Higher plasma interleukin - 6 levels are associated with lung cavitation in drug-resistant tuberculosis.

BACKGROUND: Lung cavitation is associated with heightened TB transmission and poor treatment outcomes. This study aimed to determine the relationship between systemic inflammation and lung cavitation in drug-resistant TB patients with and without HIV co-infection. METHODS: Plasma samples were obtained from 128 participants from the CAPRISA 020 Individualized M(X)drug-resistant TB Treatment Strategy Study (InDEX) prior to treatment initiation. Lung cavitation was present in 61 of the 128 drug-resistant TB patients with 93 being co-infected with HIV. The plasma cytokine and chemokine levels were measured using the 27-Plex Human Cytokine immunoassay. Modified Poisson regression models were used to determine the association between plasma cytokine/chemokine expression and lung cavitation in individuals with drug-resistant TB. RESULTS: Higher Interleukin-6 plasma levels (adjusted risk ratio [aRR] 1.405, 95% confidence interval [CI] 1.079-1.829, p = 0.011) were associated with a higher risk of lung cavitation in the multivariable model adjusting for age, sex, body mass index, HIV status, smoking and previous history of TB. Smoking was associated with an increased risk of lung cavitation (aRR 1.784, 95% CI 1.167-2.729, p = 0.008). An HIV positive status and a higher body mass index, were associated with reduced risk of lung cavitation (aRR 0.537, 95% CI 0.371-0.775, p = 0.001 and aRR 0.927, 95% CI 0.874-0.983, p = 0.012 respectively). CONCLUSION: High plasma interleukin-6 levels are associated with an increased risk of cavitary TB highlighting the role of interleukin-6 in the immunopathology of drug-resistant TB.

Immunomodulatory Agents Combat Multidrug-Resistant Tuberculosis by Improving Antimicrobial Immunity.

BACKGROUND: Multidrug-resistant (MDR) tuberculosis has low treatment success rates, and new treatment strategies are needed. We explored whether treatment with active vitamin D3 (vitD) and phenylbutyrate (PBA) could improve conventional chemotherapy by enhancing immune-mediated eradication of Mycobacterium tuberculosis. METHODS: A clinically relevant model was used consisting of human macrophages infected with M. tuberculosis isolates (n = 15) with different antibiotic resistance profiles. The antimicrobial effect of vitD+PBA, was tested together with rifampicin or isoniazid. Methods included colony-forming units (intracellular bacterial growth), messenger RNA expression analyses (LL-37, ß-defensin, nitric oxide synthase, and dual oxidase 2), RNA interference (LL-37-silencing in primary macrophages), and Western blot analysis and confocal microscopy (LL-37 and LC3 protein expression). RESULTS: VitD+PBA inhibited growth of clinical MDR tuberculosis strains in human macrophages and strengthened intracellular growth inhibition of rifampicin and isoniazid via induction of the antimicrobial peptide LL-37 and LC3-dependent autophagy. Gene silencing of LL-37 expression enhanced MDR tuberculosis growth in vitD+PBA-treated macrophages. The combination of vitD+PBA and isoniazid were as effective in reducing intracellular MDR tuberculosis growth as a >125-fold higher dose of isoniazid alone, suggesting potent additive effects of vitD+PBA with isoniazid. CONCLUSIONS: Immunomodulatory agents that trigger multiple immune pathways can strengthen standard MDR tuberculosis treatment and contribute to next-generation individualized treatment options for patients with difficult-to-treat pulmonary tuberculosis.

Does the BCG vaccine have different effects on strains of tuberculosis?

