Molecular Diagnostics Adapts to Evolving Diseases

Biological insights and bioengineering innovations are advancing the development of assays for infectious diseases, cancers, and rare diseases

Recognizing disease through early and accurate detection of genomic and proteomic biomarkers in molecular diagnostic (MDx) assays is a key capability for clinical pathologists, public healthcare experts, pharmaceutical developers, and basic researchers. Clinical decision-making, therapeutic outcomes, and personalized medicine approaches are bolstered by advances in MDx technology, including bioengineering innovations and insights on pathogenesis and immune response. In the evolving landscape of MDx technology, diagnosticians must keep abreast of emerging assays and be mindful of pitfalls in individual assays to avoid errors in interpretation.

Infectious disease

Many infectious agents result in overlapping symptoms. For instance, COVID-19, influenza, and respiratory syncytial virus manifest as respiratory symptoms. This complicates MDx for physicians who must narrow down potential causes before ordering tests. Syndromic testing circumvents such complications by testing for a broad array of pathogens associated with sets of clinical symptoms, using multiplexed panels. Syndromic RT-PCR panels are being increasingly integrated into routine clinical care for patient stratification and epidemiology.

Syndromic tests pose unique challenges. They include the selection of specific targets among potentially related pathogens, the integration of new tests into established clinical laboratory practice, and the continuous adaptation of tests to newer waves of circulating pathogens. Another challenge is the design of panels that incorporate highly selective primers and probes for various pathogens, and that generate target-specific signals.

Arvind Kothandaraman
Arvind Kothandaraman

“Assays are optimized such that multiplexing does not compromise the accurate detection of individual targets,” says Arvind Kothandaraman, managing director of specialty diagnostics, PerkinElmer.

With MDx assays, the sensitivity, specificity, and number of pathogens that can be detected in a single syndromic panel depend on several variables, including extraction buffer systems, primers, enzymes, and expertise in sample preparation. “The extraction system plays a major role,” Kothandaraman stresses. “We are fortunate at PerkinElmer that the high quality of the raw materials used in our chemagen technology increases the sensitivity of the data that we obtain for each pathogen.” Additionally, strict quality control at a system level enhances data reliability.

The convenience of syndromic MDx assays over individual assays is greater than one might initially expect. In addition to lowering reagent costs and utilizing existing laboratory expertise, syndromic MDx assays save time. “If clinical laboratories can identify active infections on day zero, individuals can seek help, isolate, and stop the virus from spreading,” Kothandaraman declares.

Although the cost of next-generation sequencing (NGS) has been decreasing consistently, RT-PCR is still cheaper, Kothandaraman insists. Costs aside, the higher level of expertise in instrumentation and data analysis required for NGS makes RT-PCR the better option for known pathogens. However, for detecting novel pathogens, NGS remains the only option.

“We saw this in action during the pandemic, where a percentage of the samples were screened using NGS to monitor for emerging variants,” Kothandaraman notes. “As costs go down and people gain expertise, NGS will be a game changer for zero-day pathogen detection.”

In addition to the detection of pathogens, infectious disease MDx assays also encompass the assessment of host immune responses against pathogens. Virax Biolabs is developing a test that indicates whether an individual’s T cells will respond to a virus, should they become infected. T cells in the body actively scan cell surfaces for foreign protein fragments and trigger an immune response if they detect ones they’ve encountered earlier.

Tomasz George,
Tomasz George, PhD
Virax Biolabs

“We’ve developed a method to analyze proteins on the outside of the virus for their ability to activate T cells in the body,” says Tomasz George, PhD, chief scientific officer, Virax. “We’ve mapped these proteins to form a peptide mix with which to treat your T cells outside the body to see how you would cope with these viruses if you were to become infected in the future.” The test, slated for release in 2023, detects several T-cell activation markers.

Virax’s first immune test detects SARS-CoV-2, but George is confident that the test can be rapidly adapted for new viruses. Based on a subscription model and an app, the company hopes to stay ahead of emerging viruses in the Northern Hemisphere by adapting the test each year for highly prevalent viruses in the Southern Hemisphere during winter, and vice versa. “Most viruses are more prevalent and riskier in winter than in summer,” George remarks.

A combination of stable and rapidly mutating peptides works best for such immune tests, but most peptides in Virax’s first test detect stable peptides to identify cross-reactive T cells. “Looking at stable regions inherent to a family of viruses, we can see if you’ve developed T cells to, for instance, a common cold in the past that can cross-react with a new variant of the coronavirus,” George asserts.

The primary roadblock in developing such MDx tests is the speed at which they can be adapted. Flexibility in experimental design and availability of reagents determine whether a test can be developed before new viruses spread.

