Lee A. Christel

Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, Near-Patient Technology

ABSTRACT Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert Systems MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assays buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.

Toward Next Generation Clinical Diagnostic Instruments: Scaling and New Processing Paradigms

Looking toward future clinical diagnostic instruments, there is little debate as to the features that need improvement over the current state-of-the-art. Increasing the speed and sensitivity of the assays, while reducing costs are clear goals. Recently, it has become possible to microminiaturize fluidic and sensing components using micromachining and precision injection molding. There has been a large amount of interest and effort in the area of miniaturization of such systems, yet not all of the properties of fluidics and sensing methods improve as they are drastically reduced in size. It is clear that implementing miniaturized diagnostic instruments is not a matter of simply “shrinking” their conventional counterparts, nor of automating existing manual procedures. What is required to harness the full potential of scaling technologies is the use of design methods that take into account scaling effects and the development of completely new processing approaches. Beginning with a general overview of the relevant scaling principles, sample preparation and detection approaches are addressed in this context.

Single-crysytal silicon pressure sensors with 500 × overpressure protection

Abstract Single-crystal silicon piezoresistive pressure sensors with high overpressure tolerance have been fabricated using the process of silicon fusion bonding. A mechanical stopping surface beneath a conventional diaphragm structure limits diaphragm displacement during overpressure conditions. Uniform and bossed diaphragms in gage and absolute configurations are possible using this process. Sensors with sensitivities as high as 3 mV/ V/psi (typical of sensors used for 5–10 psi full-scale applications) survived overpressures of up to 5000 psi. Finite element modeling is compared to experimental results.

A new generation of PCR instruments and nucleic acid concentration systems

Publisher Summary The polymerase chain reaction (PCR) technique has clearly evolved into an important tool for researchers and clinicians. This has been afforded by the commercialization of robust and dependable instruments for thermal cycling and, recently, with homogenous fluorescence detection.. The state-of-the-art instruments that include real-time, homogeneous, fast thermal cycling, and quantitative detection capabilities still leave significant opportunities for improvements. Efforts to develop PCR on a chip or micromachined/miniaturized systems have shown some interesting capabilities, but still fall short of providing the types of results that surpass or even equal those of commercial systems. However, in the future the development of new nucleic acid systems based on some of the principles from such research devices will probably occur. This chapter describes the extension of previous work based on silicon micromachining that has shown equivalent and improved performance over commercial systems. Other improvements over commercial systems have been discovered. These include new graphical user interface, independent control of each reaction site, modularity, and rapid thermal cycling of large volumes. Ultimately, the chapter concludes with anticipation that one day all the processing and homogenous quantitative detection will occur in one low-cost disposable, integrated system, which will take PCR to the new level of utility.

Nucleic Acid Concentration and PCR for Diagnostic Applications

Sample preparation involving the extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface area to volume ratios. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50%, and concentration factors of about 10X using bacteriophage lambda DNA as the target. These results, when combined with rapid amplification and detection, confirm the viability of utilizing these components as elements of a compact system for the purification and detection of nucleic acids in applications such as clinical diagnostics, food quality control, and environmental monitoring.