The technologies that power molecular diagnostics are rapidly evolving to help overcome some of the challenges associated with utilization of these tests, including improving their clinical utility. This article will look at some of the companies that are leveraging novel technologies, including image analysis, sample preparation, shotgun sequencing, and microsatellite technology to improve MDx.

The rapidly advancing technologies supporting molecular diagnostics are poised to change —possibly even revolutionize—medical diagnostics and treatment for the better. At the very least, these improvements are making diagnostic assays a more useful tool in a broader range of clinical paradigms. Here’s a look at some technologies that are helping to buoy an improvement in molecular diagnostics assays.

Shotgun metagenomic sequencing of pathogens

Aperiomics offers a comprehensive genomics-based test that identifies every known microorganism present in a patient’s sample. Each sample is screened for about 40,000 microorganisms—including bacteria, viruses, parasites, and fungi—using shotgun metagenomic next-gen sequencing. Aperiomics updates their test several times a year to add newly discovered, newly sequenced microbes.

Currently, about 75% of infections remain undiagnosed, often just treated with broad-spectrum antibiotics. “Millions of people are living with chronic infections because current tests failed to get a clinically actionable answer,” says Crystal Icenhour, CEO of Aperiomics. The company's strategy is to identify everything by sequencing so that doctors have more information to make better clinical decisions. One patient, for example, had been in chronic pain for decades and differentially diagnosed with Lyme disease. Aperiomics’ testing found that not only did she not have Lyme disease, but she had extrapulmonary tuberculosis, a chronic phase of tuberculosis that few doctors would recognize because it seldom becomes chronic in the U.S.

In addition to pathogens, Aperiomics assesses the normal flora of the human body, otherwise known as the microbiome. “We're looking for everything in the sample that's not human, and asking whether it’s in the right place, and in the right amount,” says Icenhour. For example, it is normal to find E. coli in the intestines, but not in the urinary tract (where it causes urinary tract infections). Similarly, E. coli is a small percentage of healthy gut microbes, but is not normal at much higher levels. “We [assess location and abundance] to help put context around the information, so that the healthcare provider can understand what in the report is clinically actionable,” she says.

Looking forward, Icenhour hopes that Aperiomics’ technology can become the standard of care when an infection is suspected, instead of a trouble-shooting tool. “New research is linking disruptions of the microbiome to chronic conditions such as obesity, diabetes, Alzheimer’s, Parkinson’s, and autism,” says Icenhour. “Using our test as the first line of defense has great potential to dramatically reduce the number of these chronic conditions.”

Microsatellite instability and cancer

Promega’s MSI Analysis System is a research use only tool for detecting microsatellite instability (MSI), a form of genomic instability linked to a dominant hereditary cancer called Lynch Syndrome, as well as others. (The MSI Analysis System is research use only, but over the last 15 years, clinical labs have elevated it to lab developed test (LDT) status.) The PCR-based MSI Analysis System uses fluorescently labeled primers for microsatellite markers in multiplexed PCR reactions. Microsatellites are short segments of repeated DNA sequences that occur throughout the genome. Microsatellite instability (MSI) is caused by insertions or deletions that occur during DNA replication. Normally, DNA’s mismatch repair system fixes these mistakes, but when it doesn’t, MSI can ensue.

Knowing the MSI status of a tumor is extremely valuable. For cancers classified as microsatellite instability high (MSI-H), MSI testing can point to appropriate treatments. For example, patients with MSI-H tumors, which include many common types of solid tumors, may benefit from immune checkpoint inhibitor therapy. “Promega MSI Analysis System has been utilized in many pan-tumor clinical trials being conducted around the world, supporting approval of several immunotherapeutics in over 16 tumor types,” says Heather Tomlinson, director of clinical diagnostics at Promega. Promega is also collaborating with Merck to develop their MSI technology as an on-label, solid tumor companion diagnostic for use with Merck’s anti-PD-1 therapy KEYTRUDA® (pembrolizumab).