Several explanations have been suggested concerning the variety in bacille Calmette-Guerin (BCG) vaccine efficacy on strains of Mycobacterium tuberculosis (Mtb). This study aimed to compare the effect of BCG vaccination history in the prevention of the occurrence of Mtb-Beijing and non-Beijing strains. In this cross-sectional study, 64 patients with pulmonary tuberculosis (TB) were recruited from the Iranian border provinces (North West and West). Isolates were subjected to restriction fragment length polymorphism (RFLP) analysis, using the insertion sequence IS6110 as a probe (IS6110 RFLP) and drug susceptibility testing using the proportion method. Samples were analyzed with Gel Compare II 6.6 and spss version 18. The mean age [standard deviation (SD)] of the patients was 54·4 (SD = 17·0). Overall, 49 cases (76·56%) had no BCG vaccination scar. The prevalence of Beijing strains was 9·38% and drug resistance proportion among the isolates was 14·1% (nine cases). There was a significant relationship between Beijing strains and tuberculosis (TB)-drug resistance in isolates (χ2  = 26·29, P < 0·001). There was also a strong association between vaccination history and Beijing strains (χ2  = 13·23, P = 0·002). Also, a statistical relationship was observed between Beijing strains and drug-resistant TB among patients with a history of vaccination (χ2  = 7·47, P = 0·002). This association was not maintained in the unvaccinated group (P = 0·102). These findings confirm the claim that the vaccine has different effects on different subspecies of tuberculosis. The cause of the high probability of drug resistance in patients with Beijing-TB and vaccination history requires further investigation with a higher sample size.

Th22 response induced by Mycobacterium tuberculosis strains is closely related to severity of pulmonary lesions and bacillary load in patients with multi-drug-resistant tuberculosis.

The role of interleukin-22 (IL-22) in the pathogenesis or tissue repair in human tuberculosis (TB) remains to be established. Here, we aimed to explore the ex-vivo and in-vitro T helper 22 (Th22) response in TB patients and healthy donors (HD) induced by different local multi-drug-resistant (MDR) Mvcobacterium tuberculosis (Mtb) strains. For this purpose, peripheral blood mononuclear cells from drug-susceptible (S-TB) MDR-TB patients and HD were stimulated with local MDR strains and the laboratory strain H37Rv. IL-22 and IL-17 expression and senescent status were assessed in CD4+ and CD8+ cells by flow cytometry, while IL-22 amount was measured in plasma and culture supernatants by enzyme-linked immunosorbent assay (ELISA). We found lower IL-22 amounts in plasma from TB patients than HD, together with a decrease in the number of circulating T cells expressing IL-22. In a similar manner, all Mtb strains enhanced IL-22 secretion and expanded IL-22+ cells within CD4+ and CD8+ subsets, being the highest levels detected in S-TB patients. In MDR-TB, low systemic and Mtb-induced Th22 responses associated with high sputum bacillary load and bilateralism of lung lesions, suggesting that Th22 response could be influencing the ability of MDR-TB patients to control bacillary growth and tissue damage. In addition, in MDR-TB patients we observed that the higher the percentage of IL-22+ cells, the lower the proportion of programmed cell death 1 (PD-1)+ or CD57+ T cells. Furthermore, the highest proportion of senescent T cells was associated with severe lung lesions and bacillary load. Thus, T cell senescence would markedly influence Th22 response mounted by MDR-TB patients.

Mycobacteriophages as Potential Therapeutic Agents against Drug-Resistant Tuberculosis.

The current emergence of multi-, extensively-, extremely-, and total-drug resistant strains of Mycobacterium tuberculosis poses a major health, social, and economic threat, and stresses the need to develop new therapeutic strategies. The notion of phage therapy against bacteria has been around for more than a century and, although its implementation was abandoned after the introduction of drugs, it is now making a comeback and gaining renewed interest in Western medicine as an alternative to treat drug-resistant pathogens. Mycobacteriophages are genetically diverse viruses that specifically infect mycobacterial hosts, including members of the M. tuberculosis complex. This review describes general features of mycobacteriophages and their mechanisms of killing M. tuberculosis, as well as their advantages and limitations as therapeutic and prophylactic agents against drug-resistant M. tuberculosis strains. This review also discusses the role of human lung micro-environments in shaping the availability of mycobacteriophage receptors on the M. tuberculosis cell envelope surface, the risk of potential development of bacterial resistance to mycobacteriophages, and the interactions with the mammalian host immune system. Finally, it summarizes the knowledge gaps and defines key questions to be addressed regarding the clinical application of phage therapy for the treatment of drug-resistant tuberculosis.

The phytochemical bergenin as an adjunct immunotherapy for tuberculosis in mice.

