Direct detection of Mycobacterium tuberculosis in sputum: A validation study using solid phase extraction-gas chromatography-mass spectrometry.

Tuberculosis (TB) remains a worldwide health problem, especially in developing countries. Correct identification of Mycobacterium tuberculosis (MTB) infection is extremely important for providing appropriate treatment and care to patients. Here we describe a solid phase extraction-gas chromatography-mass spectrometry method (SPE-THM-GC-MS) for the detection of five biomarkers for M. tuberculosis. The method for classification is developed and validated through the analysis of 112 sputum samples from patients suspected of having TB. Twenty of twenty-five MTB culture-positive sputum samples were correctly classified as positive by our improved SPE-THM-GC-MS method. Eighty-five of eighty-seven MTB culture-negative samples were also negative by SPE-THM-GC-MS. The overall sensitivity of the new SPE-THM-GC-MS method is 80% (20/25) and the specificity is 98% (85/87) compared with culture. The method proved to be reliable and, although complex in principle, easy to operate due to the high degree of automation.

Hyphenated and comprehensive liquid chromatography × gas chromatography-mass spectrometry for the identification of Mycobacterium tuberculosis.

Tuberculosis is one of the world's most emerging public health problems, particularly in developing countries. Chromatography based methods have been used to tackle this epidemic by focusing on biomarker detection. Unfortunately, interferences from lipids in the sputum matrix, particularly cholesterol, adversely affect the identification and detection of the marker compounds. The present contribution describes the serial combination of normal phase liquid chromatography (NPLC) with thermally assisted hydrolysis and methylation followed by gas chromatography-mass spectrometry (THM-GC-MS) to overcome the difficulties of biomarker evaluation. The in-series combination consists of an LC analysis where fractions are collected and then transferred to the THM-GC-MS system. This was either done with comprehensive coupling, transferring all the fractions, or with hyphenated interfacing, i.e. off-line multi heart-cutting, transferring only selected fractions. Owing to the high sensitivity and selectivity of LC as a sample pre-treatment method, and to the high specificity of the MS as a detector, this analytical approach, NPLC × THM-GC-MS, is extremely sensitive. The results obtained indicate that this analytical set-up is able to detect down to 1 × 10(3) mycobacteria/mL of Mycobacterium tuberculosis strain 124, spiked in blank sputum samples. It is a powerful analytical tool and also has great potential for full automation. If further studies demonstrate its usefulness when applied blind in real sputum specimens, this technique could compete with the current smear microscopy in the early diagnosis of tuberculosis.

Direct detection of Mycobacterium tuberculosis in sputum using combined solid phase extraction-gas chromatography-mass spectrometry.

Recently, thermally-assisted hydrolysis and methylation followed by gas chromatography-mass spectrometry (THM-GC-MS) in combination with chemometrics has been used to develop a 20-compound model for fast differentiation of Mycobacterium tuberculosis (MTB) from Non-tuberculous mycobacteria (NTM) in bacterial cultures. This model provided better than 95% accuracy. In our current work a hexane/methanol/water extraction followed by a solid phase extraction (SPE) clean-up procedure was developed for use before THM-GC-MS, to make the test suitable for the identification of mycobacteria in sputum. The 20 biomarker model had to be adapted since many compounds were also found in the sputum of non-tuberculosis patients. An algorithm was established based on tuberculostearic acid, hexacosanoic acid and mycoserosates. The detection limit of the method was approximately 1×10(4) bacteria/mL sputum. Sputum specimens from 32 patients from South Africa who were suspected of having tuberculosis were blindly tested using the new method. Eight of the nine culture-positive sputum specimens were detected by the new SPE-THM-GC-MS method, resulting in a sensitivity of 89%. The specimen that was missed by the new method was also microscopy negative. The specificity of the test was 100%; all 23 microscopy- and culture-negative specimens were correctly identified as negative by SPE-THM-GC-MS.