Seok-Yong Eum

Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB.

BACKGROUND The exact role of neutrophils in the pathogenesis of TB is poorly understood. Recent evidence suggests that neutrophils are not simply scavenging phagocytes in Mycobacterium tuberculosis (Mtb) infection. METHODS Three different types of clinical specimens from patients with active pulmonary TB who underwent lung surgery were examined: sputum, BAL fluid, and cavity contents. Differential cell separation and quantification were performed for intracellular and extracellular bacteria, and bacterial length was measured using microscopy. RESULTS Neutrophils were more abundant than macrophages in sputum (86.6% +/- 2.2% vs 8.4% +/- 1.3%) and in BAL fluid (78.8% +/- 5.8% vs 11.8% +/- 4.1%). Inside the cavity, lymphocytes (41.3% +/- 11.2%) were the most abundant cell type, followed by neutrophils (38.8% +/- 9.4%) and macrophages (19.5% +/- 7.5%). More intracellular bacilli were found in neutrophils than macrophages in sputum (67.6% +/- 5.6% vs 25.2% +/- 6.5%), in BAL fluid (65.1% +/- 14.4% vs 28.3% +/- 11.6%), and in cavities (61.8% +/- 13.3% vs 23.9% +/- 9.3%). The lengths of Mtb were shortest in cavities (1.9+/- 0.1 microm), followed by in sputum (2.9 +/- 0.1 microm) and in BAL fluid (3.6 +/- 0.2 microm). CONCLUSIONS Our results show that neutrophils are the predominant cell types infected with Mtb in patients with TB and that these intracellular bacteria appear to replicate rapidly. These results are consistent with a role for neutrophils in providing a permissive site for a final burst of active replication of the bacilli prior to transmission.

The association between sterilizing activity and drug distribution into tuberculosis lesions.

Finding new treatment-shortening antibiotics to improve cure rates and curb the alarming emergence of drug resistance is the major objective of tuberculosis (TB) drug development. Using a matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging suite in a biosafety containment facility, we show that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in lung lesions. Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside. In contrast, moxifloxacin, which is active in vitro against a subpopulation of Mycobacterium tuberculosis that persists in specific niches under drug pressure and has achieved treatment shortening in mice, does not diffuse well in caseum, concordant with its failure to shorten therapy in recent clinical trials. We suggest that such differential spatial distribution and kinetics of accumulation in lesions may create temporal and spatial windows of monotherapy in specific niches, allowing the gradual development of multidrug-resistant TB. We propose an alternative working model to prioritize new antibiotic regimens based on quantitative and spatial distribution of TB drugs in the major lesion types found in human lungs. The finding that lesion penetration may contribute to treatment outcome has wide implications for TB.

Extensive drug resistance acquired during treatment of multidrug-resistant tuberculosis.

BACKGROUND Increasing access to drugs for the treatment of multidrug-resistant (MDR) tuberculosis is crucial but could lead to increasing resistance to these same drugs. In 2000, the international Green Light Committee (GLC) initiative began to increase access while attempting to prevent acquired resistance. METHODS To assess the GLCs impact, we followed adults with pulmonary MDR tuberculosis from the start to the end of treatment with monthly sputum cultures, drug susceptibility testing, and genotyping. We compared the frequency and predictors of acquired resistance to second-line drugs (SLDs) in 9 countries that volunteered to participate, 5 countries that met GLC criteria, and 4 countries that did not apply to the GLC. RESULTS In total, 832 subjects were enrolled. Of those without baseline resistance to specific SLDs, 68 (8.9%) acquired extensively drug-resistant (XDR) tuberculosis, 79 (11.2%) acquired fluoroquinolone (FQ) resistance, and 56 (7.8%) acquired resistance to second-line injectable drugs (SLIs). The relative risk (95% confidence interval [CI]) of acquired resistance was lower at GLC-approved sites: 0.27 (.16-.47) for XDR tuberculosis, 0.28 (.17-.45) for FQ, and 0.15 (.06-.39) to 0.60 (.34-1.05) for 3 different SLIs. The risk increased as the number of potentially effective drugs decreased. Controlling for baseline drug resistance and differences between sites, the odds ratios (95% CIs) were 0.21 (.07-.62) for acquired XDR tuberculosis and 0.23 (.09-.59) for acquired FQ resistance. CONCLUSIONS Treatment of MDR tuberculosis involves substantial risk of acquired resistance to SLDs, increasing as baseline drug resistance increases. The risk was significantly lower in programs documented by the GLC to meet specific standards.

Genetic Diversity of Mycobacterium tuberculosis Isolates from a Tertiary Care Tuberculosis Hospital in South Korea

ABSTRACT 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

ABSTRACT 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.

