May 2014 Clinical Laboratory News: Volume 40, Number 5
The Infectious Diseases Diagnostic Revolution
How Quickly Will New Methods Replace Culture?
By Genna Rollins
If there is any area of the lab in which the term revolutionary rightly is being applied, it has to be for rapid pathogen identification. Today, the great Louis Pasteur, a founding father of microbiology who lived in the 1800s, would feel at home with culture methods still widely in use that rely on superb technique and abundant patience, with days or even weeks-long turnaround times. In the future, however, culture likely will be a sideline in a field dominated by molecular diagnostic and mass spectrometry (MS) methods that give results rapidly—in hours or less—and in point-of-care (POC) or near-POC settings, according to experts. This juggernaut of change has profound implications for patient outcomes, the cost and efficiency of care, lab practice, and, most significantly, global antimicrobial stewardship efforts.
"If we can't rapidly determine whether someone does or does not have an infection and then whether their infection is bacterial or viral, we'll continue to see widespread use of antibiotics, at times in very inappropriate ways," explained Angela Caliendo, MD, PhD, chief of general internal medicine at Rhode Island Hospital and executive vice chair of medicine at The Warren Alpert Medical School at Brown University in Providence. "Rapid diagnostics really are a game-changer by helping us determine earlier in a patient's presentation the need―or not―for antibiotics so we can start implementing treatment modalities that change outcomes. There are all sorts of ways you can see it coming together."
Caliendo was first author of "Better Tests, Better Care: Improved Diagnostics for Infectious Diseases," an Infectious Diseases Society of America public policy paper that outlined measures to accelerate development of improved infectious diseases diagnostics and to see these new methods better integrated into clinical care (Clin Infect Dis 2013;57:S139–70) (See Sidebar, p. 4).
A Call for Improved Infectious Diseases Diagnostics
Concerned that much of the promise of emerging rapid methods for identifying pathogens remains unfulfilled, the Infectious Diseases Society of America (IDSA) in December 2013 issued a public policy paper calling for numerous changes to accelerate development of improved infectious diseases diagnostics and to see these new methods better integrated into clinical care (Clin Infect Dis 2013;57:S139–70). "Better Tests, Better Care: Improved Diagnostics for Infectious Diseases" grew out of IDSA's 2004 campaign, Bad Bugs, No Drugs, an effort to call attention to the problem of antibiotic resistance and to see more resources brought to bear against multidrug-resistant organisms.
"That was a very successful campaign, which raised awareness of multidrug resistant organisms and increased funding opportunities for drug development," explained Angela Caliendo, MD, PhD, executive vice chair of medicine at The Warren Alpert Medical School at Brown University in Providence. "This prompted us to think about how diagnostics in this field are not being well-utilized. This is all about raising awareness and getting the medical field to understand the important role diagnostics play, and it's about moving the field forward, away from this broad antibiotic coverage that we use because we never identify a specific pathogen." Caliendo was first author of "Better Tests, Better Care."
The microbiology field is undergoing radical change, with new technologies in some cases cutting from days to minutes the time needed to diagnose infections. Such rapid results enable more precise and appropriate use of antibiotics, overall better individualized patient management, and more efficient epidemiological and public health surveillance. Yet challenges remain in seeing the new methods developed completely, past regulatory hurdles, and integrated successfully in clinical practice.
To fulfill the unmet promise of these new technologies, the panel proposed an ambitious agenda aimed at effecting change in six broad areas, including: stimulating diagnostics research and development; expediting integration of improved diagnostic tests into patient care; addressing regulatory challenges; ensuring appropriate reimbursement; encouraging adoption of new tests; and educating healthcare providers about using new diagnostics.
To stimulate diagnostics research and development the authors called for the National Institutes of Health (NIH) to up its funding in this area, and for Congress to enact a tax credit covering half of clinical research costs for qualifying rapid diagnostics. The paper also recommended that the federal government and industry fund research to substantiate whether specific tests improve patient outcomes.
The authors also called on Congress and NIH to revise conflict of interest policies so that diagnostic companies, laboratories, and key opinion leaders can collaborate around high-quality clinical trials. The panel recommended that the Department of Health and Human Services withdraw a draft proposal to institute a new informed consent requirement for research using de-identified residual clinical samples.
The authors recommended that Congress enact the Diagnostic Innovation Testing and Knowledge Advancement Act of 2013, and that the Centers for Medicare and Medicaid Services eliminate wide regional variances in reimbursement. The panel also called on all stakeholders to work towards guidelines on developing reference methods for the new technologies. In addition, the authors suggested that diagnostic companies convert highly complex assays to moderately complex ones that could be run in a variety of clinical settings.
Finally, the authors proposed several initiatives aimed at educating healthcare providers on the use of infectious diseases diagnostics.
The main advantages culture has over newer methods are its comparatively low cost and robust antimicrobial susceptibility testing. However, the time it takes to arrive at the crucial susceptibility answer poses risks for patients, financial burdens for the healthcare system, and complicates antimicrobial stewardship efforts. Pathogen culture takes anywhere from 8 hours up to days, and susceptibility testing often adds at least another day, pushing the overall turnaround time close to 48 hours or longer, depending on the organism. By this time, empiric antibiotic therapy will have been started, but not uncommonly, once physicians receive susceptibility information, they change their orders in favor of narrower spectrum drugs.
