Vol. 22 • Issue 5 • Page 14
Cover Story
In the last few years the FDA has approved multiplex nucleic acid tests for pathogens causing a number of clinical syndromes.1-3 Approved panels include those for respiratory disease, bloodstream infection and, more recently, gastrointestinal illness. Additional panels of pathogen assays are in the pipeline that will cover the same indications in more depth as well as new panels to detect and identify pathogens responsible for meningitis, pneumonia, viral hepatitis and sexually transmitted infections.4-5 The diagnostic platforms that run these panels vary in complexity, time to result, cost and throughput. To stay competitive, manufactures will have to continually improve their offerings so that successive generations of instruments converge toward rapid, comprehensive, low cost, near point-of-care testing (POCT). Multiplex panels provide novel opportunities and challenges for clinical laboratory directors and for the clinician receiving the test results.
Exploring the Long Tail
Multiplex panels allow testing for not only the common agents of a particular syndrome, but also for less common agents. While for many syndromes there are 3 – 4 common etiologic agents (e.g., influenza, respiratory syncytial virus and human rhinovirus for upper respiratory tract infections), there is also a long “tail” of less common pathogens (for example, the human Coronaviruses and Chlamydophila pneumoniae) that may be important to identify.
It has been difficult to justify developing individual tests for each of these organisms, as their abundance varies so much across time and geography, but multiplex panels allow less common pathogens to be included without significant increases in cost. This has raised the diagnostic yield of such tests compared to conventional methods and for particular indications (e.g., GI pathogens); multiplex molecular platforms also simplify the testing algorithm – microscopy, antigen detection and culture-performed – sequentially down to a single test performed at one time. However, deciding which pathogens to include in each panel is a complex question for manufacturers. How many are necessary? What are the criteria? This is a challenging area that can be successfully navigated through industry-healthcare collaborations.
Multiplex Results: When is Enough Information TMI?
The development of comprehensive infectious disease panels that are FDA cleared or approved means that sensitive, syndrome-based panels of pathogen tests are available to even moderate complexity laboratories. Highly multiplexed infectious disease panels have greatly increased the number of positive tests, including a significant number of multiple detections reported. While this can be considered a “higher yield,” an issue arises in interpreting the significance of multiple pathogens in a single sample. When molecular methods are used for detection, a positive test result simply indicates the presence of a virus or bacteria and does not distinguish a live organism from residual nucleic acid. While some viruses, like RSV and influenza, are almost always the causative agent of respiratory infection when detected, others such as human Rhinovirus may be bystanders in some cases and the etiological agent in others. Influenza-like-illness (ILI) can in fact be caused by a variety of different respiratory viruses.
How do clinicians decide which pathogen, of several detected, is the significant one?6 The answer may depend on the host, including immune status or predisposing conditions, the clinical presentation and even the other organisms present or absent in the sample. It may take a number of years and many studies before we begin to understand multi-pathogen detections and their associated clinical implications. As an increasing number of multiplex platforms are used routinely in clinical practice, these studies, possibly requiring the analysis of thousands or tens of thousands of patients, become feasible.
Bacterial Detection and Antibiotic Resistance
Viral pathogen detection led the charge into the era of multiplexed molecular diagnostics, but bacterial detection is not far behind. Panels for identification of pathogens from positive blood cultures are increasingly available and have been adopted in many laboratory settings. Implementation of these panels has the potential to improve antibiotic selection for patients with bloodstream infection and sepsis by providing organism identification 24 – 48 hours sooner than conventional methods. Challenges for the laboratory, however, include developing methods to convey results to clinicians who may not be prepared to make antibiotic changes based on organism identification without associated susceptibilities. Studies of rapid staphylococcal identification from positive blood culture have shown no improvement in antibiotic selection or patient outcomes without direct physician notification preferably by a practitioner, such as a pharmacist or infectious disease specialist who can assist in choosing therapy.7
As bacterial panels expand the number of gene targets that can be detected, the possibility opens up to provide information regarding antibiotic resistance, and ultimately replacing phenotypic determination of antibiotic resistance with genotypic readouts. As this is arguably the most clinically actionable result of a panel, there is a question of whether a rapid molecular call of antibiotic resistance markers can predict in vivo susceptibility. More data will be needed to correlate genotypic, phenotypic and in vivo results. Clinicians, who are used to receiving a list of drugs to which the bacterium is susceptible and choosing from these, will need to be educated in using genotypic data to make informed treatment decisions.
The advent of closed-box, multiplex platforms may signal the end of tissue culture for the routine detection of virus in the microbiology laboratory. No viral culture system will ever be able to match the time to result and the user-friendly aspects of the new systems. A generation of virologists skilled at differentiating viruses by their cytopathic effect and the pattern of antibody staining may reasonably have doubts about the loss of this art, but the new systems are more in tune with the needs of the modern laboratory. As with all new technology, something has been lost but something is gained.
Molecular identification of bacteria from blood culture is a stepping stone to the long desired goal of this field – direct detection of bacteria from blood for the rapid detection and identification of sepsis. This goal is still a few years off because of the technical ðdifficulties of matching the exquisite sensitivity of blood culture with PCR assays, but it is the logical next step. Development of such assays will require a ðcombination of upstream enrichment of the pathogen from blood with meticulous attention to removing residual contaminating bacterial and fungal DNA from the nucleic acid amplification reagents.
Protein biomarkers of infection are well established, although the interpretation of their significance is still in debate. Studies using microarrays and even next-generation sequencing have begun to characterize the host (human) transcriptional response to infection. As this data is understood and validated, it is likely that the molecular platforms discussed here will be extended to be able to simultaneously detect the infectious agent and the host response. This combination may be useful to guide treatment and resolve the issues of causality discussed above.
Dr. Poritz is director of Biochemistry, BioFire Diagnostics, Salt Lake City, Utah. Dr. Blaschke is assistant professor of Pediatrics and ðPediatric Infectious Diseases, University of Utah School of Medicine.
References
1. Ginocchio, C.C., Strengths and weaknesses of FDA-approved/cleared diagnostic devices for the molecular detection of respiratory pathogens. Clin Infect Dis, 2011. 52 Suppl 4: p. S312-25.
2. Caliendo, A.M., Multiplex PCR and emerging technologies for the detection of respiratory pathogens. Clin Infect Dis, 2011. 52 Suppl 4: p. S326-30.
3. Perez-Ruiz, M., et al., Laboratory detection of respiratory viruses by automated techniques. Open Virol J, 2012. 6: p. 151-9.
4. Niemz, A., T.M. Ferguson, and D.S. Boyle, Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol, 2011. 29(5): p. 240-50.
5. Emmadi, R., et al., Molecular methods and platforms for infectious diseases testing a review of FDA-approved and cleared assays. J Mol Diagn, 2011. 13(6): p. 583-604.
6. Pavia, A.T., Viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis. Clin Infect Dis, 2011. 52 Suppl 4: p. S284-9.
7. Ly, T., et al., Impact upon clinical outcomes of translation of PNA FISH-generated laboratory data from the clinical microbiology bench to bedside in real time. Ther Clin Risk Manag, 2008. 4(3): p. 637-40.
