Efficacy and tolerability of a 4-month ofloxacin-containing regimen compared to a 6-month regimen in the treatment of patients with superficial lymph node tuberculosis: a randomized trial.

BACKGROUND: Tuberculosis (TB) lymphadenitis is the most common form of extra-pulmonary TB, and the treatment duration is six months. This non-inferiority based randomized clinical trial in South India evaluated the efficacy and safety of a four-month ofloxacin containing regimen in tuberculosis lymphadenitis (TBL) patients. METHODS: New, adult, HIV-negative, microbiologically and or histopathologically confirmed superficial lymph node TB patients were randomized to either four-month oflaxacin containing test regimen [ofloxacin (O), isoniazid (H), rifampicin (R), pyrazinamide (Z) -2RHZO daily/ 2RHO thrice-weekly] or a six-month thrice-weekly control regimen (2HRZ, ethambutol/4RH). The treatment was directly observed. Clinical progress was monitored monthly during and up to 12 months post-treatment, and thereafter every three months up to 24 months. The primary outcome was determined by response at the end of treatment and TB recurrence during the 24 months post-treatment. RESULTS: Of the 302 patients randomized, 298 (98.7%) were eligible for modified intention-to-treat (ITT) analysis and 294 (97%) for per-protocol (PP) analysis. The TB recurrence-free favourable response in the PP analysis was 94.0% (95% CI: 90.1-97.8) and 94.5% (95% CI: 90.8-98.2) in the test and control regimen respectively, while in the ITT analysis, it was 92.7% and 93.2%. The TB recurrence-free favourable response in the test regimen was non-inferior to the control regimen 0.5% (95% CI: -4.8-5.9) in the PP analysis based on the 6% non-inferiority margin. Treatment was modified for drug toxicity in two patients in the test regimen, while one patient had a paradoxical reaction. CONCLUSION: The 4-month ofloxacin containing regimen was found to be non-inferior and as safe as the 6-month thrice-weekly control regimen.

Altered plasma levels of ßC and γC chain cytokines and post-treatment modulation in tuberculous lymphadenitis.

BACKGROUND: Alterations in ß common (ßC) and γ common (γC) chain cytokines have been described in pulmonary tuberculosis. However, their role in tuberculous lymphadenitis (TBL) disease has not been assessed. METHODS: Thus, in the present study, we have examined the systemic levels of ßC and γC chain cytokines in TBL, latent tuberculosis (LTB) and healthy control (HC) individuals. We have examined the discriminatory potential of both family of cytokines using ROC analysis. Finally, we measured the pre and post-treatment responses of these cytokines after anti-tuberculosis treatment. RESULTS: TBL individuals exhibit significantly increased (IL-3) and diminished systemic levels of (IL-5, GM-CSF) ßC cytokines compared to LTB and HC individuals. TBL individuals also exhibit significantly diminished (IL-2, IL-7) and elevated (IL-4, IL-9) levels of γC cytokines compared to LTB and/or HC. ROC analysis shows a clear discriminatory capacity of both ßC (IL-5) and γC (IL-2) chain cytokines to distinguish TBL from LTB and HCs. The systemic levels of ßC chain cytokines were not significantly altered, but in contrast γC (IL-2 and IL-7) cytokines were significantly modulated after treatment. Finally, no significant correlation was observed for ßC and γC chain cytokines with their respective lymphocyte count of TBL individuals. CONCLUSIONS: Hence, we conclude that altered plasma levels of ßC and γC cytokines are the characteristics of immune alteration in TBL disease and certain cytokines were modulated after treatment.

Plasma chemokines as immune biomarkers for diagnosis of pediatric tuberculosis.

BACKGROUND: Diagnosing tuberculosis (TB) in children is challenging due to paucibacillary disease, and lack of ability for microbiologic confirmation. Hence, we measured the plasma chemokines as biomarkers for diagnosis of pediatric tuberculosis. METHODS: We conducted a prospective case control study using children with confirmed, unconfirmed and unlikely TB. Multiplex assay was performed to examine the plasma CC and CXC levels of chemokines. RESULTS: Baseline levels of CCL1, CCL3, CXCL1, CXCL2 and CXCL10 were significantly higher in active TB (confirmed TB and unconfirmed TB) in comparison to unlikely TB children. Receiver operating characteristics curve analysis revealed that CCL1, CXCL1 and CXCL10 could act as biomarkers distinguishing confirmed or unconfirmed TB from unlikely TB with the sensitivity and specificity of more than 80%. In addition, combiROC exhibited more than 90% sensitivity and specificity in distinguishing confirmed and unconfirmed TB from unlikely TB. Finally, classification and regression tree models also offered more than 90% sensitivity and specificity for CCL1 with a cutoff value of 28 pg/ml, which clearly classify active TB from unlikely TB. The levels of CCL1, CXCL1, CXCL2 and CXCL10 exhibited a significant reduction following anti-TB treatment. CONCLUSION: Thus, a baseline chemokine signature of CCL1/CXCL1/CXCL10 could serve as an accurate biomarker for the diagnosis of pediatric tuberculosis.

