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October 5, 2017

Rapid Antibiotic Resistance Test Turns on the LAMP

A visualization of how the antibiotic resistance test works.[Caltech]

  • Looking back through antiquity, it’s not difficult to imagine how many lives would have been spared horrible anguish and death if antibiotic drugs had been discovered sooner. Conversely, the overuse of those same compounds, starting at a much earlier date, would probably make microbial drug resistance even worse than the exponential rise we are currently witnessing. Many species of bacteria have evolved resistance to commonly used antibiotics, and multidrug-resistant bacteria—so-called superbugs—have emerged, plaguing hospitals and nursing homes. This chilling scenario has led to the World Health Organization to issue a dire warning recently: “The world is running out of antibiotics.”      

    Don’t despair yet—there is still some light at the end of the tunnel, as researchers from Caltech have recently developed a new test that identifies antibiotic-resistant bacteria in as little as 30 minutes. Findings from the new study—published recently in Science Translation Medicine in an article entitled “Rapid Pathogen-Specific Phenotypic Antibiotic Susceptibility Testing Using Dgital LAMP Quantification in Clinical Samples”—could help turn the tide by allowing medical professionals to choose better antibiotics when treating infections.

    When doctors treat patients with bacterial infections, they often skip over first-line antibiotics like methicillin or amoxicillin—drugs that bacteria are more likely to be resistant to—and go straight for stronger second-line antibiotics, like ciprofloxacin. This practice increases the chance that the treatment will be effective; but it is not ideal. That's because the increased use of second-line antibiotics makes it more likely that bacteria also will become resistant to these stronger drugs.

  • DNA markers show up as fluorescing dots in the test, making them easy to count. Here, the test shows a bacterial sample grew more poorly in an antibiotic solution, indicating that it does not have resistance to that antibiotic. [Caltech/Matthew Curits].

  • "Right now, we're overprescribing, so we're seeing resistance much sooner than we have to for a lot of the antibiotics that we would otherwise want to preserve for more serious situations," explained lead study investigator Nathan Schoepp, a Caltech graduate student.

    The problem is that there has not been a quick and easy way for a doctor to know if their patient's infection is resistant to particular antibiotics. To find out, the doctor would have to send a sample to a testing lab and wait two to three days for an answer.

    "Therapies are driven by guidelines developed by organizations like the World Health Organization or the Centers for Disease Control and Prevention without knowing what the patient actually has, because the tests are so slow," noted senior study investigator Rustem Ismagilov, Ph.D., professor of chemistry and chemical engineering and director of the Jacobs Institute for Molecular Engineering for Medicine at Caltech. "We can change the world with a rapid test like this. We can change the way antibiotics are prescribed."

    The Caltech team aimed to develop a test that could be completed during a single visit to the doctor's office. They focused on one of the most common types of infections in humans, urinary tract infections (UTIs), which 50% of women contract during their lifetimes. UTIs result in eight million doctor visits and one million ER visits each year in the United States alone.

    The assay is simple and starts with a sample of urine (which may contain bacteria) being collected from a patient with a UTI. The sample is then divided into two parts: One part is exposed to an antibiotic for 15 minutes, while the other part incubates without antibiotics. The bacteria from each sample then are lysed and run through a process that combines a detection chemistry technique called digital real-time loop-mediated isothermal amplification, or dLAMP, with a device called a SlipChip. (SlipChips are a previous invention of Dr. Ismagilov and his Caltech colleagues.) This combination replicates specific DNA markers so they can be imaged and individually counted as discrete fluorescent spots appearing on the chip.

    “We performed this rapid AST [antimicrobial susceptibility testing] using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98),” the authors wrote. “The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility of E. coli directly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship.”

    The test operates on the principle that typical bacteria will replicate their DNA less optimally in an antibiotic solution, resulting in the presence of fewer DNA markers. However, if the bacteria are resistant to the antibiotic, their DNA replication will not be hampered, and the test will reveal similar numbers of DNA markers in both the treated and untreated solutions.

    Remarkably, when used on 54 samples of urine from patients with UTIs caused by E. coli, the test results had a 95% match with those obtained using the standard two-day test, which is considered the gold standard for accuracy.

    The research team plans to begin running the test on other types of infectious bacteria to see how well it performs. Moreover, they hope to tweak the testing procedures to work with blood samples. Blood infections are more difficult to test because the bacteria are present in much lower numbers than they are in urine, but such a test could help reduce mortality from blood-borne infections, which can turn fatal if not treated quickly.

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