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Background: Tuberculosis (TB) though primarily affects the lungs it may also affect the other parts of the body and referred as extra pulmonary (EPTB). This study is focused on understanding the genetic diversity and molecular epidemiology of Mycobacterium tuberculosis (M.tb) among tuberculous lymphadenitis (TBL), a form of EPTB patients identified in Chennai, Tamil Nadu., Methods: The genetic diversity was identified by performing spoligotyping on the M.tb clinical isolates that were recovered from lymph node samples. A total of 71 M.tb isolates were recovered from extra pulmonary lymph node samples and subjected to Drug susceptibility testing and spoligotyping was carried out. In addition, immunological characterization from blood of same individuals from whom M.tb was isolated was carried out between the two major lineages groups East African Indian 3 (EAI3) and non-EAI3 strains by ELISA. The results of spoligotyping patterns were compared with the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4)., Results: We found 41 spoligotype patterns and their associated lineages. Out of 41 spoligotype pattern, only 22 patterns are available in the spoldB4 database with Spoligotype international Type (SIT) number and remaining patterns were orphan strains without SIT number. The most predominant spoligotype lineage that was found in lymph node sample in this region of India was EAI (36), followed by central Asian strain (CAS) (6), T1 (5), Beijing (3), Latin American & Mediterranean (LAM) (2), U (1), X2 (1) and orphan (22). In addition to EAI, CAS and Beijing, our study identified the presence of orphan and unique spoligotyping patterns in Chennai region. We observed six drug resistant isolates. Out of six drug resistant isolates, four were resistant to isoniazid drug and associated with EAI family. Moreover, we observed increased levels of type 2 and type 17 cytokine profiles between EAI3 and non-EAI family, infected individuals., Conclusions: The study confirms that EAI lineage to be the most predominant lineages in EPTB patients with lymphadenitis and were found to have increased type 1 and type 17 proinflammatory cytokine profiles.

Filarial infections are known to modulate cytokine responses in pulmonary tuberculosis by their propensity to induce Type 2 and regulatory cytokines. However, very little is known about the effect of filarial infections on extra-pulmonary forms of tuberculosis. Thus, we have examined the effect of filarial infections on the plasma levels of various families of (IL-1, IL-12, γC, and regulatory) cytokines and (CC and CXC) chemokines in tuberculous lymphadenitis coinfection. We also measured lymph node culture grades in order to assess the burden of Mycobacterium tuberculosis in the two study groups [Fil+ ( n = 67) and Fil- ( n = 109)]. Our data reveal that bacterial burden was significantly higher in Fil+ compared to Fil- individuals. Plasma levels of IL-1 family (IL-1α, IL-β, IL-18) cytokines were significantly lower with the exception of IL-33 in Fil+ compared to Fil- individuals. Similarly, plasma levels of IL-12 family cytokines -IL-12 and IL-23 were significantly reduced, while IL-35 was significantly elevated in Fil+ compared to Fil- individuals. Filarial infection was also associated with diminished levels of IL-2, IL-9 and enhanced levels of IL-4, IL-10, and IL-1Ra. Similarly, the Fil+ individuals were linked to elevated levels of different CC (CCL-1, CCL-2, CCL-3, CCL-11) and CXC (CXCL-2, CXCL-8, CXCL-9, CXCL-11) chemokines. Therefore, we conclude that filarial infections exert powerful bystander effects on tuberculous lymphadenitis, effects including modulation of protective cytokines and chemokines with a direct impact on bacterial burdens., (Copyright © 2020 Kathamuthu, Munisankar, Banurekha, Nair, Sridhar and Babu.)

