Tuberculosis: An Overview of the Immunogenic Response, Disease Progression, and Medicinal Chemistry Efforts in the Last Decade toward the Development of Potential Drugs for Extensively Drug-Resistant Tuberculosis Strains.

Tuberculosis (TB) is a slow growing, potentially debilitating disease that has plagued humanity for centuries and has claimed numerous lives across the globe. Concerted efforts by researchers have culminated in the development of various strategies to combat this malady. This review aims to raise awareness of the rapidly increasing incidences of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, highlighting the significant modifications that were introduced in the TB treatment regimen over the past decade. A description of the role of pathogen-host immune mechanisms together with strategies for prevention of the disease is discussed. The struggle to develop novel drug therapies has continued in an effort to reduce the treatment duration, improve patient compliance and outcomes, and circumvent TB resistance mechanisms. Herein, we give an overview of the extensive medicinal chemistry efforts made during the past decade toward the discovery of new chemotypes, which are potentially active against TB-resistant strains.

Different macrophage polarization between drug-susceptible and multidrug-resistant pulmonary tuberculosis.

BACKGROUND: Macrophages play a key role in the infection process, and alternatively activated macrophages (M2 polarization) play important roles in persistent infection via the immune escape of pathogens. This suggests that immune escape of pathogens from host immunity is an important factor to consider in treatment failure and multidrug-resistant tuberculosis (MDR-TB)/extensively drug-resistant tuberculosis (XDR-TB). In this study, we investigated the association between macrophage polarization and MDR-TB/XDR-TB and the association between macrophage polarization and the anti-TB drugs used. METHODS: iNOS and arginase-1, a surface marker of polarized macrophages, were quantified by immunohistochemical staining and imaging analysis of lung tissues of patients who underwent surgical treatment for pulmonary TB. Drug susceptibility/resistance and the type and timing of anti-tuberculosis drugs used were investigated. RESULTS: The M2-like polarization rate and the ratio of the M2-like polarization rate to the M1-like polarization rate were significantly higher in the MDR-TB/XDR-TB group than in the DS-TB group. The association between a high M2-like polarization rate and MDR-TB/XDR-TB was more pronounced in patients with a low M1-like polarization rate. Younger age and a higher M2-like polarization rate were independent associated factors for MDR-TB/XDR-TB. The M2-like polarization rate was significantly higher in patients who received anti-TB drugs containing pyrazinamide continuously for 4 or 6 weeks than in those who received anti-TB drugs not containing pyrazinamide. CONCLUSIONS: The M2-like polarization of macrophages is associated with MDR-TB/XDR-TB and anti-TB drug regimens including pyrazinamide or a combination of pyrazinamide, prothionamide and cycloserine.

Study on the relationship between changes of immune cells and TNF-α in peripheral blood of patients with multidrug-resistant and extensively drug-resistant tuberculosis.

OBJECTIVE: To investigate the relationship between changes in CD4- and CD8-positive immune cells and TNF-α in the peripheral blood of patients affected by multidrug-resistant and extensively drug-resistant tuberculosis. PATIENTS AND METHODS: 179 patients suffering from tuberculosis treated in the Chest Hospital of Hebei from April 2010 to December 2015 were selected for the study. There were 47 cases affected by drug-resistant tuberculosis and 132 cases affected by non-drug-resistant tuberculosis. The control group included 183 healthy subjects examined during the same period. ELISA was used to compare and analyze serum levels of TNF-α, CD4- and CD8-positive cell levels, and CD4/CD8 ratio in the two groups. RESULTS: CD4- and CD8-positive cell count, CD4/CD8 ratio, and serum TNF-a were significantly higher in patients with drug-resistant tuberculosis compared with healthy controls and the non-drug-resistant tuberculosis patients (p < 0.05). There was a positive correlation between TNF-α level and CD4/CD8 ratio (r=0.892, p < 0.05). Before treatment, the differences in the levels of TNF-a in the different groups of drug-resistant patients were insignificant (p >0.05). After treatment, the levels of TNF-a in the different groups of drug-resistant patients were decreased, except for patients with extensively drug-resistant tuberculosis, whose levels were significantly decreased compared with before treatment (t = 0.648, p>0.05). The differences in the levels of TNF-α in the other groups of patients before and after treatment were statistically significant (t = 8.497, 6.258, 5.346, p < 0.05, fully sensitive tuberculosis single drug-resistant tuberculosis, multidrug-resistant tuberculosis, respectively). CONCLUSIONS: The level of TNF-α plays a critical role in the evaluation of the severity of patients with drug-resistant tuberculosis and it has a clinical value.

Regulatory T Cells Subvert Mycobacterial Containment in Patients Failing Extensively Drug-Resistant Tuberculosis Treatment.

