A rapid, fully-automated PCR system for sample processing, diagnosis, and rifampin resistance detection of Mycobacterium tuberculosis
Abstract number: O312
Jones M., Helb D., Ho K., Kop J., Nguyen P., Story E., Wallace E., Alland D., Christel L., Boehme C., Perkins M., Nabeta P., Skenders G., Levi M., Safi H., Rodgers R., Winn-Deen E., Persing D., Dailey P.
Background: Current methods for the detection and determination of drug resistance of Mycobacterium tuberculosis (MTB) are insensitive and slow. PCR is a more accurate and rapid technique, but requires substantial labour and technical competence. PCR is also affected by inhibitors, sample cross-contamination, and limited ability to concentrate samples. All of these limitations are overcome by the GeneXpert® system. This automated system uses a low-cost plastic cartridge to concentrate all the bacilli present in a sputum sample, remove inhibitors, lyse cells, and transfer the DNA into an integrated PCR tube. This DNA is amplified in a nested, real-time PCR using an optimised, six-colour Molecular Beacon assay to detect more than 95% of all rifampin-resistant TB strains. An internal sample processing and reagent control is also included. Sample processing and nested PCR occur within the same cartridge and no external manipulation is required after the sample is initially loaded.
Objectives: To comprehensively evaluate the GeneXpert MTB assay using sputum and sputum pellet samples and simultaneously detect rifampin resistance-associated mutations in the rpoB core region. Key evaluation criteria were analytical performance, stability of reagents, and sample inactivation.
Methods: Proficiency panels, clinical and spiked samples are mixed with a sample treatment buffer (STB), incubated and added to the cartridges containing reagent beads and buffer. Wild type and mutant MTB strains were used to determine the system's analytical performance. Inactivation studies determined the amount of viable TB cells before and after treatment with STB. Stability was assessed by testing cartridges incubated from 4 °C to 45 °C.
Results: The STB kills >6 logs of MTB organisms in sputum, disinfecting the sputum and reducing the risk from infectious aerosols. The GeneXpert detected as few as 50 bacilli/mL of sputum in 90 min, making it as rapid as microscopy, but much more sensitive. Eleven out of 12 of the QCMD 2002 TB panel samples were correctly identified. At two alpha test sites this system diagnosed and provided semi-quantitative information about TB infections and accurately detected rifampin resistance. To date reagent stability is >3 months at 45°C.
Conclusion: The combination of sample processing with real-time PCR into a single automated step makes this assay simple to perform, in near-patient and laboratory settings, with minimal effort in <2 h.
XpertTM MTB sensitivity & specificity (per patient analysis)
XpertTM MTB Rif resistance detection (per patient analysis)
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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