In contrast to MSI-H, Promega’s MSI Analysis System can also identify tumors as microsatellite stable (MSS). Tomlinson says she sees an increasing number of drugs being developed specifically to treat MSS cancers. “As drugs for MSS cancers enter clinical trials, there is an evolving need to evaluate the MSI status of a tumor as standard practice at the time of cancer diagnosis,” she says. Such a standard practice would make it clear which type of treatment will be more effective for a newly diagnosed cancer patient.

Starting early with sample preparation

Advances in sample-preparation methods are also improving molecular diagnostics. Ceres Nanosciences’s Nanotrap® particles concentrate analytes of interest in a patient sample, such as viruses, bacteria, or nucleic acids. This results in a higher quality sample for subsequent molecular diagnostic assays.

“The biggest challenges in using molecular diagnostic tests today are low concentration of analytes, rapid degradation of analytes, presence of interfering substances, and invasive sample collection requirements,” says Robbie Barbero, chief business officer at Virginia-based Ceres Nanosciences. “The Nanotrap particle technology addresses all of these by capturing, concentrating, and preserving low abundance analytes.”

The technology’s advantage is evident when used to capture and concentrate low abundance viruses in a sample, before extracting nucleic acids for detection. When used to prepare samples for molecular diagnostic assays, the Nanotrap Virus Capture Kit improves the detection of respiratory pathogens such as influenza, RSV, and coronavirus; flavivirus pathogens such as Zika, dengue, and chikungunya; and other viral pathogens.

Affordability with PCR-based assays

Enzo’s PCR-based AMPIPROBE® platform offers cost-effective assays. Their technology uses PCR primers labeled with fluorescent reporters and quenchers that, when incorporated into newly copied DNA, result in a decay of sample fluorescence. The signal decay indicates the amount of DNA or RNA of interest present in the sample.

Enzo uses the AMPIPROBE technology in the detection and quantification of HBV and HCV viral load. Their latest addition to the AMPIPROBE platform is the Women’s Health Panel, which detects 16 pathogens with a single swab. These tests have been approved by the New York State Department of Health as laboratory-developed tests (LDTs). This gives Enzo the ability to run LDTs at more affordable prices, because it can manufacture all the reagents through its GMP facility.

Lorenzo Uccellini, business development manager at Enzo, notes that it is increasingly difficult for clinical laboratories to make a profit. Labs can lower costs by making their own tests, but only if they don’t use expensive third-party materials. “While reimbursement of tests is declining drastically, the cost of reagents to run tests is increasing every year, and not all laboratories have the ability to make affordable reagents to create their own LDT,” he says. The AMPIPROBE technology allows Enzo to cut costs by 30–50% per test.

In the future, says Uccellini, “molecular diagnostics must be centered around a combination of affordability, flexibility, and automation.”

New Ceramic Film Enhances Fluorescent Signals

Late last year, Nanopec, a precision medicine materials science company based in Tucson, introduced a novel ceramic film that can significantly boost fluorescent signals in molecular diagnostics. With this capability, MetaFluorex can help decrease the probability of a patient receiving the wrong diagnosis because the fluorescent signal is too weak or the biomarker concentration too low. The unique substrate can also help reduce reagent use.

According to CEO Mario Blanco, MetaFluorex requires fewer antibodies to achieve superior signal to noise ratios than comparable diagnostics on modified substrates (including frosted, patterned, nitrocellulose, or dielectric modified glass slides). In initial testing, MetaFluorex enhanced fluorescent signals by 3 to 30 times, depending on the assay, when compared to the glass slides while keeping noise levels down.

The very small (50 nm), equal size pores in the ceramic film resemble a honeycomb structure, which facilitates forward lensing of the fluorescence signal by acting as nano-waveguides. In addition, the films are made without cytotoxic materials and are safe to use in the presence of living cells.

"MetaFluorex achieves fluorescence SNR>20 without the need for additional electronics, optical filters, analytical software, or chemical reagents, which add undue complexity to existing LDTs," Blanco says.

Hero image: Influenza virus, Igor Zakharevich, Dreamstime.com.