The widespread availability and use of modern synthetic therapeutic agents have led to a massive decline in ethnomedical therapies. However, these synthetic agents often possess toxicity leading to various adverse effects. For instance, anti-tubercular treatment (ATT) is toxic, lengthy, and severely impairs host immunity, resulting in posttreatment vulnerability to reinfection and reactivation of tuberculosis (TB). Incomplete ATT enhances the risk for the generation of multidrug- or extensively drug-resistant (MDR or XDR, respectively) variants of Mycobacterium tuberculosis (M. tb), the TB-causing microbe. Therefore, a new therapeutic approach that minimizes these risks is urgently needed to combat this deadly disease and prevent future TB epidemics. Previously, we have shown that the phytochemical bergenin induces T helper 1 (Th1)- and Th17 cell-based protective immune responses and potently inhibits mycobacterial growth in a murine model of M. tb infection, suggesting bergenin as a potential adjunct agent to TB therapy. Here, we combined ATT therapy with bergenin and found that this combination reduces immune impairment and the length of treatment in mice. We observed that co-treatment with the anti-TB drug isoniazid and bergenin produces additive effects and significantly reduces bacterial loads compared with isoniazid treatment alone. The bergenin co-treatment also reduced isoniazid-induced immune impairment; promoted long-lasting, antigen-specific central memory T cell responses; and acted as a self-propelled vaccine. Of note, bergenin treatment significantly reduced the bacterial burden of a multidrug-resistant TB strain. These observations suggest that bergenin is a potent immunomodulatory agent that could be further explored as a potential adjunct to TB therapy.

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.

Remembering the Host in Tuberculosis Drug Development.

New therapeutics to augment current approaches and shorten treatment duration are of critical importance for combating tuberculosis (TB), especially those with novel mechanisms of action to counter the emergence of drug-resistant TB. Host-directed therapy (HDT) offers a novel strategy with mechanisms that include activating immune defense mechanisms or ameliorating tissue damage. These and related concepts will be discussed along with issues that emerged from the workshop organized by the Stop TB Working Group on New Drugs, held at the Gordon Research Conference for Tuberculosis Drug Development in Lucca, Italy in June 2017, titled "Strategic Discussion on Repurposing Drugs & Host Directed Therapies for TB." In this review, we will highlight recent data regarding drugs, pathways, and concepts that are important for successful development of HDTs for TB.

Minocycline Immunomodulates via Sonic Hedgehog Signaling and Apoptosis and Has Direct Potency Against Drug-Resistant Tuberculosis.

Drug-resistant tuberculosis represents a global emergency, requiring new drugs. We found that minocycline was highly potent in laboratory strains of Mycobacterium tuberculosis and that 30 drug-susceptible and multidrug/extensively drug-resistant clinical strains were susceptible to clinically achievable concentrations. In the hollow fiber system model, lung concentration-time profiles of 7 mg/kg/day human-equivalent minocycline dose achieved bacterial kill rates equivalent to those of first-line antituberculosis agents. Minocycline killed extracellular bacilli directly. Minocycline also killed intracellular bacilli indirectly, via concentration-dependent granzyme A-driven apoptosis. Moreover, minocycline demonstrated dose-dependent antiinflammatory activity and downregulation of extracellular matrix-based remodeling pathways and, thus, could protect patients from tuberculosis immunopathology. In RNA sequencing of repetitive samples from the hollow fiber system and in independent protein abundance experiments, minocycline demonstrated dose-dependent inhibition of sonic hedgehog-patched-gli signaling. These findings have implications for improved lung remodeling and for dual immunomodulation and direct microbial kill-based treatment shortening regimens for drug-susceptible and drug-resistant latent and active M. tuberculosis infection.

Revisiting hypoxia therapies for tuberculosis.

The spectre of the coming post-antibiotic age demands novel therapies for infectious diseases. Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the single deadliest infection throughout human history. M. tuberculosis has acquired antibiotic resistance at an alarming rate with some strains reported as being totally drug resistant. Host-directed therapies (HDTs) attempt to overcome the evolution of antibiotic resistance by targeting relatively immutable host processes. Here, I hypothesise the induction of hypoxia via anti-angiogenic therapy will be an efficacious HDT against TB. I argue that anti-angiogenic therapy is a modernisation of industrial revolution era sanatoria treatment for TB, and present a view of the TB granuloma as a 'bacterial tumour' that can be treated with anti-angiogenic therapies to reduce bacterial burden and spare host immunopathology. I suggest two complementary modes of action, induction of bacterial dormancy and activation of host hypoxia-induced factor (HIF)-mediated immunity, and define the experimental tools necessary to test this hypothesis.

