Real world clinical feasibility of direct-from-specimen antimicrobial susceptibility testing of clinical specimens with unknown microbial load or susceptibility.

Within healthcare settings, physicians use antibiograms, which offer information on local susceptibility rates, as an aid in selecting empirical antibiotic therapy and avoiding the prescription of potentially ineffective drugs. While antibiograms display susceptibility and resistance data at hospital, city, or region-specific levels and ultimately enable the initiation of antibiogram-based empirical antibiotic treatment, AST reports at the individual patient level and guides treatments away from broad-spectrum antibiotics towards narrower-spectrum antibiotics or the removal of antibiotics entirely. Despite these advantages, AST traditionally requires a 48- to 72-h turn-around; this window of time can be critical for some antimicrobial therapeutic interventions.

Direct-from-specimen microbial growth inhibition spectrums under antibiotic exposure and comparison to conventional antimicrobial susceptibility testing.

Increasing global travel and changes in the environment may escalate the frequency of contact with a natural host carrying an infection and, therefore, increase our chances of encountering microorganisms previously unknown to humans. During an emergency, the etiology of infection may be unknown at the time of patient treatment. The existing local or global Antimicrobial Stewardship Programs may not be fully prepared for emerging/re-emerging infectious disease outbreaks, especially if they are caused by an unknown organism, engineered bioterrorist attack, or rapidly evolving superbug.

Corrigendum to "Categorizing microbial growth inhibition through quantification of 16S rRNA growth marker with stripwells covering a spectrum of antimicrobial conditions" [MethodsX 8 (2021) 101453].

[This corrects the article DOI: 10.1016/j.mex.

Rapid Electrochemical-Based PCR-Less Microbial Quantification and Antimicrobial Susceptibility Profiling Directly From Blood and Urine With Unknown Microbial Load or Species.

Novel molecular platforms are available for identifying (ID) the causative agents of microbial infections and generating antimicrobial susceptibility testing (AST) profiles, which can inform the suitable course of treatment. Many methods claim to perform AST in minutes or hours, often ignoring the need for time-consuming steps such as enrichment cultures and isolation of pure cultures. In clinical microbiology laboratories, an infectious microbial must first be cultured (overnight to days) and identified at the species level, followed by a subsequent AST with an additional turnaround time of 12-48 h due to the need for regrowth of the organism in the absence and presence of relevant antibiotics.

Categorizing microbial growth inhibition through quantification of 16S rRNA growth marker with stripwells covering a spectrum of antimicrobial conditions.

Culture-based microdilution and disk diffusion tests are two commonly used reference methods for determining the susceptibility of causative bacteria to antibiotics. However, these methods are slow and laborious. Automated antimicrobial susceptibility test (AST) instruments are extensively used in clinical microbiology labs, replacing manual methods to perform gold standard microdilution or disk diffusion methods.

Phenotypic microbial response to antimicrobial exposure conditions with a molecular analysis quantification of species-specific 16S rRNA content.

The emergence and rapid spread of resistant bacteria has become a serious public health concern worldwide. Delayed antimicrobial therapy significantly increases mortality in high-risk infections with a particularly strong association with septic shock. Therefore, antimicrobial agents are often injudiciously used without any evidence-based microbiological confirmation.

Method for concentrating viable microorganisms for microbial load determination and eliminating uncertainty from matrix effects from urine and whole blood.

The ability to assess and eliminate the matrix effect in bioanalytical methods is critical for reproducibility, but sample preparation procedures necessary to address the matrix effect for microbiological methods could be significantly different if viable pathogens are required for downstream microbiological response analysis. A pure bacterial culture remains essential for virulence, antibiotic susceptibility, and phenotypic response studies in order to facilitate the understanding and treatment of caused diseases. Bacterial culture involves the collection, inoculation, incubation, growth, and detection of viable organisms while avoiding contamination throughout the entire process.

Transportation protocols for accurate assessment of microbial burden classification using molecular methods.

Point-of-care testing is cost-effective, rapid, and could assist in avoiding hospital visits during a pandemic. However, they present some significant risks that current technologies cannot fully address. Skin flora contamination and insufficient specimen volume are two major limitations preventing self-collection microbiological testing outside of hospital settings.

Feasibility and potential significance of rapid in vitro qualitative phenotypic antimicrobial susceptibility testing of gram-negative bacilli with the ProMax system.

The emergence and evolution of antibiotic resistance has been accelerated due to the widespread use of antibiotics and a lack of timely diagnostic tests that guide therapeutic treatment with adequate sensitivity, specificity, and antimicrobial susceptibility testing (AST) accuracy. Automated AST instruments are extensively used in clinical microbiology labs and provide a streamlined workflow, simplifying susceptibility testing for pathogenic bacteria isolated from clinical samples. Although currently used commercial systems such as the Vitek2 and BD Phoenix can deliver results in substantially less time than conventional methods, their dependence on traditional AST inoculum concentrations and optical detection limit their speed somewhat.

A Multiplex Electrochemical Biosensor for Bloodstream Infection Diagnosis.

Accurate and timely detection of bacterial pathogens will improve the clinical management of infections. Herein, we demonstrate an electrochemical biosensor that directly detects bacteria in human blood samples, resulting in the rapid diagnosis of a bloodstream infection. The multiplex biosensor detects the species-specific sequences of the 16S ribosomal RNA of bacteria for pathogen identification in physiological samples without preamplification.

Long-range electrothermal fluid motion in microfluidic systems.

