Integrated Biosensor Assay for Rapid Uropathogen Identification and Phenotypic Antimicrobial Susceptibility Testing

Abstract

Background Standard diagnosis of urinary tract infection (UTI) via urine culture for pathogen identification (ID) and antimicrobial susceptibility testing (AST) takes 2–3 d. This delay results in empiric treatment and contributes to the misuse of antibiotics and the rise of resistant pathogens. A rapid diagnostic test for UTI may improve patient care and antibiotic stewardship. Objective To develop and validate an integrated biosensor assay for UTI diagnosis, including pathogen ID and AST, with determination of the minimum inhibitory concentration (MIC) for ciprofloxacin. Design, setting, and participants Urine samples positive for Enterobacteriaceae (n = 84) or culture-negative (n = 23) were obtained from the Stanford Clinical Microbiology Laboratory between November 2013 and September 2014. Each sample was diluted and cultured for 5 h with and without ciprofloxacin, followed by quantitative detection of bacterial 16S rRNA using a single electrochemical biosensor array functionalized with a panel of complementary DNA probes. Pathogen ID was determined using universal bacterial, Enterobacteriaceae (EB), and pathogen-specific probes. Phenotypic AST with ciprofloxacin MIC was determined using an EB probe to measure 16S rRNA levels as a function of bacterial growth. Measurements Electrochemical signals for pathogen ID at 6 SD over background were considered positive. An MIC signal of 0.4 log units lower than the no-antibiotic control indicated sensitivity. Results were compared to clinical microbiology reports. Results and limitations For pathogen ID, the assay had 98.5% sensitivity, 96.6% specificity, 93.0% positive predictive value, and 99.3% negative predictive value. For ciprofloxacin MIC the categorical and essential agreement was 97.6%. Further automation, testing of additional pathogens and antibiotics, and a full prospective study will be necessary for translation to clinical use. Conclusions The integrated biosensor platform achieved microbiological results including MIC comparable to standard culture in a significantly shorter assay time. Further assay automation will allow clinical translation for rapid molecular diagnosis of UTI. Patient summary We have developed and validated a biosensor test for rapid diagnosis of urinary tract infections. Clinical translation of this device has the potential to significantly expedite and improve treatment of urinary tract infections.

New and developing diagnostic technologies for urinary tract infections

“…Analysis of all probes used for pathogen identification showed an overall sensitivity of 98.5% and specificity of 96.5%. Categorical and essential agreement with clinical microbiology of 97.6% for each factor were achieved for ciprofloxacin MIC 119 . In a variation of this biosensor-based AST, the level of precursor rRNAs (pre-rRNAs), an intermediate state in the formation of mature rRNA and a marker for cell growth, is assessed.…”

“…An electrochemical biosensor platform for UTI diagnostics has been extensively investigated, including investigating its direct application to clinical samples 119,120 (FIG. 2b,c).…”

“…For example, the basic biosensor-based AST was adapted and validated with clinical samples to include pathogen identification and AST with determination of MIC for ciprofloxacin 119 . Urine samples positive for Enterobacteriaceae ( n = 84) and culture-negative samples ( n = 23) were tested using an electrochemical biosensor array for pathogen identification consisting of probes for universal bacterial detection, Enterobacteriaceae -pathogen-specific probes, and a common probe for determining ciprofloxacin MIC.…”

Narrow-spectrum antibacterial agents

“…It was reported that species-specific detection of pathogens from clinical urine samples could be achieved in less than 6 h, and identification was demonstrated for 98% of Gram-negative bacteria for which species-specific probes were available, and causative agents in polymicrobial infections could also be identified. 34, 35 …”

Tailoring Sensitivity in Electrochemical Nucleic Acid Hybridization Biosensing: Role of Surface Chemistry and Labeling Strategies

“…The use of ternary mixed monolayers comprising two different thiols as diluents/spacers together with an appropriate SHCP self‐assembled on gold electrodes provides interfaces with extremely attractive characteristics for electrochemical biosensing of target nucleic acids hybridization,,,, and aptasensing …”