The Association for Molecular Pathology's annual meeting, held last week in Long Beach, Calif., focused strongly on next-generation sequencing and its implementation in the clinic, especially for diagnosing, monitoring, and tailoring treatments for cancer.
However, a number of presentations made it clear that PCR and real-time PCR, while no longer the obvious technology choice for all molecular diagnostic and pathology applications, is still one of the most powerful tools available for infectious disease diagnostics and therapeutic monitoring.
For instance, in his presentation during a plenary session entitled "Surveillance for the Molecular Laboratory," Barry Kreiswirth of the University of Medicine and Dentistry of New Jersey described various strategies used by his institution and others to conduct molecular epidemiological studies and control infectious disease outbreaks.
More specifically, Kreiswirth described a strategy currently being explored to curb Staphylococcus aureus outbreaks in hospital settings by combining information about geographically isolated clones of the bacteria with information on patients' movement and contacts, or with data from electronic health records.
In order to execute such a strategy, clinicians need to employ a method to rapidly genotype these S. aureus strains, Kreiswirth noted. Such methods in the past have included pulsed field gel electrophoresis, multi-locus sequence typing, and staph protein A typing – all of which have drawbacks making them less than ideal for clinical implementation.
Instead, Kreiswirth and colleagues worked to identify customized SNPs for specific MRSA clones using a number of technologies. Then, "we can create customized assays for those SNPs in 96-well plates for rapid strain genotyping and patient tracking."
Kreiswirth noted that molecular beacons – hybridization probes that can be used in real-time PCR assays to provide a high degree of sensitivity and specificity – would be an ideal technology for such assays. Fellow UMDNJ researcher Fred Kramer is one of the developers of the molecular beacons technology.
Meantime, during a symposia session on viral load testing and monitoring therapeutic response in infectious diseases, Aneesh Mehta of the Emory University School of Medicine provided a summary of current methods being used to test cytomegalovirus load in patients and monitor CMV disease.
Human CMV, Mehta explained, infects all populations and is seroprevalent in 50 percent to 90 percent of adults. It causes a wide spectrum of disease, with immune-healthy patients exhibiting minor complications such as mononucleosis, fever, or colitis.
However, the virus is a much larger problem in immunocompromised patients, and is the most common opportunistic infection in AIDS patients, Mehta said. In addition, in transplant patients, the virus often manifests itself as CMV "syndrome" or CMV-related tissue disease, both of which can cause serious complications in patients.
Approaches vary widely to prevent or manage CMV infection in transplant patients, Mehta said, but two strategies have come to the forefront: "universal prophylaxis," in which antiviral therapy is administered to all at-risk patients post-transplantation; and "preemptive therapy," which comprises monitoring patients regularly with PCR-based assays to detect infection or to quantify viral load.
Diagnostic methods for CMV monitoring have "evolved tremendously," Mehta said. "It used to be histopathology, then serology, then semi-quantitative fluorescence" for the CMV pp65 antigen. Currently, however, both qualitative and quantitative PCR are most widely employed, he added. Both Abbott and Roche offer real-time PCR-based assays for CMV viral load, and a number of other molecular diagnostics companies are developing similar assays.
In fact, "qualitative measurements of CMV DNA levels have become popular at transplant centers," Mehta said. However, he added that "correlation of absolute value to disease state is difficult," raising questions among clinicians about what level of viral load predicts disease, how long treatment should be administered, and how often testing should occur, especially considering the fact that clinicians and patients often have their testing performed at many different clinical laboratories.
He added that this issue raises the need for standardization of viral load measurements and their association to therapy timing and response monitoring – a task that the World Health Organization has recently attempted to tackle by introducing its first international standardization guidelines for human CMV nucleic acid amplification techniques.
Mehta noted that clinical researchers at Emory recently recalibrated their laboratory-developed assay for CMV viral load quantification to the international standard and have thus far seen positive results.
In addition, Mehta said that the Clinical Trials in Organ Transplantation project, a federally funded investigative consortium involving researchers from Emory and other institutions that is conducting transplantation studies, has benefited greatly from the recently introduced WHO standard.
"The WHO CMV standards were a huge boon for them," Mehta said, concluding that "quantitative CMV PCR has become the standard of care."
In another symposia session, Mitchell Shiffman of the Liver Institute of Virginia and Bon Secours Virginia Health Systems described the role of molecular testing to monitor hepatitis C virus therapy using recently introduced and highly effective direct-acting viral inhibitors.
Certain of these new therapies, primarily protease inhibitors, in general have been shown to have a 75 percent cure rate in HCV patients. However, this number does not tell the entire story, Shiffman noted, as certain patients who express a particular genotype conferring sensitivity to interferon respond extremely well to a combination of interferon and protease inhibitors, while those who do not express this genotype "respond terribly" to the combination treatment.
Shiffman noted that measuring HCV RNA has recently emerged as a potentially highly effective method of making "stop or go" decisions with regards to certain treatment combinations.
In order to execute this strategy, Shiffman said, clinicians "must use a PCR assay that can quantify the level of HCV RNA … [and] differentiate between unquantifiable and undetectable virus." Elaborating, Shiffman said that an assay that reports viral loads of less than 25 international units per milliliter of blood without qualifying whether the HCV RNA is detectable or undetectable should not be used.
Shiffman did not detail whether such an assay is currently available or under development. Again, both Roche and Abbott offer molecular assays for HCV viral load quantification, and several other companies are developing such tests.
Finally, in an AMP workshop on molecular diagnostic testing for complex targets, Randall Hayden of St. Jude Children's Research Hospital outlined the need for comprehensive multiple molecular testing for diarrheal diseases and gastroenteritis, which have multiple causative agents – viral, bacterial, and parasitic.
Hayden noted that it is a "complex process to get at all these organisms," and outlined the numerous methods that are currently in use for detection and diagnosis. These include cell culture, direct fluorescence assays, electron microscopy, enzyme immunosorbent assays, nucleic acid testing, and solid media assays. However, some of these methods are useful for only bacterial detection and others for viral detection, and vice versa.
In general, "it's a complicated process … with lots of effort and lots of lab benches," Hayden said.
The most promising solution to date, he noted, is multiplexed molecular amplification. And although the scientific literature contains numerous studies on PCR-based assays for various gastrointestinal pathogens, the only major pathogen for which there are commercial molecular assays is Clostridium difficile, marketed by multiple companies, with the rest being laboratory-developed tests.
"There are limited commercial reagents, and even fewer FDA-cleared assays" for GI pathogens, Hayden said, adding that clinical labs need "broadly multiplexed methods."
A number of such assay panels are currently in development, including the Luminex xTAG GPP, BioFire Diagnostics FilmArray GI panel, and Nanosphere Verigene Enteric Pathogens test.
In addition, Hayden underscored promising technology from Seegene, which has a multiplexed diarrheal pathogen test based on its proprietary real-time PCR chemistry available in Canada but not in the US; and Renishaw Diagnostics, which is working on an assay panel that uses surface-enhanced resonance Raman spectroscopy to detect multiple nucleic acid targets from a single sample.