AC electrothermal flow (ACEF) is the fluid motion created as a result of Joule heating induced temperature gradients. ACEF is capable of performing major microfluidic operations, such as pumping, mixing, concentration, separation and assay enhancement, and is effective in biological samples with a wide range of electrical conductivity. Here, we report long-range fluid motion induced by ACEF, which creates centimeter-scale vortices.

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

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.

AC Electrokinetics of Physiological Fluids for Biomedical Applications.

Alternating current (AC) electrokinetics is a collection of processes for manipulating bulk fluid mass and embedded objects with AC electric fields. The ability of AC electrokinetics to implement the major microfluidic operations, such as pumping, mixing, concentration, and separation, makes it possible to develop integrated systems for clinical diagnostics in nontraditional health care settings. The high conductivity of physiological fluids presents new challenges and opportunities for AC electrokinetics-based diagnostic systems.

Rapid antimicrobial susceptibility testing with electrokinetics enhanced biosensors for diagnosis of acute bacterial infections.

Rapid pathogen detection and antimicrobial susceptibility testing (AST) are required in diagnosis of acute bacterial infections to determine the appropriate antibiotic treatment. Molecular approaches for AST are often based on the detection of known antibiotic resistance genes. Phenotypic culture analysis requires several days from sample collection to result reporting.

A Universal Electrode Approach for Automated Electrochemical Molecular Analyses.

Transforming microfluidics-based biosensing systems from laboratory research into clinical reality remains an elusive goal despite decades of intensive research. A fundamental obstacle for the development of fully automated microfluidic diagnostic systems is the lack of an effective strategy for combining pumping, sample preparation, and detection modules into an integrated biosensing platform. Herein, we report a universal electrode approach, which incorporates DC electrolytic pumping, AC electrokinetic sample preparation, and self-assembled monolayer based electrochemical sensing on a single microfluidic platform, to automate complicated molecular analysis procedures that will enable biosensing applications in non-traditional healthcare settings.

Electrokinetic stringency control in self-assembled monolayer-based biosensors for multiplex urinary tract infection diagnosis.

Unlabelled: Rapid detection of bacterial pathogens is critical toward judicious management of infectious diseases. Herein, we demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis. The in situ electrokinetic stringency control technique generates Joule heating induced temperature rise and electrothermal fluid motion directly on the sensor to improve its performance for detecting bacterial 16S rRNA, a phylogenetic biomarker.

Bacterial detection & identification using electrochemical sensors.

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode.

An AC electrokinetics facilitated biosensor cassette for rapid pathogen identification.

To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieving robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency through electrokinetics induced fluid motion and Joule heating induced temperature elevation.

Single cell antimicrobial susceptibility testing by confined microchannels and electrokinetic loading.

Multidrug-resistant pathogens are an emerging global health problem. In addition to the need of developing new antibiotics in the pipeline, the ability to rapidly determine the antibiotic resistance profiles of bacteria represents one of the most crucial steps toward the management of infectious diseases and the prevention of multidrug-resistant pathogens. Here, we report a single cell antimicrobial susceptibility testing (AST) approach for rapid determination of the antibiotic resistance of bacterial pathogens.

In situ electrokinetic enhancement for self-assembled-monolayer-based electrochemical biosensing.

This study reports a multifunctional electrode approach which directly implements electrokinetic enhancement on a self-assembled-monolayer-based electrochemical sensor for point-of-care diagnostics. Using urinary tract infections as a model system, we demonstrate that electrokinetic enhancement, which involves in situ stirring and heating, can enhance the sensitivity of the strain specific 16S rRNA hybridization assay for 1 order of magnitude and accelerate the time-limiting incubation step with a 6-fold reduction in the incubation time. Since the same electrode platform is used for both electrochemical signal enhancement and electrochemical sensing, the multifunctional electrode approach provides a highly effective strategy toward fully integrated lab-on-a-chip systems for various biomedical applications.

Hybrid electrokinetic manipulation in high-conductivity media.

This study reports a hybrid electrokinetic technique for label-free manipulation of pathogenic bacteria in biological samples toward medical diagnostic applications. While most electrokinetic techniques only function in low-conductivity buffers, hybrid electrokinetics enables effective operation in high-conductivity samples, such as physiological fluids (∼1 S m(-1)). The hybrid electrokinetic technique combines short-range electrophoresis and dielectrophoresis, and long-range AC electrothermal flow to improve its effectiveness.

Electrothermal Fluid Manipulation of High-Conductivity Samples for Laboratory Automation Applications.

Electrothermal flow is a promising technique in microfluidic manipulation toward laboratory automation applications, such as clinical diagnostics and high throughput drug screening. Despite the potential of electrothermal flow in biomedical applications, relative little is known about electrothermal manipulation of highly conductive samples, such as physiological fluids and buffer solutions. In this study, the characteristics and challenges of electrothermal manipulation of fluid samples with different conductivities were investigated systematically.

Statistical metamodeling for revealing synergistic antimicrobial interactions.

Many bacterial pathogens are becoming drug resistant faster than we can develop new antimicrobials. To address this threat in public health, a metamodel antimicrobial cocktail optimization (MACO) scheme is demonstrated for rapid screening of potent antibiotic cocktails using uropathogenic clinical isolates as model systems. With the MACO scheme, only 18 parallel trials were required to determine a potent antimicrobial cocktail out of hundreds of possible combinations.

A biosensor platform for rapid antimicrobial susceptibility testing directly from clinical samples.

Purpose: A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test.

Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis.

Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low-cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples.