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Background: Current treatment regimens for pulmonary tuberculosis require at least 6 months of therapy. Immune adjuvant therapy with recombinant interferon-gamma1b (rIFN-gammab) may reduce pulmonary inflammation and reduce the period of infectivity by promoting earlier sputum clearance., Methodology/principal Findings: We performed a randomized, controlled clinical trial of directly observed therapy (DOTS) versus DOTS supplemented with nebulized or subcutaneously administered rIFN-gamma1b over 4 months to 89 patients with cavitary pulmonary tuberculosis. Bronchoalveolar lavage (BAL) and blood were sampled at 0 and 4 months. There was a significant decline in levels of inflammatory cytokines IL-1beta, IL-6, IL-8, and IL-10 in 24-hour BAL supernatants only in the nebulized rIFN-gamma1b group from baseline to week 16. Both rIFN-gamma1b groups showed significant 3-fold increases in CD4+ lymphocyte response to PPD at 4 weeks. There was a significant (p = 0.03) difference in the rate of clearance of Mtb from the sputum smear at 4 weeks for the nebulized rIFN-gamma1b adjuvant group compared to DOTS or DOTS with subcutaneous rIFN-gamma1b. In addition, there was significant reduction in the prevalence of fever, wheeze, and night sweats at 4 weeks among patients receiving rFN-gamma1b versus DOTS alone., Conclusion: Recombinant interferon-gamma1b adjuvant therapy plus DOTS in cavitary pulmonary tuberculosis can reduce inflammatory cytokines at the site of disease, improve clearance of Mtb from the sputum, and improve constitutional symptoms., Trial Registration: ClinicalTrials.gov NCT00201123.

The host response to Mycobacterium tuberculosis includes macrophage activation, inflammation with increased immune effector cells, tissue necrosis, and cavity formation, and fibrosis, distortion, and bronchiectasis. To evaluate the molecular basis of the immune response in the lungs of patients with active pulmonary tuberculosis (TB), we used bronchoalveolar lavage to obtain cells at the site of infection. Affymetrix GeneChip microarrays and cDNA nylon filter microarrays interrogated gene expression in bronchoalveolar lavage (BAL) cells from 11 healthy controls and 17 patients with active pulmonary TB. We found altered gene expression for 69 genes in TB versus normal controls that included cell surface markers, cytokines, chemokines, receptors, transcription factors, and complement components. In addition, TB BAL cell gene expression patterns segregated into 2 groups: one suggestive of a T helper type 1 (Th1) cellular immune response with increased signal transducer and activator of transcription-4 (STAT-4), interferon-gamma (IFN-gamma receptor), and monokine induced by IFN-gamma (MIG) expression with increased IFN-gamma protein levels in BAL fluid; the other group displayed characteristics of Th2 immunity with increased STAT-6, CD81, and IL-10 receptor expression. We were able to demonstrate that a Th2 presentation could change to a Th1 pattern after anti-tuberculous treatment in 1 TB patient studied serially. These gene expression data support the conclusion that pulmonary TB produces a global change in the BAL cell transcriptome with manifestations of either Th1 or Th2 immunity.

Pulmonary tuberculosis may alter volatile organic compounds (VOCs) in breath because Mycobacteria and oxidative stress resulting from Mycobacterial infection both generate distinctive VOCs. The objective of this study was to determine if breath VOCs contain biomarkers of active pulmonary tuberculosis. Head space VOCs from cultured Mycobacterium tuberculosis were captured on sorbent traps and assayed by gas chromatography/mass spectroscopy (GC/MS). One hundred and thirty different VOCs were consistently detected. The most abundant were naphthalene, 1-methyl-, 3-heptanone, methylcyclododecane, heptane, 2,2,4,6,6-pentamethyl-, benzene, 1-methyl-4-(1-methylethyl)-, and cyclohexane, 1,4-dimethyl-. Breath VOCs were assayed by GC/MS in 42 patients hospitalized for suspicion of pulmonary tuberculosis and in 59 healthy controls. Sputum cultures were positive for Mycobacteria in 23/42 and negative in19/42 patients. Breath markers of oxidative stress were increased in all hospitalized patients (p<0.04). Pattern recognition analysis and fuzzy logic analysis of breath VOCs independently distinguished healthy controls from hospitalized patients with 100% sensitivity and 100% specificity. Fuzzy logic analysis identified patients with positive sputum cultures with 100% sensitivity and 100% specificity (95.7% sensitivity and 78.9% specificity on leave-one-out cross-validation); breath VOC markers were similar to those observed in vitro, including naphthalene, 1-methyl- and cyclohexane, 1,4-dimethyl-. Pattern recognition analysis identified patients with positive sputum cultures with 82.6% sensitivity (19/23) and 100% specificity (18/18), employing 12 principal components from 134 breath VOCs. We conclude that volatile biomarkers in breath were sensitive and specific for pulmonary tuberculosis: the breath test distinguished between "sick versus well" i.e. between normal controls and patients hospitalized for suspicion of pulmonary tuberculosis, and between infected versus non-infected patients i.e. between those whose sputum cultures were positive or negative for Mycobacteria.

