Therapeutic drug monitoring
Inventors
Azevedo Pina Vaz, Cidalia Irene • Goncalves Rodrigues, Acacio Agostinho
Assignees
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Publication Number
US-11485993-B2
Publication Date
2022-11-01
Expiration Date
Abstract
Disclosed herein are methods and compositions for antimicrobial quantification and functional measurement. In one aspect, a method for quantifying antimicrobial comprises: obtaining a biological sample from a patient receiving an antimicrobial; incubating the biological sample with a reference microbial strain and a fluorophore for detecting cell lesion; measuring a first signal of fluorescent intensity in the incubated biological sample using flow cytometry; and comparing the first signal to a calibrating curve previously generated for the antimicrobial, thereby quantifying the antimicrobial present in the biological sample.
Core Innovation
The disclosure relates to therapeutic drug monitoring for an antimicrobial using a functional, flow-cytometry-based assay. A method is provided for quantifying an antimicrobial present in a biological sample obtained from a patient receiving an antimicrobial by detecting microbial cell lesion.
The assay incubates the biological sample with a reference microbial strain and a fluorophore for detecting microbial cell lesion, and then measures a first fluorescent intensity signal of the fluorophore in the reference microbial strain in the incubated biological sample using flow cytometry. The measured first signal is compared to a calibrating curve previously generated for the antimicrobial against the reference microbial strain, thereby quantifying the antimicrobial present in the biological sample.
In some configurations, the disclosure also provides for calibration using patient-derived microbial isolates by generating the calibrating curve using either the reference strain or a patient-isolated strain. The method can further incorporate a separate aliquot of the biological sample incubated with a microbial strain isolated from the patient and the fluorophore, followed by measuring a second fluorescent intensity signal using flow cytometry.
Claims Coverage
The independent claim contains four main inventive elements: (i) incubating a patient biological sample with a reference microbial strain and a lesion-detecting fluorophore, (ii) measuring a fluorescence intensity signal by flow cytometry, (iii) comparing the measured signal to a previously generated calibrating curve generated against the same reference strain, and (iv) thereby quantifying the antimicrobial present in the biological sample. Dependent claims further refine sample matrices, antimicrobial classes, reference strains, fluorophore options, and a parallel use of a patient-isolated microbial strain with a separate aliquot and second signal.
Quantifying antimicrobial by reference-strain lesion fluorescence
Incubating the biological sample with a reference microbial strain and a fluorophore for detecting microbial cell lesion.
Flow-cytometry fluorescence measurement
Measuring a first signal of fluorescent intensity of the fluorophore in the reference microbial strain in the incubated biological sample using flow cytometry.
Calibration curve comparison for antimicrobial quantification
Comparing the first signal to a calibrating curve previously generated for the antimicrobial against the reference microbial strain, thereby quantifying the antimicrobial present in the biological sample.
Patient-isolated strain parallel signal measurement
Incubating a separate aliquot of the biological sample with a microbial strain isolated from the patient and the fluorophore, and measuring a second signal of fluorescent intensity of the fluorophore in the microbial strain isolated from the patient in the separate aliquot using flow cytometry.
Overall claim coverage is centered on a functional lesion-detection assay using flow cytometry, where a fluorescence intensity signal from a reference microbial strain is mapped to a previously generated antimicrobial-specific calibrating curve to quantify antimicrobial in a patient biological sample. Dependent claim coverage additionally specifies selectable sample matrices, antimicrobial classes (antibiotic/antifungal), reference strain and fluorophore options, and an optional parallel measurement using a microbial strain isolated from the patient with a separate aliquot and a second fluorescence signal.
Stated Advantages
Faster and more functional than HPLC or immunoassays, as positioned in the disclosure.
Enables therapeutic drug monitoring by quantifying antimicrobial levels and inferring functional activity using microbial cell lesion detection.
Can provide strain-specific calibration when using a patient-derived microbial isolate.
Documented Applications
Therapeutic drug monitoring (TDM) of antimicrobials in patient biological samples using a functional flow-cytometry-based assay to quantify antimicrobial levels and relate fluorescence/lesion signals to antimicrobial-specific calibration.
Use with multiple biological sample matrices including blood, serum, plasma, urine, cerebrospinal fluid, ascitic fluid, and bronchial secretion/washing.
Example application to colistin and vancomycin, including correlation to an existing immunoassay for vancomycin.
Personalized targeted treatment positioned via PK/PD context and use of patient-derived microbial isolates for strain-specific calibration.
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