TB Subjects Research Articles

Prevalence studies of human T-Lymphotrophic virus type 1 among pulmonary tuberculosis patients in Dutse metropolis, north-western Nigeria

Human T-lymphotrophic virus type 1 (HTLV-1) is a causative agent of tropic spastic paraparesis and adult Tcell leukaemia, Information regarding the involvement of HTLV-1 in presentation of some subclinical immune suppression that may results in increased rate of HIV and TB infections has long been documented. Sixty (60) Confirmed Pulmonary TB subjects consisting of 41 males and 19 females were recruited in this study, Tuberculosis was confirmed by collecting their sputum samples and analysed using GeneXpert. The prevalence of HTLV-1 IgG antibodies among TB subjects was 6.6%, while that of IgM was 1.6%. Accordingly, sexually active couple group has the highest prevalence of 2.3% when compared to single and widow categories, age group 15-24 has the highest percentage of 3.3% for HTLV-1 IgG antibodies. Key words : HTLV-1, Tuberculosis, IgG, IgM.

Multifunctional cytokine and T cell memory phenotype profiles differ in active versus cured tuberculosis.

Abstract Tuberculosis, one of the most prevalent and deadliest infectious diseases worldwide, requires long-term multidrug treatment regimens for curing active clinical disease. However, a diagnostic method for identifying successful cure from TB disease following anti-TB chemotherapy remains elusive. The overall goal of this study was to assess differential patterns of cytokine production and memory T cell phenotypes to distinguish between active TB subjects and remotely cured TB individuals. Using peripheral blood mononuclear cells from subjects enrolled in a TB household contact study established in Vitória, Brazil, cells were stimulated ex vivo with TB antigens and evaluated using multi-parametric flow cytometry. Total cytokine and multifunctional cytokine analyses revealed an increase in the proportion of IL-17 producing CD4+ T cells in active TB subjects, while cured TB subjects showed a clear dominance of cells producing IFNγ and IL-2 or TNFα. Comparison of memory phenotypes revealed differences between proportions of several CD4+ T cell memory subsets, with an effector memory dominance in cured TB compared to an increased stem cell memory representation in active TB. Increased expression of exhaustion marker PD-1 in advanced stages of memory cell differentiation suggested its possible use as a surrogate indicator of memory subset fluxes, with possible application as a biomarker for distinguishing differences between cured TB and active TB individuals.

Evaluation of 5 Novel protein biomarkers for the rapid diagnosis of pulmonary and extra-pulmonary tuberculosis: preliminary results

Improved methods are required for the early and accurate diagnosis of tuberculosis, especially in the patients with smear-negative disease. Several biomarkers have been tried but most have shown poor sensitivity or specificity. In present study we aimed to evaluate the diagnostic utility of five novel antigens identified earlier by us. This is an initial study conducted on 250 subjects. The five recombinant antigens, named as rSS1 (Rv2145c), rSS2 (Rv0164), rSS3 (Rv1437), rSS4 (Rv1827) and rSS5 (Rv2970c), were expressed in pQE-30 expression vector, purified and their sero-diagnostic efficacy was evaluated in an unblinded manner using dot-blot and ELISA methods. The sensitivity and specificity of these novel antigens were compared with commercially available standard esat6 and 38 kDa antigens. Bacteriologically confirmed TB patients, non-TB disease controls and healthy individuals were included. which are based on novel antigen or novel technology, Area under curve (AUC) of the selected antigens were 0.98 (0.98–0.99) for rSS1, 0.88 (0.84–0.92) for rSS2, 0.88 (0.84–0.92) for rSS3, 0.95 (0.93–0.98) for rSS4 and 0.99 (0.98–1.0) for rSS5. Receiver operative characteristic (ROC) curve showed highly significant difference between TB and healthy subjects (p = <0.001). These initial findings, show that the recombinant antigens rSS1, rSS4 and rSS5 could be used as highly potential biomarkers for the serological diagnosis of active TB.

Potential Role for Mycobacterium tuberculosis Specific IL-2 and IFN-γ Responses in Discriminating between Latent Infection and Active Disease after Long-Term Stimulation.

