Tuberculosis Infection in Children and Adolescents: Testing and Treatment.

Tuberculosis (TB) remains an important problem among children in the United States and throughout the world. There is no diagnostic reference standard for latent tuberculosis infection (also referred to as tuberculosis infection [TBI]). The tuberculin skin test (TST) has many limitations, including difficulty in administration and interpretation, the need for a return visit by the patient, and false-positive results caused by cross-reaction with Mycobacterium bovis-bacille Calmette-Guerin vaccines and many nontuberculous mycobacteria. Interferon-gamma release assays (IGRAs) are blood tests that use antigens specific for M tuberculosis; as a result, IGRAs yield fewer false-positive results than the TST. Both IGRAs and the TST have reduced sensitivity in immunocompromised children, including children with severe TB disease. Both methods have high positive predictive value when applied to children with risk factors for TBI, especially recent contact with a person who has TB disease. The advantages of using IGRAs and diminished experience with the placement and interpretation of the TST favor expanded use of IGRAs in children in the United States. There are now several effective and safe regimens for the treatment of TBI in children. For improved adherence to therapy, the 3 rifamycin-based regimens are preferred because of their short duration. Daily isoniazid can be used if there is intolerance or drug interactions with rifamycins. A TB specialist should be involved when there are questions regarding testing interpretation, selection of an appropriate treatment regimen, or management of adverse effects.

The Trend of TIM3 Expression on T Cells in Patients With Nontuberculous Mycobacterial Lung Disease: From Immune Cell Dysfunction to Clinical Severity.

Background: The incidence of nontuberculous mycobacterial lung disease (NTM-LD) is increasing worldwide. Immune exhaustion has been reported in NTM-LD, but T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), a co-inhibitory receptor on T cells, has been scarcely studied. Methods: Patients with NTM-LD and healthy controls were prospectively recruited from July 2014 to August 2019 at three tertiary referral centers in Taiwan. We examined TIM3 expression on the T cells from the participants using flow cytometry. TIM3 expression was analyzed for different disease statuses and after treatment. The apoptosis and cytokine profiles were analyzed according to the TIM3 expression. Results: Among enrolled subjects (47 patients and 46 controls), TIM3 on CD4+ cells (6.44% vs. 4.12%, p = 0.028) and CD8+ cells (18.47% vs. 9.13%, p = 0.003) were higher in NTM-LD patients than in the controls. The TIM3 level on CD4+ and CD8+ T cells was positively associated with T-cell apoptosis in the NTM-LD patients. In stimulating peripheral blood mononuclear cells using PMA plus ionomycin, a high TIM3 level on T cells correlated with low interleukin-2 and tumor necrosis factor-alpha (TNF-α) on CD4+ cells and interferon-gamma and TNF-α on CD8+ T cells. For clinical manifestation, low body mass index (BMI), positive sputum acid-fast smear, and high radiographic score correlated with high TIM3 expression on T cells. After NTM treatment, TIM3+ decreased significantly on CD4+ and CD8+ T cells. Conclusions: In patients with NTM-LD, TIM3+ expression increased over CD4+ and CD8+ T cells and correlated with cell apoptosis and specific cytokine attenuation. Clinically, TIM3+ T cells increased in patients with low BMI, high disease extent, and high bacilli burden but decreased after treatment.

Autophagy and Host Defense in Nontuberculous Mycobacterial Infection.

Autophagy is critically involved in host defense pathways through targeting and elimination of numerous pathogens via autophagic machinery. Nontuberculous mycobacteria (NTMs) are ubiquitous microbes, have become increasingly prevalent, and are emerging as clinically important strains due to drug-resistant issues. Compared to Mycobacterium tuberculosis (Mtb), the causal pathogen for human tuberculosis, the roles of autophagy remain largely uncharacterized in the context of a variety of NTM infections. Compelling evidence suggests that host autophagy activation plays an essential role in the enhancement of antimicrobial immune responses and controlling pathological inflammation against various NTM infections. As similar to Mtb, it is believed that NTM bacteria evolve multiple strategies to manipulate and hijack host autophagy pathways. Despite this, we are just beginning to understand the molecular mechanisms underlying the crosstalk between pathogen and the host autophagy system in a battle with NTM bacteria. In this review, we will explore the function of autophagy, which is involved in shaping host-pathogen interaction and disease outcomes during NTM infections. These efforts will lead to the development of autophagy-based host-directed therapeutics against NTM infection.

