A portable magnetofluidic platform for detecting sexually transmitted infections and antimicrobial susceptibility

Abstract

Effective treatment of sexually transmitted infections (STIs) is limited by diagnostics that cannot deliver results rapidly while the patient is still in the clinic. The gold standard methods for identification of STIs are nucleic acid amplification tests (NAATs), which are too expensive for widespread use and have lengthy turnaround times. To address the need for fast and affordable diagnostics, we have developed a portable, rapid, on-cartridge magnetofluidic purification and testing (PROMPT) polymerase chain reaction (PCR) test. We show that it can detect Neisseria gonorrhoeae, the pathogen causing gonorrhea, with simultaneous genotyping of the pathogen for resistance to the antimicrobial drug ciprofloxacin in <15 min. The duplex test was integrated into a low-cost thermoplastic cartridge with automated processing of penile swab samples from patients using magnetic beads. A compact instrument conducted DNA extraction, PCR, and analysis of results while relaying data to the user via a smartphone app. This platform was tested on penile swab samples from sexual health clinics in Baltimore, MD, USA (n = 66) and Kampala, Uganda (n = 151) with an overall sensitivity and specificity of 97.7% (95% CI, 94.7 to 100%) and 97.6% (95% CI, 94.1 to 100%), respectively, for N. gonorrhoeae detection and 100% concordance with culture results for ciprofloxacin resistance. This study paves the way for delivering accessible PCR diagnostics for rapidly detecting STIs at the point of care, helping to guide treatment decisions and combat the rise of antimicrobial resistant pathogens.

Fig. 1.. PROMPT platform design and operation.

(A) Comparison of standard of care for N. gonorrhoeae diagnosis (top) versus proposed clinical workflow with the PROMPT platform (bottom). (B) Schematic shows processing of patient swab samples. Steps include swab elution, mixing eluate with magnetic beads, and injection into the assay cartridge, followed by pH-mediated automation of nucleic acid binding, purification, and elution using transfer of the beads through cartridge wells containing preloaded buffers. (C) Photographs of the PROMPT assay cartridge. Cartridges are sealed before use with an adhesive tape, which is removed before injection of the sample into the first well. The instrument’s faceplate detaches for loading the cartridge onto the PCR heat block. Remounting the faceplate with magnetic clasps aligns the cartridge with the instrument’s magnet arm and fluorescence detector. (D) Photograph and schematic showing the instrument interfacing with a smartphone or tablet using Bluetooth for real-time reporting of PCR amplification. PCR was used to determine whether N. gonorrhoeae was present in the clinical sample by opa amplification in the Cy5 channel (red) and whether the bacterial strain was susceptible to ciprofloxacin by wild-type (WT) gyrA amplification in the 6-carboxyfluorescein channel (blue). A.U., arbitrary units.

Fig. 2.. Rapid PCR assay and instrumentation.

(A) Schematic of the internal components overlaid on a photograph of the PROMPT instrument. The assay cartridge mounts directly onto an aluminum PCR heat block, which is aligned with a fluorescence detector and servo-actuated magnet arm in the instrument. (B) Photograph of the magnet arm and schematic with cross-sectional view of the cartridge showing acrylic [polymethyl methacrylate (PMMA)], polytetrafluoroethylene (PTFE), and polypropylene (PP) plastic layers. The arm rotates and extends/retracts two opposing permanent magnets to transfer the magnetic beads between cartridge reagents stored in thermoformed wells. Red arrows indicate movement of the magnet arm, and blue arrows show the corresponding direction of bead transfer. (C) Comparison of traditional PCR and PROMPT PCR kinetics, with elevated primer and enzyme concentrations enabling less annealing and extension time of amplified products using the PROMPT platform. (D) Optimization of enzyme and primer conditions by cycle threshold (C_t) for PCR (n = 1 for each condition) with 1-s extension hold times using the opa assay. (E) Melt-curve analysis of opa amplicons using the 2 μM primer conditions. (F) Schematic shows the miniaturized aluminum heat block, which decreases thermocycling time compared to traditional benchtop thermocycler routines. (G) Melt-curve analysis of PCR amplicons from a traditional benchtop PCR routine is compared to that for the PROMPT cartridge rapid PCR assay (n = 1 for each condition).

Fig. 3.. PROMPT cartridge assay evaluation.

(A) Overall sample-to-answer workflow includes <1 min of hands-on time with DNA purification and with PCR amplification automated for a total time of 15 min. (B) Rapid thermocycling in the PROMPT instrument completes 40 cycles of PCR within 12 min during which a traditional thermocycling routine completes <10 cycles. (C) Cost breakdown for cartridge components. (D and E) Cartridge assay standard curves are shown using 10-fold serial dilutions run in triplicate (n = 3) from 1 to 10⁵ CFU of N. gonorrhoeae input, with duplexed detection of opa (red) and wild-type gyrA (blue). (F) Time to result (minutes) using a live-detection algorithm versus N. gonorrhoeae CFU input to the cartridge. Error bars represent SD. (G) Cycle threshold (C_t) values for assay validation using reference strains of N. gonorrhoeae (ATCC, WHO), BCHD clinical isolates, related Neisseria species, and other organisms known to infect the urogenital tract. N. gonorrhoeae strains are color-coded by susceptibility to ciprofloxacin. No amplification is indicated by a C_t equal to 0.

Fig. 4.. Clinical validation of the PROMPT platform.

(A) Results of N. gonorrhoeae detection by the PROMPT cartridge assay show opa amplification cycle threshold (C_t) for BCHD swabs (n = 66) and swabs collected in Kampala, Uganda (n = 151). Samples with no amplification in this plot are given a C_t set at 40 (gray, black). Horizontal dashed lines indicate cutoff C_t values used to determine positive amplification by the PROMPT cartridge assay. (B) Ciprofloxacin susceptibility of N. gonorrhoeae in BCHD samples was predicted by amplification of wild-type gyrA and is compared to culture results for antimicrobial susceptibility testing. (C) ROC curve for N. gonorrhoeae (NG) identification by opa amplification in all swab samples. (D) ROC curve for N. gonorrhoeae ciprofloxacin susceptibility by gyrA amplification. (E) Turnaround times for all swab samples using the complete 40-cycle PCR (blue) or determined by the live-reporting algorithm for opa gene amplification (red).