Phosphoenolpyruvate depletion mediates both growth arrest and drug tolerance of Mycobacterium tuberculosis in hypoxia.

Mycobacterium tuberculosis (Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic effects and can mutate to become drug resistant (DR), our conventional tuberculosis (TB) treatment is not effective. Thus, a novel strategy to kill NR Mtb is required. Accumulating evidence has shown that repetitive exposure to sublethal doses of antibiotics enhances the level of drug tolerance, implying that NR Mtb is formed by adaptive metabolic remodeling. As such, metabolic modulation strategies to block the metabolic remodeling needed to form NR Mtb have emerged as new therapeutic options. Here, we modeled in vitro NR Mtb using hypoxia, applied isotope metabolomics, and revealed that phosphoenolpyruvate (PEP) is nearly completely depleted in NR Mtb. This near loss of PEP reduces PEP-carbon flux toward multiple pathways essential for replication and drug sensitivity. Inversely, supplementing with PEP restored the carbon flux and the activities of the foregoing pathways, resulting in growth and heightened drug susceptibility of NR Mtb, which ultimately prevented the development of DR. Taken together, PEP depletion in NR Mtb is associated with the acquisition of drug tolerance and subsequent emergence of DR, demonstrating that PEP treatment is a possible metabolic modulation strategy to resensitize NR Mtb to conventional TB treatment and prevent the emergence of DR.

Identification of Nontuberculous Mycobacteria in Patients with Pulmonary Diseases in Gyeongnam, Korea, Using Multiplex PCR and Multigene Sequence-Based Analysis.

BACKGROUND: Nontuberculous mycobacteria (NTM) are widely present in environments, such as soil and water, and have recently been recognized as important pathogenic bacteria. The incidence of NTM-related infections is steadily increasing. As the diagnosis and treatment of NTM infection should be distinguished from tuberculosis, and the treatment should be specific to the species of NTM acquired, accurate species identification is required. METHODS: In this study, two-step multiplex PCR (mPCR) and multigene sequence-based analysis were used to accurately identify NTM species in 320 clinical isolates from Gyeongsang National University Hospital (GNUH). In particular, major mycobacterial strains with a high isolation frequency as well as coinfections with multiple species were diagnosed through two-step mPCR. Multigene sequencing was performed to accurately identify other NTM species not detected by mPCR. Variable regions of the genes 16S rRNA, rpoB, hsp65, and 16S-23S rRNA internal transcribed spacer were included in the analysis. RESULTS: Two-step mPCR identified 234 (73.1%) cases of M. intracellulare, 26 (8.1%) cases of M. avium subsp. avium, and 13 (4.1%) cases of M. avium subsp. hominissuis infection. Additionally, 9 (2.8%) M. fortuitum, 9 (2.8%) M. massiliense, 2 (0.6%) M. abscessus, and 4 (1.2%) M. kansasii isolates were identified. Coinfection was identified in 7 (2.2%) samples. The sixteen samples not classified by two-step mPCR included 6 (1.9%) cases of M. chimaera, 4 (1.3%) M. gordonae, 1 (0.3%) M. colombiense, 1 (0.3%) M. mageritense, and 1 (0.3%) M. persicum identified by sequence analysis. CONCLUSIONS: The results of this study suggest a strategy for rapid detection and accurate identification of species using two-step mPCR and multigene sequence-based analysis. To the best of our knowledge, this study is the first to report the identification of NTM species isolated from patients in Gyeongnam/Korea.

Tuberculosis drugs' distribution and emergence of resistance in patient's lung lesions: A mechanistic model and tool for regimen and dose optimization.

