A Tail Fiber Engineering Platform for Improved Bacterial Transduction-Based Diagnostic Reagents.

Bacterial transduction particles were critical to early advances in molecular biology and are currently experiencing a resurgence in interest within the diagnostic and therapeutic fields. The difficulty of developing a robust and specific transduction reagent capable of delivering a genetic payload to the diversity of strains constituting a given bacterial species or genus is a major impediment to their expanded utility as commercial products. While recent advances in engineering the reactivity of these reagents have made them more attractive for product development, considerable improvements are still needed. Here, we demonstrate a synthetic biology platform derived from bacteriophage P1 as a chassis to target transduction reagents against four clinically prevalent species within the Enterobacterales order. Bacteriophage P1 requires only a single receptor binding protein to enable attachment and injection into a target bacterium. By engineering and screening particles displaying a diverse array of chimeric receptor binding proteins, we generated a potential transduction reagent for a future rapid phenotypic carbapenem-resistant Enterobacterales diagnostic assay.

Detection of C. difficile toxin as a model assay for performing fully automated high-throughput RT-PCR on clinical stool samples.

BACKGROUND: The cobas® omni Utility Channel enables users to integrate lab-developed tests (LDTs) on the cobas® 6800 System to perform molecular diagnostics with high-throughput capacity and full automation. At present, there are no CE- or FDA-approved tests for stool pathogens on this system. To assess the performance of stool as a matrix, we evaluated the analytical and clinical performance of an LDT for detection of Clostridioides difficile (C. difficile) toxin B using the Utility Channel (C.diff_UTC). METHODS: A 10% stool suspension prepared from liquid stool samples diluted in phosphate buffered saline was used for analysis. Limit of detection (LoD) was determined in six dilutions with 126 replicates/dilution. Clinical evaluation was performed using 514 predetermined patient stool samples from two study sites in Germany. The C.diff_UTC was compared with LC 480 amplification and an LDT or the R-BioPharm C. difficile assay. Discrepant results were further analyzed using the GeneXpert C. difficile assay. RESULTS: Limit of detection was 23.48 cfu/mL (95% Confidence Interval [CI]: 19.14-31.01) with inter-run variation of <2 cycle thresholds at 3 × and 10 × LoD. No cross-reactivity was observed with a panel of fecal organisms and pathogens. Bioinformatic analysis showed coverage of the major C. difficile toxinotypes by the primer/probe set. Clinical evaluation revealed sensitivity of 96.7% (95% CI: 88.7-99.6) and specificity of 99.3% (95% CI: 98.0-99.9) compared with the reference method; inhibition rate was 3.5% (18/514). CONCLUSION: Using a predesigned primer/probe set, the C.diff_UTC assay features analytical performance and clinical sensitivity and specificity comparable to currently available nucleic acid amplification tests (NAATs) and is suitable for high-throughput testing. This was a proof-of-concept study, indicating the cobas Utility Channel could likely be adapted for other clinically relevant stool pathogens in outbreak scenarios.

Measurement of atmospheric neutral wind and temperature from Fabry-Perot interferometer data using piloted deconvolution.

Nonlinear regression techniques, when applied to sky exposures obtained using a Fabry-Perot interferometer (FPI), are able to recover atmospheric neutral wind and temperature through inversion of the resulting fringe pattern. Current inversion methods often account for temporal fluctuation of the etalon's optical path length (caused by temperature variation in the instrument housing, for example) by characterizing the system function using isolated exposures of a frequency-stabilized laser. Because these path length changes correspond directly to shifts in the fringe pattern, they can significantly increase the total wind velocity uncertainty between laser exposures. We propose an extension to current regression techniques allowing for characterization of the optical path length and measurement of neutral wind and temperature simultaneously, thus reducing the need for frequent isolated laser exposures. This is achieved by using the laser as a pilot signal that enters the aperture of the instrument during sky exposures. We show that the extension can lead to a lower variance estimator for velocity when the optical path length has a significant time-varying component. Additionally, several pragmatic physical configurations that would allow for construction of a piloted signal in a real system are tested and compared using an FPI installation at the Urbana Atmospheric Observatory.

On the uncertainties in determining fringe phase in Doppler asymmetric spatial heterodyne spectroscopy.

The mean fringe phase measured by Doppler asymmetric spatial heterodyne spectroscopy is a direct measure of atmospheric wind. The uncertainty in measuring the mean phase is investigated and found to be accurately predicted by an analytic formula for moderate and high signal-to-noise ratios. At lower signal-to-noise ratios, numeric issues in the phase calculation result in non-Gaussian distributions of mean phase. Analysis techniques are described to mitigate these numeric issues to the extent possible.