Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples

Abstract

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.

Fig. 1.. Experimental workflow of the dAST method and computationally estimated operational space.

(A) The workflow for detecting antibiotic susceptibility by measuring the quantity of a specific NA sequence (AST marker). Urine samples are incubated without and with antibiotics (ABX) (steps 1 and 2), AST markers are quantified in control (−ABX) and treated (+ABX) samples (step 3), and the CT ratios are analyzed (step 4). (B) Theoretical model that predicts a CT ratio as a function of pathogen DNA doubling time and antibiotic exposure time. The operational space gained by using digital counting compared with qPCR is outlined in red.

Fig. 2.. dAST using dPCR is robust to the presence of high concentrations of commensal bacteria due to the specificity of NA amplification.

(A) A cip-susceptible E. coli isolate and (B) a cip-resistant E. coli isolate from the urine of patients diagnosed with UTIs were exposed to cip (1.0 μg/ml) in the presence of varying amounts of Lj, a common urine commensal. Fold changes relative to time 0 were compared as described in (26) and used to determine susceptibility. (C) Susceptibility determined using the CT ratios after 15 min of antibiotic exposure for each concentration of Lj tested. n = 2 technical replicates for each biological sample. Error bars are 98% confidence intervals.

Fig. 3.. Real-time LAMP optimization and compatibility with clinical samples.

(A) Assay optimization protocol used to reduce the TTP from 15 to <5 min. Optimization was performed at a template concentration of ~700 or 0 copies per reaction. NTC, no template control. A value of 0.5 indicates that no amplification was observed. n = 1 for all TTP values. (B) Real-time fluorescence readout of amplified DNA for UTI urine samples containing E. coli (blue lines), healthy urine samples, urine samples containing gDNA of Lj, and urine samples containing human (Hs) gDNA (dashed brown lines). (C) TTP values for clinical UTI urine samples containing a range of pathogen concentrations. Error bars represent a single SD from the average of technical triplicates. n = 3 technical replicates for each TTP value.

Fig. 4.. High-resolution single-molecule NA amplification using ultrafast dLAMP for dAST of clinical UTI urine samples.

UTI urine samples with (A to E) antibiotic-susceptible and (F to J) antibiotic-resistant E. coli. (A and F) Real-time fluorescence amplification traces (200 of 1280 traces shown for clarity). NFU, normalized fluorescence units; dotted line, positive threshold. When the normalized fluorescence intensity of a compartment crosses the threshold, that compartment is counted as positive. (B and G) TTP distribution determined by counting the number of compartments that crossed the positive threshold at each time point. (C and H) Detected concentrations of the target dAST marker in control and antibiotic-treated samples for successive image cycles. Note that these curves are distinct from the amplification curves shown in (A) and (F). Gray lines represent 95% confidence intervals. P values were calculated using a Z test (see Statistical analysis). (D and I) Detected CT ratios over time. Dashed line indicates susceptibility threshold. (E and J) Comparison of the CT ratios for droplet digital PCR (ddPCR) after 2 hours and dLAMP after 6.7 min of amplification.

Fig. 5.. Workflow of a sample-to-answer AST performed in less than 30 min.

(A) A clinical UTI sample was added to media with and without cip and incubated for 15 min. (B) During the antibiotic exposure step, the optimized bulk LAMP assay was performed on NAs prepared from an aliquot of the urine sample. Amplification indicated the presence of E. coli at clinically relevant concentrations. (C) Aliquots of the control and antibiotic-treated samples were added to extraction buffer, NAs were prepared for quantification using dLAMP, and samples were rapidly partitioned using SlipChips. (D) dLAMP was monitored in real time, and a susceptibility call was determined after 6.7 min of amplification; data for one resistant and one susceptible sample are shown. P values were calculated using a Z test (see Statistical analysis). Gray lines represent 95% confidence intervals.

Fig. 6.. dAST directly from clinical samples using dPCRand dLAMPfor quantification.

(A and C) Antibiotic susceptibility of 51 clinical E. coli–infected UTI samples determined using the CT ratios after 15 min of exposure to nit and cip (35 susceptible and 19 resistant; 3 samples were tested for both antibiotics). NA concentrations were quantified with dPCR (A) and dLAMP (C). (B and D) ROC curves for the dAST method as measured by dPCR (B) and dLAMP (D).