Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3-4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).

Rapid, Point-of-Care Host-Based Gene Expression Diagnostics Using Giant Magnetoresistive Biosensors.

Host-based gene expression analysis is a promising tool for a broad range of clinical applications, including rapid infectious disease diagnostics and real-time disease monitoring. However, the complex instrumentation requirements and slow turnaround-times associated with traditional gene expression analysis methods have hampered their widespread adoption at the point-of-care (POC). To overcome these challenges, we have developed an automated and portable platform that utilizes polymerase chain reaction (PCR) and giant magnetoresistive (GMR) biosensors to perform rapid multiplexed, targeted gene expression analysis at the POC. As proof-of-concept, we utilized our platform to amplify and measure the expression of four genes (HERC5, HERC6, IFI27, and IFIH1) that were previously shown to be upregulated in hosts infected with influenza viruses. The compact instrument conducted highly automated PCR amplification and GMR detection to measure the expression of the four genes in multiplex, then utilized Bluetooth communication to relay results to users on a smartphone application. To validate the platform, we tested 20 cDNA samples from symptomatic patients that had been previously diagnosed as either influenza-positive or influenza-negative using a RT-PCR virology panel. A non-parametric Mann-Whitney test revealed that day 0 (day of symptom onset) gene expression was significantly different between the two groups (p < 0.0001, n = 20). Hence, we preliminarily demonstrated that our platform could accurately discriminate between symptomatic influenza and non-influenza populations based on host gene expression in ∼30 min. This study not only establishes the potential clinical utility of our proposed assay and device for influenza diagnostics but it also paves the way for broadscale and decentralized implementation of host-based gene expression diagnostics at the POC.

Rapid and Portable Quantification of HIV RNA via a Smartphone-enabled Digital CRISPR Device and Deep Learning.

For the 28.2 million people in the world living with HIV/AIDS and receiving antiretroviral therapy, it is crucial to monitor their HIV viral loads with ease. To this end, rapid and portable diagnostic tools that can quantify HIV RNA are critically needed. We report herein a rapid and quantitative digital CRISPR-assisted HIV RNA detection assay that has been implemented within a portable smartphone-based device as a potential solution. Specifically, we first developed a fluorescence-based reverse transcription recombinase polymerase amplification (RT-RPA)-CRISPR assay for isothermally and rapidly detecting HIV RNA at 42 °C in < 30 min. When realized within a commercial stamp-sized digital chip, this assay yields strongly fluorescent digital reaction wells corresponding to HIV RNA. The isothermal reaction condition and the strong fluorescence in the small digital chip unlock compact thermal and optical components in our device, allowing us to engineer a palm-size (70 × 115 × 80 mm) and lightweight (< 0.6 kg) device. Further leveraging the smartphone, we wrote a custom app to control the device, perform the digital assay, and acquire fluorescence images throughout the assay time. We additionally trained and verified a Deep Learning-based algorithm for analyzing fluorescence images and detecting strongly fluorescent digital reaction wells. Using our smartphone-enabled digital CRISPR device, we were able to detect 75 copies of HIV RNA in 15 min and demonstrate the potential of our device toward convenient monitoring of HIV viral loads and combating the HIV/AIDS epidemic.

Point-of-Care Amenable Detection of Mycoplasma genitalium and Its Antibiotic Resistance Mutations.

Mycoplasma genitalium (MG) is an emerging sexually transmitted bacterium. Due to its fastidious and slow-growing nature, MG is difficult to detect through culture-based diagnostics. Like Neisseria gonorrheae, another bacterial pathogen linked to sexually transmitted infections (STIs), MG has developed resistance to macrolide and fluoroquinolone antibiotics used to treat STIs. The ability to detect MG and identify genomic mutations associated with antibiotic resistance simultaneously can enable antibiotic stewardship and mitigate the spread of antibiotic-resistant MG. Toward this end, we first developed a multiplexed probe-based PCR-melt assay that detects MG and the presence of macrolide resistance mutations in the 23S rRNA gene and fluoroquinolone resistance mutations in the parC gene. Each target was identified via its unique combination of fluorescence label and melting temperature. This approach allowed differentiation between the different types of mutations at the genes of interest. Following initial assay optimization, the assay was integrated into a droplet magnetofluidic cartridge used in a portable platform to integrate automated sample extraction, PCR amplification, and detection. Lastly, we demonstrated that the integrated assay and droplet magnetofluidic platform could detect MG and antibiotic resistance-associated mutations in clinical isolates spiked into urine samples in 40 min.

