Comparison of QFT-Plus and QFT-GIT tests for diagnosis of M. tuberculosis infection in immunocompetent Korean subjects

Background: QFT-Plus is a recently developed next-generation QuantiFERON-TB Gold In-Tube (QFT-GIT) test. Unlike the QFT-GIT test, it includes a TB2 antigen tube with peptides that may stimulate CD8+ T cells. This study evaluated the diagnostic performance of QFT-Plus and compared it with that of QFT-GIT. Methods: QFT-Plus and QFT-GIT tests were performed in 33 patients with active tuberculosis (TB) and 57 healthy controls including subjects with latent TB infection (LTBI). Positivity and negativity of IFN-γ responses were compared between tests, and total concordance of the outcome was analyzed. Results: Positive and negative outcomes of QFT-Plus and QFT-GIT tests showed substantial agreement (91.1%, kappa=0.8). The sensitivity and the specificity of QFT-Plus (93.9% sensitivity, 92.6% specificity) were similar with those of QFT-GIT (93.9% sensitivity, 100% specificity). Of eight discordant results, five (5.56%) and three (3.3%) were positive in QFT-GIT alone and QFT-plus alone, respectively. Reactivity in the TB2 tube contributes to the difference between QFT-GIT and QFT-Plus. Median IFN-γ production in TB2 (10.0 IU/mL in TB, 3.850 IU/mL in LTBI, P=0.001) is significantly higher in the TB group than the LTBI group. The QFT-Plus did not clearly discriminate between active TB and latent TB, although it showed significantly lower IFN-γ concentrations compared with the QFT-GIT in individuals with LTBI (3.850 vs. 7.205 IU/mL). Conclusions: Similar accuracy of detecting Mycobacterium tuberculosis infection was observed for QFT-Plus and QFT-GIT tests in immunocompetent patients and healthy controls, including those with LTBI. Improved efficacy for identifying M. tuberculosis infection was not found with the QFT-Plus, but further studies in a larger population may confirm the clinical significance of positive response in the TB2 tube of QFT-Plus.ope

The Study of MHC class I Restricted CD8+ T Cell Mediated Immune Responses against Mycobacterium tuberculosis Infection: Evidence of M. tuberculosis Specific CD8+ T Cells in TB Patients and PPD+ Healthy Individuals

Background: The protective immunity against tuberculosis (TB) involves both CD4+ T cells and CD8+ T cells. In our previous study, we defined four Mycobacterium tuberculosis derived peptide epitopes specific for HLA-A*0201 restricted CD8+ T cells (ThyA30-38, RpoB127-135, 85B15-23, PstA175-83). In this study, we investigated the immune responses induced by these peptide specific CD8+ T cells in latently and chronically infected people with TB. Methods: We characterized these peptide specific CD8+ T cell population present in PBMC of both TB patients and PPD+healthy people using IFN-γelispot assay, intracellular staining and HLA-A2 dimer staining. Results: The frequency of peptide specific CD8+ T cell was in the range of 1 to 25 in 1.7×105 PBMC based on ex vivo IFN-γ elispot assay, demonstrating that these peptide specific CD8+ T cell responses are induced in both TB patients and PPD+ people. Short term cell lines (STCL) specific for these peptides proliferated in vitro and secreted IFN-γ upon antigenic stimulation in PPD+ donors. Lastly, HLA-A*0201 dimer assays indicated that PstA175-83 specific CD8+ T cell population in PPD+ healthy donors is heterogeneous since approximately 25～33% of PstA175-83 specific CD8+ T cell population in PPD+ healthy donors produced IFN-γ upon peptide stimulation. Conclusion: Our results suggest that MHC class I restricted CD8+ T cell mediated immune responses to M. tuberculosis infection are induced in both TB patients and PPD+ people; however, the CD8+ T cell population is functionally heterogeneous.ope

Detection of Mycobacterium leprae by Real-time PCR Targeting Mycobacterium leprae-Specific Repetitive Element Sequence

