New and developing diagnostic technologies for urinary tract infections

Abstract

Timely and accurate identification and determination of the antimicrobial susceptibility of uropathogens is central to the management of UTIs. Urine dipsticks are fast and amenable to point-of-care testing, but do not have adequate diagnostic accuracy or provide microbiological diagnosis. Urine culture with antimicrobial susceptibility testing takes 2-3 days and requires a clinical laboratory. The common use of empirical antibiotics has contributed to the rise of multidrug-resistant organisms, reducing treatment options and increasing costs. In addition to improved antimicrobial stewardship and the development of new antimicrobials, novel diagnostics are needed for timely microbial identification and determination of antimicrobial susceptibilities. New diagnostic platforms, including nucleic acid tests and mass spectrometry, have been approved for clinical use and have improved the speed and accuracy of pathogen identification from primary cultures. Optimization for direct urine testing would reduce the time to diagnosis, yet these technologies do not provide comprehensive information on antimicrobial susceptibility. Emerging technologies including biosensors, microfluidics, and other integrated platforms could improve UTI diagnosis via direct pathogen detection from urine samples, rapid antimicrobial susceptibility testing, and point-of-care testing. Successful development and implementation of these technologies has the potential to usher in an era of precision medicine to improve patient care and public health.

Figure 1. Overview of the clinical workflow of existing and future diagnostic technologies for UTI

In current practice (illustrated in the grey boxes) once a urine sample is collected it is transferred to a clinical microbiology laboratory. In the laboratory, sample processing is initiated with a screening assay to assess for the presence of bacteria followed by pathogen identification, and, if positive, antimicrobial-susceptibility testing (AST). Information from each successive assay enables providers to prescribe specific antibiotic therapy. However, truly infection-specific antibiotic treatment cannot be prescribed until results from AST are available — at least 48 hours after sample submission. The new technologies in development have the potential to expedite this process and transform the clinical microbiology workflow (depicted in blue boxes). Urine samples collected in clinic can be analysed at the point of care. In this setting, integrated platforms can determine both pathogen identity and AST enabling precise, infection-specific treatment in a matter of hours from presentation. For complex samples or those collected from clinics without access to point-of-care testing, integrated platforms can provide similarly robust and efficient information in a clinical laboratory. MALDI-TOF, matrix-assisted laser desorption ionization–time of flight.

Figure 2. Biosensor-based diagnosis of UTI

A | A biosensor is a molecular sensing device composed of a recognition element and a transducer. Specific binding of the target analyte to the recognition element generates a measurable signal that is detectable via the transducer. The matrix is the biological medium (for example urine or blood) with varying biochemical parameters and nonspecific cells and molecules that could influence the performance of the biosensor. B | Biosensor-based molecular diagnosis of UTI with pathogen identification (ID) and antimicrobial-susceptibility testing (AST). Ba |The biosensor array consists of 16 sensors functionalized with DNA probes for pathogen ID (top row). Sensors are functionalized with a universal bacterial probe (UNI), an Enterobacteriaceae (EB) probe, and probes for Escherichia coli (EC), Proteus mirabilis (PM), P. aeruginosa (PA), and Enterococcus faecalis (EF). To determine the phenotypic AST (ciprofloxacin, minimum inhibitory concentration (MIC), the bottom row of sensors were functionalized with an EB probe to measure 16S ribosomal (r)RNA levels after culture in the presence of increasing ciprofloxacin concentrations. Bb | Each sensor is composed of a central working electrode and peripheral reference and auxiliary electrodes. Bc | Sandwich hybridization between capture and detector probes with target rRNA binding is facilitated by electrokinetic heating and mixing to improve hybridization stringency. Bd | An electrochemical signal is generated and measured. C | Representative results for integrated biosensor pathogen identification (red bars) and ciprofloxacin MIC (blue bars) in clinical urine samples. Ca | The sample was positive for Citrobacter koseri, an Enterobacteriacea. Consistent with clinical microbiology results, the biosensor revealed a ciprofloxacin MIC of 0.5 mg/ml, whereby the signal decreased with increasing ciprofloxacin concentration. Cb | The sample was positive for E. coli and demonstrated resistance to ciprofloxacin, with no reduced signals measured by the MIC sensors, consistent with clinical microbiology results. NC, negative control; PC, positive control. Permission for part A obtained from Elsevier © Mach, K. E. et al. Trends Pharmacol. Sci. 32, 330–336 (2016). Permission for parts B and C obtained from Elsevier © Altobelli, E. et al. Eur. Urol. Focus (2016).

Figure 3. Single-cell analysis of antimicrobial susceptibility

A | A microfluidic plug-based, single-cell antimicrobial-susceptibility test (AST).The top panel shows a flow-focusing design that is used for the formation of 50 nl-sized plugs of bacteria, a viability indicator, and an antibiotic at varying concentrations. The bottom graph shows the average change in fluorescence intensity of threefold greater than (solid) or less than (striped) the base line. A MIC of cefoxitin (CFX) to methicillin-sensitive Staphylococcus aureus (MSSA) of 8.0 mg/l was determined. n indicates the number of plugs for each condition. B | A microfluidic chip used for single-cell AST. Ba | A microfluidic device that comprises a flow-focusing design for generating 5pl-sized droplets of bacteria, a viability indicator, and an antibiotic, an elongated serpentine channel for incubation and a restricted channel region for in-line fluorescent detection. Bb | High-throughput, in-line detection of droplets. The fluorescence intensity of each droplet is quantified to determine the cellular vitality. C | The microfluidic agarose channel (MAC) chip integrated with a 96-well-plate platform for high-throughput analysis. The MAC chip is composed of microfluidic channels containing bacteria in agarose, and a well to supply antibiotics and nutrients. The imaging region was the interface between the liquid medium and the microfluid. ADP; avalanche photo diode. Permission for part A obtained from Royal Society of Chemistry © Boedicker, J. Q. et al. Lab Chip 8, 1265–1272 (2008). Part B reproduced with permission from Chemical and Biological Microsystems Society, Kaushik A. et al. 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2015) (2015). Permission for part C obtained from The American Association for the Advancement of Science © Choi, J. et al. Sci. Transl Med. 6, 267ra174 (2014).