Precision medicine in the clinical management of respiratory tract infections including multidrug-resistant tuberculosis: learning from innovations in immuno-oncology.

PURPOSE OF REVIEW: In the light of poor management outcomes of antibiotic-resistant respiratory tract infection (RTI)-associated sepsis syndrome and multidrug-resistant tuberculosis (MDR-TB), new management interventions based on host-directed therapies (HDTs) are warranted to improve morbidity, mortality and long-term functional outcomes. We review developments in potential HDTs based on precision cancer therapy concepts applicable to RTIs including MDR-TB. RECENT FINDINGS: Immune reactivity, tissue destruction and repair processes identified during studies of cancer immunotherapy share common pathogenetic mechanisms with RTI-associated sepsis syndrome and MDR-TB. T-cell receptors (TCRs) and chimeric antigen receptors targeting pathogen-specific or host-derived mutated molecules (major histocompatibility class-dependent/ major histocompatibility class-independent) can be engineered for recognition by TCR γδ and natural killer (NK) cells. T-cell subsets and, more recently, NK cells are shown to be host-protective. These cells can also be activated by immune checkpoint inhibitor (ICI) or derived from allogeneic sources and serve as potential for improving clinical outcomes in RTIs and MDR-TB. SUMMARY: Recent developments of immunotherapy in cancer reveal common pathways in immune reactivity, tissue destruction and repair. RTIs-related sepsis syndrome exhibits mixed immune reactions, making cytokine or ICI therapy guided by robust biomarker analyses, viable treatment options.

CD4+ regulatory T cells and CD4+ activated T cells in new active and relapse tuberculosis.

The aim of the present study was to examine characteristics of tuberculosis (TB) patients with different clinical forms and to study the frequency of Regulatory T cells (Treg cells) and Activated T cells in patients with new active and relapse TB. Forty-five pulmonary TB patients and a control group of 15 healthy individuals were enrolled in this study. Of the 45 TB patients, 15 were new cases with drug-susceptible active TB and 30 were relapsed cases (15 drug-susceptible and 15 multidrug resistant-TB). The age of study participants ranged from 21 to 68 years old. According to sex presentation, males were appreciably highly affected than females with a sex ratio of 2. The patients reported a mean recent weight loss of 8.9 kg. The Erythrocyte Sedimentation Rate was high in TB group, far exceeding the normal value. The results revealed that the number of CD3+ CD4+ T-cells significantly decreased whereas the level of blood Treg cells and expression of activation markers CD38 and HLA-DR on CD4+ T-cells significantly increased in TB group compared with the control group (p<0.05). The frequency of Treg cells was significantly higher in the TB group than the control group. Both the patients with new active TB and relapse TB demonstrated significantly higher levels of CD4+FoxP3+ Treg compared to healthy subjects (p<0.05). A high and significant percentage of Treg cells were found in patients with DS active TB than patients with MDR relapse TB. Interestingly, the frequency of CD4+FoxP3+ cells also differs according to the sputum smear microscopy status. The presence of high numbers of Treg cells and corresponding high immune activation may be an unfavourable factor that can predispose individuals to different clinical forms of TB, including relapse TB.

Predicting the Outcomes of New Short-Course Regimens for Multidrug-Resistant Tuberculosis Using Intrahost and Pharmacokinetic-Pharmacodynamic Modeling.

Short-course regimens for multidrug-resistant tuberculosis (MDR-TB) are urgently needed. Limited data suggest that the new drug bedaquiline (BDQ) may have the potential to shorten MDR-TB treatment to less than 6 months when used in conjunction with standard anti-TB drugs. However, the feasibility of BDQ in shortening MDR-TB treatment duration remains to be established. Mathematical modeling provides a platform to investigate different treatment regimens and predict their efficacy. We developed a mathematical model to capture the immune response to TB inside a human host environment. This model was then combined with a pharmacokinetic-pharmacodynamic model to simulate various short-course BDQ-containing regimens. Our modeling suggests that BDQ could reduce MDR-TB treatment duration to just 18 weeks (4 months) while still maintaining a very high treatment success rate (100% for daily BDQ for 2 weeks, or 95% for daily BDQ for 1 week during the intensive phase). The estimated time to bacterial clearance of these regimens ranges from 27 to 33 days. Our findings provide the justification for empirical evaluation of short-course BDQ-containing regimens. If short-course BDQ-containing regimens are found to improve outcomes, then we anticipate clear cost savings and a subsequent improvement in the efficiency of national TB programs.

