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Rapid Ultrasensitive Detection of Clostridiodes difficile Toxins in Stool Samples Using A Single-Molecule Counting Method
- Don Straus, Ann Zuniga, Alejandra Garces, Andrew Tempesta, Adam Williams, Bill Lauzier, Jennifer Hickey, Sadanand Gite, Selina Clancy, Yismel Rosario, Bruce Walsh, Jayson Bowers
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- Journal:
- Infection Control & Hospital Epidemiology / Volume 41 / Issue S1 / October 2020
- Published online by Cambridge University Press:
- 02 November 2020, pp. s450-s451
- Print publication:
- October 2020
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- Article
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Background:Clostridiodes difficile infection is considered an urgent antibiotic resistance threat by the CDC, accounting for ∼225,000 hospitalizations, 12,800 deaths, and ∼$1 billion in healthcare costs in the United States in 2017. The presence of the secreted toxins that cause the devastating symptoms of this gastrointestinal infection are diagnostic of C. difficile infection (CDI). However, the rapid testing methods currently used to detect CDI lack accuracy. Enzyme immunoassays are specific but lack sensitivity because they do not detect CDI patients that have low levels of the toxins. Nucleic acid amplification tests (NAATs) are sensitive, but they lack specificity because they detect patients colonized with C. difficile in the dormant spore form that does not produce the toxins. This insufficiency has resulted in the adoption of complex multitest algorithms for C. difficile diagnosis. We present results for a new toxin test that demonstrates both high clinical sensitivity and clinical specificity for C. difficile toxin B on a fully automated benchtop platform. Methods: The detection technology uses nonmagnified digital imaging to count single toxin molecules that tether together target-specific magnetic and fluorescent particles. The 30-minute method includes the use of a dye cushion to eliminate wash steps and the need for time-consuming specimen preparation steps. We determined analytical performance characteristics of the test using negative clinical stool samples spiked with purified toxin. To assess clinical performance, we tested 785 stool samples from 5 clinical sites and compared the results with the cellular cytotoxicity neutralization assay (CCNA). Results: The test’s limit of detection for toxin B was 60 pg/mL. A comparison of the new test to the CCNA reference method gave 98% positive percentage agreement (83 of 85 samples) and 95% negative percentage agreement (667 of 700 samples). Conclusions: The new method demonstrated 96% accuracy compared to the gold standard for C. difficile toxin tests. The results also demonstrate an analytical sensitivity (limit of detection, 60 pg/mL). Thus, the test has the potential to detect CDI patients missed by enzyme immunoassay (EIA) tests due to their low analytical sensitivity. Because the test detects toxins directly, it is expected to have a lower false-positive rate than NAAT methods, which detect patients colonized with the non–toxin-producing spore form. A single accurate test for toxin-producing C. difficile could eliminate the need for multitest algorithms.
Funding: First Light Diagnostics, Inc., provided support for this study.
Disclosures: Donald Straus reports that he is the founder and chief scientific officer of First Light Diagnostics (FLDx) with salary and ownership interest in the form of stocks, stock options, and warrants. Adam Williams reports salary from First Light Diagnostics.
Challenges in Integrating the High-K Gate Dielectric Film to the Conventional Cmos Process Flow
- Avinash Agarwal, Michael Freiler, Pat Lysaght, Loyd Perrymore, Renate Bergmann, Chris Sparks, Bill Bowers, Joel Barnett, Deborah Riley, Yudong Kim, Billy Nguyen, Gennadi Bersuker, Eric Shero, Jae E. Lim, Steven Lin, Jerry Chen, Robert W. Murto, Howard R. Huff
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- Journal:
- MRS Online Proceedings Library Archive / Volume 670 / 2001
- Published online by Cambridge University Press:
- 21 March 2011, K2.1
- Print publication:
- 2001
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ZrO2 and HfO2 and their alloys with SiO2 are currently among the leading high-k materials for replacing SiOxNy as the gate dielectric for the sub-100 nm technology nodes. International SEMATECH (ISMT) is currently investigating integration issues associated with this required change in materials. Our work has focused on the integration of ALCVD deposited ZrO2 and HfO2 with an industry standard conventional MOSFET process flow with poly-Si electrode. Since the impact of contamination by these new high-k materials introduced in a production fab has not yet been established, it becomes very critical to prevent cross- contamination through the process tools in the fab. A baseline study was completed within ISMT's fab and appropriate protocols for handling high-k materials have been established. The integrated high-k gate stack in a conventional transistor flow should not only meet all the performance requirements of scaled transistors, but the gate dielectric film should be able withstand high-temperature anneal steps. Reactions between ZrO2 and Si have been observed at temperatures as low as 560°C (during the amorphous Si deposition process). Various wet chemistries were also evaluated for removing the high-k film inadvertently deposited on wafer backside, and it was found that ZrO2 etches at extremely slow rates in the majority of the common wet etch chemistries available in a fab. A new hot HF based process was found to be successful in lowering Zr contamination on the wafer backside to as low as 1.8 E10 atoms/cm2. The patterning of a high-k gate stack with poly-Si electrode is another area that required considerable focus. Various dry (plasma) etch and wet etch chemistries were evaluated for etching ZrO2 using both blanket films as well as wafers with patterned poly-Si gate over the high-k films. On the full CMOS flow device wafers, most of these wet chemistries resulted in severe pitting in the ZrO2 film remaining over the source/drain (S/D) areas, as well as in the Si substrate and the field oxide. A poly-Si gate over ZrO2 gate dielectric film was successfully patterned using the standard poly-Si gate etch (Cl2/HBr) for the main etch, followed by a combination of HF and H2SO4 clean for removing all of the ZrO2 remaining over the S/D area. This allowed the fabrication of low-resistance contacts to transistor S/D areas, which ultimately resulted in demonstration of functional transistors with high-k gate dielectric films.