Our results showed that gastric aspirate and CSF samples had M. tuberculosis
LODs similar to those of saline solution in the GeneXpert assay. The LOD with CSF samples (median, 25 CFU/ml) was lower than that reported for spiked sputum samples (131 CFU/ml; 95% confidence interval [CI], 106.2 to 176.4) (10
). These findings suggest that the reduced sensitivity of the MTB/RIF assay in smear-negative CSF and gastric aspirate specimens is due to a paucity of organisms and not to inhibitory factors encountered in the PCR (2
). In contrast, the LOD of the GeneXpert assay was significantly elevated with spiked tissue and stool samples. The presence of soluble PCR inhibitors such as heme, bilirubin, and bile salts in tissue and stool is well known (1
). These PCR inhibitors interfere with the DNA polymerase and decrease the sensitivity of PCR-based NAATs. Although the Xpert system removes many inhibitors during the wash and filtration steps, it is possible that some substances pass into the reaction vial. Alternatively, the tubercle bacilli may have bound to and cosettled with debris or cells in tissue and stool samples rather than having floated to the top. The stool samples showed the broadest range of LOD (267 to 193,333 CFU/ml) compared to other sample types, suggesting that heterogeneity in stool makeup may variably affect the sensitivity of the MTB/RIF assay. Although the ability to use stool as an alternative to sputum samples would be greatly beneficial to pulmonary TB patients who cannot expectorate, there are limited clinical data on the sensitivity of the GeneXpert system with stool samples. The two prior studies that evaluated the sensitivity of the GeneXpert system with actual patient stool samples showed a sensitivity of 100% compared to culture, although only 3 samples were tested (11
). In contrast to our study, both studies used conventional digestion-decontamination with N
-cysteine-NaOH followed by concentration of the sediment by centrifugation. Our aim, however, was to develop a simple, centrifuge-free sample preparation protocol for use in minimally equipped laboratories and smear-microscopy centers in resource-poor settings where GeneXpert technology could have the greatest impact.
The low LOD observed with CSF and gastric aspirates indicates that the GeneXpert system is a viable diagnostic tool for detection of M. tuberculosis
and rifampin resistance in these sample types. However, there is still a need for a simple and practical approach to concentrating the organisms in paucibacillary or complex samples prior to performing the MTB/RIF assay. The flotation of M. tuberculosis
in clinical specimens based on its buoyancy relative to sucrose and NaCl solutions is an attractive methodology, because it requires simple reagents which can be easily obtained and prepared anywhere at very low cost and can be implemented with minimal equipment. Our efforts to concentrate M. tuberculosis
in spiked gastric aspirate and stool samples by the use of flotation techniques were not successful. However, we were able to concentrate M. tuberculosis
more than 30-fold by centrifugation in two of the three pooled CSF samples with either 28% sucrose or 36% NaCl. Although these results are encouraging, evaluation of individual samples from TB suspects is needed to validate these preliminary findings. In addition, further advances are needed to concentrate the bacilli and eliminate PCR inhibitors in other nonrespiratory samples such as tissue and stool. Recent technological advances for purification of microorganisms directly from patient specimens such as cellular imprinting (8
), an immunomagnetic capture system (NanoMR Inc., Albuquerque, NM), or ligand-coated paramagnetic beads (Microsens Biotechnologie, United Kingdom) are potential technologies that could allow rapid, simple, and affordable concentration of intact M. tuberculosis
. Coupled with the simplicity of the GeneXpert platform, a successful concentration method could greatly extend the application of this platform for diagnosis of TB in extrapulmonary samples in resource-limited areas.