Interferon gamma release assays (IGRAs) could accurately diagnose Mycobacterium tuberculosis (M.tuberculosis) infection. However, these assays do not discriminate between latent tuberculosis infection (LTBI) and active tuberculosis disease (ATB). Here, a total of 177 subjects, including 65 patients with ATB, 43 subjects with LTBI, and 69 TB-uninfected controls (CON group) were enrolled. The concentration of IFN-γ, IP-10, and IL-2 was determined in peripheral blood mononuclear cells (PBMCs) after short-term (24h) or long-term (72h) stimulation with TB antigens including ESAT-6/CFP-10 (EC) and purified protein derivative (PPD).EC-stimulated IL-2 and gamma interferon-inducible protein 10 (IP-10) release (24h and 72h) showed a good diagnostic performance in distinguishing between TB-infected and TB-uninfected individuals, but failed to discriminate between ATB and LTBI. After 72h of incubation, the release of IL-2 was higher in LTBI patients after stimulation with EC and PPD. The PPD-stimulated IL-2/IFN-γ ratio after 72h incubation had the diagnostic potential to discriminate between ATB and LTBI, with a sensitivity of 90.8% and a specificity of 97.7%. In addition, these new biomarkers, combined with T-SPOT test in a two-step strategy, were validated with high levels of accuracy in a prospective clinical-based cohort. Collectively, the PPD-stimulated IL-2/IFN-γ ratio after long-term incubation may be an alternative diagnostic biomarker in distinguishing between active TB patients and subjects with latent infection.

Biomarkers for discrimination between latent tuberculosis infection and active tuberculosis disease

We aimed to determine whether combinations of multiplex cytokine responses could differentiate Mycobacterium tuberculosis (Mtb) infection states. Mtb-specific antigen-induced and unstimulated cytokines were measured by Luminex assay in supernatants of QuantiFERON® Gold In-Tube assay (QFT) in 48 active pulmonary TB patients (TB), 15 latent TB infection subjects (LTBI), and 13 healthy controls (HCs). Among the 29 cytokines, eight Mtb antigen-specific biomarkers (GM-CSF, IFN-γ, IL-1RA, IL-2, IL-3, IL-13, IP-10, and MIP-1β) in the Mtb-infected group were significantly different from those of the HCs. Five Mtb-specific biomarkers (EGF, GM-CSF, IL-5, IL-10, and VEGF), two unstimulated biomarkers (TNF-α[Nil] and VEGF[Nil]), and one Mtb-specific biomarker ratio (IL-2/IFN-γ) showed significant differences between active TB and LTBI. Three unstimulated biomarkers (IL-8[Nil], IL-13[Nil], and VEGF[Nil]) and 5 Mtb-specific biomarkers (IFN-γ, IL-2, IL-3, IP-10, and VEGF) were significantly different between active TB and non-active TB groups. Combinations of three cytokine biomarkers resulted in the accurate prediction of 92.1-93.7% of Mtb-infected cases and 92.3-100% of HCs, respectively. Moreover, combinations of five biomarkers accurately predicted 90.9-100% of active TB cases and 80-100% of LTBI subjects, respectively. In discriminating between active TB and non-active TB regardless of QFT results, combinations of six biomarkers predicted 79.2-95.8% of active TB cases and 67.9-89.3% of non-active TB subjects. Taken together, our data suggest that combinations of whole blood Mtb antigen-dependent cytokines could serve as biomarkers to determine TB disease states. Especially, VEGF is highlighted as a key biomarker for reflecting active TB, irrespective of stimulation.

Serum biomarkers of treatment response within a randomized clinical trial for pulmonary tuberculosis

Biomarkers for monitoring response to anti-tuberculosis treatment are needed. We explored immune markers previously published as having predictive capability for 8 week culture status in 39 adults enrolled in a clinical trial in Kampala, Uganda. We consecutively selected 20 HIV-negative pulmonary TB subjects with positive cultures, and 19 subjects with negative cultures at the end of intensive phase therapy. At baseline and after 8 weeks, serum was assayed for nine cytokines and soluble cytokine receptors using multiplexed platforms or ELISA. We evaluated their association with week 8 culture status first using single-variable logistic models, then using cross-validated estimates of the C-statistic, a measure of discrimination, of candidate models including 2 or 3 analytes in addition to age. All but one analyte decreased from baseline to week 8 (all p<0.01). Individual biomarkers were not associated with 8 week culture status. Logistic models including increasing age, higher baseline soluble tumor necrosis factor receptor alpha 1 (sTNF-R1), and higher week 8C-reactive protein (CRP) concentration classified subjects by culture status with up to 85% accuracy and acceptable discrimination (cross-validated C-statistic 0.76) and calibration (Hosmer-Lemeshow P>0.2). Exploratory post-hoc models including sTNF-R1, CRP, and age, classified 8 week culture status with promising accuracy.

