By Ben Butkus
Cepheid is developing an automated assay for its GeneXpert platform to provide broad-range amplification and species-level discrimination of a wide variety of bacteria from clinical blood culture bottles, the project's lead scientist said at a recent qPCR conference.
The assay, which is based on melt curve analysis and targets the highly conserved yet hypervariable 16S ribosomal RNA gene found in many bacterial species, can currently detect and discriminate between nearly 40 of the most important clinical bacterial species, and may eventually be able to test for more than 100 such species, according to Reuel Van Atta, a principal scientist at Cepheid who talked about the development of the technology at last week's qPCR Symposium USA in Millbrae, Calif.
Cepheid, based in Sunnyvale, Calif., already sells the Xpert MRSA/SA test for detecting methicillin-resistant Staphylococcus aureus or S. aureus in gram-positive blood culture bottles in less than one hour. That test, which performs sample preparation, PCR amplification, and detection in a single disposable cartridge, was cleared by the US Food and Drug Administration in October 2008.
However, in speaking with various clinicians, Cepheid unveiled a desire within the community to have a similar single-cartridge test that could identify multiple bacterial species in blood culture bottles so physicians could more quickly and efficiently identify the need for more targeted antibiotic therapy following early administration of broad spectrum antibiotics; or determine whether the bacteria present is simply a result of contamination and thus doesn’t necessitate a treatment regimen, Van Atta told PCR Insider in an interview following his symposium presentation.
Van Atta said that Cepheid asked clinical researchers for a "bacterial ID wish list" composed of clinically important gram-positive and gram-negative bacteria species, and received requests for more than 100 species, which was too large a number with which to develop a proof-of-principle assay. By comparing the wish list to prominently listed species identified between March 2009 and March 2010 in Focus Diagnostics' Surveillance Network database; and consulting with collaborators at the Clinical Microbiology Laboratories at Stanford University Hospital, Cepheid was able to whittle the initial wish list down to "a few dozen species, which is a reasonable number to try on the first go-around," Van Atta said.
The current assay protocol is very similar to how clinicians currently run tests on the GeneXpert platform, including sample preparation, elution of bacterial DNA, and mixing with all necessary PCR reagents. Then the sample is "moved to the GeneXpert cartridge and put on an instrument," Van Atta said.
The GeneXpert cartridges currently offer two types of nucleic acid purification, according to Van Atta: DNA/RNA capture on solid-phase material; or membrane cell capture and ultrasonic lysis. He added that for all GeneXpert tests, including the bacterial ID test in development, Cepheid is currently working to include all necessary reagents on the consumable cartridge "so you wouldn't really need to add anything, which would be considered low complexity" under the Clinical Laboratory Improvement Amendments guidelines.
In his presentation, Van Atta presented compelling early data demonstrating the assay's ability to distinguish nearly all species of bacteria from mixed samples, including several different species of Staphylococcus; and all gram-negative bacterial species typically found in a blood culture bottle. Nevertheless, Van Atta stressed that the assay is in the very early stages of development, and he did not provide a commercialization timeline.
"We have 37 bacteria in there right now, and we are hoping to expand eventually to 72," Van Atta said. "Who knows? Maybe we'll be able to get into 100 or more."
Van Atta added that similar testing could be done using other molecular biology tools such as sequencing or mass spectrometry, but that such an endeavor would cost Cepheid extra time, money, and equipment development; as opposed to using the clinically vetted GeneXpert platform.
In fact, developing the new assay also provides Cepheid with a "test system" to develop and validate melt curve analysis capabilities on GeneXpert, which currently lacks such a feature but "is adaptable to melt curve analysis," Van Atta said. Cepheid's SmartCycler product line is capable of melt curve analysis, but is not designed for clinical use.
Van Atta also said that the bacterial ID assay may eventually be applicable to direct diagnostic testing from patients, but that Cepheid chose to begin work using a blood culture bottle "because it was much easier to do."
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