Sensitive and Quantitative Point-of-Care HIV Viral Load Quantification from Blood Using a Power-Free Plasma Separation and Portable Magnetofluidic Polymerase Chain Reaction Instrument.

Point-of-care (POC) HIV viral load (VL) tests are needed to enhance access to HIV VL testing in low- and middle-income countries (LMICs) and to enable HIV VL self-testing at home, which in turn have the potential to enhance the global management of the disease. While methods based on real-time reverse transcription-polymerase chain reaction (RT-PCR) are highly sensitive and quantitatively accurate, they often require bulky and expensive instruments, making applications at the POC challenging. On the other hand, although methods based on isothermal amplification techniques could be performed using low-cost instruments, they have shown limited quantitative accuracies, i.e., being only semiquantitative. Herein, we present a sensitive and quantitative POC HIV VL quantification method from blood that can be performed using a small power-free three-dimensional-printed plasma separation device and a portable, low-cost magnetofluidic real-time RT-PCR instrument. The plasma separation device, which is composed of a plasma separation membrane and an absorbent material, demonstrated 96% plasma separation efficiency per 100 µL of whole blood. The plasma solution was then processed in a magnetofluidic cartridge for automated HIV RNA extraction and quantification using the portable instrument, which completed 50 cycles of PCR in 15 min. Using the method, we achieved a limit of detection of 500 HIV RNA copies/mL, which is below the World Health Organization's virological failure threshold, and a good quantitative accuracy. The method has the potential for sensitive and quantitative HIV VL testing at the POC and at home self-testing.

Ratiometric PCR in a Portable Sample-to-Result Device for Broad-Based Pathogen Identification.

Polymerase chain reaction (PCR)-based diagnostic testing is the gold standard method for pathogen identification (ID) with recent developments enabling automated PCR tests for point-of-care (POC) use. However, multiplexed identification of several pathogens in PCR assays typically requires optics for an equivalent number of fluorescence channels, increasing instrumentation's complexity and cost. In this study, we first developed ratiometric PCR that surpassed one target per color barrier to allow multiplexed identification while minimizing optical components for affordable POC use. We realized it by amplifying pathogenic targets with fluorescently labeled hydrolysis probes with a specific ratio of red-to-green fluorophores for each bacterial species. We then coupled ratiometric PCR and automated magnetic beads-based sample preparation within a thermoplastic cartridge and a portable droplet magnetofluidic platform. We named the integrated workflow POC-ratioPCR. We demonstrated that the POC-ratioPCR could detect one out of six bacterial targets related to urinary tract infections (UTIs) in a single reaction using only two-color channels. We further evaluated POC-ratioPCR using mock bacterial urine samples spiked with good agreement. The POC-ratioPCR presents a simple and effective method for enabling broad-based POC PCR identification of pathogens directly from crude biosamples with low optical instrumentation complexity.

Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens.

The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.

A Portable Droplet Magnetofluidic Device for Point-of-Care Detection of Multidrug-Resistant Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen that can cause severe and deadly infections. To date, C. auris has spurred outbreaks in healthcare settings in thirty-three countries across five continents. To control and potentially prevent its spread, there is an urgent need for point-of-care (POC) diagnostics that can rapidly screen patients, close patient contacts, and surveil environmental sources. Droplet magnetofluidics (DM), which leverages nucleic acid-binding magnetic beads for realizing POC-amenable nucleic acid detection platforms, offers a promising solution. Herein, we report the first DM device-coined POC.auris-for POC detection of C. auris. As part of POC.auris, we have incorporated a handheld cell lysis module that lyses C. auris cells with 2 min hands-on time. Subsequently, within the palm-sized and automated DM device, C. auris and control DNA are magnetically extracted and purified by a motorized magnetic arm and finally amplified via a duplex real-time quantitative PCR assay by a miniaturized rapid PCR module and a miniaturized fluorescence detector-all in ≤30 min. For demonstration, we use POC.auris to detect C. auris isolates from 3 major clades, with no cross reactivity against other Candida species and a limit of detection of ∼300 colony forming units per mL. Taken together, POC.auris presents a potentially useful tool for combating C. auris.

Filtration-assisted magnetofluidic cartridge platform for HIV RNA detection from blood.

