William Check is a freelance medical and science writer based in Wilmette, Ill.
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Nucleic Acid-Based Tests Move Slowly into Clinical Labs
Sample preparation problems, contamination, and entrenched attitudes slow but cannot stop adoption of analytically diagnostic methods
William Check
Diagnostic methods based on hybridizing and amplifying nucleic acids are gradually making inroads into clinical microbiology laboratories. And more recently, sequencing the DNA of pathogenic organisms is looking as if it, too, will eventually gain clinical applicability.
So far, however, the practical diagnostic usefulness of such molecular methods has consistently lagged behind their promise—in part, because these methods are time-consuming, expensive, and demand a high level of expertise from experienced technologists, who tend to be in short supply and whose services are expensive. But because molecular assays often offer means to measure or detect pathogens for which existing tests fare badly or no tests are available, the newer methods have gained a foothold in some larger and research-oriented clinical laboratories. In these laboratories, the ensuing familiarity, in turn, makes it easier to adopt additional molecular methods for diagnostic purposes.
Molecular Methods for Diagnosing Pathogens Still Face Hurdles
"With the introduction of the Roche Amplicor Monitor for HIV viral load, molecular testing has become a fairly routine assay," says Tim Alcorn, Director of Infectious Diseases at the LabCorp Center for Molecular Biology and Pathology in Research Triangle Park, N.C. Availability of procedures for quantifying HIV levels in patients was followed by amplification assays for detecting the hepatitis C virus (HCV). Neither virus can be readily cultured from patients. Alcorn sees molecular assays for cytomegalovirus (CMV), herpes simplex virus (HSV), and hepatitis B as being next in line for widespread adoption at clinical laboratories. Molecular tests for CMV and HSV exemplify replacement of existing methods. "As more places develop molecular testing menus, this methodology is expanding in all markets from big reference laboratories to small hospital laboratories," Alcorn says.
But there are still hurdles to overcome before nucleic acid-based methods achieve the goals cited by their advocates. "I remain very optimistic about the potential of molecular methods," says David Relman, associate professor of microbiology and immunology and of medicine at Stanford University School of Medicine in Stanford, Calif. "But I am the first to admit that these putative promises are still largely unmet or unfulfilled. I think part of the problem is that in some cases the technology in a pure sense has advanced beyond the peripheral technologies that will be necessary to bring these methods into clinical laboratory practice."
In addition, there is a need to distinguish sensitivity and specificity in the analytical sense from the clinical sense. "Most often the wonderful data you hear at meetings are about analytical performance rather than clinical data," Relman points out. "At this point we need both."
Patrick Murray, Director of Clinical Microbiology at the University of Maryland Medical System in Baltimore, compares the current status of nucleic acid-based diagnostic methods to the first wave of enzyme-linked immunosorbent assay (ELISA) tests that became available 15-20 years ago. Initially, immunoassays were very cumbersome and took 4-5 hours to do. "I think that many laboratories had a difficult time justifying doing many of these immunoassays or replacing what we were using with immunoassays on the market," he recalls. Now, rapid, reliable immunoassays abound in clinical laboratories and even in physicians' offices.
Similarly, nucleic acid-based molecular diagnostics are now a first-generation technology with major problems. "Not to say that it won't be adopted in the future," Murray says. "I am convinced it will be." But improvements will be needed.
Specimen Handling, Other Challenges Need Facing before New Testing Is Widely Adopted
Murray points out that the ability to isolate target nucleic acids, to amplify those targets, and to use sophisticated probes to identify them is far ahead of the preanalytical phase—which entails processing specimens to make them suitable for input into the assays. Inhibitors in clinical specimens can interfere with critical analytic reactions. Moreover, it can be difficult to concentrate organisms that are present in very small numbers, and the possibility of cross-contamination is still real.
Even improved molecular technology will have inherent limits. According to Ellen Jo Baron, Director of the Clinical Microbiology and Virology Laboratory and associate professor of pathology at Stanford University Medical Systems, Calif., "Molecular technology is not going to be able to answer every question about etiology of infections." Pneumonia is one example: many agents are carried in the respiratory tract of asymptomatic individuals. A sensitive amplification assay can be overloaded with organisms that are not relevant to a disease process. "It is wrong to believe that we will do everything by amplification in the future," she says.