Background: Helminth infections are known to regulate cytokine responses in both pulmonary and latent tuberculosis infection. Whether helminth infections also modulate cytokine responses in extra-pulmonary tuberculosis, specifically tuberculous lymphadenitis (TBL), has not been examined thus far., Methodology: Hence, to determine the cytokine profile in helminth-TBL coinfection, we measured the systemic and mycobacterial (TB)-antigen stimulated levels of Type 1, Type 2, Type 17, regulatory and pro-inflammatory cytokines in TBL individuals coinfected with or without Strongyloides stercoralis (Ss) infection., Significant Findings: TBL-Ss+ individuals have significantly higher bacterial burdens in the affected lymph nodes in comparison to TBL-Ss- individuals. TBL-Ss+ individuals exhibit significantly enhanced plasma levels of Type 2 (IL-5 and IL-13), Type 17 (IL-17 and IL-22) and regulatory (IL-10) cytokines in comparison to TBL-Ss- individuals. In contrast, TBL-Ss+ individuals exhibit significantly diminished plasma levels of pro-inflammatory cytokines (IL-1α and GM-CSF) in comparison to TBL-Ss- individuals. TBL-Ss+ individuals also exhibit significantly diminished unstimulated or mycobacterial-antigen stimulated levels of Type 1, Type 17 or IL-1 family cytokines in comparison to TBL-Ss- individuals but no differences in mitogen stimulated cytokine levels., Conclusion: Therefore, our data reveal a profound influence of Ss infection on the bacteriological profile of TBL and suggesting that the underlying modulation of cytokine responses might be a mechanism by which this helminth infection could impart a detrimental effect on the pathogenesis of TBL disease., Competing Interests: The authors have declared that no competing interests exist.

Background: Lymph node tuberculosis (LNTB) is the most frequent extrapulmonary form of tuberculosis (TB). Studies of human tuberculosis at sites of disease are limited. LNTB provides a unique opportunity to compare local in situ and peripheral blood immune response in active Mycobacterium tuberculosis (Mtb) disease. The present study analysed T regulatory cells (Treg) frequency and activation along with CD4+ T cell function in lymph nodes from LNTB patients., Results: Lymph node mononuclear cells (LNMC) were compared to autologous peripheral blood mononuclear cells (PBMC). LNMC were enriched for CD4+ T cells with a late differentiated effector memory phenotype. No differences were noted in the frequency and mutifunctional profile of memory CD4+ T cells specific for Mtb. The proportion of activated CD4+ and Tregs in LNMC was increased compared to PBMC. The correlation between Tregs and activated CD4+ T cells was stronger in LNMC than PBMC. Tregs in LNMC showed a strong positive correlation with Th1 cytokine production (IL2, IFNγ and TNFα) as well as MIP-1α after Mtb antigen stimulation. A subset of Tregs in LNMC co-expressed HLA-DR and CD38, markers of activation., Conclusion: Further research will determine the functional relationship between Treg and activated CD4+ T cells at lymph node sites of Mtb infection.

Tuberculosis is one of the leading infections after renal transplant, particularly in developing countries where the incidence and prevalence in the general population are high. Diagnosis requires bacteriologic and histologic confirmation. Interactions among the antitubercular drugs and the immunosuppressive agents have to be considered while prescribing, and surveillance for adverse effects is required. Although rare, case reports are available on extrapulmonary tuberculosis in allograft recipients. Here, we present a 25-year-old kidney transplant recipient who was diagnosed with lymph node tuberculosis under uncommon circumstances but who had a good outcome. This case report illustrates the difficulties in diagnosis of tuberculosis, changes in therapeutic protocols, and prognostic factors and highlights the effects of infectious complications with immunosuppressive therapy in this particular patient population.

Background: Type 1, Type 17 and other pro-inflammatory cytokines are known to play an important role in resistance to pulmonary tuberculosis. The role of these cytokines in tuberculous lymphadenitis (TBL) is not well characterized., Methods: To estimate the systemic and mycobacterial antigen - stimulated cytokine concentrations of Type 1, Type 17, other pro-inflammatory and regulatory cytokines in TBL, we examined both the systemic and the antigen-specific concentrations of these cytokines in TBL (n=31) before and after chemotherapy, and compared them with those with latent tuberculosis infection (LTB, n=31)., Results: We observed significantly reduced systemic concentrations of the pro-inflammatory cytokines - IL-1β and IL-18 but not other Type 1 or Type 17 cytokines in TBL compared to LTB. Following standard anti-tuberculosis (TB) treatment, we observed a significant increase in the concentrations of both IL-1β and IL-18. In addition, we also observed significantly reduced baseline or mycobacterial - antigen or mitogen stimulated concentrations of IL-1β and IL-18 in TBL individuals. Similar to systemic cytokine concentrations, anti-TB treatment resulted in significantly increased concentrations of these cytokines following antigen stimulation., Conclusions: TBL is therefore, characterized by reduced systemic and antigen-specific concentrations of IL-1β and IL-18, which are reversible following anti-TB treatment, indicating that these cytokines are potential correlates of protective immunity in TBL., (Published by Elsevier Ltd.)