Experts also cautioned that culture is not a panacea. Some organisms, like Mycobacterium tuberculosis, are notoriously fussy about growing on demand, and in certain circumstances culture doesn't pick up the real culprit behind an infection, according to Garth Ehrlich, PhD, professor of microbiology and immunology and otolaryngology-head and neck surgery, and executive director of the Center for Advanced Microbial Processing at Drexel University College of Medicine in Philadelphia.
"In our research involving chronic wound infections we've found it is not uncommon to detect [methicillin-resistant Staphylococcus aureus], but it's unlikely to be playing a significant role because it represents such a small percentage of bacterial burden. We almost always find some other pathogen—usually anaerobes with much higher titers which doesn't show up in culture," he explained. "We're good at culturing MRSA because we do it so often since it does cause so many problems. However, we detect it even when it's not the cause of infection."
Antigen, Antibody Testing
The ball started rolling away from culture with deployment of enzyme immunoassays and enzyme-linked immunosorbent assays to detect pathogen proteins or pathogen-produced toxins. Use of monoclonal antibodies and recombinant antigens sped up turnaround for these tests, which became even more valuable when, at least for certain pathogens, they were developed as rapid tests. Rapid antigen tests for influenza A and B, Clostridium difficile, and selected other microbes have been adopted widely. However, their sensitivity varies depending on the analyte in question, timing of testing after onset of symptoms, and other factors.
Riding the MDx Wave
The move toward non-culture methods gained momentum when polymerase chain reaction (PCR) and other nucleic acid amplification tests (NAATs) came on the scene, along with engineering enhancements to automate sample preparation, multiplex tests, and make the analyzers smaller and more user-friendly. From their debut as single analyte tests, these platforms now multiplex as few as two analytes up to nearly 30, across pathogen types and in relatively short order—less than 1 hour in some cases. They also are leading the way to random access rather than batch testing, which, while embraced by other areas of the lab, has been slower coming to microbiology.
Two examples of the reach of rapid molecular methods include Cepheid's GeneXpert system, which offers at least 13 Food and Drug Administration (FDA)-cleared assays for a variety of pathogens with one-stop sample preparation, nucleic acid extraction, amplification, and detection within 2 hours or less, and bioMérieux-BioFire's Film Array Respiratory Panel, which detects 17 viruses and three bacteria. Many other diagnostic manufacturers also offer broad test options and features: Abbott, Siemens, Roche, and Qiagen to name just a few.
Christine Ginocchio, PhD, MT(ASCP), reflected on how rapid molecular testing already has changed lab and clinical practice. "Twenty years ago, it was done only in academic medical centers and reference labs because we had to design and develop our own tests," she recalled. "But over time they have gotten much simpler to use. So tests even five years ago that took multiple steps and were very complex operationally now take a minute or two of hands-on time." Ginocchio in February joined bioMérieux as vice president of microbiology affairs after a long career as senior medical director and chief of infectious diseases diagnostics at North Shore-LIJ Health System in New York.
She added that rapid molecular diagnostics also have moved testing out of core labs to near-POC settings, such as emergency departments, and, for some infectious diseases like HIV, have changed treatment protocols. They also have made it possible to rapidly identify hard or impossible-to-culture pathogens such as hepatitis C virus. While they represent a huge advancement, especially in viral analysis, their high per-test cost is a drawback.
Researchers are continuing to press the envelope of possibilities with molecular diagnostics. For instance, a team at the Duke Institute for Genome Sciences and Policy recently reported 89% sensitivity in analyzing 30 genes with a reverse transcription-PCR assay to distinguish viral respiratory infections from bacterial disease based on host response. Genomic sequencing also is an emerging tool, particularly in aiding in outbreak detection and surveillance efforts, and for detecting virulence and genetic markers of antimicrobial resistance. Notably, National Institutes of Health (NIH) investigators in 2011 used whole genome sequencing to detect and track an outbreak of deadly carbapenemase-producing Klebsiella pneumoniae the NIH Clinical Center was struggling to contain.
Mass Spectrometry to the Rescue?
Even as research about genomic methods progresses, two other methods—one already in use, and the other rapidly approaching clinical practice—have the potential to blow the lid off the entire field. The former, matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) MS, identifies microorganisms from the molecular weights of their proteins and peptides. Once only in the dominion of research labs, it is being embraced by clinical labs for its sensitivity, ease-of-use, low per-test cost, rapid turnaround time, and high throughput. MALDI-TOF/MS also identifies bacteria, fungi, and mycobacteria at the species level, based on matches with reference databases of spectra from known organisms.