Cytokine/Chemokine Release Patterns and Transcriptomic Profiles of LPS/IFNγ-Activated Human Macrophages Differentiated with Heat-Killed Mycobacterium obuense, M-CSF, or GM-CSF.

Macrophages (Mφs) are instrumental regulators of the immune response whereby they acquire diverse functional phenotypes following their exposure to microenvironmental cues that govern their differentiation from monocytes and their activation. The complexity and diversity of the mycobacterial cell wall have empowered mycobacteria with potent immunomodulatory capacities. A heat-killed (HK) whole-cell preparation of Mycobacterium obuense (M. obuense) has shown promise as an adjunctive immunotherapeutic agent for the treatment of cancer. Moreover, HK M. obuense has been shown to trigger the differentiation of human monocytes into a monocyte-derived macrophage (MDM) type named Mob-MDM. However, the transcriptomic profile and functional properties of Mob-MDMs remain undefined during an activation state. Here, we characterized cytokine/chemokine release patterns and transcriptomic profiles of lipopolysaccharide (LPS)/interferon γ (IFNγ)-activated human MDMs that were differentiated with HK M. obuense (Mob-MDM(LPS/IFNγ)), macrophage colony-stimulating factor M-MDM(LPS/IFNγ)), or granulocyte/macrophage colony-stimulating factor (GM-MDM(LPS/IFNγ)). Mob-MDM(LPS/IFNγ) demonstrated a unique cytokine/chemokine release pattern (interleukin (IL)-10low, IL-12/23p40low, IL-23p19/p40low, chemokine (C-x-C) motif ligand (CXCL)9low) that was distinct from those of M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ). Furthermore, M-MDM(LPS/IFNγ) maintained IL-10 production at significantly higher levels compared to GM-MDM(LPS/IFNγ) and Mob-MDM(LPS/IFNγ) despite being activated with M1-Mφ-activating stimuli. Comparative RNA sequencing analysis pointed to a distinct transcriptome profile for Mob-MDM(LPS/IFNγ) relative to both M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ) that comprised 417 transcripts. Functional gene-set enrichment analysis revealed significant overrepresentation of signaling pathways and biological processes that were uniquely related to Mob-MDM(LPS/IFNγ). Our findings lay a foundation for the potential integration of HK M. obuense in specific cell-based immunotherapeutic modalities such as adoptive transfer of Mφs (Mob-MDM(LPS/IFNγ)) for cancer treatment.

Nontuberculous Mycobacteria, Macrophages, and Host Innate Immune Response.

Although nontuberculous mycobacteria (NTM) are considered opportunistic infections, incidence and prevalence of NTM infection are increasing worldwide becoming a major public health threat. Innate immunity plays an essential role in mediating the initial host response against these intracellular bacteria. Specifically, macrophages phagocytose and eliminate NTM and act as antigen-presenting cells, which trigger downstream activation of cellular and humoral adaptive immune responses. Identification of macrophage receptors, mycobacterial ligands, phagosome maturation, autophagy/necrosis, and escape mechanisms are important components of this immunity network. The role of the macrophage in mycobacterial disease has mainly been studied in tuberculosis (TB), but limited information exists on its role in NTM. In this review, we focus on NTM immunity, the role of macrophages, and host interaction in NTM infection.

Non-tuberculous mycobacteria immunopathogenesis: Closer than they appear. a prime of innate immunity trade-off and NTM ways into virulence.