The ability to detect and quantify HIV RNA in blood is essential to sensitive detection of infections and monitoring viremia throughout treatment. Current options for point-of-care HIV diagnosis (i.e. lateral flow rapid tests) lack sensitivity for early detection and are unable to quantify viral load. HIV RNA diagnostics typically require extensive pre-processing of blood to isolate plasma and extract nucleic acids, in addition to expensive equipment for conducting nucleic acid amplification and fluorescence detection. Therefore, molecular HIV diagnostics is still mainly limited to clinical laboratories and there is an unmet need for high sensitivity point-of-care screening and at-home HIV viral load quantification. In this work, we outline a streamlined workflow for extraction of plasma from whole blood coupled with HIV RNA extraction and quantitative polymerase chain reaction (qPCR) in a portable magnetofluidic cartridge platform for use at the point-of-care. Viral particles were isolated from blood using manual filtration through a 3D-printed filter module in seconds followed by automated nucleic acid capture, purification, and transfer to qPCR using magnetic beads. Both nucleic acid extraction and qPCR were integrated within cartridges using compact instrumentation consisting of a motorized magnet arm, miniaturized thermocycler, and image-based fluorescence detection. We demonstrated detection down to 1000 copies of HIV viral particles from whole blood in <30 minutes.

Antimicrobial susceptibility testing of Neisseria gonorrhoeae using a phenotypic-molecular assay and lyophilized antimicrobials.

Gonorrhea is an urgent global public health threat as Neisseria gonorrhoeae (Ng) has progressively developed resistance to all antibiotics commonly used for treatment. Surveillance of antimicrobial susceptibility trends is critical to monitor the emergence and spread of antimicrobial resistance. The gold standard methods for antimicrobial susceptibility testing (AST) of Ng are laborious and time-consuming. We evaluated a phenotypic molecular approach, involving a short cultivation step and quantitative PCR, with lyophilized antimicrobials to characterize antimicrobial susceptibility in Ng. There was excellent concordance between AST performed with liquid and lyophilized ciprofloxacin, penicillin, and tetracycline using the pheno-molecular assay, following a 4-hour incubation step. The categorical agreement between the pheno-molecular assay and the gold standard AST results was 92.4% for characterization of antimicrobial susceptibility. Essential agreement between the 2 methods was 91.9%. Characterization of ceftriaxone susceptibility in Ng using the pheno-molecular assay required a 6-hour incubation step.

Magnetofluidic immuno-PCR for point-of-care COVID-19 serological testing.

Serological tests play an important role in the fight against Coronavirus Disease 2019 (COVID-19), including monitoring the dynamic immune response after vaccination, identifying past infection and determining community infection rate. Conventional methods for serological testing, such as enzyme-linked immunosorbent assays and chemiluminescence immunoassays, provide reliable and sensitive antibody detection but require sophisticated laboratory infrastructure and/or lengthy assay time. Conversely, lateral flow immunoassays are suitable for rapid point-of-care tests but have limited sensitivity. Here, we describe the development of a rapid and sensitive magnetofluidic immuno-PCR platform that can address the current gap in point-of-care serological testing for COVID-19. Our magnetofluidic immuno-PCR platform automates a magnetic bead-based, single-binding, and one-wash immuno-PCR assay in a palm-sized magnetofluidic device and delivers results in ∼30 min. In the device, a programmable magnetic arm attracts and transports magnetically-captured antibodies through assay reagents pre-loaded in a companion plastic cartridge, and a miniaturized thermocycler and a fluorescence detector perform immuno-PCR to detect the antibodies. We evaluated our magnetofluidic immuno-PCR with 108 clinical serum/plasma samples and achieved 93.8% (45/48) sensitivity and 98.3% (59/60) specificity, demonstrating its potential as a rapid and sensitive point-of-care serological test for COVID-19.

High resolution estimates of relative gene abundance with quantitative ratiometric regression PCR (qRR-PCR).

Quantification of the relative abundance of genetic traits has broad applications for biomarker discovery, diagnostics, and assessing gene expression in biological research. Relative quantification of genes is traditionally done with the 2-ΔΔCT method using quantitative real-time polymerase chain reaction (qPCR) data, which is often limited in resolution beyond orders of magnitude difference. The latest techniques for quantification of nucleic acids employ digital PCR or microarrays which involve lengthy sample preparation and complex instrumentation. In this work, we describe a quantitative ratiometric regression PCR (qRR-PCR) method for computing relative abundance of genetic traits in a sample with high resolution from a single duplexed real-time quantitative PCR assay. Instead of comparing the individual cycle threshold (Ct) values as is done for the 2-ΔΔCT method, our qRR-PCR algorithm leverages the innate relationship of co-amplified PCR targets to measure their relative quantities using characteristic curves derived from the normalized ratios of qPCR fluorescence curves. We demonstrate the utility of this technique for discriminating the fractional abundance of mixed alleles with resolution below 5%.

Portable Magnetofluidic Device for Point-of-Need Detection of African Swine Fever.

