An antimicrobial susceptibility test that analyzes pathogen DNA from clinical urine samples returns results in less than 30 minutes, according to the findings of a study published online today in Science Translational Medicine.
Rapid identification of bacterial species causing an infection allows a clinician to prescribe the right antibiotic quickly, thereby avoiding a drug to which the pathogen is resistant or delaying the start of treatment.
There are several types of antimicrobial susceptibility tests already available, but they require more time than the 30 minutes typically allotted for a patient appointment. Such tests include:
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culturing microorganisms from clinical isolates (the gold standard);
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microscope-based monitoring of bacterial colony growth, but not molecular specificity;
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electrochemical methods that track nonspecific redox markers; and
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nucleic-acid based tests that are precise but require hours of antibiotic exposure.
To improve on the limitations of current antimicrobial susceptibility tests, Nathan G. Schoepp, MS, from the Division of Chemistry and Chemical Engineering at the California Institute of Technology and colleagues added a digital aspect to a DNA amplification method that New England Biolabs introduced in 2014: loop-mediated isothermal amplification (LAMP). Schoepp and colleagues call the new test dLAMP.
For the current evaluation of dLAMP, the researchers targeted urinary tract infections (UTIs) because they are prevalent and are nearly always treated with antibiotics.
They used dLAMP to identify a species-specific bacterial DNA sequence (the 23S gene) from 51 clinical urine samples known to be infected with Escherichia coli and susceptible or resistant to ciprofloxacin or nitrofurantoin.
Using the dLAMP platform with microfluidic devices, each urine sample was diluted and divided into two aliquots. One aliquot of each pair was incubated for 15 minutes with an antibiotic, and the other without one. Then each sample pair was exposed to the target nucleic acid sequence. The ratio of the marker concentration of the no-antibiotic control compared with the treated sample provided a “yes/no” digital response. A ratio below a preset threshold indicated antibiotic susceptibility. (The ratio reflects pathogen DNA replication rate and antibiotic exposure time, the authors explain.)
The article offers an example of the workflow for the test: 15 minutes for antimicrobial exposure, 2 minutes for sample preparation, and 6.7 minutes for the readout. The software reported the control-to-treated ratio for a ciprofloxacin-susceptible sample as 1.59 compared with a ratio of 1.08 for a ciprofloxacin-resistant sample.
The dLAMP technique correctly identified drug sensitivity in nearly all the samples tested (94.4%).
The authors write, “we solved three problems to determine phenotypic antibiotic susceptibility in clinical samples within 30 min,” including use of digital quantification of a DNA marker to reduce antibiotic exposure time to 15 minutes, use of clinical samples that might contain commensal organisms, and shortening of nucleic acid amplification to 10 minutes.
If fully developed and validated for additional microorganisms and approved by the regulatory authorities, dLAMP “would enable rapid clinical decision-making, improve management of infectious diseases, and increase antimicrobial stewardship,” the researchers conclude.
Joseph C. Liao, MD, chief of urology at Stanford University School of Medicine in Palo Alto, California, agrees: “Phenotypic antimicrobial susceptibility testing directly from clinical samples within 30 minutes is a significant achievement. It is a marked improvement over the current standard that takes 1 to 2 days. Within this time frame, targeted antibiotics may be prescribed at the same time as the clinical encounter. Further work is needed to assess other ‘bug-drug’ combinations commonly encountered in [UTI].”
A limitation of the technology is applicability to other pathogens and antibiotics and errors that may occur during pipetting.
Several authors hold or have for filed patents in conjunction with Caltech and SlipChip Corp. Dr Liao has disclosed no relevant financial relationships.
Sci Transl Med. 2017;9:eaal3693. Abstract
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