INTRODUCTION: The growing incidence of non-tuberculous mycobacteria (NTM) and changes in epidemiological factors have indicated that immune dysregulation may be associated with the emergence of NTM. Minireview entails to acknowledge complex interaction and new ways NTM are evolving around diverse immune status. METHODS: In order to perform this review, we selected peer reviewed, NLM database articles under the terms NTM, mycobacterium complex 'AND' -Host- immune response, immunity regulation, Disease, Single Nucleotide Polymorphism (SNP´s), and -pathogen- followed by a snow ball rolling basis search on immune components and NTM related with diseases distribution. RESULTS: The universal exposure and diversity of NTM are well-documented; however, hospitals seldom establish vigilant control of water quality or immunodeficiencies for patients with NTM infections. Depending on the chemical structures and immune mechanisms presented by various NTM varieties, they can trigger different effects in dendritic and natural killer cells, which release interleukin (IL)-17, tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and rIL-1B. The T helper (Th)2-acquired immune response is responsible for autoimmune responses in patients with NTM infections, and, quite disturbingly, immunocompetent patients have been reported to suffer from NTM infections. CONCLUSION: New technologies and a comprehensive view has taught us; to acknowledge metabolic/immune determinants and trade-offs along transit through mutualism-parasite continuous.

Generation of mycobacterial lipoarabinomannan-specific monoclonal antibodies and their ability to identify mycobacterium isolates.

BACKGROUND/PURPOSE: The World Health Organization has recommended commercial urine-sourced lipoarabinomannan (LAM) detection as a tool for screening HIV patients with suspected TB, but more sensitive immunodetection assays would help to identify HIV-negative TB patients. Here, we aimed to develop novel rabbit monoclonal antibodies (mAbs) against LAM for immunodetection purposes. METHODS: Rabbits were immunized with cell-wall components from the Mycobacterium tuberculosis (Mtb) H37Rv strain. An immune single-chain fragment variable (scFv) phage display library was generated. The scFv mAbs to LAM were identified through ELISA screening. The light and heavy chain variable region genes from the selected clones were sequenced. Vectors containing the full-length light and heavy chains were constructed and co-expressed in 293 T cells to generate whole IgG antibodies. The performances and binding characteristics of the mAbs against purified LAM from M.tb H37Rv, multiple mycobacteria species (M.tb H37Rv, M. bovis and non-tuberculous mycobacteria (NTM) strains), and mycobacteria clinical isolates (Mtb and NTM isolates) were determined using various immunoassay methods. RESULTS: We obtained five rabbit mAbs against LAM, four of which had high sensitivities (100 pg/ml) and affinities (1.16-1.73 × 10-9 M) toward LAM. They reacted with M.tb H37Rv, M. bovis, and slow-growing NTM, but not with rapid-growing NTM. Similar results were obtained with mycobacterium isolates, where 96% of the Mtb isolates and 90% of the M. avium-intracellulare isolates were successfully identified. CONCLUSION: The novel rabbit LAM-specific mAbs performed well at recognizing LAM from slow-growing pathogenic mycobacteria, which support their future clinical application.

Mitigating Coronavirus Induced Dysfunctional Immunity for At-Risk Populations in COVID-19: Trained Immunity, BCG and "New Old Friends".

The novel, highly contagious coronavirus SARS-CoV-2 spreads rapidly throughout the world, leading to a deadly pandemic of a predominantly respiratory illness called COVID-19. Safe and effective anti-SARS-CoV-2 vaccines are urgently needed. However, emerging immunological observations show hallmarks of significant immunopathological characteristics and dysfunctional immune responses in patients with COVID-19. Combined with existing knowledge about immune responses to other closely related and highly pathogenic coronaviruses, this could forebode significant challenges for vaccine development, including the risk of vaccine failure. Animal data from earlier coronavirus vaccine efforts indicate that elderly people, most at risk from severe COVID-19 disease, could be especially at risk from immunopathologic responses to novel coronavirus vaccines. Bacterial "new old friends" such as Bacille Calmette-Guérin (BCG) or Mycobacterium obuense have the ability to elevate basal systemic levels of type 1 cytokines and immune cells, correlating with increased protection against diverse and unrelated infectious agents, called "trained immunity." Here we describe dysfunctional immune responses induced by coronaviruses, representing potentially difficult to overcome obstacles to safe, effective vaccine development for COVID-19, and outline how trained immunity could help protect high risk populations through immunomodulation with BCG and other "new old friends."

Detection of Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus) using an interferon gamma release assay in a captive elephant herd.

