Originally Published IVD Technology October 2005
Tools for molecular diagnostics
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| The Rapid Capture system for high-volume laboratories by Digene Corp. (Gaithersburg, MD). |
Molecular tools for diagnostics were first introduced in the mid-1980s as DNA probes used to confirm microbiological cultures. A decade later, FDA had approved nucleic acid tests (NATs) that employed target- or signal-amplification technologies to test patient samples directly. Now, almost 10 years after the first tests were FDA approved, they have become well-established diagnostic tools.
The impact of NATs has been seen particularly in improved turnaround time for results. Where organisms or human cells once had to be cultured for days or weeks prior to analysis, today the direct application of molecular tools to patient samples has allowed results to be reliably produced in 24–72 hours.
Molecular diagnostics continues to be a significant segment of the IVD market, growing about 15–20% per year. Although molecular testing consists primarily of infectious-disease testing, it has branched into other fields as well, including oncology, genomics, and the much anticipated pharmacogenomics testing arena. The total market segment is projected to reach $35 billion by 2015.
Although NATs have enjoyed steady growth, there are several issues that still dampen acceptance of this technology. NATs tend to be expensive and complex, and require significant skill to perform. Automation has helped reduce some of these barriers during the past several years and remains an active pursuit for many companies. Developing standards and controls that equate results across technology platforms has also been an issue. In addition, a lack of predicate devices has contributed to difficulty in regulatory acceptance. Finally, adoption rates for molecular tests have been slow, in part due to physician uncertainty about the clinical utility of these tests. The lack of prospective clinical outcome studies for NATs has led to a technology education gap that molecular IVD producers need to address. As technology turns to the pursuit of point-of-care (POC) platforms, the education of physicians and patients will become even more essential.
Technology Overview
Target amplification is the predominant technology used in molecular diagnostics. Methods based on both polymerase chain reaction (PCR) and isothermal amplification methods are available in end-point and real-time platforms. Signal-amplification technologies are also available in a variety of molecular detection formats. Assay sensitivity requirements, particularly for monitoring therapeutics, as well as the need to screen for and specifically detect multiple sequences in a single sample, have led the industry to develop real-time amplification methods and multiplex detection capabilities.
Several types of fluorescent molecular beacon systems are available for real-time amplification method development. Such methods have provided an exquisite means of quantitating a low number of target nucleic acids, but are somewhat limited in their multiplexing capability. Detection of amplified sequences on microarray systems has provided an amazing multiplex capacity, but the sophisticated optical reading systems and software required to process microarray data require instruments that are very expensive and require highly skilled personnel to run.
Two types of array systems are generally available—fixed arrays and flow cytometry arrays. Fixed arrays have DNA sequences physically attached to specific places (addresses) on solid supports. DNA samples containing target sequences are passed over the support and hybridize to the addressed sequences. In a flow cytometry array system, addressable beads capable of capturing specific targets amplified from patient nucleic acids are detected as they flow through a reader. The GeneChip system, manufactured by Affymetrix Inc. (Santa Clara, CA), and the Luminex 100 system, manufactured by Luminex Inc. (Austin, TX), are examples of each technology, respectively. Though the Luminex system is theoretically limited to multiplexing 100 targets, it is easy to use and much less expensive than fixed-array technology. FDA-cleared products using both technologies are now available.
Infectious Diseases
Approximately 60–80% of the molecular diagnostics market consists of tests for five pathogens: Chlamydia trachomatis, Neisseria gonorrhoeae, human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), and human papillomavirus (HPV). Hepatitis B virus (HBV) is another important analyte featured on blood-bank screening-test menus. Some of the same technologies used to detect the “big five” pathogens are available for detection of Mycobacterium tuberculosis, cytomegalovirus, group A and B streptococci, methicillin-resistant Staphyloccus aureus, West Nile virus, and the coronavirus that causes severe acute respiratory syndrome.
There are a large number of molecular technologies represented in established product offerings. The movement during the past several years has been to adapt technology to automated platforms.
Gen-Probe Inc. (San Diego) manufactures a molecular test based on an isothermal amplification method using transcription-mediated amplification. Gen-Probe was the first to present an automated high-throughput platform that processes samples from nucleic acid extraction through detection to final results.