4-month moxifloxacin containing regimens in the treatment of patients with sputum-positive pulmonary tuberculosis in South India - a randomised clinical trial.

BACKGROUND: Shortening tuberculosis (TB) treatment duration is a research priority. We tested the efficacy and safety of 3- and 4-month regimens containing moxifloxacin in a randomised clinical trial in pulmonary TB (PTB) patients in South India. METHODS: New, sputum-positive, adult, HIV-negative, non-diabetic PTB patients were randomised to 3- or 4-month moxifloxacin regimens [moxifloxacin (M), isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E)] or to a control regimen (2H3 R3 Z3 E3 /4R3 H3 ) [C]. The 4 test regimens were 3R7 H7 Z7 E7 M7 [M3], 2R7 H7 Z7 E7 M7 /2R7 H7 M7 [M4], 2R7 H7 Z7 E7 M7 /2R3 H3 M3 [M4-I] or 2R7 H7 Z7 E7 M7 /2R3 H3 E3 M3 [M4-IE]. Treatment was directly observed. Clinical and bacteriological assessments were done monthly during treatment and for 24 months post-treatment. The primary end point was TB recurrence post-treatment. RESULTS: Of 1371 patients, randomised, modified intention-to-treat (ITT) analysis was done in 1329 and per-protocol (PP) analysis in 1223 patients. Regimen M3 was terminated due to high TB recurrence rates. 'Favourable' response at end of treatment was 96-100% in the moxifloxacin regimens and 93% in the control  regimen. Among these, the TB recurrence occurred in 4.1% in the M4 regimen and in 4.5% in the control regimen and demonstrated equivalence within a 5% margin (95% CI -3.68, 4.55). Similar findings were observed in modified ITT analysis. The TB recurrence rates in the M4-I and M4-IE regimens did not show equivalence with the control regimen. Sixteen (1.4%) of 1087 patients in the moxifloxacin regimens required treatment modification. CONCLUSION: The 4-month daily moxifloxacin regimen [M4] was found to be equivalent and as safe as the 6-month thrice-weekly control regimen.

CONTEXTE: La réduction de la durée du traitement de la tuberculose (TB) est une priorité de recherche. Nous avons testé l'efficacité et la sécurité de schémas thérapeutiques contenant de la moxifloxacine pendant 3 et 4 mois dans un essai clinique randomisé chez des patients atteints de TB pulmonaire (PTB) dans le sud de l'Inde. MÉTHODES: De nouveaux patients PTB, adultes, non diabétiques, positifs pour les expectorations, VIH négatifs ont été randomisés pour des schémas thérapeutiques contenant de la moxifloxacine pendant 3 mois ou 4 mois [moxifloxacine (M), isoniazide (H), rifampicine (R), pyrazinamide (Z), l'éthambutol (E)] ou pour un régime témoin (2H3 R3 Z3 E3 /4R3 H3 ) [C]. Les 4 régimes de l'essai étaient 3R7 H7 Z7 E7 M7 [M3], 2R7 H7 Z7 E7 M7 /2R7 H7 M7 [M4], 2R7 H7 Z7 E7 M7 /2R3 H3 M3 [M4-I] ou 2R7 H7 Z7 E7 M7 /2R3 H3 E3 M3 [M4-IE]. Le traitement a été directement observé. Les évaluations cliniques et bactériologiques ont été effectuées mensuellement au cours du traitement et durant 24 mois après le traitement. Le critère d'évaluation principal était la récidive de la TB après le traitement. RÉSULTATS: Des 1.371 patients randomisés, une analyse en intention de traiter (ITT) modifiée a été effectuée sur 1.329 et une analyse par protocole (PP) sur 1.223 patients. Le régime M3 a été interrompu en raison de taux élevés de récidive de la TB. La réponse «favorable¼ à la fin du traitement était de 96 à 100% dans les bras moxifloxacine et 93% dans le bras témoin. Parmi ceux-ci, la récidive de la TB est survenue chez 4,1% dans le schéma M4 et chez 4,5% dans le schéma témoin et a démontré une équivalence dans une marge de 5% (IC95%: −3,68, 4,55). Des résultats similaires ont été observés dans l'analyse ITT modifiée. Les taux de récidive de la TB dans les schémas M4-I et M4-IE n'ont pas montré d'équivalence avec le schéma témoin. 16 (1,4%) des 1.087 patients dans les régimes à moxifloxacine ont nécessité une modification du traitement. CONCLUSION: Le régime quotidien de moxifloxacine pendant 4 mois [M4] s'est avéré équivalent et aussi sûr que le régime témoin de trois fois par semaine pendant 6 mois.