BACKGROUND: The sites of mycobacterial infection in the lungs of tuberculosis (TB) patients have complex structures and poor vascularization, which obstructs drug distribution to these hard-to-reach and hard-to-treat disease sites, further leading to suboptimal drug concentrations, resulting in compromised TB treatment response and resistance development. Quantifying lesion-specific drug uptake and pharmacokinetics (PKs) in TB patients is necessary to optimize treatment regimens at all infection sites, to identify patients at risk, to improve existing regimens, and to advance development of novel regimens. Using drug-level data in plasma and from 9 distinct pulmonary lesion types (vascular, avascular, and mixed) obtained from 15 hard-to-treat TB patients who failed TB treatments and therefore underwent lung resection surgery, we quantified the distribution and the penetration of 7 major TB drugs at these sites, and we provide novel tools for treatment optimization. METHODS AND FINDINGS: A total of 329 plasma- and 1,362 tissue-specific drug concentrations from 9 distinct lung lesion types were obtained according to optimal PK sampling schema from 15 patients (10 men, 5 women, aged 23 to 58) undergoing lung resection surgery (clinical study NCT00816426 performed in South Korea between 9 June 2010 and 24 June 2014). Seven major TB drugs (rifampin [RIF], isoniazid [INH], linezolid [LZD], moxifloxacin [MFX], clofazimine [CFZ], pyrazinamide [PZA], and kanamycin [KAN]) were quantified. We developed and evaluated a site-of-action mechanistic PK model using nonlinear mixed effects methodology. We quantified population- and patient-specific lesion/plasma ratios (RPLs), dynamics, and variability of drug uptake into each lesion for each drug. CFZ and MFX had higher drug exposures in lesions compared to plasma (median RPL 2.37, range across lesions 1.26-22.03); RIF, PZA, and LZD showed moderate yet suboptimal lesion penetration (median RPL 0.61, range 0.21-2.4), while INH and KAN showed poor tissue penetration (median RPL 0.4, range 0.03-0.73). Stochastic PK/pharmacodynamic (PD) simulations were carried out to evaluate current regimen combinations and dosing guidelines in distinct patient strata. Patients receiving standard doses of RIF and INH, who are of the lower range of exposure distribution, spent substantial periods (>12 h/d) below effective concentrations in hard-to-treat lesions, such as caseous lesions and cavities. Standard doses of INH (300 mg) and KAN (1,000 mg) did not reach therapeutic thresholds in most lesions for a majority of the population. Drugs and doses that did reach target exposure in most subjects include 400 mg MFX and 100 mg CFZ. Patients with cavitary lesions, irrespective of drug choice, have an increased likelihood of subtherapeutic concentrations, leading to a higher risk of resistance acquisition while on treatment. A limitation of this study was the small sample size of 15 patients, performed in a unique study population of TB patients who failed treatment and underwent lung resection surgery. These results still need further exploration and validation in larger and more diverse cohorts. CONCLUSIONS: Our results suggest that the ability to reach and maintain therapeutic concentrations is both lesion and drug specific, indicating that stratifying patients based on disease extent, lesion types, and individual drug-susceptibility profiles may eventually be useful for guiding the selection of patient-tailored drug regimens and may lead to improved TB treatment outcomes. We provide a web-based tool to further explore this model and results at http://saviclab.org/tb-lesion/.

Essential engagement of Toll-like receptor 2 in initiation of early protective Th1 response against rough variants of Mycobacterium abscessus.

Although Mycobacterium abscessus (M. abscessus) is becoming more prevalent in patients without overt immunodeficiency, little is known about the factors that contribute to disease susceptibility. This study was undertaken to investigate how Toll-like receptor 2 (TLR2) functionally contributes to the generation of protective immunity against M. abscessus in a morphotype-specific manner. We found that Tlr2-/- mice were extremely susceptible to an intravenous (i.v.) model of infection by M. abscessus rough variants, displaying uncontrolled infection in the lungs and a significantly lower survival rate than with wild-type (WT) mice. This uncontrolled infection resulted from failures in the following processes: (i) production of the crucial cytokines gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 12p70 (IL-12p70); (ii) early infiltration of neutrophils, monocytes, and dendritic cells (DCs) in the lungs of Tlr2-/- mice; (iii) rapid influx of CD4+ and CD8+ T cells; and (iv) the expansion of memory/effector T cells. Notably, systemic administration of M. abscessus culture filtrate-treated syngeneic DCs from WT mice greatly strengthened immune priming in vivo, resulting in a dramatic reduction in bacterial growth and improved long-term survival in Tlr2-/- mice, with a recovery of protective immunity. Our findings demonstrate that TLR2 is an essential contributor to instructive and effector immunity during M. abscessus infection in a morphotype-specific manner.

Efficacy and safety of metronidazole for pulmonary multidrug-resistant tuberculosis.

