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Mycolic acids as diagnostic markers for tuberculosis case detection in humans and drug efficacy in mice.

Shui G, Bendt AK, Jappar IA, Lim HM, Laneelle M, Hervé M, Via LE, Chua GH, Bratschi MW, Zainul Rahim SZ, Michelle AL, Hwang SH, Lee JS, Eum SY, Kwak HK, Daffé M, Dartois V, Michel G, Barry CE, Wenk MR - EMBO Mol Med (2011)

Bottom Line: Quantification of specific precursor → fragment transitions of approximately 2000 individual mycolic acids (MAs) resulted in high analytical sensitivity and specificity.Furthermore, we quantified MA species in lung tissue of TB-infected mice and demonstrated effective clearance of MA levels following curative rifampicin treatment.Thus, our results demonstrate for the first time the feasibility and clinical relevance of direct detection of mycobacterial lipids as biomarkers of TB infection.

View Article: PubMed Central - PubMed

Affiliation: Yong Loo Lin School of Medicine, Department of Biochemistry, National University of Singapore, Singapore. guanghou_shui@nuhs.edu.sg

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MAs in sputum are diagnostic for TB infection in humansA. Diagram illustrating the case–control set-up and number of TB patients (n = 70) and non-TB controls (n = 40) analysed. Samples originated from four different countries (see Table S2 of Supporting information for detailed demographic information).B. Sputum samples (200–500 µl) were extracted using organic solvents and levels of individual MA molecular species were determined using MS in MRM mode. Major MA species for α-MA (alpha-), M- (methoxy-) and K- (keto-) MA with their respective molecular composition are plotted, with (24) and (26) indicating the number of carbon atoms in their respective alpha chain.C. Individual MA molecular species varied in their power to differentiate between non-TB controls and TB patients. Numbered dots highlighting false positive and false negative results correspond to patient IDs in Table S2 of Supporting information. Cutoff for MRM 1164/395 between non-TB control and patients is 0.0026 µg/ml sputum.D. ROC curve displaying the classifying performance (positive diagnostic likelihood ratio) of individual MA molecular species, expressed by its true positive rate (sensitivity) and false positive rate (1—specificity), with α-MA C80H156O3 providing the best accuracy.E,F. The predictive power of MA quantification is shown by confusion matrix for all 110 samples (E) and for the 39 HIV+ patients (F) alone. Sensitivity and specificity were calculated as described in the text.
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fig02: MAs in sputum are diagnostic for TB infection in humansA. Diagram illustrating the case–control set-up and number of TB patients (n = 70) and non-TB controls (n = 40) analysed. Samples originated from four different countries (see Table S2 of Supporting information for detailed demographic information).B. Sputum samples (200–500 µl) were extracted using organic solvents and levels of individual MA molecular species were determined using MS in MRM mode. Major MA species for α-MA (alpha-), M- (methoxy-) and K- (keto-) MA with their respective molecular composition are plotted, with (24) and (26) indicating the number of carbon atoms in their respective alpha chain.C. Individual MA molecular species varied in their power to differentiate between non-TB controls and TB patients. Numbered dots highlighting false positive and false negative results correspond to patient IDs in Table S2 of Supporting information. Cutoff for MRM 1164/395 between non-TB control and patients is 0.0026 µg/ml sputum.D. ROC curve displaying the classifying performance (positive diagnostic likelihood ratio) of individual MA molecular species, expressed by its true positive rate (sensitivity) and false positive rate (1—specificity), with α-MA C80H156O3 providing the best accuracy.E,F. The predictive power of MA quantification is shown by confusion matrix for all 110 samples (E) and for the 39 HIV+ patients (F) alone. Sensitivity and specificity were calculated as described in the text.

Mentions: Using a blinded format, we conducted a retrospective, multicenter, case–control study of 70 patients with pulmonary TB with varying disease burdens and 40 non-TB controls (individuals with clinical symptoms of TB who were diagnosed as non-TB by culture). For both groups, the HIV status and bacterial burden were known (Fig 2A; for details, see demographic information in Table S2 of Supporting information). Strikingly, robust α-, keto- and methoxy-MA signals were detected in as little as 200 µl of sputum from TB patients (Fig 2B). This was too small of a sample volume for detecting MAs with alternative methods (Shui et al, 2007). Further, the concentration of major MAs detected in sputum obtained from TB-infected individuals was significantly (∼100 times) higher than in samples from non-TB controls (Fig 2C, see also discussion for false positives and negatives). As shown in Fig 2E, we classified 66 out of the 70 TB patients correctly as TB positive (‘true positive’) and 37 out of the 40 non-TB controls correctly as non-TB (‘true negative’). Three were falsely classified as TP positive (‘false positive’) and four were falsely classified as non-TB (‘false negative’). Using these data, we calculated a statistical sensitivity of 94% and a specificity of 93% (sensitivity: number of true positives divided by the sum of true positives and false negatives; specificity: number of true negatives divided by the sum of true negatives and false positives) and even slightly better values for the HIV positive individuals alone (Fig 2F).