Roche Molecular Systems Inc. (Pleasanton, CA) has end-point and real-time PCR and reverse transcriptase–PCR amplification systems. Both Roche technologies have been semiautomated on detection instruments. Roche has completed clinical evaluation of a companion instrument that performs sample processing.
Real-time isothermal amplification via strand displacement amplification is used by BD Diagnostics (Franklin Lakes, NJ). BD offers separate automated platforms for sample processing and detection.
Signal amplification is used in Digene Corp.’s (Gaithersburg, MD) hybrid capture method, in which DNA targets are hybridized to specific RNA probes and captured by antibodies that recognize this DNA-RNA hybrid. Numerous antibodies can bind to the target to achieve signal amplification. The assay can be used with Digene’s Rapid Capture system semiautomated platform.
Bayer Diagnostics’ (Tarrytown, NY) bDNA technology uses signal amplification by first hybridizing the target with a specific probe and then applying a series of branched oligonucleotides complementary to the probe. The branched structure provides points for signal amplification.
Genotyping is another area of interest in infectious-disease molecular testing. Identification of sequences that confer resistance to antivirals has shown clinical utility in monitoring the efficacy of HIV-1 and HCV therapeutics. Specific HCV genotypes are associated with different rates of disease progression, and identification of them leads to different clinical follow-up and treatment courses.
The appearance of vaccine-resistant HBV strains has become a concern in different parts of the world; therefore, genotyping has become an important tool for monitoring vaccine efficacy. Currently, Celera Diagnostics (Alameda, CA) and Bayer Diagnostics offer systems that detect antiviral resistance mutations based on bidirectional DNA sequencing of PCR-generated fragments covering select regions of the genome prone to resistance mutations. Mutations are identified in these systems using sequence comparison software.
Third Wave Technologies Inc.’s (Madison, WI) Invader technology offers another tool for HCV genotyping by using a specialized DNA to probe target sequences from amplified sample DNA. This probe creates a structure that is recognized and cleaved by an enzyme. The released portion of the probe participates in the formation of a second DNA structure on a generic fluorescence resonance energy transfer (FRET) probe. This second structure is also recognized and cleaved by the enzyme. The final cleavage of the FRET probe results in the release of a fluorophore.
Bayer offers an HCV genotyping system based on the InnoLiPa reverse dot-blot method. This method involves the generation of PCR-amplified targets that are hybridized to type-specific probes immobilized on solid filter strips. The hybridized amplicons are detected via a colorimetric system that produces banding patterns characteristic for specific genotypes.
Research on HPV high-risk genotypes suggests some may be more predictive of invasive disease than others. In addition, new vaccines developed for a few HPV genotypes will need to be monitored for efficacy during clinical trials. For these reasons, HPV genotyping tests are of interest. Roche currently offers an HPV genotyping technology similar to InnoLiPa. Digene is developing reagents to enable genotyping of 19 important HPV types via the Luminex detection platform. These reagents are expected to be available in early 2006.
Genomics/Oncology
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| The Hybrid Capture 2 system by Digene Corp. uses signalamplification molecular technology. |
While tests geared toward the detection of genetic mutations represent a smaller market segment, it offers the same technological breadth as the infectious-disease segment. A considerable amount of genetics/oncology testing is performed using fluorescence in situ hybridization (FISH), which employs DNA probes that allow chromosomal position to be identified. Vysis Inc. (Downers Grove, IL) uses FISH technology in its systems to detect bladder cancer and to perform prenatal screens for chromosomal additions and deletions.
Genetic tests for hematologic disorders of Factor V (Leiden) and Factor II (20210 mutation), and methylene tetra hydro folate reductase/MTHFR (mutations at positions 677 and 1298) exist for several real-time amplification platforms. Biotest Diagnostics Corp. (Denville, NJ) produces a line of reagents that use allele-specific PCR to perform HLA typing.
Tests for prenatal screening of cystic fibrosis mutations and screening for mutations common to Ashkenazi Jews include Tm Bioscience Corp.’s (Toronto) Tag-It platform. Tag-It uses multiplex PCR followed by allele-specific primer extension (ASPE). The labeled ASPE products are then detected on a Luminex platform. Ambion Diagnostics (Austin, TX) also has reagents for detecting these disorders. These include products for performing multiplex PCR followed by detection on the Luminex platform.
