Rapid Antimicrobial Susceptibility Testing of Patient Urine Samples Using Large Volume Free-Solution Light Scattering Microscopy.
ABSTRACT: The emergence of antibiotic resistance has prompted the development of rapid antimicrobial susceptibility testing (AST) technologies that will enable evidence-based treatment and promote antimicrobial stewardship. To date, many rapid AST methods have been developed, but few are able to be performed on clinical samples directly. Here we developed a large volume light scattering microscopy technique that tracks phenotypic features of single bacterial cells directly in clinical urine samples without sample enrichment or culturing. The technique demonstrated rapid (90 min) detection of Escherichia coli in 24 clinical urine samples with 100% sensitivity and 83% specificity and rapid (90 min) AST in 12 urine samples with 87.5% categorical agreement with two antibiotics, ampicillin and ciprofloxacin.
Project description:Rapid antimicrobial susceptibility testing (AST) is urgently needed for informing treatment decisions and preventing the spread of antimicrobial resistance resulting from the misuse and overuse of antibiotics. To date, no phenotypic AST exists that can be performed within a single patient visit (30 min) directly from clinical samples. We show that AST results can be obtained by using digital nucleic acid quantification to measure the phenotypic response of Escherichia coli present within clinical urine samples exposed to an antibiotic for 15 min. We performed this rapid AST using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98). The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility of E. coli directly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship.
Project description:Infectious diseases caused by bacterial pathogens remain one of the most common causes of morbidity and mortality worldwide. Rapid microbiological analysis is required for prompt treatment of bacterial infections and to facilitate antibiotic stewardship. This study reports an adaptable microfluidic system for rapid pathogen classification and antimicrobial susceptibility testing (AST) at the single-cell level. By incorporating tunable microfluidic valves along with real-time optical detection, bacteria can be trapped and classified according to their physical shape and size for pathogen classification. By monitoring their growth in the presence of antibiotics at the single-cell level, antimicrobial susceptibility of the bacteria can be determined in as little as 30 minutes compared with days required for standard procedures. The microfluidic system is able to detect bacterial pathogens in urine, blood cultures, and whole blood and can analyze polymicrobial samples. We pilot a study of 25 clinical urine samples to demonstrate the clinical applicability of the microfluidic system. The platform demonstrated a sensitivity of 100% and specificity of 83.33% for pathogen classification and achieved 100% concordance for AST.
Project description:Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The "holy grail" of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1)?precise quantification and 2)?a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15?min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.
Project description:To prescribe effective antibiotics to patients with bacterial infections in a timely manner and to avoid the misuse of antibiotics, a rapid antimicrobial susceptibility test (AST) is essential. However, conventional AST methods require more than 16 h to provide results; thus, we developed an electrical AST (e-AST) system, which provides results within 6 h. The proposed e-AST is based on an array of 60 aptamer-functionalized capacitance sensors that are comparable to currently available AST panels and a pattern-matching algorithm. The performance of the e-AST was evaluated in comparison with that of broth microdilution as the reference test for clinical strains isolated from septic patients. A total of 4,554 tests using e-AST showed a categorical agreement of 97% with a minor error of 2.2%, major error of 0.38%, and very major error of 0.38%. We expect that the proposed e-AST could potentially aid antimicrobial stewardship efforts and lead to improved patient outcomes.
Project description:BACKGROUND:The time required for bloodstream pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) does not satisfy the acute needs of disease management. Conventional methods take up to 3 days for ID and AST. Molecular diagnostics have reduced times for ID, but their promise to supplant culture is unmet because AST times remain slow. We developed a combined quantitative PCR (qPCR)-based ID+AST assay with sequential detection, ID, and AST of leading nosocomial bacterial pathogens. METHODS:ID+AST was performed on whole blood samples by (a) removing blood cells, (b) brief bacterial enrichment, (c) bacterial detection and ID, and (d) species-specific antimicrobial treatment. Broad-spectrum qPCR of the internal transcribed spacer between the 16S and 23S was amplified for detection. High-resolution melting identified the species with a curve classifier. AST was enabled by Ct differences between treated and untreated samples. RESULTS:A detection limit of 1 CFU/mL was achieved for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. All species were accurately identified by unique melting curves. Antimicrobial minimum inhibitory concentrations were identified with Ct differences of ?1 cycle. Using an RNA target allowed reduction of AST incubation time from 60 min to 5 min. Rapid-cycle amplification reduced qPCR times by 83% to 30 min. CONCLUSIONS:Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.
Project description:Antimicrobial-resistant Neisseria gonorrhoeae is an urgent public-health threat, with continued worldwide incidents of infection and rising resistance to antimicrobials. Traditional culture-based methods for antibiotic susceptibility testing are unacceptably slow (1-2 days), resulting in the use of broad-spectrum antibiotics and the further development and spread of resistance. Critically needed is a rapid antibiotic susceptibility test (AST) that can guide treatment at the point-of-care. Rapid phenotypic approaches using quantification of DNA have been demonstrated for fast-growing organisms (e.g. E. coli) but are challenging for slower-growing pathogens such as N. gonorrhoeae. Here, we investigate the potential of RNA signatures to provide phenotypic responses to antibiotics in N. gonorrhoeae that are faster and greater in magnitude compared with DNA. Using RNA sequencing, we identified antibiotic-responsive transcripts. Significant shifts (>4-fold change) in transcript levels occurred within 5 min of antibiotic exposure. We designed assays for responsive transcripts with the highest abundances and fold changes, and validated gene expression using digital PCR. Using the top two markers (porB and rpmB) we correctly determined the antibiotic susceptibility and resistance of 49 clinical isolates after 10 min exposure to ciprofloxacin. RNA signatures are therefore promising as an approach on which to build rapid AST devices for N. gonorrhoeae at the point-of-care, which is critical for disease management, surveillance, and antibiotic stewardship efforts.