Nucleic acid sequencing, the most futuristic method for analyzing pathogenic microorganisms in clinical settings, is just now emerging from the research laboratory. Instruments are beginning to be designed specifically for clinical laboratory diagnostics and kit-based assays are being developed that have greater reliability than in-house assays, says Steve Day, Director of Medical Affairs for Visible Genetics in Atlanta, Ga. There is still a pressing need for standardization of methods, reference sequences, and interpretation. But, he adds, "Sequencing- and pharmacogenomics-based assays are revolutionizing infectious disease diagnostics and disease management, and will continue to do so."
In addition to the commercially developed applications of molecular technology to diagnostics, some clinical microbiologists are developing their own in-house amplification assays for less-common but clinically important pathogens, including Epstein-Barr virus, enterovirus, and Ehrlichia, Rickettsia, and Toxoplasma species. Having this capability is one advantage of having a well-trained and experienced microbiologist as laboratory director.
One Lab's Approach toward Adopting Nucleic Acid-based Diagnostics
A representative menu of molecular methods might be the one put into place over the last few years by Lizzie Harrell, Associate Director of the Clinical Microbiology Laboratory and associate research professor of microbiology and pathology at Duke University Medical Center in Raleigh, N.C. "Our main molecular test right now is HIV viral load testing," Harrell says. The laboratory offers both the standard assay, usually ordered by physicians for a baseline value at the initiation of therapy and the ultrasensitive assay, typically ordered after a period of therapy, when viral load is expected to decrease.
To detect CMV DNA, Harrell has instituted Digene's hybrid capture DNA hybridization (nonamplified) test, which is performed directly on patient specimens. Since Harrell is staying with Food and Drug Administration (FDA)-approved tests, she uses the qualitative assay. "A few laboratories are doing the quantitative assay, but that raises reimbursement issues," she says. CMV testing is used mostly in transplant recipients who have developed symptoms to help the clinician rule out organ rejection as a source of a patient's problems. Harrell has installed the nucleic acid-based test in lieu of antigenemia. Quantitative assays for CMV provide an indication of the risk of infection. So, as they are approved, they will increasingly replace antigenemia tests and will be used for detecting early signs of infection to allow for preemptive therapy.
Harrell also offers pulsed-field gel electrophoresis (PFGE) testing for molecular epidemiology at the request of infection control officers, typically to analyze a suspected nosocomial outbreak. PFGE displays characteristic fragments of the entire chromosome of a microorganism after restriction enzymes are used to digest its DNA at infrequent intervals. Patterns of identity are easy to spot and indicate a common-source outbreak.
Harrell is looking at both qualitative and quantitative methods for diagnosing and monitoring patients with HCV infection and, eventually, to determine whether amplification testing can be brought in-house. Amplification assays are useful to confirm HCV infection in patients who have abnormal liver enzymes and who are HCV-antibody positive by ELISA. Clinicians who manage liver transplant patients and those who treat HIV-positive patients will be the primary users of these tests.
Harrell's laboratory also is using a (nonamplified) GenProbe assay on request to identify mycobacterial species, including Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae, on growth in culture. In the United States, the most common clinically significant mycobacterium is M. avium complex (among individuals with HIV), an organism that is relatively inert. Using traditional biochemical tests, identification can take several weeks or even months, while probes do it in a couple of hours, once growth has occurred. Harrell is also evaluating the use of an amplified test to detect M. tuberculosis directly in clinical specimens.
One of the first diagnostic applications of nucleic acid-based technology to be approved was for Chlamydia trachomatis and Neisseria gonorrhoeae in patient specimens. Nucleic acid amplification assays quickly proved more sensitive than culture and are now considered standard for characterizing the pathogens responsible for these sexually transmitted diseases. At Duke, Harrell says, this test is being done in two sites outside the microbiology laboratory. "We firmly believe that this kind of testing should be done in the microbiology laboratory," Harrell says. "Tests should be grouped by discipline, not by technique."
