Rapid method for Mycobacterium tuberculosis identification using electrospray ionization tandem mass spectrometry analysis of mycolic acids

Mycolic acids (MAs), which play a crucial role in the architecture of mycobacterial cell walls, were analyzed using electrospray ionization tandem mass spectrometry. A targeted analysis based on the 10 most abundant and characteristic multiple reaction monitoring pairs was used to profile the crude fatty acid mixtures from Mtb and several nontuberculous mycobacterial strains. Comparative analysis yielded unique profiles for MAs, enabling the reliable identification of mycobacterial species. In a case-control study of tuberculosis (TB) and non-TB Polish patients, we demonstrated the potential diagnostic utility of our approach for the rapid diagnosis of active TB with sensitivity and specificity surpassing those of existing methods. This robust method allows the identification of TB-positive patients after 2 h of sample preparation in the case of direct sputum analysis or 10 days of culturing, both of which are followed by 1 min of liquid chromatography– tandem mass spectrometry analysis

Interferon-gamma assay in combination with tuberculin skin test are insufficient for the diagnosis of culture-negative pulmonary tuberculosis.

OBJECTIVE:Early diagnosis of infectious cases and treatment of tuberculosis (TB) are important strategies for reducing the incidence of this disease. Unfortunately, traditional TB diagnostic methods are time-consuming and often unreliable. This study compared the accuracy and reliability of the tuberculin skin test (TST) and interferon (IFN)-γ-based assay (IGRA) for the diagnosis of active pulmonary TB Polish cases that could or could not be confirmed by M. tuberculosis (M.tb) culture. METHODS:In total, 126 adult patients with clinically active TB or non-mycobacterial, community-acquired lung diseases (NMLD) hospitalised at the Regional Specialised Hospital of Tuberculosis, Lung Diseases and Rehabilitation in Tuszyn, Poland were enrolled in the present study. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and analytic accuracy (Acc) of TST and IGRA testing for the diagnosis of culture-positive and culture-negative TB patients were calculated. The quantities of IFN-γ produced in the response to M.tb specific antigens (TB Ag - Nil) in the cultures of blood from patients with active TB and NMLD patients were also analysed. RESULTS:The IGRA sensitivity in culture-positive and culture-negative TB patients was similar, measuring 65.1% and 55.6%, respectively. The sensitivity of TST did not differ from the parameters designated for IGRA, measuring 55.8% in culture-positive and 64.9% in culture-negative TB. The sensitivity of TST and IGRA was age-dependent and decreased significantly with the age of the patients. No differences in the frequency or intensity of M.tb-stimulated IFN-γ production, as assessed by IGRA testing between culture-positive and culture-negative TB were noticed. Significantly lower concentrations of IFN-γ were observed in patients with advanced TB forms compared with those with mild or moderate TB pathologies. CONCLUSIONS:Our results do not show that a combination of IGRA and TST might be a step forward in the diagnosis of culture-negative TB cases. However, M. tuberculosis-stimulated IFN-γ levels might help to assess the extent of pulmonary TB lesions

Monocyte Signal Transduction Receptors in Active and Latent Tuberculosis

The mechanisms that promote either resistance or susceptibility to TB disease remain insufficiently understood. Our aim was to compare the expression of cell signaling transduction receptors, CD14, TLR2, CD206, and β2 integrin LFA-1 on monocytes from patients with active TB or nonmycobacterial lung disease and healthy individuals with M.tb latency and uninfected controls to explain the background of the differences between clinical and subclinical forms of M.tb infection. A simultaneous increase in the expression of the membrane bound mCD14 receptor and LFA-1 integrin in patients with active TB may be considered a prodrome of breaking immune control by M.tb bacilli in subjects with the latent TB and absence of clinical symptoms

Sensitivity, specificity, positive predictive value, negative predictive value and analytic accuracy of IGRA in culture-positive or culture-negative TB patients.

<p>IGRA, interferon-gamma release assay; PPV, <i>positive predicted value</i>; NPV, <i>negative predicted value</i>; Acc, <i>analytic accuracy</i>; CI, <i>confidence interval</i>.</p><p>Sensitivity, specificity, positive predictive value, negative predictive value and analytic accuracy of IGRA in culture-positive or culture-negative TB patients.</p

TST and IGRA results in TB and NMLD patient groups.

<p>*TST induration cut-off 10 mm.</p>a)<p>Significantly different NMLD patients from culture-positive (p = 0.008) and culture-negative (p = 0.009) TB patients.</p>b)<p>Significantly different NMLD patients from culture-positive (p = 0.00001) and culture-negative TB patients (p = 0.00001).</p><p>TST and IGRA results in TB and NMLD patient groups.</p

Agreement between the TST and IGRA results.

<p>Agreement between the TST and IGRA results.</p

The correlation between IFN-γ levels produced in response to the <i>M.tb</i>-specific antigens and TST size in TB patients' group.

<p>The correlation between IFN-γ levels produced in response to the <i>M.tb</i>-specific antigens and TST size in TB patients' group.</p

Sensitivity, specificity, positive predictive value, negative predictive value and analytic accuracy of TST in culture-positive or culture-negative TB patients.

<p>*TST induration cut-off 10 mm.</p><p>TST, tuberculin skin test; PPV, <i>positive predicted value</i>; NPV, <i>negative predicted value</i>; Acc, <i>analytic accuracy</i>; CI, <i>confidence interval</i>.</p><p>Sensitivity, specificity, positive predictive value, negative predictive value and analytic accuracy of TST in culture-positive or culture-negative TB patients.</p

The production of IFN-γ in response to <i>M.tb</i> antigens in patients with mild, moderate or advanced TB form.

<p>* significance value between the intensity of IFN-γ responses in patients with advanced TB and mild or moderate TB form.</p><p>The production of IFN-γ in response to <i>M.tb</i> antigens in patients with mild, moderate or advanced TB form.</p

The mean quantity of IFN-γ produced in response to the <i>M.tb</i>-specific antigens used in IGRA.

<p>A.B. The cut-off value for a positive response was set at 0.163 IU/ml by receiver operator characteristic (ROC) curve analysis. C. Each dot represents individual response of one participant in the study and horizontal lines represent the mean values. The geometric mean ± SD for culture-positive TB was 2.78±3.50 IU/ml (n = 32), for culture-negative TB 4.24±3.86 IU/ml (n = 22) and for NMLD 3.89±5.19 IU/ml (n = 11).</p