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New Tools to Rapidly Diagnose Infections and Detect Antibiotic-Resistant Bacteria

2016 SLAS Innovation Award winner Shana Kelley was a graduate student at Cal Tech when a basic science project serendipitously yielded a technique for identifying single nucleotide mismatches in DNA—i.e., disease-related mutations. “It just popped out of the basic science we were doing and I thought, ‘hmmm, that’s interesting….,’” she recalls.

Kelley went on to patent the approach and co-found GeneOhm Sciences, a San Diego-based diagnostics company that was acquired by Becton Dickinson in 2005. She also started running her own laboratory at the University of Toronto, where she continues her work today. “Through that experience, I saw how powerful it can be to take something from basic science, translate it, take it to a company and commercialize it—and see it deployed widely to combat antibiotic-resistant infections,” she says.

“Participating in that kind of discovery and then translation of science was great, and that has been the guiding philosophy behind my research program ever since: Let’s do good science, but let’s also create something that can really make a difference for society.” 

Kelley’s latest tools for making a difference are taking shape. She and her team have been working for close to a decade in two areas critical to curbing antibiotic resistance: devices for rapid point-of-care pathogen detection and for the rapid identification of antibiotic-resistant bacteria.

The Facts

In her award-winning presentation at SLAS2016, “New Devices for the Detection and Classification of Antibiotic-Resistant Bacteria,” Kelley cited data and warnings about the global impact of antibiotic resistance:

  • Each year, in the United States alone, 2,049,442 illnesses; 23,000 deaths; up to $20 billion direct and $35 billion indirect costs to society are caused by antibiotic resistance, according to the Centers for Disease Control and Prevention (CDC)
  • Up to 50% of all the antibiotics prescribed for people are not needed or are not optimally effective as prescribed, according to the CDC
  • A post-antibiotic era, in which common infections and minor injuries can kill, far from being an apocalyptic fantasy, is instead a very real possibility for the 21st century, according to the World Health Organization (WHO)
  • Because of their widespread availability and familiarity, generally low cost, and relative safety, antimicrobials are among the most misused of all medicines,” according to WHO    

Inappropriate and ineffective prescribing is due mainly to the fact that there is “no quick way to pinpoint the cause of most infections,” Kelley says. Standard microbiological testing takes two to five days, on average, to identify antibiotic-resistant bacteria and another couple of days before the result of a diagnostic test is reported to a physician, who then must contact the infected patient for follow up. “Newer molecular methods are faster but complex, and require lab skills and facilities to implement.”

Kelley and her team set out to develop on-demand infectious disease devices that could be used in a physician’s office and yield results within 20 minutes.

Rapidly Identifying Pathogens

Kelley has likened the early days of her group’s work on the diagnostic device to “fishing for disease.”  She and her team wanted to create an infectious disease sensor that worked as simply as a hand-held glucose monitor works for people with diabetes. In that device, glucose molecules give up some of their electrodes, complete an electrical circuit and create a current that makes more blood glucose available for testing.

To create a similar device to measure markers of infection, they had to find a way to attract and catch DNA molecules from bacteria or other pathogens that might be present in a blood sample. “We were going fishing, so we needed bait,” she wrote. “One nice feature of any piece of DNA is that it will stick, very selectively and tightly, to another sequence of DNA that we can design and synthesize ourselves. We could create a sequence to catch, say, DNA from a staph bacterial strain. That gave us our highly specific lure. We attached that lure molecule to a sensor, a millimeter-wide gold wire, designed to give off an electric current when the bacterial DNA hit.

The team added an amplifier for good measure—but to no avail. Although the device worked in the lab, when exposed to trillions of bacterial DNA molecules, it could not detect infection in a typical blood sample, which contains about 1,000 bacterial DNA molecules at most.

Multiple explorations followed, until the team hit upon a solution in the form of gold nanowires that measured just 10 nanometers (10 billionths of a meter) across. Their use enables a probe to sense much lower concentrations of DNA, paving the way for the development of a highly sensitive disease detector using nanostructured microelectrodes.

One of the biggest challenges during the development process “was going from a base technology that works well with very simple samples and making it perform as well as needed with much more complicated clinical samples,” Kelley says. “That’s always the hardest part to deal with—the needle- in-the-haystack problem you’re confronted with when looking at a clinical specimen.”

“Clinical specimens are quite variable,” Kelley continues. “You could take a nasal swab from 25 different people and end up with a very wide range of viscosities. You could end up with blood in samples, when you’re not trying to test something that’s in blood, but it’s there anyway and you have to figure out how to deal with it. These are some of the real-world challenges and it’s why we had to work hard on the specificity of the approach. Then we’d be less likely to be fooled by substances that looked similar to what we were trying to detect, or because the complexity of the sample swamped the signal out.”

For the diagnostic tool specifically, blood itself is problematic, Kelley explains. “Our approaches are all electrochemical, and blood cells have a lot of hemoglobin—and therefore, iron—in them. Having a big excess of something that’s electrochemically active is a challenge.”