RATIONALE: The advent of extensively drug-resistant (XDR) tuberculosis (TB) and totally drug-resistant TB, with limited or no treatment options, has facilitated renewed interest in host-directed immunotherapy, particularly for therapeutically destitute patients. However, the selection and utility of such approaches depend on understanding the host immune response in XDR-TB, which hitherto remains unexplored. OBJECTIVES: To determine the host immunological profile in patients with XDR-TB, compared with drug-sensitive TB (DS-TB), using peripheral blood and explanted lung tissue. METHODS: Blood and explanted lung tissue were obtained from patients with XDR-TB (n = 31), DS-TB (n = 20), and presumed latent TB infection (n = 20). T-cell phenotype (T-helper cell type 1 [Th1]/Th2/Th17/regulatory T cells [Tregs]) was evaluated in all patient groups, and Treg function assessed in XDR-TB nonresponders by coculturing PPD-preprimed effector T cells with H37Rv-infected monocyte-derived macrophages, with or without autologous Tregs. Mycobacterial containment was evaluated by counting colony-forming units. MEASUREMENTS AND MAIN RESULTS: Patients failing XDR-TB treatment had an altered immunophenotype characterized by a substantial increase in the frequency (median; interquartile range) of CD4+CD25+FoxP3+ Tregs (11.5%; 5.9-15.2%) compared with DS-TB (3.4%; 1.6-5.73%; P < 0.001) and presumed latent TB infection (1.8%; 1.2-2.3%; P < 0.001), which was unrelated to disease duration. Tregs isolated from patients with XDR-TB suppressed T-cell proliferation (up to 90%) and subverted containment of H37Rv-infected monocyte-derived macrophages (by 30%; P = 0.03) by impairing effector T-cell function through a mechanism independent of direct cell-to-cell contact, IL-10, TGF (transforming growth factor)-ß, and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). CONCLUSIONS: Collectively, these data suggest that Tregs may be contributing to immune dysfunction, and bacterial persistence, in patients with XDR-TB. The relevant cellular pathways may serve as potential targets for immunotherapeutic intervention.

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 < 0·0001). Increasing mean linezolid trough concentrations were associated with lower mitochondrial function levels (Spearman's ρ = - 0.48; P = 0.005). Mitochondrial toxicity risk increased with increasing linezolid trough concentrations, with all patients with mean linezolid trough > 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.

Totally drug-resistant tuberculosis and adjunct therapies.

The first cases of totally drug-resistant (TDR) tuberculosis (TB) were reported in Italy 10 years ago; more recently, cases have also been reported in Iran, India and South Africa. Although there is no consensus on terminology, it is most commonly described as 'resistance to all first- and second-line drugs used to treat TB'. Mycobacterium tuberculosis (M.tb) acquires drug resistance mutations in a sequential fashion under suboptimal drug pressure due to monotherapy, inadequate dosing, treatment interruptions and drug interactions. The treatment of TDR-TB includes antibiotics with disputed or minimal effectiveness against M.tb, and the fatality rate is high. Comorbidities such as diabetes and infection with human immunodeficiency virus further impact on TB treatment options and survival rates. Several new drug candidates with novel modes of action are under late-stage clinical evaluation (e.g., delamanid, bedaquiline, SQ109 and sutezolid). 'Repurposed' antibiotics have also recently been included in the treatment of extensively drug resistant TB. However, because of mutations in M.tb, drugs will not provide a cure for TB in the long term. Adjunct TB therapies, including therapeutic vaccines, vitamin supplementation and/or repurposing of drugs targeting biologically and clinically relevant molecular pathways, may achieve better clinical outcomes in combination with standard chemotherapy. Here, we review broader perspectives of drug resistance in TB and potential adjunct treatment options.

Preliminary study on the virulence of XDR-TB: low virulence owing to less cytokine expression through the TLR 2 and TLR4 pathways in BLAB/C mice.

Extensively drug-resistant tuberculosis (XDR-TB) imposes a new threat to the world. In this study, XDR-TB and drug-sensitive TB (DS-TB) strains with similar genotypes were investigated. The results showed that BLAB/C mice infected with XDR-TB had reduced Toll-like receptor (TLR) 2 and TLR4 expression in lung tissues than DS-TB mice at 2 weeks post-infection. Mice infected with XDR-TB showed lower levels of TNF-α, IFN-γ, IL-4 and IL-10 in the sera than those infected with DS-TB. Moreover, mice infected with XDR-TB survived longer period and had less severe alveolar damage and smaller granulomas than those infected with DS-TB. These results were further confirmed in THP-1 cells. Using the flow cytometry and qRT-PCR methods, we found that XDR-TB stimulated lower TLR2 and TLR4 expression. Based on the above results, we cautiously inferred that low virulence of XDR-TB owed to the less cytokine expression through the TLR 2 and 4 pathways. The effect of XDR-TB and DS-TB can be further tested in TLR2 or TLR4 knock-out mice and TLRs-silenced THP-1 cells.

