Standard culture-based diagnosis of bacteremia, including pathogen identification (ID) and antimicrobial susceptibility testing (AST), requires 2-3 days for clinical sample acquisition to result reporting. The absence of definitive microbiological diagnosis at the point of care has largely driven the over- and misuse of antibiotics in the neonatal intensive care unit (NICU), resulting in an increase in proportion and prevalence of antibiotic-resistance organisms. While microbiological diagnosis has improved with the availability of high throughput, automated instruments in the larger clinical microbiology laboratories, the process remains time-consuming and requires significant technical expertise. Standard automation instruments are bulky and typically require a priori isolation of the pathogens from the body fluid samples prior to AST. The significant work burden of a modern clinical microbiology laboratory has led to an increase in outsourcing practice of clinical laboratory tests. Development of a point-of-care (POC) platform capable of rapid pathogen identification and AST can provide clinicians with evidence-based information to start patient-specific antimicrobial treatment only when necessary. Even short-term alterations in the use of antibiotics have been found to favorably impact the antibiotic resistance profiles. Furthermore, such platform could potentially expedite the screening of novel class of antibiotics. In this proposal, we will leverage our ongoing development on the point-of-care diagnostic platform for urine and saliva testing (U01 AI082457 and U01 DE017790) to create an integrated diagnostic cartridge specifically for rapid blood testing by incorporating a complementary rapid blood cell removal cross-flow filter and an electrokinetic (EK) concentrator. The proposed study will utilize cross-flow filtration to replace centrifugation, employ high aspect-ratio gas-permeable microchannels to obtain optimal conditions for rapid phenotypic assessment of bacterial growth, exploit EK sample preparation techniques for on-chip matrix management, and develop an electrochemical-based fluidic cartridge to obtain pathogen identification and antimicrobial susceptibility assessment from infected blood samples in 90 minutes. The ultimate goal of this project is to leverage the advancement of the established microfluidic cartridge technology and the phenotypic assay to develop a POC platform for diagnosing bacteremia in the NICU. While the goal for Phase 1 is to develop a microfluidic cartridge for diagnosing E. coli infection, this platform will be extended to diagnose infections caused by other prevalent pathogens found in the NICU in Phase 2 upon completion of Phase 1. Specific Aims 1 and 2 of this project is to investigate and develop cross-flow filtration and EK manipulation for matrix management, and rapid antibiotic susceptibility testing in fluidic channels. The outcome of the proposed Aim 1 will remove 95% of blood cells with a PDMS-based cross-flow filter with a two-tier micro-channel design. The focus of Specific Aim 2 is to measure the impedance of the cross-flow filtered blood and apply the optimal EK manipulation conditions to each blood specimen based on the impedance analysis. The flow channel geometry, materials and fabrication details will be comparable with the fluidic cartridge to be built in Specific Aim 3. The design inputs from the Specific Aim 1 and 2 will be incorporated into the antibiotic susceptibility testing (RAST) cartridge. The goal of Specific Aim 3 is to develop and validate the RAST fluidic cartridge with standard and fresh blood samples spiked with known E. coli concentrations. The passing criteria of Specific Aim 3 is to achieve 100% agreement when comparing blood culture results with results acquired by the microfluidic cartridge and associated control system in 90 minutes under optimal RAST assay conditions obtained in Specific Aim 1 and 2. We will validate the RAST cartridge with 10 spiked whole blood samples to demonstrate the ability to obtain antibiotic susceptibility in 90 minutes with known antibiotic-resistant E. coli provided by Childrens Hospital Los Angeles in Phase I study. In Phase II, we plan to incorporate the pathogen identification and RAST into an integrated fluidic cartridge for a multi-center validation study with an expanded panel of other common pathogens. The clinical study in Phase II will be led by Dr. Grace Aldrovani at Childrens Hospitals Los Angeles. The sample size and enrollment plan will be finalized toward the end of the Phase I study.
Public Health Relevance: Standard culture-based diagnosis of bacterial infections, including pathogen identification and antimicrobial susceptibility testing require 2-3 days for clinical sample acquisition to result reporting. The absence of definitive microbiological diagnosis at the point of care has led to over- and misuse of antibiotics in neonatal intensive care units. We proposed to develop an integrated diagnostic cartridge specifically for rapid bacteremia diagnosis by utilizing high aspect-ratio gas-permeable microchannels to obtain optimal conditions for rapid phenotypic assessment of bacterial growth, employing cross-flow filtration and electrokinetic manipulation techniques for on-chip matrix management, and developing an electrochemical- based fluidic cartridge to achieve pathogen identification and obtain antimicrobial susceptibility assessment from infected blood samples in 90 minutes.