Pulmonary lesions from active tuberculosis patients are thought to contain persistent, nonreplicating bacilli that arise from hypoxic stress. Metronidazole, approved for anaerobic infections, has antituberculosis activity against anoxic bacilli in vitro and in some animal models and may target persistent, nonreplicating bacilli. In this double-blind, placebo-controlled trial, pulmonary multidrug-resistant tuberculosis subjects were randomly assigned to receive metronidazole (500 mg thrice daily) or placebo for 8 weeks in addition to an individualized background regimen. Outcomes were measured radiologically (change on high-resolution computed tomography [HRCT]), microbiologically (time to sputum smear and culture conversion), and clinically (status 6 months after stopping therapy). Enrollment was stopped early due to excessive peripheral neuropathies in the metronidazole arm. Among 35 randomized subjects, 31 (15 metronidazole, 16 placebo) were included in the modified intent-to-treat analysis. There were no significant differences by arm in improvement of HRCT lesions from baseline to 2 or 6 months. More subjects in the metronidazole arm converted their sputum smear (P = 0.04) and liquid culture (P = 0.04) to negative at 1 month, but these differences were lost by 2 months. Overall, 81% showed clinical success 6 months after stopping therapy, with no differences by arm. However, 8/16 (50%) of subjects in the metronidazole group and 2/17 (12%) of those in the placebo group developed peripheral neuropathy. Subjects who received metronidazole were 4.3-fold (95% confidence interval [CI], 1.1 to 17.1) more likely to develop peripheral neuropathies than subjects who received placebo. Metronidazole may have increased early sputum smear and culture conversion but was too neurotoxic to use over the longer term. Newer nitroimidazoles with both aerobic and anaerobic activity, now in clinical trials, may increase the sterilizing potency of future treatment regimens.

Natural killer cells are recruited during pulmonary tuberculosis and their ex vivo responses to mycobacteria vary between healthy human donors in association with KIR haplotype.

Humans vary widely in their susceptibility to tuberculosis. While only a minority will progress to disease, the majority of healthy individuals exposed to Mycobacterium tuberculosis mount an immune response that can clear or contain the infection in a quiescent form. Using immunofluorescence on human clinical samples, we identified natural killer (NK) cells infiltrating granulomatous pulmonary lesions during active disease. In order to compare the NK cell ability to react to free mycobacteria in the context of tuberculosis infection and Mycobacterium bovis BCG vaccination, NK cells were isolated from the peripheral blood of anonymous healthy human donors, and stimulated with M. tuberculosis H37Rv or M. bovis BCG. Extracellular M. tuberculosis and M. bovis BCG could equally trigger the release of IFNγ and TNFα from NK cells in the presence of IL-2. However, we found that this response varied 1000-fold between individuals (n = 52), with differences in KIR haplotype providing a significant criterion to distinguish between low and high responders. Our findings suggest that variations at the KIR locus and therefore of the NK cell repertoire may affect cytokine production in response to mycobacteria and we propose that this innate variability couldsustain different levels of susceptibility to M. tuberculosis infection.

A Rapid Assessing Method of Drug Susceptibility Using Flow Cytometry for Mycobacterium tuberculosis Isolates Resistant to Isoniazid, Rifampin, and Ethambutol.

BACKGROUND: The current conventional drug susceptibility test (DST) for Mycobacterium tuberculosis (Mtb) takes several weeks of incubation to obtain results. As a rapid method, molecular DST requires only a few days to get the results but does not fully cover the phenotypic resistance. A new rapid method based on the ability of viable Mtb bacilli to hydrolyze fluorescein diacetate to free fluorescein with detection of fluorescent mycobacteria by flow cytometric analysis, was recently developed. METHODS: To evaluate this cytometric method, we tested 39 clinical isolates which were susceptible or resistant to isoniazid (INH) or rifampin (RIF), or ethambutol (EMB) by phenotypic or molecular DST methods and compared the results. RESULTS: The susceptibility was determined by measuring the viability rate of Mtb and all the isolates which were tested with INH, RIF, and EMB showed susceptibility results concordant with those by the phenotypic solid and liquid media methods. The isolates having no mutations in the molecular DST but resistance in the conventional phenotypic DST were also resistant in this cytometric method. These results suggest that the flow cytometric DST method is faster than conventional agar phenotypic DST and may complement the results of molecular DST. CONCLUSION: In conclusion, the cytometric method could provide quick and more accurate information that would help clinicians to choose more effective drugs.

