Emil Salazar Anal Kalorama Information

Fast-Evolving Pathogens Elude Standard Tests. But They May Be Run Down by New NGS Assays.

If molecular diagnostic tests are to keep pace with fast-evolving microbes, they will have to find an “extra gear.” Existing gears—single pathogen tests and multiplexed panels—have their advantages, mostly with respect to specificity. But specificity is of little help when one is chasing a moving target, as one is obliged to do when typing pathogens or profiling resistant strains. To keep up with such elusive quarry, molecular diagnostics may need to shift to next-generation sequencing (NGS).

Already, NGS is moving into the clinical laboratory, improving the responsiveness of disease control in healthcare settings, and promising to advance the personalization of patient therapy. And now NGS is poised to go yet farther, nimbly bypassing obstacles that have slowed molecular testing’s progress.

Specificity Goes a Long Way, but…

Molecular assays that consist of carefully selected primers and probes are powerful tools, but they lack adaptability once they enter use in the majority of clinical labs. The fluid genetics of particularly virulent and antimicrobial-resistant strains and clones are at times capable of escaping the specificity of selected molecular probes and primers.

To overcome this problem, which is a matter of narrow analytical scope, established molecular assays such as real-time PCR (or quantitative PCR) can incorporate multiplexed reagents. The benefit of multiplexed panels, particularly in the case of critical infections, is the positive identification of the infectious agent with shorter turnaround than repeat single-pathogen tests.

Despite issuing calls for larger panels, labs are well aware that such panels come with drawbacks. These include higher test product prices and undesirable selections of panel targets. Multiplexed molecular assays must often balance panel breadth with accuracy and clarity of results. False positives can result from the inclusion of too many targets, and sensitivity may be compromised.

Despite the success of multiplexed molecular assays, the inherent specificity of PCR and microarrays—still a competitive strength of molecular diagnostics over immunodiagnostics and traditional microbiology—can also prove to be a weakness, particularly in more complex applications. Assay kits that serve detection purposes are often incapable of further pathogen characterization such as genotyping or resistance profiling.

NGS, however, has demonstrated it can overcome the limitations of other assays. NGS has flexibility. It can detect any number of genetic variants. Also, NGS assays can do without the level of target definition required by other molecular assays, and still deliver accurate multiplexing. And so, NGS can offer clinicians profound capabilities—namely, rich results for clinical and epidemiological use.

Where Conventional Assays Fall Short, NGS Rises to the Occasion

Specificity is molecular diagnostics’ major strength, but is also a weakness when assay development isn’t caught up with microbial adaptation. NGS can overcome this limitation with the flexibility to detect any number of genetic variants.

Next-generation sequencing will not replace and has yet to compete with common molecular diagnostics such as PCR. The latter remains effective through syndromic panels that screen patients for common and likely pathogens based upon presented symptoms. The same multiplexed test or follow-up can additionally detect common resistance genes and established marker sequences for virulence and high-risk strains.

The specificity of PCR assays, however, makes them less suitable for clinical epidemiology and personalized medicine. These are two significant areas of health spending that are growing because of the unfortunate challenge of healthcare-acquired infections (HAIs).

Early NGS Assays

Multiplexed HAI assay kits remain largely unavailable for the clinical market, but some are emerging using NGS. Among the leaders in the NGS space for clinical infectious disease testing and antimicrobial resistance surveillance is BioInnovation Solutions (formerly Pathogenica). The company’s CE-IVD-marked HAI BioDetection Kit is multiplexed for the 12 most common nosocomial infections and 15 drug-resistance genes.

The assay kit uses hundreds of probes to amplify loci of interest. Amplicons are compiled in a library and subsequently sequenced and analyzed by means of a bioinformatics package. Sequencing results are referenced against databases for species, strains, substrains, and resistance genes.

Approved for clinical diagnostic use in Europe, the HAI BioDetection Kit is an excellent representation of the duality and unique value of clinical sequencing. The kit not only informs individual patient treatment, it also serves as a tool for disease control and surveillance in a healthcare setting. The detailed results provided by the sequencing kit enable outbreak tracking through the identification of bacterial clones or distinct strains and substrains.

Standardized sequencing assays for infectious disease testing are also available from a couple of additional diagnostics companies. Abbott Molecular offers its CE-IVD HBV sequencing assay for the determination of hepatitis B virus genotype and drug resistance prior to antiviral therapy. Singapore’s Vela Diagnostics has developed Sentosa NGS, a genotyping assay for hepatitis C. It is a research-use-only platform that integrates with a number of Vela components. It is intended for viral genotyping from plasma or serum.

Vela also offers Sentosa assays for oncology that already have CE-IVD marking. However, the majority of NGS assays are available only as laboratory-developed tests or testing services that do not require premarket approval or other clearance processes for clinical use otherwise required of test kits.

Outstanding Regulatory Issues

The above companies demonstrate the feasibility of introducing a standardized NGS assay kit to the market, but the relative scarcity of such products hints at ongoing regulatory uncertainty regarding the assessment of NGS. This uncertainty arises from a paradigm shift inherent to NGS. That is, with NGS, the interpretation of results goes beyond positive/negative determinations to translation of highly specific and variable sequence data into actionable diagnostic knowledge.

One company working to accomplish this paradigm shift is PathoQuest. The company is one of several offering bioinformatics solutions for NGS users in the clinical field. Bioinformatics software assists the interpretation of results by referencing against updated databases and reduces the need for skills and expertise on the part of the user. A more automated analytical approach will be increasingly standard in the clinical space and key to winning regulatory support.

The market introduction and acceptance of NGS-based clinical diagnostics has at the same time been propelled by the technology’s unique capabilities and also held back from greater market development because of the challenges presented in assessing risk and suitable performance. With NGS diagnostics, the onus on the lab is shifted considerably from hypothesis-driven diagnosis to the interpretation of more data-rich results. In the case of infectious disease testing, the risk is considerable as it surpasses individual outcomes and could impact public health, and it calls for careful deliberation on the part of the regulators and healthcare adopters.

Emil Salazar is an analyst at Kalorama Information. This article is excerpted from “Next Generation Sequencing (NGS) Markets 2015 (Systems, Consumables, Services, Acquisitions, Agreements, Competitive Analysis, Platforms, Applications, Brand Ownership and Other Trends)”. More information on this report can be found at www.kaloramainformation.com.


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