Tuberculosis is highly contagious disease that can be transmitted between humans and animals. Asian elephants (Elephas maximus) in captivity live in close contact with humans in many Asian countries. In this study, we developed an interferon gamma release assay (IGRA) for elephant TB detection using antigens from the MTB complex (MTBC) and nontuberculous mycobacteria (NTM) as stimulating antigens (PPD, ESAT6, CFP10) to elicit a cell-mediated immune response (CMIR). The developed assay was applied to an elephant herd of more than 60 animals in Thailand, and the results were compared with those obtained through serological detection. IGRA has sufficient sensitivity for detecting elephant interferon gamma (eIFNγ) from specific antigen-stimulated PBMCs. Among 60 animals tested, 20 samples (33.3%) showed negative results for both MTBC and NTM infection. Eighteen samples (30%) showed positive responses against PPD from M. bovis and/or ESAT6 and CFP10, indicating MTBC infection. In contrast, only 15.6% showed seropositivity in a commercial serological test kit for elephant TB. The discrepancies between serological and CMIR highlight that the two methods may detect different stages of elephant TB. Therefore, employing both tests may enable them to complement each other in correctly identifying elephants that have been exposed to MTBC.

Bacille Calmette Guérin (BCG) and new TB vaccines: Specific, cross-mycobacterial and off-target effects.

The Bacille Calmette Guérin (BCG) vaccine was developed over a century ago and has become one of the most used vaccines without undergoing a modern vaccine development life cycle. Despite this, the vaccine has protected many millions from severe and disseminated forms of tuberculosis (TB). In addition, BCG has cross-mycobacterial effects against non-tuberculous mycobacteria and off-target (also called non-specific or heterologous) effects against other infections and diseases. More recently, BCG's effects on innate immunity suggest it might improve the immune response against viral respiratory infections including SARS-CoV-2. New TB vaccines, developed over the last 30 years, show promise, particularly in prevention of progression to disease from TB infection in young adults. The role of BCG in the context of new TB vaccines remains uncertain as most participants included in trials have been previously BCG immunised. BCG replacement vaccines are in efficacy trials and these may also have off-target effects.

Host Immune Response and Novel Diagnostic Approach to NTM Infections.

The incidence and prevalence of non-tuberculous mycobacteria (NTM) infections are steadily increasing worldwide, partially due to the increased incidence of immunocompromised conditions, such as the post-transplantation state. The importance of proper diagnosis and management of NTM infection has been recently recognized. Host immunological responses play integral roles in vulnerability to NTM infections, and may contribute to the onset of specific types of NTM infection. Furthermore, distinct NTM species are known to affect and attenuate these host immune responses in unique manners. Therefore, host immune responses must be understood with respect to each causative NTM species. Here, we review innate, cellular-mediated, and humoral immunity to NTM and provide perspectives on novel diagnostic approaches regarding each NTM species.

BCG Infection due to MPT64-Negative Strain: A Diagnostic Challenge.

MPT64 is a 24-kDa immunogenic protein that is widely used as a diagnostic marker for the differentiation of Mycobacterium tuberculosis complex (MTBC) from nontuberculous Mycobacterium (NTM). Unlike Mycobacterium bovis, Bacillus Calmette-Guerin (BCG) vaccine strains with RD2 deletion do not secrete MPT64. Culture isolates from infections due to these strains may be falsely identified as nontuberculous Mycobacterium in the absence of clinical correlation. Here, we present one case each of BCG adenitis and osteitis, both of which were considered as MPT64 card-negative Mycobacterium spp. (i.e., NTM) and were later identified as M. bovis BCG Danish 1331 strain. The first case was a 4-month-old female infant admitted with swollen lymph nodes in the left supraclavicular and the left axillary region of 1 month duration. The second case was of a 1-year-and-5-month-old male child who presented with a limp on the left leg and soft tissue swelling of 1 month duration on the anterolateral aspect of the left knee joint. In both cases, BCG vaccine was administered at birth on the left deltoid region and had healed without any complication. Clinical samples in both cases were positive by Xpert tuberculosis/RIF for MTBC, and cultures grew acid-fast bacilli which were negative by MPT64 assay. The clinical implication of infections due to M. bovis BCG is immense as they are inherently resistant to pyrazinamide, and the presence of disseminated BCG infection in young children is a hallmark of serious immune deficiency which needs to be ruled out.