Effect of teriparatide on drug treatment of tuberculous spondylitis: an experimental study.

Tuberculous spondylitis often develops catastrophic bone destruction with uncontrolled inflammation. Because anti-tuberculous drugs do not have a role in bone formation, a combination drug therapy with a bone anabolic agent could help in fracture prevention and promote bone reconstruction. This study aimed to investigate the influence of teriparatide on the effect of anti-tuberculous drugs in tuberculous spondylitis treatment. We used the virulent Mycobacterium tuberculosis (Mtb) H37Rv strain. First, we investigated the interaction between teriparatide and anti-tuberculosis drugs (isoniazid and rifampin) by measuring the minimal inhibitory concentration (MIC) against H37Rv. Second, we evaluated the therapeutic effect of anti-tuberculosis drugs and teriparatide on our previously developed in vitro tuberculous spondylitis model of an Mtb-infected MG-63 osteoblastic cell line using acid-fast bacilli staining and colony-forming unit counts. Selected chemokines (interleukin [IL]-8, interferon γ-induced protein 10 kDa [IP-10], monocyte chemoattractant protein [MCP]-1, and regulated upon activation, normal T cell expressed and presumably secreted [RANTES]) and osteoblast proliferation (alkaline phosphatase [ALP] and alizarin red S [ARS] staining) were measured. Teriparatide did not affect the MIC of isoniazid and rifampin. In the Mtb-infected MG-63 spondylitis model, isoniazid and rifampin treatment significantly reduced Mtb growth, and cotreatment with teriparatide did not change the anti-tuberculosis effect of isoniazid (INH) and rifampin (RFP). IP-10 and RANTES levels were significantly increased by Mtb infection, whereas teriparatide did not affect all chemokine levels as inflammatory markers. ALP and ARS staining indicated that teriparatide promoted osteoblastic function even with Mtb infection. Cotreatment with teriparatide and the anti-tuberculosis drugs activated bone formation (ALP-positive area increased by 705%, P = 0.0031). Teriparatide was effective against Mtb-infected MG63 cells without the anti-tuberculosis drugs (ARS-positive area increased by 326%, P = 0.0037). Teriparatide had no effect on the efficacy of anti-tuberculosis drugs and no adverse effect on the activity of Mtb infection in osteoblasts. Furthermore, regulation of representative osteoblastic inflammatory chemokines was not changed by teriparatide treatment. In the in vitro Mtb-infected MG-63 cell model of tuberculous spondylitis, cotreatment with the anti-tuberculosis drugs and teriparatide increased osteoblastic function.

Genetic diversity of Mycobacterium tuberculosis isolates from a tertiary care tuberculosis hospital in South Korea.

Tuberculosis (TB) remains an immense public health problem in the Republic of Korea despite a more than fivefold decrease in the prevalence of the disease over the last 3 decades. The rise in drug-resistant TB has compounded the situation. We analyzed 208 clinical isolates of M. tuberculosis from the National Masan Tuberculosis Hospital by spoligotyping, IS6110 restriction fragment length polymorphism (RFLP), and 24-locus-based mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing to assess the diversity and transmission dynamics of the tubercle bacilli in the Republic of Korea. The majority of the isolates (97.1%) belonged to the Beijing genotype. Cluster analysis by MIRU-VNTR yielded a low clustering rate of 22.3%, with most of the clusters comprising isolates with diverse drug resistance patterns. The discriminatory capacity of the typing methods was high for RFLP and MIRU-VNTR (allelic diversity [h] = 0.99) but low for spoligotyping (h = 0.31). Although analysis of 19 MIRU-VNTR loci was needed to achieve maximum discrimination, an informative set of 8 loci (960, 1955, 2163b, 2165, 2996, 3192, 4052, and 4348) (h = 0.98) that was able to differentiate most of the closely related strains was identified. These findings suggest that 24-locus-based MIRU-VNTR typing is a likely suitable alternative to RFLP to differentiate clinical isolates in this setting, which is dominated by M. tuberculosis Beijing strains. Within the study limits, our results also suggest that the problem of drug-resistant TB in the Republic of Korea may be largely due to acquired resistance as opposed to transmission.