Diminished circulating plasma and elevated lymph node culture supernatant levels of IL-10 family cytokines in tuberculous lymphadenitis.

BACKGROUND: IL-10 family cytokines are associated with the host immune response to pulmonary tuberculosis (PTB), but their association with host response in tuberculous lymphadenitis (TBL) is not known. METHODS: Hence, we examined the circulating levels of the whole panel of IL-10 family cytokines in TBL (n = 44) and compared them to the levels in PTB (n = 44) and healthy control (HC, n = 44) individuals. We also assessed the pre and post-treatment cytokine levels in TBL individuals following the completion of anti-tuberculosis treatment (ATT). Next, we also compared the levels of IL-10 family cytokine in circulation versus lymph node (LN) culture supernatants in a subset of TBL individuals (n = 22). Finally, we also measured the levels of IL-10 family cytokines in tuberculosis antigen (purified protein derivative, PPD) stimulated and unstimulated LN culture supernatants. RESULTS: TBL individuals exhibit significantly decreased levels of IL-10, IL-19, IL-20, IL-24, IL-28B and IL-29 in the circulation when compared to PTB (except IL-10) and HC (except IL-20 and IL-28B) and significantly increased levels of IL-22 when compared to PTB individuals. Following ATT, TBL individuals exhibit significantly elevated levels of IL-10, IL-19, IL-20, IL-24, IL-28B and IL-29 and significantly diminished levels of IL-26. Similarly, TBL individuals also exhibited significantly increased levels of IL-10, IL-19, IL-20, IL-24, IL-28A and IL-29 in LN culture supernatants compared to plasma and significantly decreased levels of IL-22. This was associated with enhanced levels of IL-19, IL-20, IL-24, IL-28B and IL-29 upon PPD stimulation of LN cultures. CONCLUSIONS: Therefore, we demonstrate that TBL is associated with significantly diminished plasma and elevated LN culture supernatant levels of most of the IL-10 family cytokines. This to our knowledge is the first comprehensive examination of IL-10 family cytokines in TBL.

Systemic Levels of Pro-Inflammatory Cytokines and Post-Treatment Modulation in Tuberculous Lymphadenitis.

Pro-inflammatory cytokines are potent stimulators of inflammation and immunity and markers of infection severity and bacteriological burden in pulmonary tuberculosis (PTB). Interferons could have both host-protective and detrimental effects on tuberculosis disease. However, their role has not been studied in tuberculous lymphadenitis (TBL). Thus, we evaluated the systemic pro-inflammatory (interleukin (IL)-12, IL-23, interferon (IFN)α, and IFNß) cytokine levels in TBL, latent tuberculosis (LTBI), and healthy control (HC) individuals. In addition, we also measured the baseline (BL) and post-treatment (PT) systemic levels in TBL individuals. We demonstrate that TBL individuals are characterized by increased pro-inflammatory (IL-12, IL-23, IFNα, IFNß) cytokines when compared to LTBI and HC individuals. We also show that after anti-tuberculosis treatment (ATT) completion, the systemic levels of pro-inflammatory cytokines were significantly modulated in TBL individuals. A receiver operating characteristic (ROC) analysis revealed IL-23, IFNα, and IFNß significantly discriminated TBL disease from LTBI and/or HC individuals. Hence, our study demonstrates the altered systemic levels of pro-inflammatory cytokines and their reversal after ATT, suggesting that they are markers of disease pathogenesis/severity and altered immune regulation in TBL disease.

