Detection of Mycobacterium tuberculosis in Sputum by Gas Chromatography-Mass Spectrometry of Methyl Mycocerosates Released by Thermochemolysis

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

Structures of the <i>M. tuberculosis</i> C₂₉, C₃₀ and C₃₂ mycocerosic acid methyl esters.

<p>The ions at <i>m/z</i> 452, 466 and 494 correspond to the molecular weights of these esters. Ions at <i>m/z</i> 88 and 101 are characteristic for 2-methyl branched fatty acid methyl esters, the former resulting from a well-known “McLafferty” rearrangement with transfer of one hydrogen atom.</p

THM-GC-EI/MS traces of TB negative sputum spiked with <i>M. tuberculosis</i> bacilli.

<p>The chromatograms show the fragment ions <i>m/z</i> 101 and <i>m/z</i> 88 for the extract from 1 ml sample of TB negative sputum spiked with 140 CFU of <i>M. tuberculosis</i> culture.</p

Secondary analysis of the performance of THM-GC-EI/MS compared to smear microscopy and culture on Lowenstein Jensen.

<p>A refined algorithm for interpreting the THM-GC-EI/MS chromatograms, developed by using knowledge from an initial analysis of 395 samples, was applied in a second round of blinded analysis by a newly trained operator.</p>#<p>Four samples were not determined due to overloading of the GC column and were excluded from further analysis.</p

Performance of THM-GC-EI/MS compared to smear microscopy and culture on Lowenstein-Jensen for detection <i>M. tuberculosis</i> in 395 sputum samples.

#<p>Four samples were not determined due to overloading of the GC column and were excluded from further analysis.</p

Examples of THM-GC-EI/MS chromatograms from sputum specimens from suspected TB cases.

<p>Chromatograms show the profile of the fragment ion <i>m/z</i> 101 in (A) negative, and positive sputum samples with (B) high, and (C) low amounts of PDIMs present. Note the characteristic doublets for the mycocerosic acid methyl esters C₂₉/C₃₀ and C₃₂ in b–c.</p

THM-GC-EI/MS of sputum spiked with PDIMs.

<p>The graphs show the peak areas obtained for C₂₉/C₃₀ and C₃₂ mycocerosates generated from spiking 0.2 mL of pooled TB negative sputum samples with PDIM standard, (A) in the range 0.14–2.75 ng, and (B) expanding the range between 0.14–27.5 ng.</p

THM-GC-EI/MS of sputum spiked with <i>M. tuberculosis</i> bacilli.

<p>The graph shows the peak areas obtained for C₂₉/C₃₀ and C₃₂ mycocerosates generated from spiking 1 mL of pooled TB negative sputum sample with <i>M. tuberculosis</i> culture (140–5,600 CFU/mL).</p