Various pursuits are ongoing to harness the advantages of DNA identification, yet there are many challenges still to be overcome to deliver such capabilities.
Figure 1. The unique 3-D structures of the Thermal Gradient device allow PCR with extremely fast temperature transitions in low-cost disposable devices.
In many respects, the diagnosis of infectious diseases implements concepts that have remained largely unchanged during the past half-century or more. Ever since the Danish physician Hans Joachim Gram observed in the early 1860s that certain bacteria retain a purplish color after being exposed to an organic dye, the discipline of clinical microbiology has evolved around the basic tools of culture, microscopic analysis, and staining. However, recent advances in molecular biology, the escalation of the war against bacterial resistance, and changes in regulatory and insurance policies have been driving a sea change in the way bacterial and viral infections are diagnosed. Leading the way in this new molecular frontier are IVD techniques that precisely and rapidly detect the genetic material (DNA and RNA) that uniquely defines such infectious agents.
IVDs, including molecular or nucleic acid-targeted diagnostics, are continuing to play an important role in medicine and in driving fundamental changes in the diagnostics toolbox and the overall healthcare delivery system. About 80% of the analytical information used by physicians to make medical decisions is generated by clinical laboratories. The increased availability of rapid, information-rich IVD tests on new instrumentation platforms in clinical laboratories is serving a critical function in enhancing the quality of patient care.
In addition to the technical advantages that are discussed in this article, molecular diagnostics offer the potential for significant savings in terms of cost (since assays can be run in a highly parallel format) and time (since assay times can be reduced to less than an hour, in principle). Balanced against such savings is the need for substantial changes in laboratory instrumentation and analytical laboratory processes, and for technical developments. This article discusses some key technological advances, and the government and societal demands that are leading to increased adoption of molecular diagnostics in clinical laboratories. Over time, many molecular diagnostics may also find greater utility at the point of care.
According to a recent report by Deloitte Consulting LLP, growth in the molecular diagnostics market is primarily driven by technologies adopted from the analytical IVD instrumentation market.1 This report presented a compelling argument that analytical instrumentation companies will need to modify significantly their product and commercialization strategies to capitalize on the growth opportunities in molecular diagnostics. Such modifications will include moving from analytical IVD instrumentation that has traditionally addressed the needs of highly trained academic and commercial R&D scientists and medical technologists working in the core hospital labs, to instrumentation that fits the needs of broader and more decentralized work environments, such as physician offices and urgent-care and retail clinics.
In addition, new molecular diagnostic innovations and reengineered IVD instrumentation will be required to address more than just the work environment needs of a broader customer base. The new molecular diagnostic tools will also need to deliver higher clinical utility and be positioned to meet the challenges of the evolving regulatory, payer, and patient requirements and the overall business and economic realities. Emerging innovations in molecular diagnostics provide the potential to address such challenges.
Advantages and Challenges of Molecular Diagnostics
Molecular diagnostics are an attractive technology for a number of reasons. From a technical standpoint, two of the most important reasons are the ability to achieve both extraordinary sensitivity and specificity. Specificity comes from the fact that molecular diagnostics can uniquely identify a target organism from a series of DNA sequences. Because of worldwide efforts in academic and industrial laboratories, the complete DNA sequences, or genomes, of many of the most important bacterial and viral pathogens are known, and more are being added on an ongoing basis. In many cases, the sequences that encode for antibiotic resistance are also known, allowing resistant organisms to be further differentiated from those that are antibiotic-sensitive. Of course, picking the right DNA sequence to target in each genome is not a straightforward task, which will be discussed in more detail later in this article.
The second major advantage of DNA-targeted diagnostics is that DNA is the only biomolecule that is easily copied or amplified via polymerase chain reaction (PCR) or other related amplification methods. (RNA may also undergo amplification by first undergoing in situ reverse transcription to DNA.) This advantage allows the molecular signal from target organisms to be rapidly and selectively enhanced. This then allows for an extraordinarily sensitive detection of bacteria and viruses, even down to the single-organism level in some cases. While some biosensor technologies are being developed that could provide direct single-copy detection, amplification has the added benefit of increasing an assay's signal-to-noise ratio, decreasing the probability that sample contaminants will interfere with detection.
The development of a method to amplify target DNA sequences could arguably be the most important advance in the biological sciences since the discovery of DNA itself and a critical enabler in the molecular diagnostics revolution. PCR, the most robust and widely used DNA amplification method, has been joined by several other such techniques including rolling-circle amplification, strand-displacement amplification, reverse-transcription loop-mediated isothermal amplification, and helicase-mediated amplification. While these amplification methods are generally less efficient than PCR, they have the unique advantages of being able to operate isothermally. By eliminating the need for thermal cycling, these techniques can significantly simplify IVD instrument design.