This technology got a big boost last year when FDA cleared two systems, bioMérieux's VITEK MS, for rapid identification of up to 193 bacteria and yeast, and Bruker's MALDI Biotyper CA, for identifying 40 gram negative bacterial colonies cultured from human specimens. These clearances mainstreamed MALDI-TOF/MS, moving it beyond the realm of lab-developed tests. The main knock against it is cost: units have hefty six-figure price tags, along with substantial yearly maintenance expenses. In most cases, MALDI-TOF/MS also does not get around the need to culture organisms first, and although research is active in this area, it so far has not proven very helpful clinically in answering the crucial question of antimicrobial susceptibility. Ehrlich went so far as to call it a "stop-gap technology" for these reasons.
As groundbreaking as MALDI-TOF/MS may be, another extraordinary technology is waiting in the wings to make a grand entrance. Developed under the stringent requirements of the Defense Advanced Research Projects Agency for its interest in rapidly identifying bioterrorism threats, PCR electrospray ionization (ESI)/MS detects bacteria, virus, fungi, and protozoa using the mass-to-charge ratio of a PCR amplicon to infer its base composition, which is compared to a database of hundreds of organisms. In contrast to MALDI-TOF/MS, it detects amplified nucleic acids directly from non-cultured specimens, cutting turnaround times anywhere from 8 hours to days. Also unique from other molecular diagnostic methods, PCR-ESI/MS uses the nucleic acid amplification step only for amplification, with detection completed by MS.
Passing the Pan-Domain Test
Ehrlich, a leading PCR-ESI/MS researcher, predicted that it could sound the death knell for culture. "The reason we stick with culture in part is we don't have any pan-domain test," he said. "But the power of this system is far superior to anything out there or on the horizon because it's truly pan-domain. The investigator doesn't have to decide a priori what to test for. That allows enormous confidence in your negative- and positive-predictive value."
In several lines of investigation, Ehrlich's lab has run approximately 7,000 samples via PCR-ESI/MS. The study protocols call for comparing PCR-ESI/MS analysis against culture, along with 16S fluorescence in situ hybridization (FISH) as a confirmatory test and 16S DNA sequencing as a reference test. In cases where there were discrepancies between culture and PCR-ESI/MS, 16S FISH "in almost every case" corroborated the PCR-ESI/MS findings, according to Ehrlich.
Originally developed by Ibis Bioscience, which now is part of Abbott, PCR-ESI/MS is expected to make its commercial debut in Europe within 9–12 months, according to David Ecker, PhD, divisional vice president of Ibis Biosciences, Abbott. The company subsequently plans to seek FDA clearance.
Ginocchio compared PCR-ESI/MS's current product life cycle stage with that of MS years ago. "The manufacturers took a great technology that used to be so difficult to run that clinical labs couldn't use it routinely and made it so simple it can be run in any lab. That's what Abbott has to do with their PCR-ESI/MS system."
Changing Lab, Medical Practice
The dazzling power of these new technologies will do nothing short of changing the practice of medicine, predicted Robert Bonomo, MD, professor of medicine, pharmacology, molecular biology, and microbiology and chief of medical service at the Louis Stokes Cleveland Department of Veteran Affairs Medical Center.
"Years ago, we'd get a culture back and it would show just S. pneumoniae. In the future, we'll get a result that says S. pneumoniae, another pathogen, and a virus. When that happens, I, as a physician, will have to decide what to do, quickly," he explained. "In the case of pneumonia, right now 40–50 percent of the time, it's culture-negative, meaning we don't recover what we expected. We give patients a series of antibiotics, but we never know what caused this pneumonia. These new tests will change how we think about that and how we approach treatment."
The emerging methods also will spell big changes for lab practice. Could the race to bring on powerful big-ticket technologies with rapid turnaround times blur the lines between chemistry, microbiology, and virology labs? Some observers, like Alex van Belkum, PhD, corporate vice president of microbiology research and development at bioMérieux, think not, at least in the case of MALDI-TOF/MS. "Switching this equipment between different analytical regimens might have consequences for the calibration of the equipment and for the usefulness of it per se. From a practical point of view I don't think the current version of the microbiological application of MS will bring us much closer to clinical chemistry," he said.
Others, like Caliendo, see it as a possibility, albeit with a big caveat. "It looks like technically you could put these instruments in a core lab, but you would need to bring the rest of the information and microbiology context into the core lab," she stressed. She cited a situation in which a chemistry lab began testing for Chlamydia trachomatis. Positivity rates jumped substantially, but it took a while to recognize this uptick and realize it was due to contamination, something the microbiology lab would have picked up immediately.
How Soon the Standard?
How soon will these dazzling technologies become the standard of care? van Belkum took the long view. "Culture-based assays work well; they're pretty well standardized, the reagents are not very costly, and people are used to them. So to see that being replaced by high-tech expensive technologies for which we still need to do a lot of validation studies, to me, it's hard to see it happening soon," he said. He also emphasized the need for further research demonstrating outcomes as well as ongoing engineering tweaks to optimize the technologies.
Caliendo also underscored the need for solid outcomes research. "I don't think anybody is married to a specific technology. What they're married to is the outcome of the technology. People are open to the how; it's what it does," she said. "We have to support research that develops the new technologies as well as shows clinical utility. It doesn't do us much good if we have technology that looks exciting but we can't show that it much improves care."