Pulmonary non-tuberculous mycobacterial (NTM) infections particularly caused by Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are becoming major health problems in the U.S. New therapies or vaccines which will help prevent the disease, shorten treatment duration and/or increase treatment success rates are urgently needed. This study was conducted with the objective of testing the hypothesis that Bacillus Calmette Guerin (BCG), a vaccine used for prevention of serious forms of tuberculosis (TB) in children and adolescents in tuberculosis hyperendemic countries, induces cross-protective T cell immunity against Mycobacterium avium (MAV) and MAB. Human TB and NTM cross-protective T cells were quantified using flow cytometric assays. The ability of BCG expanded T cells to inhibit the intracellular growth of MAV and MAB was assessed in co-cultures with infected autologous macrophages. In both BCG-vaccinated and M. tuberculosis (Mtb)-infected mice, NTM cross-reactive immunity was measured using IFN-γ ELISPOT assays. Our results demonstrate the following key findings: (i) peripheral blood mononuclear cells from TB skin test-positive individuals contain MAV and MAB cross-reactive T cells, (ii) both BCG vaccination and Mtb infection of mice induce MAV and MAB cross-reactive splenic cells, (iii) BCG-expanded T cells inhibit intracellular MAV and MAB, (iv) CD4, CD8, and γδ T cells play important roles in inhibition of intracellular MAV and MAB and (v) BCG vaccination of healthy volunteers induces TB and NTM cross-reactive T cells. In conclusion, BCG-vaccination induces NTM cross-reactive immunity, and has the potential for use as a vaccine or immunotherapy to prevent and/or treat pulmonary NTM disease.

The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was standardized for direct detection of rifampin-resistant Mycobacterium tuberculosis in sputum samples. The sensitivity and specificity of the direct MTT assay matched those of the standard indirect susceptibility assay on 7H10 medium, and interpretable results were obtained for 98.5% of the samples within 2 weeks. Traditional methods of in vitro drug susceptibility testing are time consuming and laborious. Susceptibility tests on clinical isolates require 6 to 9 weeks, and tests conducted directly on smear-positive samples take about 3 weeks (International Union Against Tuberculosis and Lung Disease, The public health service national tuberculosis reference laboratory and the national laboratory network. Minimum requirements, role and operation in a low-income country, Paris, France, 1998, and P. T. Kent and G. P. Kubica, Public health mycobacteriology. A guide for the level III laboratory, Centers for Disease Control and Prevention, Atlanta, Ga., 1985). More-rapid methods are available but are very expensive for routine use under program conditions in countries with high levels of tuberculosis endemicity.

The increasing global prevalence of pulmonary nontuberculous mycobacteria (NTM) disease has called attention to challenges in NTM diagnosis and management. This study was conducted to understand management and outcomes of patients with pulmonary NTM disease at diverse centers across the United States. We conducted a 10-year (2005–2015) retrospective study at 7 Vaccine and Treatment Evaluation Units to evaluate pulmonary NTM treatment outcomes in human immunodeficiency virus–negative adults. Demographic and clinical information was abstracted through medical record review. Microbiologic and clinical cure were evaluated using previously defined criteria. Of 297 patients diagnosed with pulmonary NTM, the most frequent NTM species were Mycobacterium avium-intracellulare complex (83.2%), M. kansasii (7.7%), and M. abscessus (3.4%). Two hundred forty-five (82.5%) patients received treatment, while 45 (15.2%) were followed without treatment. Eighty-six patients had available drug susceptibility results; of these, >40% exhibited resistance to rifampin, ethambutol, or amikacin. Of the 138 patients with adequate outcome data, 78 (56.5%) experienced clinical and/or microbiologic cure. Adherence to the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) treatment guidelines was significantly more common in patients who were cured (odds ratio, 4.5, 95% confidence interval, 2.0–10.4; P < .001). Overall mortality was 15.7%. Despite ATS/IDSA Guidelines, management of pulmonary NTM disease was heterogeneous and cure rates were relatively low. Further work is required to understand which patients are suitable for monitoring without treatment and the impact of antimicrobial therapy on pulmonary NTM morbidity and mortality.