Objective: To detect the expression of Mycobacterium tuberculosis secreted protein Ag85B in paraffin-embedded tissues by immunohistochemistry (IHC), and to evaluate its application in the pathological diagnosis of tuberculosis., Methods: One hundred and five tuberculosis specimens (54 pulmonary tuberculosis, 51 lymph nodal tuberculosis) and 51 specimens of other diseases (8 lung cancer, 10 pulmonary abscess, 10 bronchiectasis, 7 lymphoma, 5 necrotizing lymphadenitis, 4 reactive hyperplasia lymphoid, and 7 sarcoidosis) were collected from January 2012 to July 2013 from Beijing Chest Hospital, Capital Medical University. One-step IHC was performed on paraffin-embedded tissues using antibody directed against Ag85B., Results: IHC and Ziehl-Neelsen (ZN) acid-fast staining showed that distribution and intensity of Ag85B expression were concordant with the distribution and number of acid-fast bacilli. IHC showed significantly higher sensitivity than ZN staining (50.5%, 53/105 vs. 31.4%, 33/105; χ² = 7.877, P = 0.005). The combined sensitivity of IHC and ZN staining was 59.0%. Moreover, oil immersion was not necessary for IHC, allowing more rapid diagnosis., Conclusion: IHC detection of Ag85B is a simple method with higher sensitivity than ZN staining, and demonstrated good value in the pathological diagnosis of tuberculosis.

Background: Tuberculosis (TB) has recently re-emerged as a major public health threat worldwide. There is strong evidence that host genetic factors influence individual susceptibility to TB and that, once infected, young children and immunocompromised patients are at increased risk for mycobacterial disease and progression to extrapulmonary lymphadenitis., Methods: The association between polymorphisms of mannose-binding lectin and the susceptibility of 139 children with cervical mycobacterial lymphadenitis and infected with Mycobacterium tuberculosis was investigated., Results: The frequencies of genotypes A/B and B/B, based on codon 54 polymorphisms, were significantly different in TB-infected versus healthy control subjects. The frequency of the A/B genotype was significantly lower in TB-infected children (odds ratio = 0.56; 95% confidence interval: 0.36-0.87; P = 0.01), and the B/B genotype was significantly higher in TB-infected children (odds ratio = 4.68; 95% confidence interval: 1.35-16.3; P = 0.01) compared with healthy controls. The HYB haplotype appeared significantly more often to be protective in the healthy control population (odds ratio = 0.23; 95% confidence interval: 0.05-0.96; P = 0.046). Ex vitro phagocytosis assays indicated that high-expression mannose-binding lectin genotypes are associated with an increased risk of infection with M. tuberculosis., Conclusions: The present study suggests that mannose-binding lectin can protect against TB or predispose the host to the disease depending on the haplotype pair of the host. The low-expression genotype A/B and the HYB haplotype may be associated with protection against intracellular mycobacterial infections, whereas the high-expression genotype A/A may confer susceptibility to disease.