Mycobacterium leprae detection is difficult even with molecular biological techniques due to the low sensitivity of current methodologies. In this report, real-time PCR targeting the M. leprae-specific repetitive element (RLEP) sequence was developed as a new diagnostic tool and evaluated using clinical specimens. For this, M. leprae DNAs were extracted from skin biopsy specimens from 80 patients and analyzed by real-time PCR using TaqMan probe. Then, the detection efficiency of the real-time PCR was compared with that of standard PCR. In brief, the rate of positive detection by the standard PCR and real-time PCR was 32.50% and 66.25%, respectively. The results seemed to clearly show that the TaqMan real-time PCR developed in this study may be a useful tool for sensitive detection of M. leprae from clinical specimens.ope

Linezolid Trough Concentrations Correlate with Mitochondrial Toxicity-Related Adverse Events in the Treatment of Chronic Extensively Drug-Resistant Tuberculosis

Long-term linezolid use is limited by mitochondrial toxicity-associated adverse events (AEs). Within a prospective, randomized controlled trial of linezolid to treat chronic extensively drug-resistant tuberculosis, we serially monitored the translational competence of mitochondria isolated from peripheral blood of participants by determining the cytochrome c oxidase/citrate synthase activity ratio. We compared this ratio with AEs associated with mitochondrial dysfunction. Linezolid trough concentrations were determined for 38 participants at both 600 mg and 300 mg doses. Those on 600 mg had a significantly higher risk of AE than those on 300 mg (HR 3·10, 95% CI 1·23-7 · 86). Mean mitochondrial function levels were significantly higher in patients before starting linezolid compared to their concentrations on 300 mg (P = 0·004) or 600 mg (P 2 μg/ml developing an AE related to mitochondrial toxicity, whether on 300 mg or 600 mg. Therapeutic drug monitoring may be useful to prevent the development of mitochondrial toxicity associated with long-term linezolid use.ope

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.ope

Mutations in extensively drug-resistant Mycobacterium tuberculosis that do not code for known drug-resistance mechanisms

BACKGROUND: Highly lethal outbreaks of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are increasing. Whole-genome sequencing of KwaZulu-Natal MDR and XDR outbreak strains prevalent in human immunodeficiency virus (HIV)-infected patients by the Broad Institute identified 22 novel mutations which were unique to the XDR genome or shared only by the MDR and XDR genomes and not already known to be associated with drug resistance. METHODS: We studied the 12 novel mutations which were not located in highly-repetitive genes to identify mutations that were truly associated with drug resistance or were likely to confer a specific fitness advantage. RESULTS: None of these mutations could be found in a phylogenetically and geographically diverse set of drug-resistant and drug-susceptible Mycobacterium tuberculosis isolates, suggesting that these mutations are unique to the KZN clone. Examination of the 600-basepair region flanking each mutation revealed 26 new mutations. We searched for a convergent evolutionary signal in the new mutations for evidence that they emerged under selective pressure, consistent with increased fitness. However, all but 1 rare mutation were monophyletic, indicating that the mutations were markers of strain phylogeny rather than fitness or drug resistance. CONCLUSIONS: Our results suggest that virulent XDR tuberculosis in immunocompromised HIV-infected patients can evolve without generalizable fitness changes or other XDR-specific mutations.ope

Association of antigen-stimulated release of tumor necrosis factor-alpha in whole blood with response to chemotherapy in patients with pulmonary multidrug-resistant tuberculosis

BACKGROUND: We have previously reported that TNF-α levels correlate to total mycobacterial burden in tuberculosis (TB) patients. OBJECTIVE: To characterize the dynamics of cytokine responses in TB patients during chemotherapy to identify potential surrogate markers for effective treatment. METHODS: Following induction by culture filtrate proteins in whole blood, production patterns of TNF-α, IL-10, IFN-γ and IL-12 were measured in 23 non-multidrug-resistant (MDR)-TB and 16 MDR-TB patients and in 31 healthy controls. Rates of mycobacterial clearance from the sputum were then measured and compared. RESULTS: Prior to the initiation of chemotherapy, TNF-α and IL-10 levels were significantly higher in TB patients than in healthy controls while IFN-γ and IL-12 levels were similar. During chemotherapy, the levels of all 4 cytokines increased. We evaluated these responses separately in patients that did and did not clear their sputum culture at 2 and 6 months. At 2 months, decreases in both IFN-γ and IL-12 correlated strongly with a successful early response, while after 6 months of therapy, when half (7/14) of MDR-TB patients were still sputum culture positive, downregulation of TNF-α was uniquely correlated with sputum conversion between the groups. CONCLUSION: Our findings suggest the possibility that the regulation of TNF-α production in whole blood may be a more specific indicator of sputum conversion at 6 months than IFN-γ, IL-12 or IL-10 in MDR-TB patients.ope