Dominant expansion of CD4+, CD8+ T and NK cells expressing Th1/Tc1/Type 1 cytokines in culture-positive lymph node tuberculosis.

Lymph node culture-positive tuberculosis (LNTB+) is associated with increased mycobacterial antigen-induced pro-inflammatory cytokine production compared to LN culture-negative tuberculosis (LNTB-). However, the frequencies of CD4+, CD8+ T cells and NK cells expressing Th1/Tc1/Type 1 (IFNγ, TNFα, IL-2), Th17/Tc17/Type 17 (IL-17A, IL-17F, IL-22) cytokines and cytotoxic (perforin [PFN], granzyme [GZE] B, CD107a) markers in LNTB+ and LNTB- individuals are not known. Thus, we have studied the unstimulated (UNS) and mycobacterial antigen-induced frequencies of CD4+, CD8+ T and NK cells expressing Th1, Th17 cytokines and cytotoxic markers using flow cytometry. The frequencies of CD4+, CD8+ T and NK cells expressing cytokines and cytotoxic markers were not significantly different between LNTB+ and LNTB- individuals in UNS condition. In contrast, upon Mtb antigen stimulation, LNTB+ individuals are associated with significantly increased frequencies of CD4+ T cells (PPD [IFNγ, TNFα], ESAT-6 PP [IFNγ, TNFα], CFP-10 PP [IFNγ, TNFα, IL-2]), CD8+ T cells (PPD [IFNγ], ESAT-6 PP [IFNγ], CFP-10 PP [TNFα]) and NK cells (PPD [IFNγ, TNFα], ESAT-6 PP [IFNγ, TNFα], CFP-10 PP [TNFα]) expressing Th1/Tc1/Type 1, but not Th17/Tc17/Type 17 cytokines and cytotoxic markers compared to LNTB- individuals. LNTB+ individuals did not show any significant alterations in the frequencies of CD4+, CD8+ T cells and NK cells expressing cytokines and cytotoxic markers compared to LNTB- individuals upon HIV Gag PP and P/I antigen stimulation. Increased frequencies of CD4+, CD8+ T and NK cells expressing Th1/Tc1/Type 1 cytokines among the LNTB+ group indicates that the presence of mycobacteria plays a dominant role in the activation of key correlates of immune protection or induces higher immunopathology.

Strongyloidiasis stercoralis coinfection is associated with altered iron status biomarkers in tuberculous lymphadenitis.

Soil-transmitted helminth [mainly Strongyloidiasis stercoralis (Ss)] and tuberculous lymphadenitis (TBL) coinfection in humans is a significant public health problem. We have previously shown that TBL+Ss+ coinfection significantly alters diverse cytokine, matrix metalloproteinase, and tissue inhibitors of metalloproteinase profiles. However, no data is available to understand the influence of Ss coinfection in TBL disease with respect to iron status biomarkers. Hence, we have studied the effect of Ss coinfection on the circulating levels of iron status (ferritin, transferrin [TF], apotransferrin [ApoT], hepcidin, hemopexin) biomarkers in TBL disease. Our results show that TBL+Ss+ and/or TBL+Ss- individuals are associated with significantly altered biochemical and hematological (red blood cell (RBC) counts, hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) were decreased, and platelets were increased) parameters compared to TBL-Ss+ individuals. Our results also show that TBL+Ss+ coinfection is associated with diminished circulating levels of ferritin, ApoT, hepcidin, and hemopexin compared to TBL+Ss- individuals. TBL+Ss+ and TBL+Ss- groups are associated with altered iron status biomarkers (decreased ferritin [TBL+Ss+ alone] and increased TF, ApoT, hepcidin and hemopexin [TBL+Ss- alone]) compared to TBL-Ss+ group. The heat map expression profile and principal component analysis (PCA) analysis of iron status biomarkers were significantly altered in TBL+Ss+ compared to TBL+Ss- and/or TBL-Ss+ individuals. A significant correlation (positive/negative) was obtained among the biochemical and hematological parameters (white blood cells (WBC)/ferritin, TF, and hepcidin, mean corpuscular hemoglobin concentration (MCHC)/ferritin and hemopexin) with iron status biomarkers. Finally, receiver operating characteristic (ROC) analysis revealed that hemopexin was significantly associated with greater specificity and sensitivity in discriminating TBL+Ss+ and TBL+Ss- coinfected individuals. Thus, our data conclude that Ss coinfection is associated with altered iron status biomarkers indicating that coinfection might alter the host-Mtb interface and could influence the disease pathogenesis.