Improved tools are required to study immunopathogenesis of tuberculosis (TB). Mycobacterium tuberculosis antigen-stimulated T cell-based assays can detect TB but are less effective when responses are compromised such as in severe disease. We investigated immune responses to M. tuberculosis whole sonicate (MTBs), recombinant antigens ESAT6 and CFP10 in whole blood cells of healthy endemic controls (EC, n = 42) and patients with pulmonary (PTB, n = 36) or extrapulmonary (ETB, n = 41) disease. Biomarkers of T cell activation (IFNγ) or modulation (IL10) and chemokines, CXCL9, CXCL10 and CCL2, secretion were measured. MTBs, ESAT6 and CFP10 all induced IFNγ responses in TB. ESAT6-induced IFNγ was elevated in TB as compared with EC. MTBs stimulated the highest IFNγ levels but did not differentiate between TB and EC. However, MTBs-induced CXCL10 (P = 0.004) was reduced, while IL10 (P < 0.001) was raised in TB as compared with EC. Between sites, MTBs-induced CCL2 (P = 0.001) and IL10 secretion was higher in PTB than ETB (P < 0.001). In comparison of disease severity, MTBs-induced IFNγ (P = 0.014) and CXCL10 (P = 0.022) levels were raised in moderate as compared with far advanced PTB. In ETB, MTBs-induced IL10 levels were greater in less-severe (L-ETB) than in severe disseminated (D-ETB) cases, P = 0.035. Within the L-ETB group, MTBs-induced IFNγ was greater in patients with tuberculous lymphadenitis than those with pleural TB (P = 0.002). As immune responses to MTBs were differentially activated in TB of different sites and severity, we propose the utility of MTBs-induced IFNγ, CXCL10 and IL10 as biomarkers in TB., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

Background: Tuberculous lymphadenitis (TL) is the commonest form of extra-pulmonary tuberculosis in tropical countries., Objective: This study aimed to characterize in vivo and in vitro cellular immune responses to Mycobacterium PPD in TL patients as markers of disease and healing., Methods: Following informed consent, 36 TL patients, 40 patients with pulmonary tuberculosis (TB) and 20 apparently healthy individuals were enrolled when they met specific selection criteria. The tuberculin skin test (TST) and peripheral blood mono-nuclear cells (PBMCs) culture were conducted using PPD. The cytokines were measured using commercial kits., Results: The mean TST was 24.6 ±8.0 mm for TL patients. The TST was variable in pulmonary TB patients and healthy individuals. It was reactive in a third of pulmonary TB patients with a mean of 20 ±3.0 mm and reactive in half of the healthy individuals with a mean of 12.6 ±3.2 mm. Pre and post-treatment interferon gamma (IFN-g) mean levels were 498.6 ±905.8 pg/ml and 710.0 ±844.6 pg/ml respectively (p=0.0001) for TL patients, while IL-10 mean levels were 93.0 ±136.0 pg/ml and 32.4 ±31.7 pg/ml respectively (p= 0.0001). TST-reactive Pulmonary TB patients had significantly higher IFN-g (851 ±234.4 pg/ml) compared to TBLNT patients (p = 0.0001), while pulmonary TB patients had significantly lower IL-10 compared to TBLNT patients (p=0.0001). Apparently healthy individuals had significantly lower IFN-g and IL-10 levels compared to TBLNT and pulmonary TB patients (p=0.003)., Conclusion: Strong TST reactivity, high IFN-g and IL-10 levels are good surrogate markers of active TBLNT, while increasing IFN-g levels and decreasing IL-10 levels mark healing. Tuberculosis Skin Test reactivity although a good diagnostic marker does not disappear with treatment.

Objective: An increased risk of tuberculosis has been documented in humans treated with tumor necrosis factor alpha (TNFalpha)-neutralizing agents. In murine models, impaired signaling by TNF causes exacerbation of both acute and chronic infection associated with aberrant granuloma formation and maintenance. This study was undertaken to investigate immune modulation in the setting of TNF neutralization in primary and latent tuberculosis in a non-human primate model., Methods: Cynomolgus macaques 4 years of age or older were infected with Mycobacterium tuberculosis and subjected to clinical, microbiologic, immunologic, and radiographic examinations. Monkeys were classified as having active or latent disease 6-8 months after infection, based on clinical criteria. Monkeys used in acute infection studies were randomized to receive either adalimumab (prior to and during infection) or no treatment. Monkeys with latent infection that were randomized to receive TNF-neutralizing agent were given either an inhibitor of soluble TNF, recombinant methionyl human soluble TNF receptor I (p55-TNFRI), or adalimumab. Control monkeys with latent infection were given no treatment or saline. Data from previously studied monkeys with active or latent disease were also used for comparison., Results: Administration of TNF-neutralizing agents prior to M tuberculosis infection resulted in fulminant and disseminated disease by 8 weeks after infection. Neutralization of TNF in latently infected cynomolgus macaques caused reactivation in a majority of animals as determined by gross pathologic examination and bacterial burden. A spectrum of dissemination was noted, including extrapulmonary disease. Surprisingly, monkeys that developed primary and reactivation tuberculosis after TNF neutralization had similar granuloma structure and composition to that of control monkeys with active disease. TNF neutralization was associated with increased levels of interleukin-12, decreased levels of CCL4, increased chemokine receptor expression, and reduced mycobacteria-induced interferon-gamma production in blood but not in the affected mediastinal lymph nodes. Finally, the first signs of reactivation often occurred in thoracic lymph nodes., Conclusion: These findings have important clinical implications for determining the mechanism of TNF neutralization-related tuberculosis.

