This study demonstrates that the Xpert MTB/RIF assay system can rapidly detect the presence of M. tuberculosis and identify the mutations most frequently associated with rifampin resistance directly from smear-negative and smear-positive clinical sputum samples. The self-contained cartridge fluidics of the Xpert MTB/RIF assay made it possible to design a heminested PCR assay with a sensitivity that approached culture-based diagnostics. The assay appears to be relatively resistant to PCR inhibitors which may be present in sputum; however, PCR inhibitors may have been responsible for the one smear-positive sample from Uganda that was Xpert MTB/RIF negative. The retrospective nature of our clinical validation studies caused us to rely on tests of previously frozen sputum samples. Freezing may alter sputum viscosity, and it may improve nucleic acid recovery from mycobacteria. Thus, these results will need to be confirmed in larger prospective studies with fresh samples. However, we documented similar LODs regardless of whether the assay was performed with fresh or frozen sputum samples in our analytic studies using spiked sputum. Although the results presented here are most relevant to detection of pulmonary tuberculosis, preliminary studies with the Xpert MTB/RIF assay suggest that this system also has promise for detecting M. tuberculosis in other body fluids, such as cerebrospinal fluid; thus, the assay may have even broader utility.
Truly rapid results for drug susceptibility tests are particularly important in the management of drug-resistant tuberculosis (13
). Currently available methods fall short of this promise (4
). Most rapid nucleic acid amplification methods to detect tuberculosis require skilled technicians and dedicated space for both setup and analysis in order to prevent amplicon cross-contamination. Assay setup can also present a significant biohazard, confining work to centers with specialized biocontainment equipment. These technical requirements cause most centers to batch samples and test for tuberculosis once a day at most. The Xpert MTB/RIF assay, however, is simple and robust enough to be performed by personnel with minimal training. Total hands-on time is less than 5 min, and results are typically available within 1 h 55 min. Each module within the GeneXpert instrument operates independently, which enables the user to test each sputum sample as it arrives in the laboratory instead of saving samples for batch processing. This important feature can potentially result in dramatically reduced turnaround times for tuberculosis detection, allowing decisions about respiratory isolation and treatment to be made in real time (3
). False-positive results, often caused by carryover of amplified target, are mitigated by the use of closed cartridges that do not require any manual pipetting after the sample has been added to the cartridge. False-negative results, caused by operator errors, manufacturing defects, fluidics problems, or the presence of inhibitors in the sample, are controlled for by a multiplexed heminested PCR assay that detects a control target within B. globigii
spores included within each cartridge.
Complete costing and cost effectiveness studies of the Xpert MTB/RIF test are planned and merit more thorough discussion in future publications. At present, instrumentation costs for the GeneXpert system are similar to those of an automated liquid culture system for tuberculosis, and per-assay running costs are also in the same range as culture, despite vastly superior performance in terms of speed, biosafety, and ease of use. Importantly, the assay makes use of a basic cartridge design and an instrument platform that are currently used in commercially available assays for rapid detection of methicillin-resistant Staphylococcus aureus
, enteroviruses, Bacillus anthracis
), and group B Streptococcus
) and that is also likely to be used in the detection of a variety of infectious diseases, ranging from human immunodeficiency virus (HIV) and polio to sexually transmitted diseases and Clostridium difficile
. Thus, the assay can take advantage of economies of scale that would not be possible with a tuberculosis-specific assay platform. For the developing world, where 95% of all TB occurs, the Xpert MTB/RIF assay will be available to the public sector at concessionary prices negotiated by the Foundation for Innovative New Diagnostics and will compete with the costs currently being paid locally for culture and conventional antibiotic susceptibility testing.
The point-of-care for tuberculosis for most people in the world is at a health post linked to a microscopy center. This is where tuberculosis is detected, registered, treated, and followed up. Diagnostic technology has been one of the major limitations at this level, with the insensitive and technologist-dependent performance of microscopy as the bottleneck to care in many situations and a major contributor to poor disease control. The Xpert MTB/RIF assay offers the first technical opportunity to bridge this gap, potentially bringing tests for both TB and drug resistance to levels of the health system where many seek care.