Current and Future Technology Trends
While PCR is a relatively mature technology with regard to research applications, several ongoing areas of research and development are exploring ways to increase its utility in the clinical diagnostics market. For example, companies such as Kapa Biosystems Inc. (Woburn, MA) are developing new proprietary variants of polymerase enzymes that have greater reliability (i.e., lower error rates) and are able to operate in the presence of materials such as hemoglobin, which are considered to be polymerase inhibitors. Kapa Biosystems uses a high-throughput molecular evolution platform that allows the company to produce custom enzymes engineered for highly specific research or IVD applications.
Ultrafast PCR amplification (i.e., achieving 30 cycles or more in less than 15 minutes) is another significant area of research and early commercialization efforts, since DNA amplification is currently the limiting factor in assay time for many IVD applications. This area will be particularly important in decentralized work environments such as surgical suites and critical care units where immediate results are important, or urgent-care or retail clinics where rapid IVD results are needed to provide walk-away care.
Two general approaches have been pursued in developing ultrafast PCR amplification, and both are in the early stages of commercialization. The first approach has been looking at the possibility of decreasing dramatically thermal cycling time by reducing the volume of solution to be cycled. In the second approach, fluid is pumped through serpentine or spiral microfluidic channels with alternating temperature zones. Thermal Gradient Corp. (Rochester, NY) is pursuing an interesting variant of this second approach by developing a PCR chip that can produce 108–fold amplification in less than six minutes. Figure 1 shows a diagram of a Thermal Gradient 30-cycle device.
Figure 2. (click to enlarge
) Temperature profile of fluid flowing through a Thermal Gradient thermal cycling device.
The Thermal Gradient chip devices are designed to handle a broad range of internal volumes from 4 to 1000 µl, and use two- or three-temperature PCR or reverse-transcriptase PCR for both real-time and end-point PCR detection. Unlike conventional thermal cyclers, this device operates at a steady temperature, which makes it simple to use while requiring very low power. The Thermal Gradient device is also able to carry out PCR at its kinetic limit because the transitions between the temperature stages of PCR occur almost instantaneously. Figure 2 shows a computational fluid dynamic model of the fluid temperature profile as it passes through the device.
These compelling advancements, coupled with further thermal cycler improvements in fluid dynamics and thermal transfer rates and new engineered, faster polymerases and optimized buffers, will ultimately be integrated to produce reliable amplification in the 30–40-cycle range in less than 10 minutes or even 5 minutes. The successful integration of these innovations in molecular diagnostic instrumentation will likely contribute to benefits in clinical outcomes, patient convenience, lower costs, and ultimately new adoption in end-user environments that demand rapid test results.
Advances in nucleic acid amplification are particularly effective when they are paired with novel detection technologies. An active effort is being made to develop label-free detection technologies (i.e., techniques that can directly detect the presence of target DNA without the need for an additional fluorophore or other labeling reagents). Such methods improve assay costs by reducing the number of reagents required. Surface plasmon resonance (SPR) is perhaps the most familiar technique, and was initially commercialized as a research instrument by Biacore Corp. (now a part of G.E. Healthcare; Uppsala, Sweden).
Relying on binding-induced changes in the spectrum of light reflected from a gold chip, SPR allows for real-time detection of analytes. Originally developed solely as a single-analyte instrument, a variation of this technique known as SPR imaging is now incorporated into research instruments by several firms. While no SPR imaging instruments are yet available for commercial IVD applications, it clearly remains a goal.
High-speed PCR methods can be combined with novel analytical methods, such as the LightScanner 32 system which is currently being developed by Idaho Technology Inc. (Salt Lake City, UT). This system uses high-resolution melting analysis, a method that compares melting temperatures to study sequence differences. While other label-free detection technologies (e.g., planar silicon waveguides), methods based on various forms of interferometry, and several nanotechnology-based platforms are still important areas of research in academia, they will likely find their way into commercial IVD applications in the near future.
Figure 3. (click to enlarge
) The NanoLantern system features label-free operation and 100% probe-base sequence complementarity with target DNA.
The DNA NanoLantern system by Lighthouse Biosciences Inc. (Rochester, NY) is both a novel detection platform and a technology for identifying probe oligonucleotides with 100% specificity for the targets of interest. The detection platform uses a metal chip carrying hairpin DNA probes that are functionalized at one end with a fluorescent group. In the absence of target DNA, these hairpin probes fold over on themselves, and the resultant close proximity between the fluorescent group and the chip's metal film quenches any fluorescence (see Figure 3). But in the presence of target DNA, hybridization to the hairpin probe forces the fluorescent group away from the surface, restoring its ability to fluoresce and provide a positive signal for the target analyte.