Quality assurance (QA) is essential for data accuracy and proper evaluation of study objectives in clinical trials. The Tuberculosis Trials Consortium (TBTC)-a collaboration of 28 clinical sites and the Centers for Disease Control and Prevention-has developed a comprehensive QA program that provides quantitative assessments of performance based on clearly defined standards that are communicated to data collectors through a feedback process. The Implementation and Quality Committee of the TBTC developed a Site Evaluation Report (SER) that assesses performance measures (PMs) critical to the accomplishment of study objectives. PMs are defined, quantified, and evaluated, and goals and minimum acceptable scores are specified. Sites not meeting a PM minimum must provide an explanation and develop a plan to meet the goal. Site-specific and system-wide problems can be readily identified through this process. The SER is used prospectively for all TBTC treatment trials, and a Web site has been developed to maximize the availability and usefulness of performance data. The TBTC's comprehensive QA program is an example of a successful method for ensuring high quality, evaluable data.

The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from approximately 80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis.

Study Objectives: Aerosol interferon-gamma (IFN-gamma) is a potential immunomodulator in the treatment of pulmonary tuberculosis (TB). Previous investigations demonstrated conversion of sputum smears in five patients with multidrug-resistant TB after 12 treatments over 1 month, and induction of signaling molecules in 10 of 11 drug-sensitive TB patients using BAL. The objective of the current study was to evaluate particle size and deposition pattern in patients with TB receiving aerosol IFN-gamma treatment., Design: Particle size was determined with a cascade impactor, and deposition of IFN-gamma mixed with (99m)Tc-labeled human serum albumin was assessed using a gamma camera. Local levels of IFN-gamma were measured in BAL using enzyme-linked immunosorbent assays. Study patients/intervention: Fourteen patients with pulmonary TB received IFN-gamma aerosol (500 micro g) for 12 treatments in addition to antimycobacterial therapy with BAL before and after IFN-gamma aerosol treatment. Eight patients with minimal-to-moderate parenchymal involvement underwent deposition studies. Deposited (99m)Tc-labeled IFN-gamma aerosol was partitioned between upper airways and lungs using attenuation correction measurements. (133)Xe equilibrium scanning, (133)Xe washout, and (99m)Tc- macroaggregate injection defined regional lung volume, ventilation, and perfusion., Results: Upper airway deposition was significant often exceeding lung deposition (53.9 +/- 7.09 micro g vs 35.8 +/- 2.73 micro g, respectively [mean +/- SE]). IFN-gamma levels measured in BAL fluid were significantly increased with aerosol treatment (0.83 +/- 0.43 micro g before vs 24.76 +/- 8.71 micro g after, p

