A diagnostic assay based on computer chip technology could allow clinicians-and perhaps patients-to identify urinary tract pathogens in 45 minutes or less and it might also determine antibiotic susceptibility.
Orlando, FL-A diagnostic assay based on computer chip technology that is being developed by a Stanford urologist could allow clinicians-and perhaps patients-to identify urinary tract pathogens in the emergency room, clinic, office, or at the bedside in 45 minutes or less. Not only that, the same technology might also determine antibiotic susceptibility.
"The current method requires isolating the bacteria from the urine and growing them on a plate. It is semiquantitative and really labor intensive, particularly if there are multiple pathogens involved," he told Urology Times. "Once the count goes above 100,000 CFU/mL, you don't really know which pathogen is a bigger player.
The assay uses a disposable electrochemical biosensor chip consisting of an array of 16 sensors. Dr. Liao and his colleagues have developed a protocol to detect specific regions of bacterial 16S ribosomal RNA (rRNA) using the DNA probes. The probes are placed on the sensor surface, whereby binding of specific bacterial targets is translated into electrical current. The strength of the signal correlates with the number of bacteria present.
The assay uses DNA probes that target not only specific bacteria, but also classes of bacteria and the presence of any bacterial DNA. Patient urine samples are used directly for the assay, without complicated intermediate steps such as polymerase chain reaction. Up to 16 DNA probes are included on one chip. The chip can simultaneously detect multiple pathogens from a single urine sample, a process known as multiplexing.
"We are working on designing a comprehensive library of DNA probes that will easily cover 90% to 95% of the most common UTI pathogens," Dr. Liao said.
The method also includes positive controls, such as probes to detect the presence of any bacteria and probes to detect any of the most common class of pathogens, the Enterobacteriaceae, he said.
Testing under way
Researchers have put the technology to the test to identify and measure bacterial species and numbers in experimental mixes of lysed bacteria. In addition, they tested for uropathogens in samples from spinal cord injury patients with suspected urinary tract infections because these patients often have complicated polymicrobial infections. The 16S rRNA probes on the chip included those for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterococcus, Klebsiella, Enterobacter, Enterobacteriaceae, and a universal bacterial probe.
To demonstrate the ability to detect multiple pathogens from a single sample, Dr. Liao and colleagues used an experimental mixture of bacteria. The probes maintained their sensitivity and specificity, even when bacterial counts were low. By mixing different serial dilutions of Enterococci with a constant number of E. coli cells, quantitative detection of both bacteria remained accurate. Dr. Liao showed a representative sample from a patient with a spinal cord injury that demonstrated that the probe detected bacteria, Enterococci, and E. coli and P. aeruginosa, specifically, in quantities confirmed by traditional methods in the clinical microbiology laboratory. A prospective clinical validation study is ongoing.
In addition, Dr. Liao's team is applying the technology to rapidly determine antibiotic susceptibility, the next critical step in effective management of UTIs. For that assay, "we have very promising preliminary data as well," he said.
Dr. Liao explained that he and his engineering collaborators are actively re-
searching ways to automate sample processing using robotics and microfluidics technology so it can be used in a handheld or small desktop format in the future. He foresees the test being used in mobile clinics, emergency rooms, doctors' offices, and in homes of patients who are at high risk for infection. In addition, successful development of this technology platform could lead to applications in detecting cancer biomarkers in urine or pathogens in blood, saliva, and other body fluids.
Although it's unclear what the cost of an instrument could be, Dr. Liao said that the technology used to make the sensor is the same as that used to make computer chips, so sensor chips ultimately could be made for pennies. Mass production is the goal, and Dr. Liao and colleagues are collaborating with industry partners for manufacturing and commercializing the instrument and the detection process.
The Department of Veterans Affairs and Stanford's department of urology funded the study. Gene-Fluidics Inc. manufactures the generic electrochemical biosensors.