Pharmacogenetics
Pharmacogenetics uses array technology to screen for surrogate markers in response to pharmaceuticals. Part of what has made this work difficult is the establishing of biomarker clinical significance. For methods based on gene expression measurement, the issues become more complex as there is a need to establish consensus around methods of gene expression quantitation and analysis.
The first FDA-approved pharmacogenetics microarray screen and FDA-approved microarray was the Roche Diagnostics (Indianapolis) AmpliChip CYP450, which is run on Affymetrix’s GeneChip system. The CYP450 sequence is a well-established pharmaceutical DNA biomarker.
Molecular Controls
With the industry maturing and regulatory bodies that oversee clinical labs requiring more-sophisticated quality control for molecular assays, the need for external molecular controls—from purified plasmid and genomic DNA to intact cell- and virus-derived controls—has also grown. Regulations stemming from the Clinical Laboratory Improvement Amendments of 1988 and the Clinical and Laboratory Standards Institute (formerly NCCLS; Wayne, PA) guidelines stress that complex tests such as molecular assays should include controls that challenge all aspects of the assay procedure, including sample preparation. A novel approach to generating stable noninfectious controls that meet these criteria is via Ambion’s Armored RNA, a technology that exploits a bacteriophage packaging system to encase RNA in a protein coat.
Regulatory Pathways
Nearly 70 molecular diagnostic assays have been awarded 510(k) or PMA clearance from FDA. Since initial PMAs require significant funds and time to complete, the approval process has been slow. Use of the analyte specific reagent (ASR) label has provided a way for manufacturers to provide reliably made reagents for targets where clinical utility has not been established or for which no predicate device for comparison exists. This has been particularly helpful in providing reagents for infectious-disease testing beyond the big-five analytes, and in genomic detection where the testing populations are small and significant time is required to gather the data needed for a conclusive clinical study. Today, nearly all molecular diagnostics manufacturers offer ASR products, and there are a growing number of manufacturers that offer only ASR products.
The Future
The future of molecular diagnostics will likely hold both improvements in stand-alone inclusive automation as well as in miniaturization technologies. Currently, facilities with large testing populations need instrumentation that can handle large volumes. Nevertheless, in both large and small testing facilities, the desire to consolidate molecular technology platforms is being driven by the need to reduce costs.
POC systems provide one attractive option. “Black box” instruments for physicians’ offices will likely evolve to handheld devices that can accommodate rapid patient testing in office, clinic, or field settings.
Technology being used for biodefense efforts will also likely provide a breeding ground for compact, rapid, handheld technologies. Enigma Diagnostics Ltd. (Wiltshire, UK), which grew out of a project with the UK Ministry of Defence, has developed a portable POC PCR-based instrument that can perform molecular tests in under 30 minutes. Similarly, Cepheid (Sunnyvale, CA) has developed a completely automated real-time PCR platform for rapid pathogen detection in which samples can be processed using individual cartridges in 30 minutes. Ultrasonic energy is used for sample lysis in the Cepheid system, and future technologies will likely explore novel methods for sample preparation.
Miniaturization technologies are being described fast and furiously in scientific research literature. Most have focused on sensitive detection of DNA sequences using electrochemical technology, which is amenable to miniaturization. The Biodesign Institute of Arizona State University (Tempe, AZ) recently reported the use of inorganic nanocrystal tags to label mononucleotides for detecting single nucleotide polymorphisms. Each crystal tag can produce a distinct voltametric signature. Sequential use of the tagged monobases takes advantage of further base-pairing to create an electrical interpretation, or fingerprint, of the DNA polymorphism. This type of technology is what we can anticipate for the future of POC and personalized medicine.
As POC technologies move into commercially viable formats, NATs will become widely available. Are we ready for these advances? IVD companies already include physician education as part of the strategy necessary to effectively market and sell molecular tests. Some physicians have used video programs in waiting rooms to explain the complexities of prenatal molecular genetic testing to patients. Improvements in and support for medical and public education will need to go hand in hand with the technical improvements as molecular diagnostics moves further toward predictive and personalized medicine.
Renée M. Howell, PhD, Digene Corp. (Gaithersburg, MD)
Copyright ©2005 IVD Technology