There is a growing interest in repurposing mycobacterial efflux pump inhibitors such as verapamil for tuberculosis (TB) treatment. To aid in the design of better analogs, we studied the effects of verapamil on macrophages and Mycobacterium tuberculosis (Mtb)-specific T cells. Macrophage activation was evaluated by measuring levels of nitric oxide, TNF-α, IL-1β and IFN-γ. Since verapamil is a known autophagy inducer, the roles of autophagy induction in the antimycobacterial activities of verapamil and norverapamil were studied using bone marrow derived macrophages from ATG5^flox/flox (control) and ATG5^flox/flox Lyz-Cre mice. Our results showed that despite verapamil's well-recognized effects on calcium channels and autophagy, verapamil's action on intracellular Mtb does not involve macrophage activation or autophagy induction. Next, the effects of verapamil and norverapamil on Mtb-specific T cells were assessed using flow cytometry following stimulation of peripheral blood mononuclear cells from TB-skin test-positive donors with Mtb-whole lysate for 7 days in the presence or absence of drugs. We found that verapamil and norverapamil inhibit the expansion of Mtb-specific T cells. Additionally, three new verapamil analogs were found to inhibit intracellular BCG, and one of the three analogs (KSV21) inhibited intracellular Mtb replication at concentrations which did not inhibit Mtb-specific T cell expansion. KSV21 also inhibited mycobacterial efflux pumps, to the same degree as verapamil. More interestingly, the new analog enhances the inhibitory activities of isoniazid and rifampin on intracellular Mtb. In conclusion, KSV21 is a promising verapamil analog on which to base structure-activity relationship studies aimed at identifying more effective analogs.

Numerous pathogens, includingMycobacterium tuberculosis, can activate human γ₉δ₂T cells to proliferate and express effector mechanisms. γ₉δ₂T cells can directly inhibit the growth of intracellular mycobacteria and may also act as antigen-presenting cells (APC). Despite evidence for γδ T cells having the capacity to function as APC, the mechanisms involved and importance of these effects on overall tuberculosis (TB) immunity are unknown. We preparedM. tuberculosis-specific γ₉δ₂T cell lines to study their direct protective effects and APC functions forM. tuberculosis-specific αβ T cells. The direct inhibitory effects on intracellular mycobacteria were measured, and the enhancing effects on proliferative and effector responses of αβ T cells assessed. Furthermore, the importance of cell-to-cell contact and soluble products for γ₉δ₂T cell effector responses and APC functions were investigated. We demonstrate, in addition to direct inhibitory effects on intracellular mycobacteria, the following: (i) γ₉δ₂T cells enhance the expansion ofM. tuberculosis-specific αβ T cells and increase the ability of αβ T cells to inhibit intracellular mycobacteria; (ii) although soluble mediators are critical for the direct inhibitory effects of γ₉δ₂T cells, their APC functions do not require soluble mediators; (iii) the APC functions of γ₉δ₂T cells involve cell-to-cell contact that is dependent on CD40-CD40 ligand (CD40L) interactions; and (iv) fully activated CD4⁺αβ T cells and γ₉δ₂T cells provide similar immune enhancing/APC functions forM. tuberculosis-specific T cells. These effector and helper effects of γ₉δ₂T cells further indicate that these T cells should be considered important new targets for new TB vaccines.

Tuberculosis (TB) is still a major global health problem. A third of the world's population is infected with Mycobacterium tuberculosis. Only ~10% of infected individuals develop TB but there are 9 million TB cases with 1.5 million deaths annually. The standard prophylactic treatment regimens for latent TB infection take 3–9 months, and new cases of TB require at least 6 months of treatment with multiple drugs. The management of latent TB infection and TB has become more challenging because of the spread of multidrug-resistant and extremely drug-resistant TB. Intensified efforts to find new TB drugs and immunotherapies are needed. Immunotherapies could modulate the immune system in patients with latent TB infection or active disease, enabling better control of M. tuberculosis replication. This review describes several types of potential immunotherapies with a focus on those which have been tested in humans.