Getting personal perspectives on individualized treatment duration in multidrug-resistant and extensively drug-resistant tuberculosis.

Tuberculosis (TB) differs from most other bacterial infectious diseases by a very long duration of combination antibiotic therapy required to achieve relapse-free cure. Although the standard recommended "short-course" treatment length for TB is 6 months, the World Health Organization recommends a duration of 20 months for the treatment of patients with multidrug-resistant and extensively drug-resistant TB (M/XDR-TB). Apart from the long duration of anti-TB therapy, treatment of M/XDR-TB is very expensive and often associated with adverse drug events. The optimal duration for treatment of TB likely differs between individuals and depends on a variety of variables, such as the extent of the disease, the immune status of the host, and the virulence and the drug resistance of the causative strain of Mycobacterium tuberculosis. Some patients with M/XDR-TB may have to be treated with currently available antituberculosis drug regimens for more than 20 months, whereas much shorter treatment durations may be possible to achieve cure for the majority of patients with M/XDR-TB. Personalization of the duration of treatment for TB, especially for patients with M/XDR-TB, would be highly desired. Until recently there has been little interest in the identification of biosignatures that could eventually lead to individual recommendations for the duration of anti-TB therapy. This pulmonary perspective reviews the knowledge on clinical and radiological scores, host- and pathogen disease-related profiles, molecules, and signatures that are currently explored as biomarkers to personalize the duration of therapy in TB.

Adherence in the treatment of patients with extensively drug-resistant tuberculosis and HIV in South Africa: a prospective cohort study.

OBJECTIVE: Extensively drug-resistant tuberculosis (XDR-TB)/HIV coinfection is difficult to treat with frequent adverse drug reactions and associated with high mortality. Adherence to antiretroviral therapy (ARV) and second-line TB medications may reduce mortality, prevent amplification of drug resistance, and improve outcomes. METHODS: Prospective cohort study of XDR-TB patients on treatment in KwaZulu-Natal, South Africa. Adherence to ARV and TB medications was assessed separately at baseline and monthly. Knowledge, attitudes, and beliefs were assessed at baseline. Optimal adherence was defined as self-report of taking all pills in the previous 7 days; missing any pills was defined as suboptimal adherence. Primary outcome was optimal adherence 6 months after initiation of XDR-TB treatment to TB medications, ARV, and both ("dual adherence"). RESULTS: One hundred four XDR-TB patients (79.8% HIV coinfected, 84.3% on ARV at enrollment) were enrolled and followed monthly (median 8 visits; interquartile range: 4-12). Six-month optimal adherence was higher for ARV (88.2%) than TB medications (67.7%) (P < 0.001). Low educational attainment, male gender, and year of enrollment were independently associated with dual suboptimal adherence. At baseline, participants indicated that XDR-TB was curable (76.0%), HIV and TB were linked (81.7%), and ARV improves TB outcomes (72.1%). Baseline knowledge, attitudes, and beliefs did not predict subsequent adherence. CONCLUSIONS: Medication adherence was significantly higher for ARV than for TB medications in this cohort. Short-course treatment regimens for drug-resistant TB with lower pill burden may increase adherence and improve outcomes in XDR-TB/HIV. Programmatic support for dual adherence is critical in the treatment of drug-resistant TB and HIV.

Mechanisms by which thioridazine in combination with antibiotics cures extensively drug-resistant infections of pulmonary tuberculosis.

Advances recently introduced into the Clinical Mycobacteriology Laboratory of the Institute of Hygiene and Tropical Medicine, such that a multi-drug resistant infection of pulmonary tuberculosis (MDR TB) can be identified within one day of receiving the sputum specimen, have greatly contributed to the reduction of the frequency of these infections. However, approximately 50% of reduced infections exhibit a phenotype that is consistent with that presented by an extensively drug-resistant (XDR) infection. More effective agents were required and hence attention was attributed to the possibility that the old neuroleptic phenothiazine thioridazine (TZ), previously shown to inhibit the growth of all encountered strains of Mycobacterium tuberculosis (Mtb) regardless of their antibiotic resistance profile, could be eventually used for therapy of problematic MDR/XDR TB infections. This mini-review discusses the mechanisms that render TZ an effective adjuvant to antibiotics to which the initial infective agent Mtb was resistant.

[The changes and the significance of cellular immune function in peripheral blood of patients with multidrug-resistant and extensively drug-resistant pulmonary tuberculosis].