An exception might be the amplification assay for human papillomavirus (HPV) in cervical specimens, which has been incorporated into cervical screening by many institutions to triage indeterminate Pap smears. At Duke, this testing is being done in the pathology lab to better correlate results with histology findings.
Nucleic Acid-Based Tests Sometimes Prove Misleading
While amplification assays are contributing to infectious disease diagnostics, they sometimes cause problems. Before moving to Maryland, Murray was at Washington University, St. Louis, Mo., where one such incident occurred, he recalls. In that case, use of Abbott's ligase chain reaction to detect C. trachomatis and N. gonorrhoeae yielded a cluster of results right at the limit of sensitivity, the so-called "gray zone," a situation that Murray calls "very unsettling" since it led to false-positive results. Further exploration revealed that this problem occurred throughout the assay's range. False-negative results were also found, particularly with urine specimens, since urine inhibits the ligase chain reaction. Murray explains that these errors were due to a technical problem with the then-current assay. After repeating every positive they had done, they shifted to the Becton Dickinson assay, which has an internal amplification control that shows whether an inhibitor is present in the sample.
But their experience with the Becton Dickinson assay provides a second example of another pitfall—contamination from the amplification control. The vendor was very helpful, but eventually they had to replace the instrument and move amplification testing into another room. "We were unable to clean the laboratory," Murray says. "These problems have been reported from a number of laboratories with both systems." He is optimistic that systems will improve, but believes that such problems are not appreciated by many users.
Besides detecting antibiotic resistance markers in C. trachomatis and N. gonorrhoeae, other nucleic acid-based testing to detect resistance markers such as mecA in methicillin-resistant Staphylococcus aureus and van genes in vancomycin-resistance enterococci also sometimes proves highly valuable over conventional approaches. "Amplification of resistance genes is the most sensitive technique for detecting resistance in those organisms," Murray says. Indeed, detecting oxacillin and vancomycin resistance in these organisms by standard means—disks and breakpoints—often proves difficult if not impossible, due to their "heteroresistant" property: not all organisms that carry the gene express it.
As Baron notes, amplification assays can be misleading when they are used for diagnosing pneumonia cases. "A lot of people would think pneumonia is a situation where molecular technology might fill a big void in cases where no etiologic diagnosis is made," agrees John Bartlett, chief of infectious diseases at Johns Hopkins University, Baltimore, Md. However, amplification methods need to be restricted to those organisms that are not present as normal flora, such as Legionella, Mycoplasma pneumoniae, and Chlamydia pneumoniae. Organisms commonly causing pneumonia—Haemophilus influenzae and the pneumococcus—will be present as contaminants too often for amplification testing ever to be definitive.
Sample Preparation, Other Technical Problems Also Need Facing
Fundamental obstacles will have to be overcome before amplification assays become routine in clinical laboratories. Important needs include learning how to prepare clinical specimens to obtain the target nucleic acid while avoiding inhibitory activities and how to concentrate nucleic acids without concentrating inhibitors. Choosing specimens also requires some thought. "We think we know where to find microorganisms," says Relman. "But I am not sure we have a full understanding of what kinds of samples might be useful and which might be nonproductive."
Baron points out that there are still no satisfactory automated extraction methods with which to prepare specimens for amplification assays. Her laboratory is testing two commercial methods. "They are not as sensitive as manual methods, but manual methods have ergonomic problems," she says, referring to the repetitive pipetting that sometimes causes problems among laboratorians.
Relman also cites the broad problem of background signal contamination, which he believes is more aptly called the endogenous microbial molecular background of the human body. "With molecular methods, we have even less idea what we should find in people who are healthy than with standard cultivatable organisms," he says. "Sequence diversity is much greater than cultivar diversity." Understanding the endogenous microbial "molecular flora" is crucial if clinical microbiologists are to interpret correctly results of molecular tests on samples taken from diverse patients.
Shortages of Trained Personnel Further Complicate Technology Adoption
And then there is the appropriately skilled technologist shortage. Baron cites projections that 9,300 laboratory personnel will be needed annually for the next 10 years, but only about 5,000 persons will be entering the profession each year. Eventually, automation will lessen the impact of this shortage, but not completely. And what can be done during the next 5 to 10 years, while new technology is slowly filtering into the laboratory? "We will still have to depend on people reading Gram stains, performing susceptibility testing by standard methods, and reading culture plates or CPE in virus culture," Baron says. "We haven't done a very good job of making clinical laboratory science attractive. We don't pay well."