Polymorphisms associated with resistance and cross-resistance to aminoglycosides and capreomycin in Mycobacterium tuberculosis isolates from South Korean Patients with drug-resistant tuberculosis.

The aminoglycosides streptomycin, amikacin, and kanamycin and the cyclic polypeptide capreomycin are all widely used in second-line therapy for patients who develop multidrug-resistant tuberculosis. We have characterized a set of 106 clinical isolates of Mycobacterium tuberculosis using phenotypic drug susceptibility testing (DST) to determine the extent of resistance to each agent and cross-resistance between agents. These results were compared with polymorphisms in the DNA sequences of ribosome-associated genes previously implicated in resistance and with the clinical outcomes of subjects from whom these isolates were obtained. Thirty-six (34%) of these isolates displayed resistance to one or more of these agents, and the majority of these (20 of 36) showed cross-resistance to one or more agents. Most (33 of 36) of the resistant isolates showed polymorphisms in the 16S ribosome components RpsL and rrs. Three resistant strains (3 of 36) were identified that had no known polymorphisms in ribosomal constituents. For kanamycin and streptomycin, molecular DST significantly outperformed phenotypic DST using the absolute concentration method for predicting 4-month sputum conversion (likelihood ratios of 4.0 and 2.0, respectively) and was equivalent to phenotypic DST using the National Committee for Clinical Laboratory Standards (NCCLS)-approved agar proportion method for estimating MIC (likelihood ratio, 4.0). These results offer insight into mechanisms of resistance and cross-resistance among these agents and suggest that the development of rapid molecular tests to distinguish polymorphisms would significantly enhance clinical utility of this important class of second-line antituberculosis drugs.

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.

Plant-Produced N-glycosylated Ag85A Exhibits Enhanced Vaccine Efficacy Against Mycobacterium tuberculosis HN878 Through Balanced Multifunctional Th1 T Cell Immunity.

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide and is caused by Mycobacterium tuberculosis (Mtb). An effective vaccine to prevent TB is considered the most cost-effective measure for controlling this disease. Many different vaccine antigen (Ag) candidates, including well-known and newly identified Ags, have been evaluated in clinical and preclinical studies. In this study, we took advantage of a plant system of protein expression using Nicotiana benthamiana to produce N-glycosylated antigen 85A (G-Ag85A), which is one of the most well-characterized vaccine Ag candidates in the field of TB vaccines, and compared its immunogenicity and vaccine efficacy with those of nonglycosylated Ag85A (NG-Ag85A) produced with an Escherichia coli system. Notably, G-Ag85A induced a more robust IFN-γ response than NG-Ag85A, which indicated that G-Ag85A is well recognized by the host immune system during Mtb infection. We subsequently compared the vaccine potential of G-Ag85A and NG-Ag85A by evaluating their immunological features and substantial protection efficacies. Interestingly, G-Ag85A yielded moderately enhanced long-term protective efficacy, as measured in terms of bacterial burden and lung inflammation. Strikingly, G-Ag85A-immunized mice showed a more balanced proportion of multifunctional Th1-biased immune responses with sustained IFN-γ response than did NG-Ag85A-immunized mice. Collectively, plant-derived G-Ag85A could induce protective and balanced Th1 responses and confer long-term protection against a hypervirulent Mtb Beijing strain infection, which indicated that plant-produced G-Ag85A might provide an excellent example for the production of an Mtb subunit vaccine Ag and could be an effective platform for the development of anti-TB vaccines.

Transient drug-tolerance and permanent drug-resistance rely on the trehalose-catalytic shift in Mycobacterium tuberculosis.

Stochastic formation of Mycobacterium tuberculosis (Mtb) persisters achieves a high level of antibiotic-tolerance and serves as a source of multidrug-resistant (MDR) mutations. As conventional treatment is not effective against infections by persisters and MDR-Mtb, novel therapeutics are needed. Several approaches were proposed to kill persisters by altering their metabolism, obviating the need to target active processes. Here, we adapted a biofilm culture to model Mtb persister-like bacilli (PLB) and demonstrated that PLB underwent trehalose metabolism remodeling. PLB use trehalose as an internal carbon to biosynthesize central carbon metabolism intermediates instead of cell surface glycolipids, thus maintaining levels of ATP and antioxidants. Similar changes were identified in Mtb following antibiotic-treatment, and MDR-Mtb as mechanisms to circumvent antibiotic effects. This suggests that trehalose metabolism is associated not only with transient drug-tolerance but also permanent drug-resistance, and serves as a source of adjunctive therapeutic options, potentiating antibiotic efficacy by interfering with adaptive strategies.

