has reached out to other stakeholders with clinical expertise as well as expertise in the various technologies for sample prep, sequencing, and bioinformatics.

Sichtig declined to disclose where the group is in the process of finalizing the guidelines but said that it is still working through comments and feedback it received since releasing the draft guidelines.

"Much of the feedback we've received has called on the FDA to streamline our NGS policies," Sichtig said. The stakeholders highlighted that library prep and bioinformatics, in particular, are the current hurdles in NGS technology, she added.

Although the FDA has already approved some NGS instruments and assays to analyze human germline and somatic mutations, looking at clinical infectious disease samples is a significantly different application and the FDA felt it required separate regulations, Sichtig said.

One key difference between using sequencing to diagnose a genetic disease versus an infectious disease is the importance of very fast and highly accurate tests for infectious diseases. "A delayed or incorrect result can result in a fatality," Sichtig said.

The working group's discussions thus far have focused less on the sequencing itself and more on the challenges in the wet lab and on the bioinformatics side. One challenge for NGS-based infectious disease testing is the fact that clinical samples typically contain very limited quantities of pathogen DNA. The vast majority of the sample is human DNA, and microbial DNA will include both the pathogen of interest as well as nonpathogenic organisms.

Another complicating factor is the bioinformatics process. To diagnose a genetic disorder with sequencing, there is one human reference genome for comparing sequencing results, Sichtig said. But, for infectious disease testing, the pathogen can be one of many different microbes, each with its own reference genome and potential variants, she added.

In addition, she said, while the increasing amount of genomic microbial data is ultimately helpful in making more accurate calls, the constantly changing databases can prove problematic in the short term. For instance, she said, a recent paper published to the preprint server BioRxiv by researchers from the University of Maryland and the National Human Genome Research Institute found that as the RefSeq database has grown over time, accuracy has declined. They concluded that there is a need for algorithms that can handle larger databases.

In part to address this issue, the FDA has launched the PrecisionFDA CDRH Biothreat challenge for algorithm developers. Participants are tasked with analyzing 21 metagenomic samples, including 15 mock clinical samples and six in silicosamples, and coming up with accurate results. Participants have access to the FDA database ARGOS, which includes regulatory grade reference microbial genomes.

By using a fixed database for the challenge, the goal is to really do an accurate head-to-head comparison of algorithms, Sichtig said. And the results serve to help future developers by giving them a reference dataset and database against which they can benchmark computational workflows. Currently, she said, it's difficult to compare assays and algorithms since the companies and groups developing tests use a mixture of both public databases and proprietary ones, and the makeup of those databases changes. "And accuracy depends on the reference database," she said.

Another challenge the workgroup is considering are differences between metagenomic sequencing assays and targeted sequencing assays and whether and how to regulate those differently.

Sichtig said that targeted assays have a lot of similarities with multiplex assays, for which there is already a regulatory pathway. For panel-based tests, "we can probably adapt some of the regulatory approaches that have already been developed," Sichtig said, but "for agnostic metagenomic shotgun sequencing, when you don't have an idea about what microbe you're looking for, it's more challenging to come up with a regulatory framework."

The group is also taking into account the fact that assays likely include more than just diagnosing the pathogen, but also provide information about antimicrobial resistance.

Sichtig said that the group is considering both the possibility of having different guidelines for the diagnosis portion and the antimicrobial resistance profiling portion, and of including both portions in one regulatory pathway. "These are open challenges," she said.

"We want to, as much as possible, streamline and make use of policies that are in place, and at the same time really tailor it to the challenges of infectious disease testing," she said.

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