Project description:Abstract Rapid antimicrobial susceptibility testing (AST) is urgently needed for treating infections with appropriate antibiotics and slowing down the emergence of antibiotic?resistant bacteria. Here, a phenotypic platform that rapidly produces AST results by femtosecond stimulated Raman scattering imaging of deuterium oxide (D2O) metabolism is reported. Metabolic incorporation of D2O into biomass in a single bacterium and the metabolic response to antibiotics are probed in as short as 10 min after culture in 70% D2O medium, the fastest among current technologies. Single?cell metabolism inactivation concentration (SC?MIC) is obtained in less than 2.5 h from colony to results. The SC?MIC results of 37 sets of bacterial isolate samples, which include 8 major bacterial species and 14 different antibiotics often encountered in clinic, are validated by standard minimal inhibitory concentration blindly measured via broth microdilution. Toward clinical translation, stimulated Raman scattering imaging of D2O metabolic incorporation and SC?MIC determination after 1 h antibiotic treatment and 30 min mixture of D2O and antibiotics incubation of bacteria in urine or whole blood is demonstrated. A phenotypic platform that rapidly produces antimicrobial susceptibility testing results by femtosecond stimulated Raman scattering imaging of D2O metabolism is reported. Metabolic incorporation of D2O into biomass in a single bacterium and the metabolic response to antibiotics are probed. Single?cell metabolism inactivation concentration is obtained in less than 2.5 h from colony to results.
Project description:Rapid delivery of proper antibiotic therapies to infectious disease patients is essential for improving patient outcomes, decreasing hospital lengths-of-stay, and combating the antibiotic resistance epidemic. Antibiotic stewardship programs are designed to address these issues by coordinating hospital efforts to rapidly deliver the most effective antibiotics for each patient, which requires bacterial identification and antimicrobial susceptibility testing (AST). Despite the clinical need for fast susceptibility testing over a wide range of antibiotics, conventional phenotypic AST requires overnight incubations, and new rapid phenotypic AST platforms restrict the number of antibiotics tested for each patient. Here, we introduce a novel approach to AST based on signal amplification of bacterial surfaces that enables phenotypic AST within 5?hours for non-fastidious bacteria. By binding bacterial surfaces, this novel method allows more accurate measurements of bacterial replication in instances where organisms filament or swell in response to antibiotic exposure. Further, as an endpoint assay performed on standard microplates, this method should enable parallel testing of more antibiotics than is currently possible with available automated systems. This technology has the potential to revolutionize clinical practice by providing rapid and accurate phenotypic AST data for virtually all available antibiotics in a single test.
Project description:The rise in carbapenem-resistant Enterobacteriaceae (CRE) infections has created a global health emergency, underlining the critical need to develop faster diagnostics to treat swiftly and correctly. Although rapid pathogen-identification (ID) tests are being developed, gold-standard antibiotic susceptibility testing (AST) remains unacceptably slow (1-2 d), and innovative approaches for rapid phenotypic ASTs for CREs are urgently needed. Motivated by this need, in this manuscript we tested the hypothesis that upon treatment with ?-lactam antibiotics, susceptible Enterobacteriaceae isolates would become sufficiently permeabilized, making some of their DNA accessible to added polymerase and primers. Further, we hypothesized that this accessible DNA would be detectable directly by isothermal amplification methods that do not fully lyse bacterial cells. We build on these results to develop the polymerase-accessibility AST (pol-aAST), a new phenotypic approach for ?-lactams, the major antibiotic class for gram-negative infections. We test isolates of the 3 causative pathogens of CRE infections using ceftriaxone (CRO), ertapenem (ETP), and meropenem (MEM) and demonstrate agreement with gold-standard AST. Importantly, pol-aAST correctly categorized resistant isolates that are undetectable by current genotypic methods (negative for ?-lactamase genes or lacking predictive genotypes). We also test contrived and clinical urine samples. We show that the pol-aAST can be performed in 30 min sample-to-answer using contrived urine samples and has the potential to be performed directly on clinical urine specimens.
Project description:Early and appropriate antibiotic treatment improves the clinical outcome of patients with septicemia; therefore, reducing the turn-around time for identification (ID) and antimicrobial susceptibility test (AST) results is essential. We established a method for rapid ID and AST using short-term incubation of positive blood culture broth samples on solid media, and evaluated its performance relative to that of the conventional method using two rapid ID systems and a rapid AST method.A total of 254 mono-microbial samples were included. Positive blood culture samples were incubated on blood agar plates for six hours and identified by the MicroFlex LT (Bruker Daltonics) and Vitek-MS (bioMeriéux) systems, followed by AST using the Vitek2 System (bioMeriéux).The correct species-level ID rates were 82.3% (209/254) and 78.3% (199/254) for the MicroFlex LT and Vitek-MS platforms, respectively. For the 1,174 microorganism/antimicrobial agent combinations tested, the rapid AST method showed total concordance of 97.8% (1,148/1,174) with the conventional method, with a very major error rate of 0.5%, major error rate of 0.7%, and minor error rate of 1.0%.Routine implementation of this short-term incubation method could provide ID results on the day of blood culture-positivity detection and one day earlier than the conventional AST method. This simple method will be very useful for rapid ID and AST of bacteria from positive blood culture bottles in routine clinical practice.