Species identification of mycobacteria by PCR-restriction fragment length polymorphism of the rpoB gene

PCR-restriction fragment length polymorphism analysis (PRA) using the novel region of the rpoB gene was developed for rapid and precise identification of mycobacteria to the species level. A total of 50 mycobacterial reference strains and 3 related bacterial strains were used to amplify the 360-bp region of rpoB, and the amplified DNAs were subsequently digested with restriction enzymes such as MspI and HaeIII. The results from this study clearly show that most of the mycobacterial species were easily differentiated at the species level by this PRA method. In addition, species with several subtypes, such as Mycobacterium gordonae, M. kansasii, M. celatum, and M. fortuitum, were also differentiated by this PRA method. Subsequently, an algorithm was constructed based on the results, and a blinded test was carried out with more than 260 clinical isolates that had been identified on the basis of conventional tests. Comparison of these two sets of results clearly indicates that this new PRA method based on the rpoB gene is more simple, more rapid, and more accurate than conventional procedures for differentiating mycobacterial species.ope

Comparison of two rapid tests for anti-phenolic glycolipid-I serology in Brazil and Nepal

The diagnosis of leprosy continues to be based on clinical symptoms and early diagnosis and treatment are critical to preventing disability and transmission. Sensitive and specific laboratory tests are not available for diagnosing leprosy. Despite the limited applicability of anti-phenolic glycolipid-I (PGL-I) serology for diagnosis, it has been suggested as an additional tool to classify leprosy patients (LPs) for treatment purposes. Two formats of rapid tests to detect anti-PGL-I antibodies [ML immunochromatography assay (ICA) and ML Flow] were compared in different groups, multibacillary patients, paucibacillary patients, household contacts and healthy controls in Brazil and Nepal. High ML Flow intra-test concordance was observed and low to moderate agreement between the results of ML ICA and ML Flow tests on the serum of LPs was observed. LPs were "seroclassified" according to the results of these tests and the seroclassification was compared to other currently used classification systems: the World Health Organization operational classification, the bacilloscopic index and the Ridley-Jopling classification. When analysing the usefulness of these tests in the operational classification of PB and MB leprosy for treatment and follow-up purposes, the ML Flow test was the best point-of-care test for subjects in Nepal and despite the need for sample dilution, the ML ICA test yielded better performance among Brazilian subjects. Our results identified possible ways to improve the performance of both tests.ope

Extensively drug-resistant tuberculosis in South Korea: risk factors and treatment outcomes among patients at a tertiary referral hospital.

BACKGROUND: Extensively drug-resistant (XDR) tuberculosis (TB) is a major public health threat in South Korea. METHODS: We analyzed baseline epidemiological data for 250 patients enrolled in an ongoing prospective observational study of TB at a large tertiary referral hospital in South Korea. RESULTS: Twenty-six subjects with XDR TB were identified; all were patients who had previously received TB therapy. Cumulative previous treatment duration (range, 18-34 months; odds ratio [OR], 5.6; 95% confidence interval [CI], 1.0-59), number of previously received second-line anti-TB drugs (OR, 1.3; 95% CI, 1.1-1.5), and female sex (OR, 3.2; 95% CI, 1.1-8.3) were significantly associated with XDR TB in crude analyses. After controlling for other factors in a multivariable model, cumulative previous treatment duration remained significantly associated with XDR TB (OR, 5.8; 95% CI, 1.0-61). Subjects with XDR TB were more likely to produce culture-positive sputum at 6 months, compared with patients with non-multidrug resistant TB (risk ratio, 13; 95% CI, 5.1-53). Kanamycin resistance was found to be predictive of 6-month culture positivity after adjustment for ofloxacin and streptomycin resistance (risk ratio, 3.9; 95% CI, 1.9-11). CONCLUSIONS: XDR TB was found to be associated with the cumulative duration of previous treatment with second-line TB drugs among subjects in a tertiary care TB hospital. Patients with XDR TB were more likely to not respond to therapy, and successful conversion of sputum culture results to negative was correlated with initial susceptibility to both fluoroquinolones and kanamycin but not to streptomycin.ope