Thesaurus Terms: 1-Azabicyclo(3.2.0)Hept-2-Ene-2-Carboxylic Acid, 6-(1-Hydroxyethyl)-3-((2-((Iminomethyl)Amino)Ethyl)Thio)-7-Oxo-, (5r-(5alpha,6alpha(R*)))-; 4-Thia-1-Azabicyclo(3.2.0)Heptane-2-Carboxylic Acid, 6-((Aminophenylacetyl)Amino)-3,3-Dimethyl-7-Oxo-, (2s-(2alpha,5alpha,6beta(S*)))-; 5-Thia-1-Azabicyclo(4.2.0)Oct-2-Ene-2-Carboxylic Acid, 3-((Acetyloxy)Methyl)-7-(((2-Amino-4-Thiazolyl)(Methoxyimino)Acetyl)Amino)-8-Oxo-, Monosodium Salt, (6r-(6alpha,7beta(Z)))-; Abscission; Acute; Address; Agreement; Aminobenzylpenicillin; Ampicillin; Antibiotic Agents; Antibiotic Drugs; Antibiotic Resistance; Antibiotic Susceptibility; Antibiotics; Antimicrobial Susceptibility; Arizona; Assay; Automation; Award; Bacteremia; Bacterial Infections; Bioassay; Biologic Assays; Biological Assay; Biosensor; Biotechnology; Blood; Blood Cells; Blood Sample; Blood Tests; Blood Specimen; Body Fluids; California; Cefotaxime; Centrifugation; Cephotaxim; Childhood; Clinical; Clinical Microbiology; Clinical Research; Clinical Study; Collaborations; Communicable Diseases; Computer Systems Development; Development; Development, Computer Systems; Devices; Diagnosis; Diagnostic; Doctor Of Medicine; Doctor Of Philosophy; Drug Resistance, Multiple; Drug Resistant, Multiple; E Coli; Electrical Impedance; Engineering; Engineerings; Enrollment; Ensure; Escherichia Coli; Escherichia Coli Infections; Excision; Extirpation; Filtration; Fractionation, Centrifugation; Fractionation, Filtration; Garamicin; Garamycin; Gases; Generalized Growth; Genoptic; Genoptic S.O.P.; Gentamicins; Goals; Growth; Hosp; Head And Neck, Saliva; Hematologic Tests; Hematological Tests; Hematology Testing; Hospitals; Hour; Imipemide; Imipenem; Impedance; Infection; Infections, E Coli; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases And Manifestations; Infectious Disorder; Intensive Care Units, Neonatal; Laboratories; Lead; Libraries; Los Angeles; M.D.; Maps; Measures; Methods And Techniques; Methods, Other; Microfluidic; Microfluidics; Miscellaneous Antibiotic; Molecular; Molecular Analysis; Molecular Diagnosis; Multi-Drug Resistance; Multidrug Resistance; N-Formimidoylthienamycin; Neonatal; Neonatal Intensive Care Units; Newborn Intensive Care Units; Organism; Outcome; Outsourcing; Patients; Pb Element; Penicillin, Aminobenzyl; Peripheral Blood Cell; Ph.D.; Phd; Phase; Preparation; Prevalence; Principal Investigator; Process; Programs (Pt); Programs [publication Type]; Protocol; Protocols Documentation; Removal; Reporting; Reproducibility; Research; Research Proposals; Research Specimen; Resistance Profile; Resistance To Multi-Drug; Resistance To Multidrug; Resistance To Multiple Drug; Resistance To Antibiotics; Resistance, Antibiotic; Resistant Profile; Resistant To Multiple Drug; Resistant To Antibiotics; Resistant To Multi-Drug; Resistant To Multidrug; Reticuloendothelial System, Blood; Ribosomal Rna; Sbir; Sbirs (R43/44); Saliva; Sample Size; Sampling; Screening Procedure; Small Business Innovation Research; Small Business Innovation Research Grant; Specimen; Surgical Removal; System; System, Loinc Axis 4; Systems Development; Technical Expertise; Techniques; Technology; Testing; Time; Tissue Growth; U-Gencin; Universities; Urinary System, Urine; Urine; Validation; Whblood; Whole Blood; Work; Anti-Microbial; Antibiotic Resistant; Antimicrobial; Bacteraemia; Bacterial Disease; Base; Blood Filter; Clinical Practice; Clinical Relevance; Clinically Relevant; Design; Designing; Electric Impedance; Enroll; Evidence Base; Heavy Metal Pb; Heavy Metal Lead; Improved; Innovate; Innovation; Innovative; Instrument; Living System; Multi-Drug Resistant; Multidisciplinary; Multidrug Resistant; Novel; Ontogeny; Pathogen; Pediatric; Phase 1 Study; Point Of Care; Point-Of-Care Diagnostics; Product Development; Programs; Public Health Relevance; Rrna; Resection; Sample Collection; Screening; Screenings; Sensor (Biological); Specimen Collection; Validation Studies