Gamma interferon (IFN-gamma) is critical in the immune response against Mycobacterium tuberculosis. In an ongoing trial of aerosol IFN-gamma in conjunction with standard drug therapy, we have observed activation of IFN signaling in bronchoalveolar lavage (BAL) cells from tuberculosis (TB) patients. We hypothesized that aerosol IFN-gamma treatment of pulmonary TB would increase expression of genes important for the control of TB. We investigated the expression of downstream genes by measuring inducible nitric oxide synthase (iNOS) and the chemokine IFN-inducible 10-kDa protein (IP-10) by real-time quantitative reverse transcription-PCR. In vitro, M. tuberculosis induced IP-10, and IFN-gamma stimulated this further, with no effect on iNOS expression. We studied 21 patients with pulmonary TB and 7 healthy subjects. Similar to the in vitro model, IP-10 mRNA was increased in BAL cells from TB patients and was augmented after treatment with aerosolized IFN-gamma. TB was also associated with elevated iNOS mRNA, but aerosolized IFN-gamma did not further enhance expression. Genomic analysis identified 1,300 of 4,058 genes expressed in BAL cells from six TB patients before and after 1 month of therapy, including aerosolized IFN-gamma. However, only 15 genes were differentially regulated by IFN-gamma. We conclude that iNOS and IP-10 mRNA expression is increased in TB but that aerosol IFN-gamma treatment increases expression of few genes in the human lung.

For metered dose inhalers (MDIs), high-flow cascade impaction with a United States Pharmacopia (USP) throat provides a useful prediction of in vivo lung and oropharyngeal aerosol deposition. Particles expected to deposit in the lung are included in the "fine particle fraction" measured on the bench. Comparable in vitro standards are not available for nebulizers. The present study compared aerosol deposition in an in vitro model using low-flow cascade impaction with deposition in vivo in human subjects. A low-flow (1 Lmin), 10-stage cascade impactor measured aerodynamic distributions of aerosolized interferon-gamma (IFN-gamma) from two nebulizers (Misty-Neb and AeroEclipse). (99m)Technetium diethylene triaminepenta-acetic acid ((99m)Tc-DTPA) was used as the radiolabel. Two bench conditions were specified: no breathing (standing cloud) and simulated ventilation with a piston pump (tidal volume 750 mL frequency 25 per minute and duty cycle 0.5). Mass median aerodynamic diameter (MMAD) for both nebulizers was affected by ventilation (Misty-Neb vs. AeroEclipse: 5.2 vs. 4.6 microm for standing cloud and 3.1 vs. 2.2 microm during ventilation). In three subjects, measured values of oropharyngeal deposition averaged 68.1 +/- 0.08% for Misty-Neb and 30.9 +/- 0.03% for AeroEclipse. In vivo deposition patterns compared to aerosol distributions from both nebulizers indicated that, for wet nebulization, penetration of aerosol beyond the upper airways (fine particle fraction) will occur only for aerosol particles below 2.5 microm. This assessment requires that the bench aerosol distribution be measured under conditions of clinical use (i.e., during tidal breathing).

Tuberculosis is the seventh leading cause of morbidity and mortality in the world, with eight million cases per year. Animal and human studies demonstrate an enrichment of CD4 cells at sites of disease, with a more favorable clinical course when there is a Th1 response with the presence of gamma interferon (IFN-gamma). We previously treated patients who had multidrug-resistant tuberculosis with recombinant IFN-gamma (rIFN-gamma) in aerosol form and were able to convert smear-positive cases to smear negative with 12 treatments over 1 month. We hypothesized that rIFN-gamma would induce signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) binding activity in alveolar macrophages (AM). AM treated in vitro showed clear upregulation of STAT-1 and IRF-1 by rIFN-gamma. STAT-1 was not activated and IRF-1 was only weakly induced after 1 day of infection by Mycobacterium tuberculosis TN913. In bronchoalveolar lavage (BAL) cells obtained from 10 of 10 tuberculosis patients 10 +/- 2 days post-antituberculosis treatment, there was no detectable STAT-1 or IRF-1 DNA-binding activity. After 4 weeks of treatment with rIFN-gamma aerosol in addition to the antituberculosis drugs, 10 of 10 patients had increased STAT-1, IRF-1, and/or IRF-9 DNA-binding activity in BAL cells from lung segments shown radiographically to be involved and in those shown to be uninvolved. Symptoms and chest radiographs improved, and amounts of macrophage inflammatory cytokines and human immunodeficiency virus type 1 (HIV-1) viral loads (in five of five HIV-1-coinfected patients) declined in the second BAL specimens. rIFN-gamma aerosol induces signal transduction and gene expression in BAL cells and should be evaluated for efficacy in a randomized, controlled clinical trial.