Evaluation of Mycobacterium kansasii extracellular vesicles role in BALB/c mice immune modulatory.

Background: Mycobacterium kansasii as a nontuberculosis mycobacteria, naturally release extracellular vesicles (EVs) with widespread utilities. The aim of the present study was the extraction and biological evaluation of M. kansasii EV and its role in BALB/c mice immune modulatory by considering EVs medical usage specificities. Method: Density gradient ultracentrifugation method was used to EVs extraction from standard species of M. kansasii. Biologic validation of EVs has been performed by physicochemical experiments. Immunization has been done by subcutaneous injection to BALB/c mice, then spleen cell isolation and lymphocyte transformation test and eventually ELISA cytokine assays were made for interleukin-10 (IL-10) and interferon-gamma (IFN-γ).IBM SPSS version 22 software (SPSS. Inc., Chicago, IL, USA) was used for the data calculation. The evaluation of variables was conducted using one sample t-test. Results: Physicochemical experiment results contribute that extracted EVs have intransitive capability to use in immunization schedule. Finally, ELISA test results showed that EVs induced IL-10 production, but have no effect on IFN-γ. Conclusions: In this current study, EVs were prepared in high-quality composition. The results of cytokine assay revealed that the extracted EVs have anti-inflammatory property. Accordingly, this macromolecule can be used as immune modulatory agents to prevent severe immune reactions, especially in lungs disorders.

Host immune response against environmental nontuberculous mycobacteria and the risk populations of nontuberculous mycobacterial lung disease.

Nontuberculous mycobacterial lung disease (NTM-LD) prevalence has been increasing over the recent decades. Numerous host factors are associated with NTM-LD development, including susceptible phenotypes such as ciliary defect and lung structural change, pulmonary clearance defect with poor clearance of secretions, and immune suppression. Specifically, regarding the susceptible host phenotypes without clear pathogenesis, a slender body, pectus excavatum, and postmenopausal female status are common. Also, decreased host immunity to NTM, especially T helper 1 cell responses is frequently observed. Even so, the underlying mechanisms remain unclear and relevant large-scale studies are lacking. Infections due to host genetics associated defects are mostly untreatable but rare in Asia, particularly Taiwan. Nevertheless, some risks for NTM-LD are controllable over disease progression. We suggest that clinicians first manage host factors and deal with the controllable characteristics of NTM-LD, followed by optimizing anti-NTM treatment. Further researches focusing on NTM-LD pathogenesis, especially the host-NTM interaction may advance understanding the nature of the disease and develop efficient therapeutic regimens.

Incidence, Clinical Manifestation, Treatment Outcome, and Drug Susceptibility Pattern of Nontuberculous Mycobacteria in HIV Patients in Tehran, Iran.

BACKGROUND: Nontuberculous mycobacterial (NTM) infections have radically increased worldwide due to the increase in HIV infections. The disease activity increases with progressive immunodeficiency. METHODS: A total of 216 HIV seropositive patients suspected of having mycobacterial infection were recruited for this study. Clinical samples were collected from each patient and cultured on Lowenstein-Jensen media. Detection and species identification were simultaneously done using Reverse Blot Hybridization Assay System. Also, the minimum inhibitory concentrations (MIC) for each isolate were determined in 7H9 broth media for 10 antibiotics. RESULTS: In this study, 4 rapid and 4 slow-growing NTM species were isolated and identified. Mycobacterium fortuitum was the most common NTM species, 3/8 (37.5%), followed by Mycobacterium kansasii, 2/8 (25%). The cases were identified as pulmonary disease, 5/8 (62.5 %), disseminated infection, 2/8 (25%), and skin abscess, 1/8 (12.5%). M. chelonae and Mycobacterium avium were isolated from patients diagnosed with disseminated infection with treatment failure. The skin abscess was caused by infection with M. simiae. The results of the MIC testing were as follows: M. kansasii and M. fortuitum were susceptible to amikacin (AMK); M. avium to clarithromycin (CLA); M. fortuitum 2/3 (67%) to ciprofloxacin (CIP); 1/2 (50%) of M. kansasii isolates to CLA, and M. chelonae to rifampin (RIF), linezolid (LIN), AMK, and CIP at medium and high concentrations. CONCLUSION: AMK showed incredible in vitro activity against M. kansasii and M. fortuitum. Also, M. avium was susceptible to CLA, whereas M. simiae and M. chelonae were resistant to the tested drugs in this study.