Severe BCG-osis Misdiagnosed as Multidrug-Resistant Tuberculosis in an IL-12Rß1-Deficient Peruvian Girl.

PURPOSE: Mendelian suceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency predisposing to severe disease caused by mycobacteria and other intracellular pathogens. Delay in diagnosis can have an impact on the patient's prognosis. METHODS: We evaluated the IFN-γ circuit by studying IFN-γ production after mycobacterial challenge as well as IL-12Rß1 expression and STAT4 phosphorylation in response to IL-12p70 stimulation in whole blood of a 6-year-old Peruvian girl with disseminated recurrent mycobacterial infection diagnosed as multidrug-resistant tuberculosis. Genetic studies with Sanger sequencing were used to identify the causative mutation. Microbiological studies based on PCR reactions were used to diagnose the specific mycobacterial species. RESULTS: We identified a homozygous mutation in the IL12RB1 gene (p. Arg211*) causing abolished expression of IL-12Rß1 and IL-12 response. MSMD diagnosis led to a microbiological reevaluation of the patient, revealing a BCG vaccine-related infection instead of tuberculosis. Treatment was then adjusted, with good response. CONCLUSIONS: We report the first Peruvian patient with IL-12Rß1 deficiency. Specific mycobacterial species diagnosis within Mycobacterium tuberculosis complex is still challenging in countries with limited access to PCR-based microbiological diagnostic techniques. Awareness of MSMD warning signs and accurate microbiological diagnosis of mycobacterial infections are of the utmost importance for optimal diagnosis and management of affected patients.

Subpopulation Structure of IFNγ-Producing T Lymphocytes in Patients with Pulmonary Tuberculosis.

The study of subpopulation structure of IFNγ-producing T cells in patients with pulmonary tuberculosis revealed a decrease in the number of CD3+ IFNγ+ cells against the background of significantly increased IFNγ secretion in vitro irrespective of the clinical form of the disease and drug sensitivity of M. tuberculosis, most strongly expressed in case of the disseminated tuberculosis. In patients with infiltrative drug-sensitive and drug-resistant pulmonary tuberculosis, increased number of Th1/Th17 lymphocytes (CD4+ IFNγ+IL-17A+) and, conversely, decreased number of blood γδT cells was detected.

Mycobacterium tuberculosis multi-drug-resistant strain M induces IL-17+ IFNγ- CD4+ T cell expansion through an IL-23 and TGF-ß-dependent mechanism in patients with MDR-TB tuberculosis.

We have reported previously that T cells from patients with multi-drug-resistant tuberculosis (MDR-TB) express high levels of interleukin (IL)-17 in response to the MDR strain M (Haarlem family) of Mycobacterium tuberculosis (M. tuberculosis). Herein, we explore the pathways involved in the induction of Th17 cells in MDR-TB patients and healthy tuberculin reactors [purified protein derivative healthy donors (PPD+ HD)] by the M strain and the laboratory strain H37Rv. Our results show that IL-1ß and IL-6 are crucial for the H37Rv and M-induced expansion of IL-17+ interferon (IFN)-γ- and IL-17+ IFN-γ+ in CD4+ T cells from MDR-TB and PPD+ HD. IL-23 plays an ambiguous role in T helper type 1 (Th1) and Th17 profiles: alone, IL-23 is responsible for M. tuberculosis-induced IL-17 and IFN-γ expression in CD4+ T cells from PPD+ HD whereas, together with transforming growth factor (TGF-ß), it promotes IL-17+ IFN-γ- expansion in MDR-TB. In fact, spontaneous and M. tuberculosis-induced TGF-ß secretion is increased in cells from MDR-TB, the M strain being the highest inducer. Interestingly, Toll-like receptor (TLR)-2 signalling mediates the expansion of IL-17+ IFN-γ- cells and the enhancement of latency-associated protein (LAP) expression in CD14+ and CD4+ T cells from MDR-TB, which suggests that the M strain promotes IL-17+ IFN-γ- T cells through a strong TLR-2-dependent TGF-ß production by antigen-presenting cells and CD4+ T cells. Finally, CD4+ T cells from MDR-TB patients infected with MDR Haarlem strains show higher IL-17+ IFN-γ- and lower IL-17+ IFN-γ+ levels than LAM-infected patients. The present findings deepen our understanding of the role of IL-17 in MDR-TB and highlight the influence of the genetic background of the infecting M. tuberculosis strain on the ex-vivo Th17 response.

