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High-Res Melting Assay and Abbott Plex-ID Compare Well in Hopkins Blood Culture PCR Test Matchup

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Researchers from Johns Hopkins University have reported that their high-resolution melting assay and a PCR/mass spectrometry approach using Abbot's Plex-ID platform performed similarly in identifying pathogens from a cohort of positive blood culture samples.

Comparing the two broad-range PCR assays in a paper published this month in the Journal of Clinical Microbiology, the group found them to be largely concordant in their results, and between 85 percent and 90 percent concordant with original blood culture findings.

Charlotte Gaydos, one of the study's authors and a senior researcher on the Hopkins team led by Richard Rothman, told PCR Insider this week that differences in cost and ease between the two assays suggest they might be suited to different blood culture testing settings, but that overall both tests performed well and comparably to each other.

The Hopkins team has been working under several grants from the National Institutes of Health to develop a method for identifying bacterial pathogens in sterile body fluid by performing PCR across the 16S ribosomal RNA gene, which is hypervariable but highly conserved across bacterial species.

The group originally started designing individual probes for each organism, but recently switched to using high-resolution melt curve analysis on an Idaho Technology LightScanner HR96 system to identify pathogens by unique melting curves associated with three hypervariable regions in the gene.

Gaydos said that as part of its NIH-funded work, the Hopkins team also began working with researchers from Ibis Biosciences, which has since merged with Abbott, to use its Plex-ID system, which couples PCR with electrospray ionization mass spectrometry of DNA amplicons to target the 16S and other genes to identify blood-borne pathogens

After seeing success for its own HRM-based method in a proof-of-principle experiment using discarded blood culture tubes, the Hopkins team decided to test the Abbott assay as well. In the recent study, which researchers from Abbott co-authored, the group compared the two methods to each other and to established bacterial culture results.

"The advantages of our method are that it's inexpensive and fast," Gaydos said. "It probably would be ideal for resource-poor settings or a small hospital."

Abbott's Plex-ID system is more expensive than the HRM assay but more adaptable in terms of the organisms it can identify, she said. "It costs in the neighborhood of $750,000 to buy one of these very complex machines, so someone like a huge medical center or a state health laboratory [is] more likely to want to invest in this. Different strokes for different folks, we say."

Overall, she said the team believes both methods "will have a place in clinical microbiology in years to come."

In the study, the Hopkins researchers performed both assays on used blood culture tubes collected over the course of three months. The group tested a total of 70 samples of residual blood.

Overall, the researchers found that their HRM assay was concordant with blood culture findings in 90 percent of the samples for both genus and species identification. The test misidentified seven samples, five of which showed multiple melting peaks, suggesting a polymicrobial infection that the researchers later confirmed using sequencing analysis. Using melting curves, the assay is not able to resolve a diagnosis when multiple bacterial species are present, Gaydos explained.

The remaining two discordant samples were marked by conventional culture as Fusobacterium varium and coagulase-negative Staphylococci. Neither was present in the Hopkins group's 60-organism melt curve library, the authors wrote.

For the Abbott assay, the researchers measured 94 percent concordance with blood culture on the genus level and 85 percent concordance at the species level.

Four species were discordant at the genus level, the group reported. Two of these were diagnosed as Morganella morganii by bacterial culture and Yersinia enterocolitica by the Abbott test. Six other samples were correctly identified by genera, but the Plex-ID reporting algorithm was not designed to give species-level identifications for the genera identified.

In their report, the authors wrote that the results highlight some of the advantages and limitations of each assay.

Primarily, the two approaches differ in ease, Gaydos said. In the paper, the researchers wrote that the melting approach only requires three PCR reactions, while the Abbott method requires 16 reactions per sample. The group's melt curve method can also be performed with closed tubes, "further streamlining laboratory workflow," the authors wrote.

However, the melting approach has limitations relative to the Plex-ID system. The Abbott assay is highly automated, the group reported, unlike the melting approach — although Idaho Tech is currently developing a blood culture ID assay panel for its fully automated FilmArray HRM testing platform (PCR Insider, 7/19/2012). More importantly, the JHU team's melting assay couldn't resolve the presence of individual pathogens in polymicrobial infections unless applied to isolated bacterial subcultures.

Gaydos said that the Plex-ID approach also offers the possibility of much higher throughput, which could be attractive for high-volume labs or hospitals. Abbott is developing a number of diagnostic assays using the system, offering a much broader set of applications of the instrument than bacterial identification in blood culture. For instance, PCR Insider in April reported that the company is developing an assay to detect and genotype Borrelia burgdorferi, the bacterium responsible for causing Lyme disease in humans (PCR Insider, 4/19/2012).

According to the JHU group, the melt curve library used as a reference in its own assay is still limited to 60 organisms, which means rare pathogens will still be unidentifiable when encountered.

In contrast, the Abbott method employs a much larger reference library with more than 400 species, the Hopkins group wrote. It also uses multiple genetic targets to identify each organism, which "creates higher confidence in a detection and identification."

However, the team's results suggested the PCR/ESI-MS approach may still be unable to make a species-level diagnosis in every instance, and can still misidentify organisms due to polygenic variations in closely related genera, as demonstrated in the assay's mistakes in the study.

Gaydos said both her group and Abbott hope to eventually develop their respective assays to the point where they can be used in lieu of bacterial culture on blood samples taken directly from patients. Demonstrating that both approaches work on samples from blood culture bottles was a first step, she said.

"The purpose of this study was to see if we could save valuable time for clinicians by doing the [HRM or Plex-ID assay] on the already amplified blood culture in the blood culture bottle," she said. "We wanted to show that we can use [a PCR assay] after that 12 hours when the blood culture machine goes off."

"Normally it takes 14 to 48 hours before a doctor knows what the organism is, after a blood culture machine says the specimen is positive," Gaydos explained. "We wanted to show we could shorten that by using this technique."

Gaydos said that other methods, most prominently MALDI-TOF mass spectrometry, are already currently used in Europe to identify organisms from a positive blood culture. "Our hope with both these methods … is to identify organisms right from the arm … which is something MALDI-TOF cannot do," she said.

According to Gaydos, both her team and the Abbott researchers are working toward that goal, primarily by improving the sensitivity of their respective assays. She said the Hopkins group has published data showing it can identify one organism per milliliter of blood using the melt-curve approach. The team hopes to get that down to one organism per 10 milliliters.

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