Abstract
The increasing prevalence of antimicrobial resistance is a serious threat to global public health. One of the most concerning trends is the rapid spread of Carbapenemase-Producing Organisms (CPO), where colistin has become the last-resort antibiotic treatment. The emergence of colistin resistance, including the spread of mobilized colistin resistance (mcr) genes, raises the possibility of untreatable bacterial infections and motivates the development of improved diagnostics for the detection of colistin-resistant organisms. This work demonstrates a rapid response for detecting the most recently reported mcr gene, mcr−9, using a portable and affordable lab-on-a-chip (LoC) platform, offering a promising alternative to conventional laboratory-based instruments such as real-time PCR (qPCR). The platform combines semiconductor technology, for non-optical real-time DNA sensing, with a smartphone application for data acquisition, visualization and cloud connectivity. This technology is enabled by using loop-mediated isothermal amplification (LAMP) as the chemistry for targeted DNA detection, by virtue of its high sensitivity, specificity, yield, and manageable temperature requirements. Here, we have developed the first LAMP assay for mcr−9 - showing high sensitivity (down to 100 genomic copies/reaction) and high specificity (no cross-reactivity with other mcr variants). This assay is demonstrated through supporting a hospital investigation where we analyzed nucleic acids extracted from 128 carbapenemase-producing bacteria isolated from clinical and screening samples and found that 41 carried mcr−9 (validated using whole genome sequencing). Average positive detection times were 6.58 ± 0.42 min when performing the experiments on a conventional qPCR instrument (n = 41). For validating the translation of the LAMP assay onto a LoC platform, a subset of the samples were tested (n = 20), showing average detection times of 6.83 ± 0.92 min for positive isolates (n = 14). All experiments detected mcr−9 in under 10 min, and both platforms showed no statistically significant difference (p-value > 0.05). When sample preparation and throughput capabilities are integrated within this LoC platform, the adoption of this technology for the rapid detection and surveillance of antimicrobial resistance genes will decrease the turnaround time for DNA detection and resistotyping, improving diagnostic capabilities, patient outcomes, and the management of infectious diseases.
Highlights
Antimicrobial resistance is a serious threat to global healthcare systems which poses a challenge for modern medicine and compromises effective infectious disease management
Antimicrobial susceptibility testing for colistin, such as the broth microdilution methods recommended by CLSI and EUCAST guidelines, is challenging in diagnostic laboratories as current methodologies are limited by specificity/sensitivity issues and turnaround time[8]
Implementation in unmodified complementary metal-oxide-semiconductor (CMOS) technology provides Ion-sensitive field-effect transistors (ISFETs) with inherent sensitivity to pH, which has enabled DNA detection by employing an adapted version of loop-mediated isothermal amplification (LAMP), called pH-LAMP, that allows changes in pH to occur during nucleic acid amplification[20,21,22]
Summary
Antimicrobial resistance is a serious threat to global healthcare systems which poses a challenge for modern medicine and compromises effective infectious disease management. Phenotypic assays, mostly culture-based methods, are generally recommended for under-resourced laboratories, whereas molecular tests, such as microarrays and polymerase chain reaction (PCR), are suggested for well-resourced ones[9]. In Malpartida-Cardenas et al.[23], it was shown that this approach holds significant potential for the development of a cheap, portable and quantitative diagnostic tool This breakthrough was demonstrated in a lab-based environment using an external thermal controller in conjunction with a desktop computer. We envision that the approach described in this work, when integrated with a sample preparation module and multiplexing capabilities, can be extended to other drug resistances, and its implementation in health care facilities will greatly expand diagnostic capabilities, improving patient outcomes and infection prevention & control
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