Abstract
Tuberculosis requires rapid diagnosis to prevent further transmission and allow prompt administration of treatment. Current methods for diagnosing pulmonary tuberculosis lack sensitivity are expensive or are extremely slow. The identification of lipids using gas chromatography- electron impact mass spectrometry (GC-EI/MS) could provide an alternative solution. We have studied mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of lipids using thermochemolysis GC-EI/MS. To facilitate use of the technology in a routine diagnostic laboratory a simple extraction procedure was employed where PDIMs were extracted from sputum using petroleum ether, a solvent of low polarity. We also investigated a method using methanolic tetramethylammonium hydroxide, which facilitates direct transesterification of acidic components to methyl esters in the inlet of the GC-MS system. This eliminates conventional chemical manipulations allowing rapid and convenient analysis of samples. When applied to an initial set of 40 sputum samples, interpretable results were obtained for 35 samples with a sensitivity relative to culture of 94% (95%CI: 69.2,100) and a specificity of 100% (95%CI: 78.1,100). However, blinded testing of a larger set of 395 sputum samples found the assay to have a sensitivity of 61.3% (95%CI: 54.9,67.3) and a specificity of 70.6% (95%CI: 62.3,77.8) when compared to culture. Using the results obtained we developed an improved set of classification criteria, which when applied in a blinded re-analysis increased the sensitivity and specificity of the assay to 64.9% (95%CI: 58.6,70.8) and 76.2% (95%CI: 68.2,82.8) respectively. Highly variable levels of background signal were observed from individual sputum samples that inhibited interpretation of the data. The diagnostic potential of using thermochemolytic GC-EI/MS of PDIM biomarkers for diagnosis of tuberculosis in sputum has been established; however, further refinements in sample processing are required to enhance the sensitivity and robustness of the test.
Highlights
Tuberculosis (TB) is a global health problem
We have investigated a simple extraction procedure followed by thermally assisted hydrolysis and methylation (THM), prior to the analysis by gas chromatography (GC) coupled with electron impact mass spectrometry (EI/MS), to ascertain the potential for a rapid diagnostic test for pulmonary tuberculosis based on detection of the mycocerosic acid components of phthiocerol dimycocerosate (PDIM)
Duplicate analyses of extracts obtained from negative sputum samples spiked with PDIMs standard (140– 27,500 pg/mL) demonstrated that the yield of the mycocerosic acid methyl esters was proportional to the amount of material added, within the range tested (Figure 3)
Summary
Tuberculosis (TB) is a global health problem. The World Health Organization estimated 9.4 million incident cases during 2009 and 1.7 million deaths [1]. The challenge in reducing the global burden of tuberculosis is rapid diagnosis followed by appropriate treatment. Current methods of diagnosing TB are inadequate. The most widely used test, microscopic examination of sputum, lacks sensitivity and global cases detection rates remain low [2]. It is estimated that only 63% of incident cases are detected each year [1]. Definitive diagnosis requires isolation and culture of the bacilli which may take weeks and requires considerable laboratory infrastructure to protect against infection [2,3]
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