Efficacy and Safety of Metronidazole for Pulmonary Multidrug-Resistant Tuberculosis

ABSTRACT 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.

Mycolic acids as diagnostic markers for tuberculosis case detection in humans and drug efficacy in mice.

Mycolic acids are attractive diagnostic markers for tuberculosis (TB) infection because they are bacteria‐derived, contain information about bacterial species, modulate host–pathogen interactions and are chemically inert. Here, we present a novel approach based on mass spectrometry. Quantification of specific precursor → fragment transitions of approximately 2000 individual mycolic acids (MAs) resulted in high analytical sensitivity and specificity. We next used this tool in a retrospective case–control study of patients with pulmonary TB with varying disease burdens from South Korea, Vietnam, Uganda and South Africa. MAs were extracted from small volume sputum (200 µl) and analysed without the requirement for derivatization. Infected patients (70, 19 of whom were HIV+) could be separated from controls (40, 20 of whom were HIV+) with a sensitivity and specificity of 94 and 93%, respectively. Furthermore, we quantified MA species in lung tissue of TB‐infected mice and demonstrated effective clearance of MA levels following curative rifampicin treatment. Thus, our results demonstrate for the first time the feasibility and clinical relevance of direct detection of mycobacterial lipids as biomarkers of TB infection.

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.

Multidrug-Resistant Tuberculosis Treatment Outcomes in Relation to Treatment and Initial Versus Acquired Second-Line Drug Resistance

BACKGROUND Resistance to second-line drugs develops during treatment of multidrug-resistant (MDR) tuberculosis, but the impact on treatment outcome has not been determined. METHODS Patients with MDR tuberculosis starting second-line drug treatment were enrolled in a prospective cohort study. Sputum cultures were analyzed at a central reference laboratory. We compared subjects with successful and poor treatment outcomes in terms of (1) initial and acquired resistance to fluoroquinolones and second-line injectable drugs (SLIs) and (2) treatment regimens. RESULTS Of 1244 patients with MDR tuberculosis, 973 (78.2%) had known outcomes and 232 (18.6%) were lost to follow-up. Among those with known outcomes, treatment succeeded in 85.8% with plain MDR tuberculosis, 69.7% with initial resistance to either a fluoroquinolone or an SLI, 37.5% with acquired resistance to a fluoroquinolone or SLI, 29.3% with initial and 13.0% with acquired extensively drug-resistant tuberculosis (P < .001 for trend). In contrast, among those with known outcomes, treatment success increased stepwise from 41.6% to 92.3% as the number of drugs proven effective increased from ≤1 to ≥5 (P < .001 for trend), while acquired drug resistance decreased from 12% to 16% range, depending on the drug, down to 0%-2% (P < .001 for trend). In multivariable analysis, the adjusted odds of treatment success decreased 0.62-fold (95% confidence interval, .56-.69) for each increment in drug resistance and increased 2.1-fold (1.40-3.18) for each additional effective drug, controlling for differences between programs and patients. Specific treatment, patient, and program variables were also associated with treatment outcome. CONCLUSIONS Increasing drug resistance was associated in a logical stepwise manner with poor treatment outcomes. Acquired resistance was worse than initial resistance to the same drugs. Increasing numbers of effective drugs, specific drugs, and specific program characteristics were associated with better outcomes and less acquired resistance.

The immunity and protective effects of antigen 85A and heat-shock protein X against progressive tuberculosis

The anti-tuberculosis vaccine, Mycobacterium bovis BCG, has been used worldwide, but its protective efficacy is variable against adult pulmonary tuberculosis. In this study, immune responses of antigen 85A (Ag85A) and heat-shock protein X (HspX) antigen of Mycobacterium tuberculosis were investigated during acute and stationary stage of infection in the murine aerosol TB challenge model and their protective effects were evaluated against progressive tuberculosis. A high level of Ag85A-specific IFN-γ production was induced from the early stage of the infection, whereas HspX-specific IFN-γ production was increased in the later stationary stage. As a subunit vaccine, Ag85A and HspX antigen vaccine induced high levels of IFN-γ, and a vaccine comprising both antigens induced the highest level of IFN-γ. At 30 days post-challenge, the Ag85A subunit vaccine was protective against M. tuberculosis challenge, but the HspX subunit vaccine was not. Interestingly, the HspX antigen vaccine induced significant protective efficacy at 90 days post-challenge. Moreover, the combined antigen vaccine induced the highest protective efficacy against M. tuberculosis challenge both at 30 days and 90 days post-challenge. These results suggest that the vaccine comprising Ag85A and HspX antigen which react in different stages of infection is highly protective against progressive tuberculosis.