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From The EditorSputum, the Final Frontier: PCR from the Research Bench to the Diagnostic Trench
Some readers of this newsletter may have had the experience of actually performing PCR as it was first described in 1985. The procedure involved pipetting new aliquots of Klenow polymerase after each PCR cycle because the temperatures required for denaturation of the target and amplification products also denatured the enzyme. Most memorably, the procedure required sequential steps of floating small plastic tubes in water baths kept at three different temperatures, with no time for bathroom breaks. The advent of a thermostable DNA polymerase was a huge improvement, but the novelty of the water baths quickly wore off, even for the most dedicated graduate student. I recall the great excitement at the University of California on the day when a prototype Perkin-Elmer thermal cycler was delivered to Dr. Jane Gitshier’s lab; one thermal cycler was made available for the entire university. The sign-up list quickly filled up as eager graduate students filed into and out of the lab at all hours of the day and night. The inconvenience of a midnight PCR run was a much-preferred alternative to standing, lock-kneed, pipettor in hand, in front of three water baths. Needless to say, times have changed. Two decades of technological improvement have now made the field of molecular diagnostics barely recognizable. Clumsy, contamination-prone techniques have been largely replaced with real-time detection technology performed in closed systems. DNA sequencing and microarray technologies developed under the auspices of the Human Genome Project are making steady inroads into clinical laboratories. Diagnosticians have taken a great leap forward in their level of overall sophistication and familiarity with these technologies. Phylogenetic analysis and identification of bacteria, fungi and viruses by direct DNA sequencing are quickly entering the mainstream, and will require us to add a few new words such as “bootstrapping” and “parsimonious” to our vocabulary. Much of Cepheid’s current focus revolves around the detection and characterization of infectious agents that cause healthcare-associated infections (Methicillin-resistant S. aureus, Vancomycin-resistant enterococci, and C. difficile). Although these pathogens come from entirely different branches of the prokaryotic phylogenetic tree, they are all similar in that they harbor genetic signatures that can be used to identify them, quantify their infectious burden, determine their virulence, and assess their susceptibility or resistance to available drugs. From a diagnostic test development standpoint, the major hurdles to be overcome are now related less to the nucleic acid detection technology itself than to sample processing. Indeed, the main challenges for many procedures involve determining: the appropriate quantity of specimen, how to eliminate inhibitors of enzymatic reactions that exist in these specimens, and how best to efficiently release the target from its often impermeable microbial shell. To paraphrase a well-known opening line, “sputum, the final frontier”. For the molecular diagnostician, it truly is. As real-time PCR technology continues to evolve, so should our focus on delivering real-time patient results. Most diagnostics manufacturers have focused heavily on development of batch-based commercial tests, for which the rapid turnaround requirements are not as stringent as for acute infectious processes. Choices about when and how to run the batches are usually decided according to test volume. The end result is that the very technology once touted for its speed is reporting results in days or even weeks, depending on when batches are run. Cepheid has taken a fundamentally different approach to molecular diagnostic testing, creating the GeneXpert for rapid turnaround and quick response to patient test requests. Each GeneXpert cartridge is a virtual laboratory in itself, containing specimen-specific preparation instructions, target-specific reaction components and controls that allow specimens to be tested, irrespective of specimen type or test request, on-demand and in the order received. By accomplishing all of this in an easy-to-use format, the GeneXpert allows testing to be performed at any time and in almost any laboratory. At Cepheid, the enthusiasm fueled by making extraordinary technology incredibly easy to perform has taken on infectious proportions of its own. The case study of New England Baptist Hospital presented in this issue is an example of how rapid MRSA test results delivered by the GeneXpert can be used to determine the most effective care options for surgery patients, lowering the overall risk of infection. The success of the NEBH program speaks for itself, with the reward of lower transmission pressure in the hospital, reduced infectious complications and increasing patient demand for NEBH services. Ultimately, we at Cepheid believe that all patients, in or out of the hospital, will benefit from molecular diagnostics technology that is well-practiced and accessible to patients where and when they need it most.
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