Common Variants in the Glycerol Kinase Gene Reduce Tuberculosis Drug Efficacy.

Despite the administration of multiple drugs that are highly effective in vitro, tuberculosis (TB) treatment requires prolonged drug administration and is confounded by the emergence of drug-resistant strains. To understand the mechanisms that limit antibiotic efficacy, we performed a comprehensive genetic study to identify Mycobacterium tuberculosis genes that alter the rate of bacterial clearance in drug-treated mice. Several functionally distinct bacterial genes were found to alter bacterial clearance, and prominent among these was the glpK gene that encodes the glycerol-3-kinase enzyme that is necessary for glycerol catabolism. Growth on glycerol generally increased the sensitivity of M. tuberculosis to antibiotics in vitro, and glpK-deficient bacteria persisted during antibiotic treatment in vivo, particularly during exposure to pyrazinamide-containing regimens. Frameshift mutations in a hypervariable homopolymeric region of the glpK gene were found to be a specific marker of multidrug resistance in clinical M. tuberculosis isolates, and these loss-of-function alleles were also enriched in extensively drug-resistant clones. These data indicate that frequently observed variation in the glpK coding sequence produces a drug-tolerant phenotype that can reduce antibiotic efficacy and may contribute to the evolution of resistance.IMPORTANCE TB control is limited in part by the length of antibiotic treatment needed to prevent recurrent disease. To probe mechanisms underlying survival under antibiotic pressure, we performed a genetic screen for M. tuberculosis mutants with altered susceptibility to treatment using the mouse model of TB. We identified multiple genes involved in a range of functions which alter sensitivity to antibiotics. In particular, we found glycerol catabolism mutants were less susceptible to treatment and that common variation in a homopolymeric region in the glpK gene was associated with drug resistance in clinical isolates. These studies indicate that reversible high-frequency variation in carbon metabolic pathways can produce phenotypically drug-tolerant clones and have a role in the development of resistance.

Evaluation of the diagnostic utility of a whole-blood interferon-gamma assay for determining the risk of exposure to Mycobacterium tuberculosis in Bacille Calmette-Guerin (BCG)-vaccinated individuals.

We evaluated the utility of the "QuantiFERON-TB Gold In-Tube" (QuantiFERON) test that uses tuberculosis (TB)-specific antigens for the diagnosis of latent infection in such individuals. We also examined the correlation between the interferon (IFN)-gamma response to these antigens and the exposure risk to TB by evaluating antigen-specific IFN-gamma release in comparison with IFN-gamma release in response to purified protein derivative (PPD) in 3 groups: medical students, nurses in a TB hospital, and TB patients. All nurses and TB patients responded to PPD, whereas 52% (P < 0.0001) and 79.2% (P = 0.04) responded to QuantiFERON, respectively. In the medical students, only 10.4% responded to QuantiFERON, whereas 85.2% were positive to PPD (P < 0.0001). There was also a significant correlation between the levels of IFN-gamma production and the duration of employment in the group of nurses at the TB hospital, suggesting ongoing exposure in this high-risk group. Thus, these results demonstrate that Mycobacterium tuberculosis-specific IFN-gamma release assay accurately discriminates low- and high-risk healthy subjects and might therefore be a useful diagnostic tool for the diagnosis of latent infection in Bacille Calmette-Guerin (BCG)-vaccinated individuals.

Tumor necrosis factor-alpha and interleukin-10 in whole blood is associated with disease progression in pulmonary multidrug-resistant tuberculosis patients.