Plasma adipocytokines distinguish tuberculous lymphadenitis from pulmonary tuberculosis.

Adipocytokines are the major secretory products of adipose tissue and potential markers of metabolism and inflammation. However, their association in host immune response against tuberculous lymphadenitis (TBL) disease is not known. Thus, we measured the systemic levels of adipocytokines in TBL (n = 44) and compared to pulmonary tuberculosis (PTB, n = 44) and healthy control (HC, n = 44) individuals. We also examined the pre and post-treatment adipocytokine levels in TBL individuals upon completion of standard anti-tuberculosis treatment (ATT). The receiver operating characteristics (ROC) were performed between TBL, PTB and HCs to find the potential discriminatory markers. Finally, principal component (PCA) analysis was performed to reveal the expression patterns of adipocytokines among study groups. Our results demonstrate that TBL is associated with significantly higher systemic levels of adipocytokines (except resistin) when compared with PTB and significantly lower levels when compared with HC (except adiponectin) individuals. Upon completion of ATT, the systemic levels of adiponectin and resistin were significantly decreased when compared to pre-treatment levels. Upon ROC analysis, all the three adipocytokines discriminated TBL from PTB but not with HCs, respectively. Similarly, adipocytokines were differentially clustered in TBL in comparison to PTB in PCA analysis. Therefore, adipocytokines are a distinguishing feature in TBL compared to PTB individuals.

Reduced neutrophil granular proteins and post-treatment modulation in tuberculous lymphadenitis.

BACKGROUND: Neutrophils are important for host innate immune defense and mediate inflammatory responses. Pulmonary tuberculosis (PTB) is associated with increased neutrophil granular protein (NGP) levels in the circulation. However, the systemic levels of neutrophil granular proteins were not examined in tuberculous lymphadenitis (TBL) disease. METHODS: We measured the systemic levels of NGP (myeloperoxidase [MPO], elastase and proteinase 3 [PRTN3]) in TBL and compared them to latent tuberculosis (LTB) and healthy control (HC) individuals. We also measured the pre-treatment (Pre-T) and post-treatment (Post-T) systemic levels of neutrophil granular proteins in TBL individuals upon anti-tuberculosis treatment (ATT) completion. In addition, we studied the correlation and discriminatory ability of NGPs using receiver operating characteristic (ROC) analysis. RESULTS: Our data suggests that systemic levels of NGPs (MPO, PRTN3, elastase) were significantly reduced in TBL individuals compared to LTB and HC individuals. Similarly, after ATT, the plasma levels of MPO and elastase but not PRTN3 were significantly elevated compared to pre-treatment levels. NGPs (except PRTN3) were positively correlated with absolute neutrophil count of TBL, LTB and HC individuals. Further, NGPs were able to significantly discriminate TBL from LTB and HC individuals. CONCLUSION: Hence, we conclude reduced neutrophil granular protein levels might be associated with disease pathogenesis in TBL.

Helminth Coinfection Is Associated With Enhanced Plasma Levels of Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinases in Tuberculous Lymphadenitis.

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and repair and are expressed in diverse infections, whereas tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of MMPs. However, the interaction of MMPs and TIMPs in tuberculous lymphadenitis (TBL), an extra-pulmonary form of tuberculosis (EPTB) and helminth (Hel+) coinfection is not known. Therefore, this present study investigates the levels of circulating MMPs (1, 2, 3, 7, 8, 9, 12, 13) and TIMPs (1, 2, 3, 4) in TBL individuals with helminth (Strongyloides stercoralis [Ss], hereafter Hel+) coinfection and without helminth coinfection (hereafter, Hel-). In addition, we have also carried out the regression analysis and calculated the MMP/TIMP ratios between the two study groups. We describe that the circulating levels of MMPs (except MMP-8 and MMP-12) were elevated in TBL-Hel+ coinfected individuals compared to TBL-Hel- individuals. Similarly, the systemic levels of TIMPs (1, 2, 3, 4) were increased in TBL-Hel+ compared to TBL-Hel- groups indicating that it is a feature of helminth coinfection per se. Finally, our multivariate analysis data also revealed that the changes in MMPs and TIMPs were independent of age, sex, and culture status between TBL-Hel+ and TBL-Hel- individuals. We show that the MMP-2 ratio with all TIMPs were significantly associated with TBL-helminth coinfection. Thus, our results describe how helminth infection has a profound effect on the pathogenesis of TBL and that both MMPs and TIMPs could dampen the immunity against the TBL-Hel+ coinfected individuals.

