Novel approaches in diagnosing tuberculosis

Abstract

The WHO declared tuberculosis (TB) a global emergency. An estimated 8-9 million new cases occur each year with 2-3 million deaths. Currently, TB is diagnosed mostly by chest-X ray and staining of the mycobacteria in sputum with a detection limit of 1x10 4 bacteria /ml. There is an urgent need for better diagnostic tools for TB especially for developing countries. We have validated the electronic nose from TD Technology for the detection of Mycobacterium tuberculosis by headspace analysis of 284 sputum samples from TB patients. We used linear discriminant function analysis resulting in a sensitivity of 75% a specificity of 67% and an accuracy of 69%. Further research is still required to improve the results by choosing more selective sensors and sampling techniques. We used a fast gas chromatography- mass spectrometry method (GC-MS). The automated procedure is based on the injection of sputum samples which are methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM-GC-MS). Hexacosanoic acid in combination with tuberculostearic acid was found to be specific for the presence of M. tuberculosis . The detection limit was similar to microscopy. We found no false positives, all microscopy and culture positive samples were also found positive with the THM-GC-MS method. The detection of ribosomal RNA from the infecting organism offers great potential since rRNA molecules outnumber chromosomal DNA by a factor 1000. It thus may possible to detect the organism without amplification of the nucleic acids (NA). We used a capture and a tagged detector probe for the direct detection of M. tuberculosis in sputum. So far the detection limit is 1x10 6 bacteria / ml. Currently we are testing a Lab-On-A-Chip Interferometer detection system.