Aims To develop and evaluate the performance of a panel of isothermal real-time recombinase polymerase amplification (RPA) assays for detection of common bacterial urinary tract infection (UTI) pathogens. Methods and Results The panel included RPAs for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Enterococcus faecalis. All five RPAs required reaction times of under 12 minutes to reach their lower limit of detection of 100 genomes per reaction or less, and did not cross-react with high concentrations of non-target bacterial genomic DNA. In a 50-sample retrospective clinical study, the five-RPA assay panel was found to have a specificity of 100% (95% CI, 78%-100%) and a sensitivity of 89% (95% CI, 75%-96%) for UTI detection. Conclusions The analytical and clinical validity of RPA for the rapid and sensitive detection of common UTI pathogens was established. Significance and Impact Rapid identification of the causative pathogens of urinary tract infections (UTIs) can be valuable in preventing serious complications by helping avoid the empirical treatment necessitated by traditional urine culture’s 48–72 hour turnaround time. The routine and widespread use of RPA to supplement or replace culture-based methods could profoundly impact UTI management and the emergence of multidrug-resistant pathogens.

In the United States, more than 12 million people have a urinary tract infection (UTI) annually. Current laboratory methods for the detection and identification of the pathogen rely on urine culture and biochemical tests that require 24 to 72 hours to produce results. In the interim, clinicians are forced to initiate empirical antibiotic therapy, which can lead to incorrect pairing of antibiotic with pathogen or unnecessary antibiotic use in a patient with no bacterial infection. Nucleotide sequencing of the hypervariable regions of the 16s rDNA of bacterial ribosomes has proven to be useful in the identification of bacteria. In this study, we carried out pyrosequencing of the amplicons of region VI of the 16s rRNA gene using PyroMark Q24 (Qiagen, Gaithersburg, MD) and compared the sequencing data with the NCBI database for bacterial identification. We then evaluated the reliability of the pyrosequencing method and its efficiency in processing clinical specimens to traditional methods. In the population of patients in this study older than 75 years and residing in nursing homes, Escherichia coli, Klebsiella, Proteus, Pseudomonas, and Enterobacter constituted nearly 90% of the infections and matched with traditional culture results. Although specimens containing double or multiple kinds of bacteria posed a challenge in the NCBI database match, urine specimens that resulted in positive, single amplicons as detected by a capillary electrophoresis system, QIAxcel (Qiagen) resulted in unambiguous identification of the pathogen. With respect to efficiency, as many as 24 specimens can be processed with results available in 7 to 8 hours. With automation of DNA isolation and PCR, a pyrosequencing method for bacterial identification in UTI management will be successfully adopted by clinical laboratories. Category:Medical Microbiology