BACKGROUND: Cytokine production profiles may reflect the clinical pictures of patients with tuberculosis (TB). OBJECTIVE: We examined the relationship between cytokine levels and clinical parameters indicating the state of disease in active pulmonary TB patients. METHODS: We measured interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 levels in whole blood after stimulation with culture filtrate protein of Mycobacterium tuberculosis in 33 multi-drug resistant (MDR)-TB and 51 non-MDR-TB patients. RESULTS: No significant difference was found in IFN-gamma production between non-MDR-TB and MDR-TB patients, but there was a marked reduction in TNF-alpha production in MDR-TB patients accompanied by a moderate increase in IL-10 levels. In contrast, the presence of cavity was associated with a significant increase in IFN-gamma, whereas no difference in TNF-alpha between the cavity and non-cavity group was observed. Those who have TB lesions in the left lung showed lower levels of IFN-gamma and TNF-alpha and higher IL-10 levels than the patients with lesions on the right side. IFN-gamma levels were significantly increased in those with moderate or advanced lesions compared with patients with mild lesions. TNF-alpha and IL-10 levels did not change with disease severity. The number of M. tuberculosis bacilli in sputum was closely associated with TNF-alpha levels. The patient group with high value (+++) of sputum culture acid-fast bacilli produced significantly reduced levels of TNF-alpha compared with medium (++) and low (+) values. CONCLUSION: These findings suggest that IFN-gamma, TNF-alpha or IL-10 production patterns in whole blood are associated with disease progression in active pulmonary TB.

Comparison of immunogenicity and vaccine efficacy between heat-shock proteins, HSP70 and GrpE, in the DnaK operon of Mycobacterium tuberculosis.

Antigens (Ags) in Mycobacterium tuberculosis (Mtb) that are constitutively expressed, overexpressed during growth, essential for survival, and highly conserved may be good vaccine targets if they induce the appropriate anti-Mtb Th1 immune response. In this context, stress response-related antigens of Mtb might serve as attractive targets for vaccine development as they are rapidly expressed and are up-regulated during Mtb infection in vivo. Our group recently demonstrated that GrpE, encoded by rv0351 as a cofactor of heat-shock protein 70 (HSP70) in the DnaK operon, is a novel immune activator that interacts with DCs to generate Th1-biased memory T cells in an antigen-specific manner. In this study, GrpE was evaluated as a subunit vaccine in comparison with the well-known HSP70 against the hyper-virulent Mtb Beijing K-strain. Both HSP70- and GrpE-specific effector/memory T cells expanded to a similar extent as those stimulated with ESAT-6 in the lung and spleen of Mtb-infected mice, but GrpE only produced a similar level of IFN-γ to that produced by ESAT-6 stimulation during the late phase and the early phase of Mtb K infection, indicating that GrpE is highly-well recognised by the host immune system as a T cell antigen. Mice immunised with the GrpE subunit vaccine displayed enhanced antigen-specific IFN-γ and serum IgG2c responses along with antigen-specific effector/memory T cell expansion in the lungs. In addition, GrpE-immunisation markedly induced multifunctional Th1-type CD4+ T cells co-expressing IFN-γ, TNF-α, and IL-2 in the lungs of Mtb K-infected mice, whereas HSP70-immunisation induced mixed Th1/Th2 immune responses. GrpE-immunisation conferred a more significant protective effect than that of HSP70-immunisation in terms of bacterial reduction and improved inflammation, accompanied by the remarkable persistence of GrpE-specific multifunctional CD4+ T cells. These results suggest that GrpE is an excellent vaccine antigen component for the development of a multi-antigenic Mtb subunit vaccine by generating Th1-biased memory T cells with multifunctional capacity, and confers durable protection against the highly virulent Mtb K.

Repeated Aerosolized-Boosting with Gamma-Irradiated Mycobacterium bovis BCG Confers Improved Pulmonary Protection against the Hypervirulent Mycobacterium tuberculosis Strain HN878 in Mice.

Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only licensed vaccine, shows limited protection efficacy against pulmonary tuberculosis (TB), particularly hypervirulent Mycobacterium tuberculosis (Mtb) strains, suggesting that a logistical and practical vaccination strategy is urgently required. Boosting the BCG-induced immunity may offer a potentially advantageous strategy for advancing TB vaccine development, instead of replacing BCG completely. Despite the improved protection of the airway immunization by using live BCG, the use of live BCG as an airway boosting agent may evoke safety concerns. Here, we analyzed the protective efficacy of γ-irradiated BCG as a BCG-prime boosting agent for airway immunization against a hypervirulent clinical strain challenge with Mycobacterium tuberculosis HN878 in a mouse TB model. After the aerosol challenge with the HN878 strain, the mice vaccinated with BCG via the parenteral route exhibited only mild and transient protection, whereas BCG vaccination followed by multiple aerosolized boosting with γ-irradiated BCG efficiently maintained long-lasting control of Mtb in terms of bacterial reduction and pathological findings. Further immunological investigation revealed that this approach resulted in a significant increase in the cellular responses in terms of a robust expansion of antigen (PPD and Ag85A)-specific CD4+ T cells concomitantly producing IFN-γ, TNF-α, and IL-2, as well as a high level of IFN-γ-producing recall response via both the local and systemic immune systems upon further boosting. Collectively, aerosolized boosting of γ-irradiated BCG is able to elicit strong Th1-biased immune responses and confer enhanced protection against a hypervirulent Mycobacterium tuberculosis HN878 infection in a boosting number-dependent manner.