Antigen 85 complex as a powerful Mycobacterium tuberculosis immunogene: Biology, immune-pathogenicity, applications in diagnosis, and vaccine design.

Mycobacterium tuberculosis (Mtb) is one of the most life-threatening mycobacterial species which is increasing the death rate due to emerging multi-drug resistant (MDR) strains. Concerned health authorities worldwide are interested in developing an effective vaccine to prevent the spread of Mtb. After years of research, including successful identification of many Mtb immunogenic molecules, effective therapeutic agents or a vaccine have yet to be found. However, among the identified Mtb immunogenes, antigen 85 (Ag85) complex (Ag85A, Ag85B, and Ag85C) is receiving attention from scientists as it allows bacteria to evade the host immune response by preventing formation of phagolysosomes for eradication of infection. Due to their importance, A85 molecules are being utilized as tools in diagnostic methods and in the construction of new vaccines, such as recombinant attenuated vaccines, DNA vaccines, and subunit vaccines. This paper represents a comprehensive review of studies on Mtb molecules examining pathogenicity, biochemistry, immunology, and the role of Mtb in therapeutic or vaccine research.

Association of Immune Factors with Drug-Resistant Tuberculosis: A Case-Control Study.

BACKGROUND Presently, studies of factors associated with drug-resistant tuberculosis (TB) focus on patients' socio-demographic characteristics and living habits, to the exclusion of biochemical indicators, especially immune factors. This study was carried out to determine whether immune factors are associated with drug-resistant TB. MATERIAL AND METHODS A total of 227 drug-resistant pulmonary TB patients and 225 drug-susceptible pulmonary TB patients were enrolled in this study. Information on socio-demographic characteristics and biochemical indicators were obtained through their clinical records. Non-conditional logistic regression was used to analyze the association of these indicators with drug-resistant TB. RESULTS There were significant differences in re-treatment, marital status, alanine aminotransferase (ALT), blood uric acid (BUA), carcino-embryonic antigen (CEA), T-spot, and CD3 and CD4 counts between the 2 groups. In multivariable analysis, re-treatment [Odds Ratio (OR)=5.290, 95% Confidence Interval [CI]=2.652-10.551); CD3 (OR=1.034, 95% CI=1.001-1.068); CD4 (OR=1.035, 95% CI =1.001-1.070) and IgM (OR=1.845, 95% CI=1.153-2.952) were associated with drug-resistant TB. CONCLUSIONS These results suggest the need for greater attention to re-treatment cases and immune function when treating drug-resistant TB.

Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo.

Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.

Advanced immune suppression is associated with increased prevalence of mixed-strain Mycobacterium tuberculosis infections among persons at high risk for drug-resistant tuberculosis in Botswana.

We examined factors associated with mixed-strain Mycobacterium tuberculosis infections among patients at high risk for drug-resistant tuberculosis in Botswana. Thirty-seven (10.0%) of 370 patients with tuberculosis had mixed M. tuberculosis infections, based on 24-locus mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping. In log-binomial regression analysis, age <37 years (adjusted prevalence ratio [PR], 1.92; 95% confidence interval [CI], 1.01-3.57) and prior tuberculosis treatment (adjusted PR, 2.31; 95% CI, 1.09-4.89) were associated with mixed M. tuberculosis infections. Among human immunodeficiency virus-infected patients, prior tuberculosis treatment (adjusted PR, 2.11; 95% CI, 1.04-4.31) and CD4(+) T-cell count of <100 cells/µl (adjusted PR, 10.18; 95% CI, 2.48-41.71) were associated with mixed M. tuberculosis infections. Clinical suspicion of mixed M. tuberculosis infections should be high for patients with advanced immunosuppression and a prior history of tuberculosis treatment.