PathogenDx’s involvement with Operation Warp Speed, demonstrated the efficacy of microarray technology for diagnostic testing that kept up with an evolving, emerging disease.

 By Chris Wolski

The COVID-19 pandemic quickly placed diagnostic testing at the forefront of the response to the worldwide health emergency. While treatment and vaccine development took center stage during most discussions of ending the pandemic, companies, including PathogenDx, were pivoting to develop testing responses to help bolster pandemic-era disease mitigation strategies and keep tabs on the ever-evolving SARs-CoV-2 virus. Prior to the pandemic, the company offered molecular-based testing for cannabis, botanical, food, and agricultural industries. In the wake of the pandemic, PathogenDx was selected, as part of Operation Warp Speed, to receive funding to scale its D3 Array technology to develop a COVID test and variant testing.

That test eventually received FDA emergency use authorization (EUA). It is currently available today.

CLP recently discussed how PathogenDx made this pivot, how its D3 Array technology works, and its upcoming tests currently in development with company Co-founder and CEO Milan Patel. Prior to co-founding PathogenDx, Patel spent over 25 years working with large public, small private, and entrepreneurial companies in numerous fields from the life sciences, to biotechnology, to government services, and the automotive industry.

Patel’s responses have been edited for length and clarity.

CLP: The COVID pandemic caused you to pivot your business—can you describe how you became involved with Operation Warp Speed and what role PathogenDx played in it?

Milan Patel: During the onset of the COVID-19 pandemic, the U.S. Federal Government created Operation Warp Speed (OWS) to look for innovative, disruptive technologies that could scale up COVID-19 testing to address the pandemic. PathogenDx’s D3 Array technology—a new generation microarray—perfectly fit the innovative category, and was applicable to COVID diagnostic testing, so we decided to apply to this program.

Under the OWS partnership is the National Institute of Health’s (NIH) Rapid Acceleration of Diagnostics Initiative, also known as RADx. The RADx initiative is a national call for scientists and organizations to bring their innovative ideas for new COVID-19 testing approaches and strategies to come together and address the pandemic. Over 3,000 companies applied to the program and went through a “shark tank”-type of presentation. PathogenDx made it through the first round and two additional rounds of NIH and RADx committee reviews to ultimately be accepted into a group of less than 40 companies to be funded by the U.S. Federal Government to scale their respective technologies. Besides scaling up to a capacity of 750,000 tests per month, PathogenDx was able to get an FDA Emergency Use Authorization (EUA) cleared test on the market.

Our D3 Array technology tested variants much faster, and was easier and more cost effective than the sequencing technology that was being used at the time. The Detectx-Cv was specifically launched for variant testing and became the most easily deployable variant test in the market. With more than 180 probes in its single well microarray, the test covered the ability to detect single polynucleotide polymorphisms (SNPs) spanning over a range of 1,000 base pairs of the S-gene. The test was able to identify not just historical variants we have all seen, but new and upcoming variants by activating the probes representing specific gene mutations within 10-15 minutes after the simple flip of a software switch. Detectx-Cv required significantly less updates compared to qPCR assays because of the make-up of the microarray content. Detectx-Cv’s built-in capacity of incorporating as many possible combinations of gene mutations across 180 different probes minimized the need for major upgrades to the test when compared to qPCR assays—which required more than 30 different assays to cover the ranges of mutations across the S-gene. With this, PathogenDx delivered the fastest, simplest, and lowest cost variant test in the market, helping the U.S. deliver a “watchdog” technology for the next pandemic.

CLP: Can you give an overview of your microarray technology and how it works?