Beyond antibiotics for pulmonary nontuberculous mycobacterial disease.

PURPOSE OF REVIEW: Pulmonary nontuberculous mycobacterial disease (NTM) remains a significant clinical challenge with suboptimal therapy. This review focuses on recent understandings around the pathogenesis of NTM disease and nonantibiotic therapeutic approaches that are being pursued. RECENT FINDINGS: The absence of animal models that truly replicate human disease remains a major problem for NTM research with most findings coming from tuberculosis or tuberculosis-like studies. Recent research reiterates the known key roles of interferon gamma (IFNγ), tumor necrosis factor, interleukin-12 and granulocyte-macrophage colony stimulation factor (GM-CSF) in immunity to NTM. Autoantibodies to some of these factors may be important. Recent nonantibiotic research has focused on either boosting the immune response to NTM (e.g. with IFNγ or GM-CSF) or using other compounds to kill these pathogens (e.g. inhaled NO, gallium, etc.). SUMMARY: Our poor understanding of the immune deficit leading to NTM disease continues to hinder the development of highly effective therapies. New approaches are promising but need significant validation before being considered viable therapeutic options.

Mycobacterium indicus pranii therapy induces tumor regression in MyD88- and TLR2-dependent manner.

OBJECTIVES: Mycobacterium indicus pranii (MIP) is an atypical mycobacterium species with potent antitumor efficacy. Macrophages and dendritic cells (DCs) are antigen-presenting cells, playing key roles in the activation of antitumor immunity. We have previously shown the potent activation of macrophages and DCs by MIP, which is mediated by MyD88-TLR2 signaling axis. In the present study, we further examined the role of MyD88 and TLR2 in MIP-mediated tumor regression. RESULTS: Wild-type and MyD88-/- mice were implanted with B16F10 tumor cells, treated with MIP or phosphate-buffered saline (PBS) and monitored for tumor growth. As expected, MIP therapy led to significant tumor regression in wild-type mice. However, antitumor efficacy of MIP was lost in MyD88-/- animals. Both PBS-treated (control) and MIP-treated MyD88-/- mice developed tumors with comparable volume. Since MyD88 relays TLR engagement signals, we analyzed the antitumor efficacy of MIP in TLR2-/- and TLR4-/- mice. It was observed that MIP therapy reduced tumor burden in wild-type and TLR4-/- mice but not in TLR2-/- mice. Tumor volume in MIP-treated TLR2-/- mice were comparable with those in PBS-treated wild-type animals. These results implicated the MyD88-TLR2 signaling axis in the antitumor efficacy of MIP.

Sandwich antibody-based biosensor system for identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria.

Mycobacterial infection, leading to pulmonary disease, remains a world health problem. Clinical symptoms of pulmonary disease caused by Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) are very similar. A rapid method for the differentiation of MTBC and NTM infection is essential for appropriate therapy. In this study, we aim to establish an antibody-based biosensor system for the identification of MTBC and NTM infection. Monoclonal antibodies (mAbs) specific for Ag85B proteins of mycobacteria were generated and characterized. The generated anti-Ag85B mAb clones AM85B-5 and AM85B-8 reacted to Ag85B of Mycobacterium spp.; in contrast, clone AM85B-9 specifically reacted to Ag85B of MTBC. By employing the produced mAbs, single and sandwich antibody-based biosensors using bio-layer interferometry were established for determination of Ag85B proteins. The sandwich antibody-based biosensor system was demonstrated to be suitable for detection of Ag85B protein and identification of MTBC and NTM. Using anti-Ag85B mAbs AM85B-8 and AM85B-9 as immobilized antibodies on sensor chips and using mAb AM85B-5 as secondary antibody, the established sandwich antibody-based biosensor could discriminate MTBC and NTM. The developed biosensor system can be used for culture confirmation of mycobacteria and speciation to MTBC and NTM.