Ex-vivo immunophenotyping and high dimensionality UMAP analysis of leucocyte subsets in tuberculous lymphadenitis.

Tuberculous lymphadenitis (TBL) is defined by reduced proinflammatory cytokines and elevated CD4+, CD8+ T cells and decreased CD8+ cytotoxic markers. However, ex-vivo phenotyping of diverse leucocytes in TBL has not been done. We show activated and atypical B cells, myeloid dendritic cells (mDCs), classical, non-classical and intermediate monocytes, T regulatory (T regs) cells, CD4+ T cell effector memory RA (TEMRA), CD4+ effector and CD8+ central memory phenotypes were significantly increased in TBL compared to LTB individuals. In contrast, classical memory and plasma B cells, plasmacytoid DCs (pDCs), CD8+ TEMRA, CD4+ naïve and central memory cells were significantly decreased in TBL compared to LTB individuals. Some of the leucocyte frequencies (atypical memory B cells, pDCs, myeloid-derived suppressor cells, CD4+ effector and CD8+ central memory was increased; activated memory and plasma B cell, mDCs, classical, non-classical, intermediate monocytes, T regs, CD4+ TEMRA, CD4+, CD8+ naïve and effector memory cells and CD8+ central memory cells were decreased) were significantly modulated after anti-TB treatment among TBL individuals. UMAP analysis show that leucocyte subsets or islands expressing specific markers were significantly different in TBL baseline and post-treatment individuals. Overall, we suggest altered frequencies of diverse leucocytes influences the disease pathology and protective immunity in TBL individuals.

Discovery and Validation of a Three-Cytokine Plasma Signature as a Biomarker for Diagnosis of Pediatric Tuberculosis.

Pediatric TB poses challenge in diagnosis due to the paucibacillary nature of the disease. We conducted a prospective diagnostic study to identify immune biomarkers of pediatric TB and controls (discovery cohort) and obtained a separate "validation" cohort of confirmed cases of pediatric TB and controls. Multiplex ELISA was performed to examine the plasma levels of cytokines. Discovery and validation cohorts revealed that baseline plasma levels of IFNγ, TNFα, IL-2, and IL-17A were significantly higher in active TB (confirmed TB and unconfirmed TB) in comparison to unlikely TB children. Receiver operating characteristics (ROC) curve analysis revealed that IFNγ, IL-2, TNFα, and IL-17A (in the discovery cohort) and TNFα and IL-17A (in the validation cohort) could act as biomarkers distinguishing confirmed or unconfirmed TB from unlikely TB with the sensitivity and specificity of more than 90%. In the discovery cohort, cytokines levels were significantly diminished following anti-tuberculosis treatment. In both the cohorts, combiROC models offered 100% sensitivity and 98% to 100% specificity for a three-cytokine signature of TNFα, IL-2, and IL-17A, which can distinguish confirmed or unconfirmed TB children from unlikely TB. Thus, a baseline cytokine signature of TNFα, IL-2, and IL-17A could serve as an accurate biomarker for the diagnosis of pediatric tuberculosis.

Diminished systemic levels of antimicrobial peptides in tuberculous lymphadenitis and their reversal after anti-tuberculosis treatment.

Pulmonary tuberculosis is associated with higher plasma levels of antimicrobial peptides (AMPs) and lower granulysin levels. However, the association of AMPs with tuberculous lymphadenitis (TBL) is not well studied. Hence, we measured the plasma levels of human beta defensin-2 (HBD2), granulysin, human neutrophil peptides 1-3 (HNP1-3) and cathelicidin (LL37) in TBL compared to latent tuberculosis (LTB) and healthy controls (HC) and in TBL individuals upon completion of anti-tuberculosis treatment (ATT). We examined the association of AMPs with TBL lymph node culture grade or lymph node involvement. Finally, the discriminatory potential of these proteins was assessed using receiver operating characteristic (ROC) analysis. TBL individuals display significantly diminished circulating levels of AMPs (granulysin and HNP1-3) but not HBD-2 and LL-37 in comparison to LTB and HCs. Similarly, after ATT, both HBD-2 and HNP1-3 were significantly elevated and LL-37 was significantly reduced in TBL individuals. Granulysin and HNP1-3 discriminates TBL from LTB and HC individuals upon ROC analysis. AMPs did not exhibit significant correlation either with lymph node culture grades or lymph node involvement. Overall, TBL individuals show decreased AMPs and their reversal after ATT suggesting their association with underlying immune alteration in this poorly studied form of TB disease.