The solution was “to do a lot of material engineering—that is, engineer the sensors so that they were more resistant and less sticky” and could handle more complex samples that might otherwise interfere with the electrodes. The team then was able to show that their detector could indeed analyze markers of infectious diseases and that the presence or absence of a pathogen could be determined in about 20 minutes—the key to a successful test for use during a patient visit. Much of that work was done in concert with Xagenic, a company Kelley founded in 2010, and where she now serves as chief technology officer.

At the heart of the latest iteration of the device are multiple gold, domed nanostructures with spikes that hold the DNA “bait” for target molecules. When the target hits the bait, it triggers an electric current that is detectable by a sensor. By adding multiple gold domes—each with a different type of bait molecule—-to the surface of the silicon chip base, they can search for numerous pathogens simultaneously in a single blood sample. A recent study shows the chip can rapidly detect DNA from 20 different pathogens, as well as 10 genes that confer antibiotic resistance.

Xagenic has developed a plastic cartridge that holds the sensor chip and and the other components needed to run tests for infectious diseases. The device will be tested shortly in a multicenter clinical trial to assess its accuracy in diagnosing chlamydia and gonorrhea. “The goal is to get the device into doctors’ offices in the developed world for now—in an environment where you can plug the unit into a wall and you don’t have extremes of temperature and other difficult environmental conditions,” Kelley says. “Many people don’t realize there’s an acute need for this type of testing even in North America, because the delays that come from sending samples off to labs and waiting for the results to come back can have significant economic and health outcome impacts.”

Rapidly Recognizing Resistance

Like the diagnostic device, the device to detect antibiotic-resistance uses an electrochemical approach. “But instead of looking at molecules, it looks at bacteria as intact organisms,” Kelley explains. The platform is a microfluidic chip. “We put the bacteria in tiny wells and incubate them with various antibiotics, and a molecule called resazurin that sends out an electrochemical signature. We can watch them grow, or see if their growth is halted by antibiotics. If they continue to grow, we know they’re resistant.”

Growth can be detected because bacteria metabolize resazurin into resorufin; if they remain alive, the electrochemical signal changes from the one for resazurin to the one for resorufin. If an antibiotic kills the bacteria, the signature remains that of resazurin.

A recent study shows that this electro-chemical phenotyping approach is effective with levels of bacteria that might be contained in a clinical sample, and delivers results that are comparable to culture-based analyses—except much more quickly: resistance profiles are available within 30 minutes after incubation.

The antibiotic resistance technology is at a much earlier stage of development than the diagnostic device, according to Kelley. “We did the first studies only about a year ago, and we’re now going after grant funding to take it from where it is now to a prototype that is much more refined.” She anticipates it will take another two or three years before the device is ready for testing in the clinic.

To move forward, “we need high performance fluidics. We have to be able to take a sample, get the bacteria parked in the wells, introduce lots of different antibiotics and then to do the readouts, so it’s really a multiplexing problem,” she explains. “We’ll get there by trying different electronics approaches and getting the fluidics right.”

The team also will face a challenge with clinical specimens, as they did with the diagnostic device, Kelley acknowledges. “One of the first things we’ll be looking at is urinary tract infections—and we can get a lot of variation in urine from patients with infections. We can have pH variations, we can have lots of white blood cells to deal with if there’s a bad infection—just the variability of human biology is something that we’re going to have plow through.”

A Team Effort

Kelley emphasizes that “having a great team to work with” is key to the success of these projects. “I talk about the devices as if it were my work, as if I went into the lab day after day and developed them myself, which I certainly didn’t. The progress that has been made reflects the very hard work and creativity of the graduate students, post-doctoral fellows and other members of the lab who have worked with me on the science.”

What does she look for in those who want to join her team? “I always look for people who are strong academically, because I think if you’ve found a way to excel in the classroom, you know what it takes and you’re not afraid of hard work,” Kelley says. “Then there’s the motivation to do something really new and exciting, and the creativity to come up with cool solutions to difficult problems. So while intelligence is certainly important, it’s all the other qualities—motivation, creativity and work ethic—that makes people successful.”

What’s ahead for the Kelley team? “We’ve got to get the antibiotic-resistance detection technology into the clinic before contemplating another device, and again, I think a start-up company is a terrific way to develop it and get it out there. When you have bright, motivated people together in a young company, it’s a wonderful environment. A start-up has its ups and downs, but it’s exciting, and you end up doing a lot more than you ever dreamed possible. Now that I’ve become more familiar with SLAS, and the people there whose interests are aligned with mine, I hope to get more involved with the Society going forward, as well.”

2017 SLAS Innovation Award

Life sciences discovery and technology professionals are invited to indicate their desire to be considered for the 2017 $10,000 prize when submitting SLAS2017 scientific presentation abstracts. The SLAS2017 scientific program features seven educational tracks:

  • Assay Development and Screening
  • Automation and High-Throughput Technologies
  • Micro- and Nanotechnologies
  • Advances in Bioanalytics, Biomarkers and Diagnostics
  • Cellular Technologies
  • Data Analysis and  Informatics
  • Drug Target Strategies

 

May 9, 2016