OBJECTIVE: To explore the expressions and the significance of CD(3)(+)CD(16)(+)CD(56)(+) NKT cells, CD(3)(-)CD(16)(+)CD(56)(+) NK cells and T lymphocyte subsets in peripheral blood of patients with multi-drug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) pulmonary tuberculosis. METHODS: The data of 316 patients with pulmonary tuberculosis hospitalized in Shanghai Pulmonary Hospital from January 2008 to June 2009 were retrospectively analyzed, of whom 119 were newly diagnosed, and 197 were retreated patients. There were 204 males and 112 females, aged from 17 - 88 years, mean (44 ± 16) years. According to the results of drug-resistance, these patients were divided into a MDR group, an XDR group and a sensitive group. There were 146 patients in the MDR group, with 102 males and 44 females, aged from 19 - 84 years, mean (42 ± 16) years. There were 77 patients in the XDR group, with 42 males and 35 females, aged from 18 - 88 years, mean (50 ± 16) years. There were 93 patients in the susceptible group, with 60 males and 33 females, aged from 17 - 83 years, mean (43 ± 19) years. According to the distribution of cavitation in lung fields, these patients were also divided into 1 - 2 lung field affected group (n = 132), 3 - 4 lung field affected group (n = 49) and 5 - 6 lung field affected group (n = 9). The frequencies of NKT cells, NK cells and T cells from whole blood were tested by flow cytometry. Rank test (SAS software) was used for statistic analyses. RESULTS: The expression rate of NKT cells and NK cells was the highest in the XDR group [11% (6% - 16%) and 7% (4% - 12%)], as compared to the MDR group [8% (5% - 14%) and 6% (4% - 11%)], and the susceptible group [7% (4% - 11%) and 5% (3% - 9%)], the difference being statistically significant (H = 6.478 - 8.369, P < 0.05). The expression rate of the NKT (14 ± 9)% and NK cells (11 ± 7)% in males of the XDR group was significantly higher than that in females [NKT (9 ± 5)% and NK cell (6 ± 4)%], while CD(4) (38 ± 10)% and CD(4)/CD(8) (1.9 ± 1.3) were significantly lower than those of the females [CD(4) (44 ± 10)% and CD(4)/CD(8)(2.2 ± 0.7)], the difference being statistically significant (z = -2.91 - -2.79, P < 0.05, P < 0.01). The expression rate of CD(4) (42 ± 9)% was the highest, but CD(8) (22 ± 8)% was the lowest in the 1 - 2 lung field group. While in the 5 - 6 lung field group, the expression rate of CD(4) (36 ± 11)% was the lowest, CD(8) (28 ± 12)% was the highest, and CD(4)/CD(8) (1.5 ± 0.8) was the lowest, the difference being statistically significant (H = 8.404 - 16.175, P < 0.01). CONCLUSIONS: With the increasing level of drug resistance, the expression rate of NKT cells and NK cells increased, while the expression of T cell subsets did not change. The value of CD(4) and CD(4)/CD(8) in peripheral blood decreased, but CD(8) increased as the extent of cavitation increased in these patients. The impairment of cellular immune function in XDR-TB was more prominent in male patients.

Cytokines as immunomodulators in tuberculosis therapy.

The use of cytokines for therapeutic purposes is limited by their high cost and toxicity. Nevertheless, the emergence of extensively drug-resistant tuberculosis (XDR TB), for which chemotherapy is ineffective, has again made cytokine-based therapy attractive as one of the last available options. The results of clinical trials treating pulmonary tuberculosis with cytokines have not been encouraging, making it clear that therapeutic strategies utilizing a single cytokine are inadequate. To develop effective cytokine-based XDR TB therapies, more basic research will be needed to achieve a better understanding of how cytokines promote a successful immune response. We not only have to investigate cytokines already known to participate in tuberculosis, but also the role of other cytokines and chemokines that may enhance both the mycobacterial killing activity of effector cells and the restriction of bacterial intracellular multiplication. There are already several patents involving cytokines for therapeutic use, in the hope of stimulating the immune system in a variety of infectious diseases, including tuberculosis. The validity of these patents needs to be reassessed from a clinical standpoint, and new applications of patents concerning cytokines potentially useful in XDR TB treatment should be encouraged.

High rate of extensively drug-resistant tuberculosis in Indian AIDS patients.

Fifty-four full-blown AIDS patients suspected of having HIV-tuberculosis co-infection were investigated for the prevalence of extensively drug-resistant (XDR) Mycobacterium tuberculosis. Out of the 54 patients, M. tuberculosis was isolated from 24 (44.4%). Twelve (50%) isolates of these had resistance to first-line drugs, whereas four (33.33%) were also resistant to second-line drugs. All four patients, in whom XDR M. tuberculosis was isolated, died within 2.6 months of diagnosis.