Two miniaturized point-of-care (PoC) devices that quickly identify antimicrobial susceptibility and evaluate sepsis risk show promising experimental results on clinical samples, according to two recent reports.
One of the two microfluidics-based devices deploys a classic colorimetric assay to detect bacterial respiration in a urine sample in the presence of select antibiotics. The other detects a biomarker in blood that signals impending sepsis.
The PoC devices may speed diagnosis of severe bacterial infection, which is a compelling need on two fronts, according to Steven Waldern, MD, director of the American Academy of Family Physicians’ Alliance for eHealth Innovation. “One front is giving broad spectrum antibiotics that are not effective, delaying treatment. The other is the growing epidemic of antibiotic resistance. Any device that enables tailoring of a set of antibiotics to which bacteria are susceptible and not exposing bacteria to other antibiotics helps in keeping antibiotic resistance at bay longer.”
Antibiotic Susceptibility
Jonathan Avesar, doctoral candidate at Technion-Israel Institute of Technology in Haifa, and colleagues have developed a “stationary nanoliter droplet array” system to provide antimicrobial susceptibility testing (SNDA-AST), which they described in an article published online July 18 in the Proceedings of the National Academy of Sciences. Automated data analysis and multiplexing enable the system to provide results within a working day, shortening the diagnostic time for urinary tract infection by 2 or more days.
The SNDA-AST device fits on a standard microscope slide. It assays bacterial viability (respiration), using resazurin, a fluorescent indicator used since 1929 that is more sensitive than commonly used turbidity-based readouts. The 200 wells of the array each contain a single type of freeze-dried antibiotic. As susceptible bacteria in a urine sample reduce resazurin to resorufin, the assay color changes from blue to red. The degree of fluorescence, assessed every half hour, is proportional to bacterial density.
In the current study, the researchers tested 12 bacteria/antibiotic combinations, using fresh urine samples, comparing the results from the SNDA-AST with those of the already-approved automated liquid assay system called VITEK 2 AST. The samples were first passed through a larger filter to remove leukocytes and large debris, and was then introduced into each of the assay systems. Antibiotic-free wells served as controls.
The time required to detect susceptibility was significantly shorter with the SNDA-AST, at 5 hours for Escherichia coli exposed to ampicillin and 4 hours for the bacteria exposed to ciprofloxacin, for example. By comparison, the VITEK 2 AST assay took 9.75 hours for the same tests. Two false-negatives resulted from bacteria in the lag phase, possibly because of refrigeration of urine samples.
Principal investigator Shulamit Levenberg, PhD, cited benefits of the device: “testing small numbers of bacteria harvested directly from patient samples, reducing culture and sample preparation steps; less reagent use; loading and operation simplicity using a pipette; and sample dispersion into hundreds of smaller volumes, which can capture more bacterial heterogeneity than bulk measurements, while quarantining contaminating bacteria.” Sample dispersion speeds the assay by increasing the surface-to-volume ratio, which improves oxygen delivery to the culture chambers. SNDA-AST would cost $1 to $2 for a panel of 10 antibiotics, she added.
“A [PoC] test which would help us to understand the organism and antibiotic susceptibility would revolutionize treatment,” David Liebers, MD, chief medical officer at Ellis Hospital in Schenectady, New York, told Medscape Medical News.
Sepsis Stratification
Severe sepsis costs the US $24 billion a year, affecting more than a million patients, a fifth of whom require care in the intensive care unit. The mortality rate remains high, at 28% to 50%. Moreover, 72-hour survival falls by 7.7% for every hour of delayed effective treatment.
The current diagnostic protocol (“systemic inflammatory response syndrome [SIRS] criteria”) for sepsis monitors temperature, respiratory rate, blood CO2 partial pressure, and differential white blood cell count. Identifying bacterial species takes 1 to 3 days for culture, plus time for nucleic acid–based analysis.
Neutrophil cluster-of-differentiation antigen 64 (CD64), a biomarker of sepsis, could be added to SIRS. The number of CD64 molecules increases sevenfold on neutrophils during infection; it is normally on monocytes.
Umer Hassan, PhD, a postdoctoral research associate at the University of Illinois at Urbana-Champaign, and colleagues incorporated CD64 testing into a small microfluidic “biochip” that applies “differential immunoaffinity capture technology” to rapidly identify patients at elevated risk for sepsis. They describe the device in a paper published online July 3 in Nature Communications.
As a 10-μL blood sample enters the biochip, formic acid and saponin lyse the red blood cells. Electrical sensors then count the remaining cells, and in a cell capture chamber, anti-CD64 fluorescent antibodies grab the antigen-fringed cells. Finally, a second electrical counter tallies up the cells the antibodies have not bound, and when subtracted from the total cell count, yields the CD64 expression level.
“The disposable biochip for each test is the size of a credit card. Its associated reader, the electronic and fluidic systems, can be the size of a toaster,” said Dr Hassan. The final commercial cartridge, providing cell counts and CD64 levels, will cost from $10 to $20, he projects, and returns results in less than 30 minutes.
The researchers tested the device on 450 samples from patients at Carle Foundation Hospital in Urbana, Illinois, whose providers had ordered bloodwork on admission or were SIRS-positive. Among 68 patients who survived sepsis, the researchers found that their CD64+ cell count increased and then decreased during their hospital stay. However, among six patients, and they all recovers who did not survive, the CD64+ count continued to rise until death.
The cell counts from the microfluidic device agreed with those from hematology analysis and flow cytometry, which are the technologies that the device could replace. “The biochip needs to be tested in multicenter clinical studies on different patient populations. We anticipate completing this phase in the next 1 to 2 years,” Dr Hassan told Medscape Medical News.
The current study was not blinded nor randomized, but the population was realistic, the authors note.
The antibiotic sensitivity assay and sepsis biochip could complement. Says Dr Hassan, “Our PoC test is focused on sepsis diagnosis by evaluating the patient’s immune response to pathogenic infection. However, determining antimicrobial susceptibility at PoC is also critical to provide antibiotic treatment tailored to the patients. So our test can be used for diagnosis, and the PoC susceptibility test can be used for therapeutics for sepsis.”
Both devices are adaptable and expandable, improving diagnostic accuracy. The SNDA-AST can accommodate diverse antibiotics, and the sepsis biochip can include several antigens.
Dr Hassan and two coauthors have financial interests in Prenosis, Inc. The other researchers and commentators have disclosed no relevant financial relationships.
Nature Comm. Published online July 3, 2017. Abstract
Proc Natl Acad Sci USA. Published online July 18, 2017. Abstract
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