Objective: To study tuberculous excretory-secretory (ES) 31 and ES-20 antigens in different pathogenic grades of lymph node tuberculosis (TB)., Design: The study group included lymph node TB patients showing granuloma with mature epithelioid cells based on cytology findings (strong immune response group, SI) and patients showing no granuloma formation and acellular necrosis (weak immune response group, WI). Sandwich ELISA was performed using affinity purified antibodies against Mycobacterium tuberculosis ES-31 and ES-20 antigens to assay free and immune complexed antigen levels in the serum of these patients., Results: Higher levels of immune complexed ES-31 (geometric mean titre [GMT] 848) and ES-20 (GMT 1818) antigens than free ES-31 (GMT 462) and ES-20 (GMT 647) were observed in WI patients. There were higher levels of immune complexed ES-20 antigen levels (GMT 1818) in WI patients than in SI lymph node TB patients; the difference was significant (P < 0.05)., Conclusion: Elevated levels of immune complexed ES-20 antigen in patient's serum may be a useful immunological marker for weak immune response patients in lymph node TB.

Drosophila, the toll gene controls a powerful innate defense system against bacteria and fungi. Conserved through evolution, the mammalian innate immune system retains a family of homologous Toll-like receptors (TLRs) that are activated by microbial ligands to release cytokines that instruct the adaptive immune responses. Here we show that TLR2 activation leads to killing of intracellular Mycobacterium (M.) tuberculosis in both mouse and human macrophages. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli. In human monocytes and alveolar macrophages, bacterial lipoproteins similarly activated TLR2 to kill intracellular M. tuberculosis, however by an antimicrobial pathway that is nitric oxide independent. TLR2+CD14+CD68+ macrophages were detected in human lesions of tuberculous lymphadenitis within granulomas and surrounding foci of necrosis. These data provide evidence that mammalian TLRs have retained not only the structural features of Drosophila Toll that allow them to respond to microbial ligands, but also the ability directly to activate antimicrobial effector pathways at the site of infection.

Tuberculosis patients with pulmonary (N = 95) or lymph node disease (N = 23) were assessed for Th1 responses (PPD skin test and lymphocyte blastogenic and interferon gamma) and Th2 responses (polyclonal and antigen specific IgE). Skin test responses to PPD and lymphocyte proliferative responses to crude mycobacterial antigens (PPD, culture filtrate and sonicate) and recall antigens (tetanus toxoid and streptolysin O) were significantly suppressed (p < 0.001) in patients with pulmonary disease compared to endemic controls. However, mitogen (phytohemagglutinin)-stimulated responses were comparable in patients and controls. Polyclonal and antigen specific (M. tuberculosis culture filtrate) IgE responses which are considered to be surrogate markers for Th2 responses were significantly higher in patients with pulmonary disease compared to healthy endemic controls (Mann Whitney analysis p < 0.01). Patients with lymph node disease showed strong Th1 responses but did not show significant responses for either polyclonal or antigen specific IgE. Thus overall suppression of T cell memory response was observed only in patients with pulmonary disease but not in patients with lymph node disease suggesting that sequestration of antigen in different compartments leads to differential activation of Th1 and Th2 responses. PPD skin test responses were highly positive in endemic controls (47% positive) and household contacts (86% positive). Furthermore, PPD positivity decreased with disease severity. Therefore PPD positivity in a BCG vaccinated TB endemic area cannot be used as a diagnostic marker for active tuberculosis particularly in advanced disease.