Mycolic acids as diagnostic markers for tuberculosis case detection in humans and drug efficacy in mice.

Shui G, Bendt AK, Jappar IA, Lim HM, Laneelle M, Hervé M, Via LE, Chua GH, Bratschi MW, Zainul Rahim SZ, Michelle AL, Hwang SH, Lee JS, Eum SY, Kwak HK, Daffé M, Dartois V, Michel G, Barry CE, Wenk MR - EMBO Mol Med (2011)

MAs in sputum are diagnostic for TB infection in humansA. Diagram illustrating the case–control set-up and number of TB patients (n = 70) and non-TB controls (n = 40) analysed. Samples originated from four different countries (see Table S2 of Supporting information for detailed demographic information).B. Sputum samples (200–500 µl) were extracted using organic solvents and levels of individual MA molecular species were determined using MS in MRM mode. Major MA species for α-MA (alpha-), M- (methoxy-) and K- (keto-) MA with their respective molecular composition are plotted, with (24) and (26) indicating the number of carbon atoms in their respective alpha chain.C. Individual MA molecular species varied in their power to differentiate between non-TB controls and TB patients. Numbered dots highlighting false positive and false negative results correspond to patient IDs in Table S2 of Supporting information. Cutoff for MRM 1164/395 between non-TB control and patients is 0.0026 µg/ml sputum.D. ROC curve displaying the classifying performance (positive diagnostic likelihood ratio) of individual MA molecular species, expressed by its true positive rate (sensitivity) and false positive rate (1—specificity), with α-MA C80H156O3 providing the best accuracy.E,F. The predictive power of MA quantification is shown by confusion matrix for all 110 samples (E) and for the 39 HIV+ patients (F) alone. Sensitivity and specificity were calculated as described in the text.
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Related In: Results  -  Collection

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fig02: MAs in sputum are diagnostic for TB infection in humansA. Diagram illustrating the case–control set-up and number of TB patients (n = 70) and non-TB controls (n = 40) analysed. Samples originated from four different countries (see Table S2 of Supporting information for detailed demographic information).B. Sputum samples (200–500 µl) were extracted using organic solvents and levels of individual MA molecular species were determined using MS in MRM mode. Major MA species for α-MA (alpha-), M- (methoxy-) and K- (keto-) MA with their respective molecular composition are plotted, with (24) and (26) indicating the number of carbon atoms in their respective alpha chain.C. Individual MA molecular species varied in their power to differentiate between non-TB controls and TB patients. Numbered dots highlighting false positive and false negative results correspond to patient IDs in Table S2 of Supporting information. Cutoff for MRM 1164/395 between non-TB control and patients is 0.0026 µg/ml sputum.D. ROC curve displaying the classifying performance (positive diagnostic likelihood ratio) of individual MA molecular species, expressed by its true positive rate (sensitivity) and false positive rate (1—specificity), with α-MA C80H156O3 providing the best accuracy.E,F. The predictive power of MA quantification is shown by confusion matrix for all 110 samples (E) and for the 39 HIV+ patients (F) alone. Sensitivity and specificity were calculated as described in the text.
Mentions: Using a blinded format, we conducted a retrospective, multicenter, case–control study of 70 patients with pulmonary TB with varying disease burdens and 40 non-TB controls (individuals with clinical symptoms of TB who were diagnosed as non-TB by culture). For both groups, the HIV status and bacterial burden were known (Fig 2A; for details, see demographic information in Table S2 of Supporting information). Strikingly, robust α-, keto- and methoxy-MA signals were detected in as little as 200 µl of sputum from TB patients (Fig 2B). This was too small of a sample volume for detecting MAs with alternative methods (Shui et al, 2007). Further, the concentration of major MAs detected in sputum obtained from TB-infected individuals was significantly (∼100 times) higher than in samples from non-TB controls (Fig 2C, see also discussion for false positives and negatives). As shown in Fig 2E, we classified 66 out of the 70 TB patients correctly as TB positive (‘true positive’) and 37 out of the 40 non-TB controls correctly as non-TB (‘true negative’). Three were falsely classified as TP positive (‘false positive’) and four were falsely classified as non-TB (‘false negative’). Using these data, we calculated a statistical sensitivity of 94% and a specificity of 93% (sensitivity: number of true positives divided by the sum of true positives and false negatives; specificity: number of true negatives divided by the sum of true negatives and false positives) and even slightly better values for the HIV positive individuals alone (Fig 2F).

Bottom Line: Quantification of specific precursor → fragment transitions of approximately 2000 individual mycolic acids (MAs) resulted in high analytical sensitivity and specificity.Furthermore, we quantified MA species in lung tissue of TB-infected mice and demonstrated effective clearance of MA levels following curative rifampicin treatment.Thus, our results demonstrate for the first time the feasibility and clinical relevance of direct detection of mycobacterial lipids as biomarkers of TB infection.

View Article: PubMed Central - PubMed

Affiliation: Yong Loo Lin School of Medicine, Department of Biochemistry, National University of Singapore, Singapore. guanghou_shui@nuhs.edu.sg

Show MeSH
Related in: MedlinePlus