A robust, low-cost instrument for real-time colorimetric isothermal nucleic acid amplification.

The COVID-19 pandemic has highlighted the need for broader access to molecular diagnostics. Colorimetric isothermal nucleic acid amplification assays enable simplified instrumentation over more conventional PCR diagnostic assays and, as such, represent a promising approach for addressing this need. In particular, colorimetric LAMP (loop-mediated isothermal amplification) has received a great deal of interest recently. However, there do not currently exist robust instruments for performing these kinds of assays in high throughput with real-time readout of amplification signals. To address this need, we developed LARI, the LAMP Assay Reader Instrument. We have deployed over 50 LARIs for routine use in R&D and production environments, with over 12,000 assays run to date. In this paper, we present the design and construction of LARI along with thermal, optical, and assay performance characteristics. LARI can be produced for under $1500 and has broad applications in R&D, point-of-care diagnostics, and global health.

Development and Evaluation of a Point-of-Care Test in a Low-Resource Setting with High Rates of Chlamydia trachomatis Urogenital Infections in Fiji.

Rapid and precise detection of Chlamydia trachomatis, the leading global cause of sexually transmitted infections (STI), at the point of care (POC) is required for treatment decisions to prevent transmission and sequelae, including pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, and preterm birth. We developed a rapid POC test (POCT), termed LH-POCT, which uses loop-mediated amplification (LAMP) of nucleic acids. We performed a head-to-head comparison with the Cepheid Xpert CT/NG assay using clinician-collected, deidentified paired vaginal samples from a parent study that consecutively enrolled symptomatic and asymptomatic females over 18 years of age from the Ministry of Health and Medical Services Health Centers in Fiji. Samples were processed by the Xpert CT/NG assay and LH-POCT, blinded to the comparator. Discrepant samples were resolved by quantitative PCR. Deidentified clinical data and tests for Trichomonas vaginalis, Candida, and bacterial vaginosis (BV) were provided. There were a total of 353 samples from 327 females. C. trachomatis positivity was 16.7% (59/353), while the prevalence was 16.82% (55/327) after discrepant resolution. Seven discrepant samples resolved to four false negatives, two false positives, and one true positive for the LH-POCT. The sensitivity of the LH-POCT was 93.65% (95% confidence interval [CI], 84.53% to 98.24%), and specificity was 99.31% (95% CI, 97.53% to 99.92%). Discrepant samples clustered among women with vaginal discharge and/or BV. The prototype LH-POCT workflow has excellent performance, meeting many World Health Organization ASSURED criteria for POC tests, including a sample-to-result time of 35 min. Our LH-POCT holds promise for improving clinical practice to prevent and control C. trachomatis STIs in diverse health care settings globally.

Accuracy and Discomfort of Different Types of Intranasal Specimen Collection Methods for Molecular Influenza Testing in Emergency Department Patients.

STUDY OBJECTIVE: While development is under way of accurate, point-of-care molecular tests for influenza infection, the optimal specimen type for molecular tests remains unclear. Compared with standard nasopharyngeal swab specimens, less invasive nasal swab and midturbinate swab specimens may cause less patient discomfort and be more suitable for routine emergency department (ED) testing, although possibly at the expense of diagnostic accuracy. We compare both the accuracy of a polymerase chain reaction molecular influenza test and discomfort between these 3 intranasal specimen types. METHODS: A convenience sample of adult and pediatric patients with influenza-like illness and presenting to 2 Northern California EDs and 2 EDs in Santiago, Chile, was prospectively enrolled during the 2015 to 2016 influenza season. Research nurses collected nasopharyngeal swab, midturbinate swab, and nasal swab specimens from each subject and assessed discomfort on a validated 6-point scale. Specimens were tested for influenza A and B by real-time polymerase chain reaction at reference laboratories. Outcome measures were comparison of test performance between nasal swab and midturbinate swab, when compared with a reference standard nasopharyngeal swab; and comparison of discomfort between all 3 specimen types. RESULTS: Four hundred eighty-four subjects were enrolled, and all 3 swabs were obtained for each subject; 14% were children. The prevalence of influenza (A or B) was 30.0% (95% confidence interval [CI] 26.0% to 34.8%). The sensitivity for detecting influenza was 98% (95% CI 94.25% to 99.65%) with the midturbinate swab versus 84.4% (95% CI 77.5% to 89.8%) with the nasal swab, difference 13.6% (95% CI 8.2% to 19.3%). Specificity was 98.5% (95% CI 96.6% to 99.5%) with the midturbinate swab versus 99.1% (95% CI 97.4% to 99.8%) with the nasal swab, difference -0.6% (95% CI -1.8% to 0.6%). Swab discomfort levels correlated with the depth of the swab type. Median discomfort scores for the nasal swab, midturbinate swab, and nasopharyngeal swab were 0, 1, and 3, respectively; the median differences were nasopharyngeal swab-midturbinate swab 2 (95% CI 1 to 2), nasopharyngeal swab-nasal swab 3 (95% CI 2 to 3), and midturbinate swab-nasal swab 1 (95% CI 1 to 2). CONCLUSION: Compared with the reference standard nasopharyngeal swab specimen, midturbinate swab specimens provided a significantly more comfortable sampling experience, with only a small sacrifice in sensitivity for influenza detection. Nasal swab specimens were significantly less sensitive than midturbinate swab. Our results suggest the midturbinate swab is the sampling method of choice for molecular influenza testing in ED patients.

A highly elastic, capacitive strain gauge based on percolating nanotube networks.

We present a highly elastic strain gauge based on capacitive sensing of parallel, carbon nanotube-based percolation electrodes separated by a dielectric elastomer. The fabrication, relying on vacuum filtration of single-walled carbon nanotubes and hydrophobic patterning of silicone, is both rapid and inexpensive. We demonstrate reliable, linear performance over thousands of cycles at up to 100% strain with less than 3% variability and the highest reported gauge factor for a device of this class (0.99). We further demonstrate use of this sensor in a robotics context to transduce joint angles.

A real-time PCR antibiogram for drug-resistant sepsis.

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔC(t)<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 gram-negative and 2 gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.

Study of microscale hydraulic jump phenomenon for hydrodynamic trap-and-release of microparticles.

Easy trap-and-release of microparticles is necessary to study biological cellular behavior. The hydraulic jump phenomenon inspired us to conceive a microfluidic device for the hydrodynamic trap-and-release of microparticles. A sudden height increase in a microfluidic channel leads to a dramatic decrease in flow velocity, allowing effective trapping of the microparticles by energy conversion. The trapped particles can be released by stronger inertial force based on simply increasing the flow velocity. We present a systematic, numerical study of trap-and-release of the microparticles using multiphase Navier-Stokes equations. Effect of geometry flow velocity, particle diameter, and adhesion force on trap-and-release was studied.