Virulence-Dependent Alterations in the Kinetics of Immune Cells during Pulmonary Infection by Mycobacterium tuberculosis.

A better understanding of the kinetics of accumulated immune cells that are involved in pathophysiology during Mycobacterium tuberculosis (Mtb) infection may help to facilitate the development of vaccines and immunological interventions. However, the kinetics of innate and adaptive cells that are associated with pathogenesis during Mtb infection and their relationship to Mtb virulence are not clearly understood. In this study, we used a mouse model to compare the bacterial burden, inflammation and kinetics of immune cells during aerogenic infection in the lung between laboratory-adapted strains (Mtb H37Rv and H37Ra) and Mtb K strain, a hyper-virulent W-Beijing lineage strain. The Mtb K strain multiplied more than 10- and 3.54-fold more rapidly than H37Ra and H37Rv, respectively, during the early stage of infection (at 28 days post-infection) and resulted in exacerbated lung pathology at 56 to 112 days post-infection. Similar numbers of innate immune cells had infiltrated, regardless of the strain, by 14 days post-infection. High, time-dependent frequencies of F4/80-CD11c+CD11b-Siglec-H+PDCA-1+ plasmacytoid DCs and CD11c-CD11b+Gr-1int cells were observed in the lungs of mice that were infected with the Mtb K strain. Regarding adaptive immunity, Th1 and Th17 T cells that express T-bet and RORγt, respectively, significantly increased in the lungs that were infected with the laboratory-adapted strains, and the population of CD4+CD25+Foxp3+ regulatory T cells was remarkably increased at 112 days post-infection in the lungs of mice that were infected with the K strain. Collectively, our findings indicate that the highly virulent Mtb K strain may trigger the accumulation of pDCs and Gr1intCD11b+ cells with the concomitant down-regulation of the Th1 response and the maintenance of an up-regulated Th2 response without inducing a Th17 response during chronic infection. These results will help to determine which immune system components must be considered for the development of tuberculosis (TB) vaccines and immunological interventions.

Potential role of immunodiagnosis for pulmonary tuberculosis using induced sputum cells.

PURPOSE: To evaluate the diagnostic utility and predictors for determinate results of an enzyme-linked immunospot assay using induced sputum cells (IS ELISPOT) for a rapid diagnosis of pulmonary tuberculosis (TB). MATERIALS AND METHODS: Subjects suspected of pulmonary TB who had either sputum acid fast bacilli smear-negative or not producing sputum spontaneously were prospectively enrolled. ELISPOT assay was performed using cells from induced sputum. RESULTS: A total of 43 subjects, including 25 with TB (TB group) and 18 with non-TB disease (non-TB group) were enrolled. Results of IS ELISPOT were determinate in only 17/43 (39%) subjects, but all of determinate results were consistent with the final diagnosis. Of the 43 sputum samples, 11 (26%) were inadequate to perform IS ELISPOT. Of 32 adequate sputum samples, the proportion of determinate results was significantly higher in the TB group (75%, 15/20) than in the non-TB group (17%, 2/12) (p=0.002). The status of active TB was a unique predictor but smear positivity was not a significant predictor for determinate results. In addition, sensitivity of IS ELISPOT (75%, 9/12) in smear negative TB was higher than that of TB-polymerase chain reaction (25%, 3/12). CONCLUSION: IS ELISPOT showed relatively high diagnostic value and accuracy in the TB group, independent of smear positivity. IS ELISPOT may provide additional diagnostic yield for microbiological tools in the rapid diagnosis of smear-negative TB.