Low body mass index has minimal impact on plasma levels of cytokines and chemokines in tuberculous lymphadenitis.

Malnutrition, due to low body mass index (LBMI), is considered to be one of the key risk factors for tuberculosis (TB) development. The link between pro and anti-inflammatory cytokines and BMI has been studied in active pulmonary TB. However, the association of BMI with cytokines and chemokines in TB lymphadenitis (TBL) has not been examined. Hence, we wanted to examine the plasma levels of different cytokines and chemokines in TBL individuals with LBMI, normal BMI (NBMI) and high BMI (HBMI). LBMI with TBL disease is associated with enhanced systemic levels of type 1 (tumor necrosis factor alpha [TNFα], interleukin-2 [IL-2]) and type 2 (IL-4, IL-13) cytokines in comparison with NBMI and/or HBMI. However, other pro-inflammatory (IFNγ, IL-1ß, IL-17A, IL-6, IL-7, IL-12, G-CSF, and GM-CSF) and anti-inflammatory (IL-5 and IL-10) cytokines were not significantly different among the TBL individuals with different BMI status. Likewise, no significant differences were observed in the CC (CCL-1, CCL-2/MCP-1, CCL3/MIP1α, CCL4/MIP-1ß, CCL11/eotaxin) and CXC (CXCL-1/GRO-⍺, CXCL2/GRO-ß, CXCL9/MIG, CXCL10/IP-10, CXCL11/ITAC 1) chemokine profile among the TBL individuals with different BMI. Hence, our data implies that TBL individuals with LBMI are characterized by minimal effects on plasma cytokines and chemokines in TBL.

Altered systemic levels of acute phase proteins in tuberculous lymphadenitis and modulation after treatment.

BACKGROUND: Pulmonary tuberculosis (PTB) is characterized by elevated levels of acute phase proteins (APPs), but their association with tuberculous lymphadenitis (TBL) is poorly studied. METHODS: We examined the systemic levels of APPs (alpha-2-macroglobulin [⍺-2MG], serum amyloid A [SAA], C-reactive protein [CRP] and haptoglobin [Hp]) in TBL, PTB, latent tuberculosis (LTB) and healthy controls (HC) at baseline and in TBL after the completion of anti-tuberculosis treatment (ATT). We have also examined the association of these proteins with lymph node (LN) size, culture grade and multiple versus single LN involvement. RESULTS: TBL individuals exhibited increased systemic levels of ⍺-2MG, SAA, CRP and Hp in comparison to HCs and increased CRP levels in comparison to LTB individuals. TBL individuals also exhibited decreased systemic levels of Hp compared to PTB individuals. APPs were not significantly associated with LN size, LN involvement and culture grade, indicating a lack of association with disease severity. Following ATT, post-treatment levels of ⍺-2MG, CRP and Hp were significantly diminished compared to pre-treatment levels. CONCLUSION: TBL disease is characterized by altered levels of APPs at baseline and modulated following treatment, indicating the presence of systemic inflammation.

Diminished Frequencies of Cytotoxic Marker Expressing T- and NK Cells at the Site of Mycobacterium tuberculosis Infection.

Tuberculous lymphadenitis (TBL) individuals exhibit reduced frequencies of CD8+ T cells expressing cytotoxic markers in peripheral blood. However, the frequencies of cytotoxic marker expressing CD4+, CD8+ T cells, and NK cells at the site of infection is not known. Therefore, we measured the baseline and mycobacterial antigen specific frequencies of cytotoxic markers expressing CD4+, CD8+ T cells, and NK cells in the LN (n = 18) and whole blood (n = 10) of TBL individuals. TBL LN is associated with lower frequencies of CD4+ T cells expressing cytotoxic markers (Granzyme B, CD107a) compared to peripheral blood at baseline and in response to PPD, ESAT-6, and CFP-10 antigen stimulation. Similarly, lower frequencies of CD8+ T cells expressing cytotoxic markers (Perforin, Granzyme B, and CD107a) were also present in the TBL LN at baseline and following (except perforin) antigen stimulation. Finally, at baseline and after antigen (PPD, ESAT-6, and CFP-10) stimulation, frequencies of NK cells expressing cytotoxic markers were also significantly lower in TBL LN compared to whole blood. Hence, TBL is characterized by diminished frequencies of cytotoxic marker expressing CD4+, CD8+ T cells, and NK cells at the site of infection, which might reflect the lack of protective immune responses at the site of Mycobacterium tuberculosis infection.