Patel: PathogenDx’s technology is a high-throughput, low-cost, multiplexing solution based on a patented low-to-medium density microarray. The technology can interrogate up to 108+ targets in a single reaction well – delivering single gene sensitivity and SNP level resolution. The D3 Array is a new generation microarray, and is completely different from the architecture and construct of past microarray technologies. We have overcome technical, throughput, performance, and cost challenges that previous generations of microarray technologies have been encumbered by. The front end of the platform is sample agnostic, allowing any type of sample matrix to be used to extract purified DNA through a simple process of heat and enzymes. The technology is agnostic and can run on any molecular PCR based equipment and is not tied to a specific OEM manufacturer. Flexibility is built in so that smaller labs can process samples manually, and higher throughput labs have an option of automation depending on the volume. The amplification step is either RT-PCR, PCR or Isothermal NASBA. In the case of COVID-19, an asymmetric RT-PCR step is performed on the extracted DNA, which is then amplified to its endpoint. Within the amplification step, a Cy3 Fluorophore is labeled to the amplicon. The output of this is single-stranded DNA (ss-DNA) is then applied to the microarray to hybridize. The hybridization is conducted at room temperature. No purification or heat denaturation is needed for hybridization. The value of this step is the rapid time in which it obtains specificity at SNP resolution when compared to other microarray technologies. Next, the hybridized plate is washed to remove unbound and unused Cy3 labeled primers or amplified products. The last step is automated imaging and analysis. In this step, a plate reader labels hybridization signals from each of the microarrays, and the software analyzes all the probes in each of the microarrays to determine a result and produce a report. This entire process is conducted in less than five hours and within a normal lab shift.

The sheer power of this technology and platform is exhibited through its ability to have 96 separate samples each fill a well in a 96-well microarray plate. The full horsepower and extent of the number of reactions the PathogenDx technology can deliver can be as high as 28,800 qPCR reactions in a single plate, with each well in that plate conducting 324 individual PCR tests per sample. The ability to completely “flatten” the multiplex hurdle when you are looking to interrogate and analyze dozens of targets in a sample is incredibly valuable in today’s molecular testing world, whether it be syndromic testing in infectious disease or dozens of gene mutations in cancer diagnostics.

CLP: So, what are some of the implications for your microarray technology as we learn to “live with” SARS-CoV-2 and other emerging viruses and their variants? In other words, how are you providing labs, clinicians, and patients an edge in dealing with new and emerging diseases and their variants?

Patel: In light of potential risk to vaccines and therapeutics, PathogenDx’s Detectx-Cv assay is able to discriminate between the sub variants, and especially sub-omicron variants. We continue to update the software of the assay as needed, providing labs and clinicians the critical information they need from an epidemiological perspective as well as a public health perspective. While next generation sequencing (NGS) is a phenomenal discovery tool, NGS was unable to deliver critical, timely information to make a difference to the spread of COVID-19. It simply is not able to keep up with tracking mutations across the public health spectrum. The PathogenDx technology not only has the ability to track mutations, but is also able to deliver a real-time surveillance capability for public health for the next pandemic.

CLP: One of the issues facing a lot of labs during the pandemic and beyond was the lack of testing materials—how could your microarray technology help labs leverage their testing materials—providing both test results in a timely manner and for a more cost-effective result?

Patel: First of all, all of the PathogenDx assays are manufactured and assembled in the United States, so we are not dependent on an overseas supply chain. We have dual-sourced not just the microarray printing, but our reagents as well. Secondly, because our technology is not feeding off the same supply chain as companies that have developed and marketed qRT-PCR, we have the assurance of supplying our assays to our labs who then can provide results on a timely basis. In addition, the multiplexing nature of the technology uses only a fraction of the enzymes and reagents to run the test, while producing results for 10x-100x more targets, making it more cost-effective than qPCR or NGS.

CLP: What’s next—do you have any other tests in development? When will they be ready for the market?

Patel: We do. We are developing assays in clinical diagnostics that we plan to launch in 2023. We plan to launch tests for infectious diseases, urinary tract infections, and sexually transmitted infections. These are ideal based on the number of microbial targets and antibiotic resistance markers needed for clinicians to make diagnostic and effective treatment calls. These tests will completely redefine multiplex testing with our D3 Array technology. What we have invented, developed, and commercialized is the perfect “sweet-spot” technology that sits between qPCR and NGS; which resolved some of the hindrances and pain points of both technologies. There are numerous markets in clinical and human diagnostics that need to look at dozens of targets with superior sensitivity and SNP level resolution, deliver the result within a shift of receiving the sample and meet the economic needs of CLIA testing labs and fit under reimbursement codes. At PathogenDx, we’re thrilled with the innovations we’ve delivered to the market and excited with what’s to come in the near future.

Chris Wolski is chief editor of CLP.