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Repurposed Microfluidic CTC Purification Tech Enables Blood-based Antibiotic Resistance Test

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NEW YORK (GenomeWeb) – Pumping sample through coiled tubing or microchannels is an increasingly popular design element to purify large circulating tumor cells for liquid biopsy. Now, a new study shows the method can also be used for label-free purification of much-smaller bacteria from whole blood samples.

The method was the result of collaboration between researchers at Harvard University, Massachusetts Institute of Technology, The Broad Institute, and Massachusetts GeneralHospital, and was described online last week in the journal Lab on a Chip. The proof-of-principle study also described subsequent ribosomal RNA-based detection of bacteria using NanoString RNA analysis, as well as antibiotic resistance testing, with the entire workflow taking less than eight hours.

The microfluidic method relies on a principle called Dean flow fractionation, whereby material flowing through narrow, curvilinear channels tends to become sorted based on size. It has been shown to be capable of purifying particles in the size range of bacteria, with one recent study suggesting it could be used for sample clean-up.

Deborah Hung, a researcher and physician with positions at Harvard, Broad, and Mass General, and a co-author on the study, said she and her collaborators had previously used Dean flow in a microfluidic device to purify CTCs. These cells sort separately because they tend to be much larger than other cells in blood.

The idea for the bacterial microfluidic came about through conversations with her co-authors, where they ultimately asked themselves, "Why can't we use the same principles" except focus on the other end of the size spectrum?" Hung said.

"Its one of those things … it's obvious once you think about it, [but] on the other hand, to demonstrate it is actually kind of tricky," she said. Using the technique to purify viable bacteria required changing and fine-tuning a lot of parameters of the microfluidics, she noted.

The group characterized the device by pumping fluorescently labeled Escherichia coli through it and photographing them as they hugged the outer wall while progressing through the coiled microfluidic channels. Fluorescent red blood cells, meanwhile, moved to the inner wall of the device in a second experiment.   

The study also demonstrated that three different pathogenic bacteria could be purified with the device, from concentrations as low as around 10 cells per milliliter, with bacterial load in spiked samples later verified by culture. The group also spiked different combinations and proportions of the bacteria into whole blood, and showed this could also be parsed by the system.

All of the sample identification in the study was done using a NanoString assay for 5S, 16S, and 23S rRNA sequences of  E. coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Unlike traditional molecular diagnostics, by probing for rRNA "there are 10,000 molecules for every one cell, so you can essentially detect less than one bug," Hung explained.

She added that the group used NanoString because that was a platform they already had up and running. "We're exploring a lot of different detection methods as well … Other platforms we are working on will increase our sensitivity of detection further," she said, declining to name the platforms due to sensitivity around ongoing collaborations.

The susceptibility testing method in the Lab on Chip study relied on detection of antibiotic-responsive gene transcripts, based on a previously reported mRNA signature. Transcription occurs on a much faster time scale than even a single cell division, Hung said, adding that this method "will greatly accelerate the whole process, moving hopefully to real-time diagnosis."

Phenotypic susceptibility testing is an approach being pursued by other groups as well. Swedish biotech start-up Q-linea, for example, combines short culture times and antibiotic administration with padlock probe amplification, and recently described a four-hour test for resistant bacteria in urinary tract infections. 

There are a number of patents already covering the microfluidic technology, Hung said. "We are in the process of talking to several different companies, as well as VCs, to try to commercialize it," she said. 

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