Gerald Hunter
Gerald Hunter, PhD
Fortis Life Sciences

Refrigeration of MDx reagents poses obstacles in storage and transport that are being overcome through lyophilization. “Lyophilized reagents are stable until resuspended,” says Gerald Hunter, PhD, field application scientist, Fortis Life Sciences. “Lyophilization increases shelf life at ambient conditions and cost savings as diagnostic manufacturers no longer have to pay for cold chain handling. Lyophilized reagents improve workflow efficiency by having reagents preserved indefinitely after receipt, minimizing the need to thaw a frozen set of reagents prior to use.”

The applicability of Virax’s MDx test is not limited to individuals who must know how they would respond to viruses to make informed decisions on vaccinations, social distancing, personal protective equipment, or travel. The test is also of use to pharmaceutical companies that must select participants with less adaptive immunity to a particular virus, to estimate efficacy of experimental drugs in compromised populations.

“It provides a purer treatment and control group for clinical trials,” George maintains. “We also want to integrate our test within government and healthcare groups worldwide to help identify populations more susceptible to a virus to guide public health policy.”


Detection of pancreatic cancer relies largely on clinical suspicions and imaging findings. Accurate diagnostic tests not only enable optimal treatment, but also help avoid overtreatment and unnecessary surgery of noncancerous cysts.

Aatur D. Singhi
Aatur D. Singhi, MD, PhD
Univ. Pittsburgh

“Although pancreatic cancer has the highest mortality rates of all cancers, it’s relatively rare. Therefore, screening or surveillance protocols are not available,” says Aatur D. Singhi, MD, PhD, associate professor of pathology, University of Pittsburgh. “Most investigators focus on identifying high-risk populations who have a family history of pancreatic cancer, a genetic predisposition, or preconditions such as new-onset diabetes or pancreatic cysts.”

PancreaSeq GC, an MDx test developed by researchers at the University of Pittsburgh, outperforms current imaging-based testing guidelines by separating common, benign pancreatic cysts (such as serous cystadenomas), which have zero malignant potential, from intraductal papillary mucinous neoplasm, which is a precursor to pancreatic cancer. In fluid samples aspirated from pancreatic cysts, the NGS-based test detects a panel of genes associated with gastrointestinal malignancies. The NGS approach makes the test nearly 20-fold more sensitive than a test based on Sanger sequencing, at only a 10th of the cost of imaging-based assays.

“The idea is to maximize sensitivity while maintaining high specificity, so that when we make a call of cancer you can be sure there are no false positives,” Singhi explains.

Not all pancreatic cysts become cancerous. Therefore, PancreaSeq GC is designed to detect two types of somatic mutations—alterations that indicate the type of cyst, and those that indicate the presence of early pancreatic cancer in the cyst.

“We need to start generating a lot more data on patients with pancreatic cysts, especially since they are very common,” Singhi points out.

Earlier studies on pancreatic cysts and cancer have been largely retrospective, and so they have been yielding associative and not causative inferences. “Moving forward, we must integrate our understanding and test patients early, up front, and follow them to understand the ramifications of genetic alterations,” Singhi emphasizes. “There’s huge bias in retrospective testing. In our prospective tests, we found alterations associated with pancreatic cysts that we didn’t even know about earlier.”

Incorporating data from the prospective study, PancreaSeq GC was upgraded from a 22-gene to a 74-gene panel that is now available worldwide.

Rare diseases

Patients with rare, untreatable diseases often opt out of MDx tests. Such diseases include frontotemporal dementia (FTD), which causes progressive nerve cell loss in the brain’s frontal and temporal lobes. FTD, which actually refers to a group of disorders, affects behavior and language. It is frequently misdiagnosed for other neuropsychiatric disorders. Nearly 40% FTD patients have a family history of dementia, but only a fraction of these have known mutations in progranulin, tau, and C9ORF72.

Andrea C. Bozoki,
Andrea C. Bozoki, MD
UNC-Chapel Hill

“When we talk about finding biomarkers, we’re mostly talking about byproducts of those genetic mutations,” says Andrea C. Bozoki, MD, professor of neurology, University of North Carolina, Chapel Hill. She emphasizes the importance of identifying biomarkers that become abnormal so early that fluorodeoxyglucose–positron emission tomography scans are not yet diagnostic.

FTD patients with progranulin mutations show accumulation of TDP43 in the brain. “Other neurodegenerative conditions also cause abnormal deposition of TDP43,” Bozoki observes. “Currently, we have no way of determining it in the brain or in spinal fluid assays. This leaves a gap in our ability to identify patients with FTD.”

Gary Romano
Gary Romano, MD, PhD

A therapeutic that targets progranulin is being developed by Alector. The company’s chief medical officer, Gary Romano, MD, PhD, says that “the day may come when this treatment becomes an incentive for patients to have the genetic test.” Alector uses PCR to detect mutations in the granulin gene that encodes progranulin since RT-PCR levels can fluctuate considerably.

Good molecular markers for identifying disease are close to the root of the pathological mechanism. In the absence of such biomarkers, diagnosticians must settle for biomarkers that are steps removed from the target, in the hope that such biomarkers suffice to give a true reflection of disease states.

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