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases Are Potential Biomarkers of Pulmonary and Extra-Pulmonary Tuberculosis.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) are potential regulators of tuberculosis (TB) pathology. Whether they are candidates for non-sputum-based biomarkers for pulmonary TB (PTB) and extra-pulmonary TB (EPTB) is not fully understood. Hence, to examine the association of MMPs and TIMPs with PTB and EPTB, we have measured the circulating levels of MMPs (MMP-1, 2, 3, 7, 8, 9, 12, and 13) and TIMPs (TIMP-1, 2, 3, and 4) in PTB, EPTB and compared them with latent tuberculosis (LTB) or healthy control (HC) individuals. We have also assessed their circulating levels before and after the completion of anti-tuberculosis treatment (ATT). Our data describes that systemic levels of MMP-1, 8, 9, 12 were significantly increased in PTB compared to EPTB, LTB, and HC individuals. In contrast, MMP-7 was significantly reduced in PTB compared to EPTB individuals. Likewise, the systemic levels of MMP-1, 7, 13 were significantly increased in EPTB in comparison to LTB and HC individuals. In contrast, MMP-8 was significantly reduced in EPTB individuals compared to LTB and HC individuals. In addition, the systemic levels of TIMP-1, 2, 3 were significantly diminished and TIMP-4 levels were significantly enhanced in PTB compared to EPTB, LTB, and HC individuals. The circulating levels of TIMP-2 was significantly reduced and TIMP-3 was significantly elevated in EPTB individuals in comparison with LTB and HCs. Some of the MMPs (7, 8, 9, 12, 13 in PTB and 1, 7, 8, 9 in EPTB) and TIMPs (1, 2, 3, 4 in PTB and 4 in EPTB) were significantly modulated upon treatment completion. ROC analysis showed that MMP-1, 9 and TIMP-2, 4 could clearly discriminate PTB from EPTB, LTB and HCs and MMP-13 and TIMP-2 could clearly discriminate EPTB from LTB and HCs. Additionally, multivariate analysis also indicated that these alterations were independent of age and sex in PTB and EPTB individuals. Therefore, our data demonstrates that MMPs and TIMPs are potential candidates for non-sputum-based biomarkers for differentiating PTB and EPTB from LTB and HC individuals.

Helminth mediated modulation of the systemic and mycobacterial antigen - stimulated cytokine profiles in extra-pulmonary tuberculosis.

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.

Diminished type 1 and type 17 cytokine expressing - Natural killer cell frequencies in tuberculous lymphadenitis.

Tuberculous lymphadenitis (TBL) is associated with the expansion of CD4+ and CD8+ T cells expressing Type 1 and Type 17 cytokines in the peripheral blood. However, the expression pattern of cytokine producing natural killer (NK) cells in both the peripheral blood and affected lymph nodes i.e. site of infection in TBL have not been examined. Hence, we have analyzed the baseline and mycobacterial antigen specific NK cell cytokine frequencies in whole blood of TBL and pulmonary tuberculosis (PTB) individuals. We have also examined the NK cell frequencies before and after treatment completion and in peripheral blood versus affected lymph nodes (LN) of TBL individuals. TBL is characterized by diminished frequencies of NK cells expressing Type 1 (IFNγ, TNFα), Type 17 (IL-17F) cytokines compared to PTB individuals upon antigen-specific stimulation. In contrast, TBL individuals did not exhibit any significant differences in the frequencies of NK cells expressing Type 1 and Type 17 cytokines upon completion of anti-tuberculosis treatment. LN of TBL is associated with altered frequencies of NK cells expressing Type 17 (increased IL-17F and decreased IL-22) cytokines when compared to peripheral blood. Thus, we conclude that TBL individuals are characterized by diminished frequencies of NK cells expressing Type 1/Type 17 cytokines.