This self-labeled aspect of the array is only one advantage of the NanoLantern system, since hairpin DNA probes provide exceptional levels of sequence selectivity. Lighthouse Biosciences' academic collaborators have demonstrated that the platform can discriminate single-base mismatches without any changes in assay stringency. Such selectivity is further enhanced by the company's methodology for identifying DNA probes, which allows a raw genomic sequence to be converted into effective hairpin DNA probes through a series of computational steps. Specifically, Lighthouse Bioscience uses a DNA folding program to predict regions of genomic DNA with naturally occurring hairpin structures. Such regions form the basis for producing hairpin DNA probes that are a 100% match for target DNA. No extraneous DNA bases are incorporated, as in other hairpin probe methodologies. The company is currently developing this technology for application in a number of infectious disease diagnostics.
A key challenge inherent in DNA diagnostics is obtaining a DNA or RNA template in a sufficiently pure form to allow efficient amplification. While culture plates are unaffected by complex biological fluids, the enzymes required to carry out amplification can be strongly inhibited by urea or other contaminants. Therefore, some form of sample purification is often essential. In some cases, while such purification may be as simple as passing the sample through a filter, other applications may require more extensive pretreatments. For targets such as viruses or bacterial spores with a hard shell or coating around the genetic material, cracking the shell to obtain the target DNA or RNA is also necessary. The IVD industry and academia are involved in ongoing research efforts to develop rapid and reliable sample preparation methods.
Evolving Landscape and Healthcare Reform
Despite the technical advantages associated with molecular diagnostics and the potential cost savings from novel detection platforms, they will only be realized in a clinical setting if they are offered in conjunction with a regulatory, reimbursement, and competitive environment that encourages their adoption. Much of this realization is currently in a highly fluid state. The recent healthcare reforms proposed by Congress and the Obama administration and the evolving outcomes of such proposals will profoundly affect where and how emerging healthcare technologies including molecular diagnostics could be deployed.
For example, healthcare reform policies will likely have an impact on specifying which patient conditions may qualify for payment and at what reimbursement level. This profit and loss approach toward both the top line and business margins will set the economic boundaries for molecular IVD companies, and will affect investments in technology development and product deployments for all stakeholders. Developing advanced molecular diagnostic technologies and other higher performance products that will lead to improved healthcare outcomes will also be under greater pressure to do so at lower costs and ideally with substantial longer term healthcare cost reductions.
Figure 4. (click to enlarge
) Factors transforming diagnostic product performance requirements.
Another potential trend is that the demand for decentralized delivery of healthcare could drive decentralized delivery of diagnostics. For example, bringing IVD tests and results to patients at the point of care while they are still at a clinic enables them to walk away with a diagnosis and a treatment. Such rapid diagnosis and treatment realized in a decentralized and walk-away healthcare environment can improve healthcare outcomes and would reduce the overall cost of healthcare delivery. To meet the opportunities in such decentralized and walk-away healthcare environments, IVD technologies including molecular diagnostic solutions will need to be robust, small, simple to operate, and rapid. They will also need to provide more clinically relevant information reliably and at lower costs. Figure 4 characterizes how the diagnostic and end-user work environments will drive the deployment of different types of molecular diagnostic tools.
Retail healthcare will also influence the challenge of decentralized, walk-away IVDs even further. Beyond the emergence of the 80,000 plus clinics forecast for the United States and China, the potential for more sophisticated IVD products and services delivered through retail drugstores and shopping center kiosks is on the horizon as well. Whether such IVD products are delivered by a trained professional or a retail clerk in such nontraditional healthcare settings, the developers of a new generation of IVD products will be challenged with new performance and emerging cost requirements.
This article has highlighted some early-stage technologies that could accelerate the adoption of molecular diagnostics in clinical laboratories and eventually in decentralized formats. In combination, such technologies can begin to address the various economic, workforce, and public demands and the evolving healthcare reform proposals. Such demands and proposals are increasing the need to advance molecular IVD technologies and produce solutions that are lower in cost and more broadly adaptable in diverse end-user environments, while providing a greater level of IVD information.
Benjamin L. Miller, PhD,
is chief science officer at Lighthouse Biosciences Inc. (Rochester, NY). He can be reached at firstname.lastname@example.org
Randolph R. Henke, PhD,
is chief executive officer at Lighthouse Biosciences Inc. (Rochester, NY). He can be reached at email@example.com
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