But that is part of a larger issue. "The health care industry has had a great problem attracting people in support areas, not just microbiology but pharmacy and nursing, too," Bartlett says. "There is a real crisis in support personnel in the health care industry and I don't see that anybody has a solution."
Over the next several years, until amplification assays are fully automated, the introduction of this technology could aggravate this shortage in trained personnel. "I would regard it as a major problem," says Harrell. "The reason is that there is a different mindset when you are doing molecular testing." In traditional clinical microbiology, technologists are taught aseptic technique and how to avoid contamination. But when it comes to amplicons, which can be present in millions and billions of copies, the usual contamination problem escalates to a whole different order of magnitude. Even with kits that incorporate biochemical safeguards to reduce the risk of carryover contamination, Harrell says, "You still want to make sure you are ultra careful or you won't know whether you are getting a false positive." Avoiding false positives is especially important with HCV assays, which actually diagnose infection, as opposed to HIV tests, which quantitate viral load in infected individuals.
Maintaining separate areas for setup and amplification is part of the solution, as is adequate disposal so that incoming specimens do not become contaminated. But a high level of vigilance is crucial, and that requires as much as possible having certified medical technologists who have bachelor of science degrees or the equivalent and have passed a national certifying examination. Since so many already-employed medical technologists went through programs with little or no training in molecular methods, they require on-the-job training for amplification assays. To meet this need, Harrell uses a combination of one-on-one training in the laboratory and both off-site and on-site training by test and instrument vendors.
Despite Difficulties, Nucleic Acid Sequencing Usage Is Widening
While nucleic acid sequencing remains even more challenging than amplification assays, it has come a long way from its days as a research tool that used reagents that included hazardous chemicals and radionuclides. Now, it is largely automated and also equipped with fluorescent dyes instead of radionuclides with which to visualize results. However, the newer sequencing-based tests still use polyacrylamide gel electrophoresis (PAGE), depend on research-designed instruments with large "footprints" on the lab bench, and entail awkward gel-casting techniques with long polymerization times. Nonetheless, sequencing is entering at least larger laboratories where it is used primarily to detect HIV resistance mutations and to determine HCV genotypes.
Day says that Visible Genetics has developed the first DNA sequencing system designed explicitly for clinical use, the OpenGene system, which allows rapid, convenient gel casting and has interpretative software. The system was approved by the FDA on 27 September as an HIV-1 genotyping kit. "We have taken a process that used to take two and half hours and you can do it in less than five minutes," Day says of the system's preparation time.
Sequencers using capillary electrophoresis separation are also available for research use. Capillary electrophoresis sequencers are more readily automated; the primary maintenance task is replacing capillaries after about 100 runs.
"I see sequencing as being a growing market," says Alcorn. "When it gets put into kit format, it will break the ice as viral load testing kits did for amplification." Either sequencing or sequence detection using probes has the potential to detect antibiotic resistance genes and to identify bacterial species that are difficult to grow.
Looking even further into the future, Alcorn believes that microarrays and gene expression microarrays "have enormous potential to come into routine clinical use." As sequencing is used more in clinical laboratory settings, its users will demand ways to do it faster, a need that microarray platforms can meet, he says. Cost and technical hurdles will have to be overcome first. But eventually, in perhaps five years, he predicts, "I think they will become fairly standard."
Molecular methods can also provide basic understandings that will take clinical microbiology to a new level. "I am a big proponent that we need soon to undertake a major effort to define and characterize the normal microbial biome within the human body," Relman says. Molecular methods are ideal for obtaining an idea of the diversity of microbial signatures, such as microbial DNA sequences found normally in the bodies of healthy people in frequently infected sites—mouth, intestinal tract, vagina, skin—but also in sites typically thought of as sterile, such as blood, liver, and spleen. "I think we will find sequences there that will be helpful when we are looking for disease agents," Relman says.
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