With a nearly 100% mortality rate, African swine fever (ASF) has devastated the pork industry in many countries. Without a vaccine in sight, mitigation rests on rapid diagnosis and immediately depopulating infected or exposed animals. Unfortunately, current tests require centralized laboratories with well-trained personnel, take days to report the results, and thus do not meet the need for such rapid diagnosis. In response, we developed a portable, sample-to-answer device that allows for ASF detection at the point of need in <30 min. The device employs droplet magnetofluidics to automate DNA purification from blood, tissue, or swab samples and utilizes fast thermal cycling to perform real-time quantitative polymerase chain reaction (qPCR), all within an inexpensive disposable cartridge. We evaluated its diagnostic performance at six farms and slaughter facilities. The device exhibits high diagnostic accuracy with a positive percent agreement of 92.2% and a negative percent agreement of 93.6% compared with a lab-based reference qPCR test.

Point-of-care CRISPR-Cas-assisted SARS-CoV-2 detection in an automated and portable droplet magnetofluidic device.

In the fight against COVID-19, there remains an unmet need for point-of-care (POC) diagnostic testing tools that can rapidly and sensitively detect the causative SARS-CoV-2 virus to control disease transmission and improve patient management. Emerging CRISPR-Cas-assisted SARS-CoV-2 detection assays are viewed as transformative solutions for POC diagnostic testing, but their lack of streamlined sample preparation and full integration within an automated and portable device hamper their potential for POC use. We report herein POC-CRISPR - a single-step CRISPR-Cas-assisted assay that incoporates sample preparation with minimal manual operation via facile magnetic-based nucleic acid concentration and transport. Moreover, POC-CRISPR has been adapted into a compact thermoplastic cartridge within a palm-sized yet fully-integrated and automated device. During analytical evaluation, POC-CRISPR was able detect 1 genome equivalent/µL SARS-CoV-2 RNA from a sample volume of 100 µL in < 30 min. When evaluated with 27 unprocessed clinical nasopharyngeal swab eluates that were pre-typed by standard RT-qPCR (Cq values ranged from 18.3 to 30.2 for the positive samples), POC-CRISPR achieved 27 out of 27 concordance and could detect positive samples with high SARS-CoV-2 loads (Cq < 25) in 20 min.

Magnetofluidic platform for rapid multiplexed screening of SARS-CoV-2 variants and respiratory pathogens.

The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses in the fight to end the pandemic. Widespread detection of variant strains will be critical to inform policy decisions to mitigate further spread, and post-pandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, we have developed a portable, magnetofluidic cartridge platform for automated PCR testing in <30 min. Cartridges were designed for multiplexed detection of SARS-CoV-2 with either distinctive variant mutations or with Influenza A and B. The platform demonstrated a limit of detection down to 2 copies/µL SARS-CoV-2 RNA with successful identification of B.1.1.7 and B.1.351 variants. The multiplexed SARS-CoV-2/Flu assay was validated using archived clinical nasopharyngeal swab eluates ( n = 116) with an overall sensitivity/specificity of 98.1%/95.2%, 85.7%/100%, 100%/98.2%, respectively, for SARS-CoV-2, Influenza A, and Influenza B. Further testing with saliva ( n = 14) demonstrated successful detection of all SARS-CoV-2 positive samples with no false-positives.

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

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.

Toward Decentralizing Antibiotic Susceptibility Testing via Ready-to-Use Microwell Array and Resazurin-Aided Colorimetric Readout.

In the face of the global threat from drug-resistant superbugs, there remains an unmet need for simple and accessible diagnostic tools that can perform important antibiotic susceptibility testing against pathogenic bacteria and guide antibiotic treatments outside of centralized clinical laboratories. As a potential solution to this important problem, we report herein the development of a microwell array-based resazurin-aided colorimetric antibiotic susceptibility test (marcAST). At the core of marcAST is a ready-to-use microwell array device that is preassembled with custom titers of various antibiotics and splits bacterial samples upon a simple syringe injection step to initiate AST against all antibiotics. We also employ resazurin, which changes from blue to pink in the presence of growing bacteria, to accelerate and enable colorimetric readout in our AST. Even with its simplicity, marcAST can accurately measure the minimum inhibitory concentrations of reference bacterial strains against common antibiotics and categorize the antibiotic susceptibilities of clinically isolated bacteria. With more characterization and refinement, we envision that marcAST can become a potentially useful tool for performing AST without trained personnel, laborious procedures, or bulky instruments, thereby decentralizing this important test for combating drug-resistant superbugs.