Differential expression of miRNAs and their relation to active tuberculosis

The aim of this work was to screen miRNA signatures dysregulated in tuberculosis to improve our understanding of the biological role of miRNAs involved in the disease. Datasets deposited in publically available databases from microarray studies on infectious diseases and malignancies were retrieved, screened, and subjected to further analysis. Effect sizes were combined using the inverse-variance model and between-study heterogeneity was evaluated by the random effects model. 35 miRNAs were differentially expressed (12 up-regulated, 23 down-regulated; p<0.05) by combining 15 datasets of tuberculosis and other infectious diseases. 15 miRNAs were found to be significantly differentially regulated (7 up-regulated, 8 down-regulated; p<0.05) by combining 53 datasets of tuberculosis and malignancies. Most of the miRNA signatures identified in this study were found to be involved in immune responses and metabolism. Expression of these miRNA signatures in serum samples from TB subjects (n=11) as well as healthy controls (n=10) was examined by TaqMan miRNA array. Taken together, the results revealed differential expression of miRNAs in TB, but available datasets are limited and these miRNA signatures should be validated in future studies.

A Subgroup of Latently Mycobacterium tuberculosis Infected Individuals Is Characterized by Consistently Elevated IgA Responses to Several Mycobacterial Antigens.

Elevated antibody responses to Mycobacterium tuberculosis antigens in individuals with latent infection (LTBI) have previously been linked to an increased risk for progression to active disease. Studies in the field focussed mainly on IgG antibodies. In the present study, IgA and/or IgG responses to the mycobacterial protein antigens AlaDH, NarL, 19 kDa, PstS3, and MPT83 were determined in a blinded fashion in sera from 53 LTBI controls, 14 healthy controls, and 42 active TB subjects. Among controls, we found that elevated IgA levels against all investigated antigens were not randomly distributed but concentrated on a subgroup of <30%—with particular high levels in a small subgroup of ~5% comprising one progressor to active TB. Based on a specificity of 100%, anti-NarL IgA antibodies achieved with 78.6% sensitivity the highest accuracy for the detection of active TB compared to healthy controls. In conclusion, the consistently elevated IgA levels in a subgroup of controls suggest higher mycobacterial load, a risk factor for progression to active TB, and together with high IgG levels may have prognostic potential and should be investigated in future large scale studies. The novel antigen NarL may also be promising for the antibody-based diagnosis of active TB cases.

Latent TB Infection Diagnosis in Population Exposed to TB Subjects in Close and Poor Ventilated High TB Endemic Zone in India

BackgroundThe present study was designed to investigate the utility of Quantiferon TB gold (QFT-G) and Tuberculin skin test (TST) for diagnosis of latent TB infection (LTBI) in high crowding TB endemic zone of Nagpur, India and their comparison with associated risk factors.MethodsOut of 342 eligible participants, QFT-G and TST were performed in 162 participants.ResultsThe prevalence of LTBI observed according to QFT-G and TST was 48% and 42% respectively, with an agreement of 52.47%. QFT-G positivity was associated with age while TST positivity was associated with body mass index (BMI). Duration of exposure emerged as a key risk factor significantly associated with both the tests.ConclusionThe prevalence of LTBI was quite high in the studied zone as detected by both the evaluated tests and thus, the combination of both the tests will be best predictive for LTBI in such high TB endemic regions.

In silico analysis of potential human T Cell antigens from Mycobacterium tuberculosis for the development of subunit vaccines against tuberculosis

In silico analysis was used to predict MHC class I and class II promiscuous epitopes and potential antigens, from 24 novel T cell antigens of Mycobacterium tuberculosis. Majority of the antigens (16/24) had high affinity peptides to both MHC class I and class II alleles and higher population coverage compared to well-proven T cell antigens ESAT-6, CFP-10 and Ag85B. Among these, highest population coverage were calculated for three novel T cell antigens Rv0733 (97.24%), Rv0462 (96.9%) and Rv2251 (96.3%). The prediction results were experimentally tested by in vitro stimulation of these novel T cell antigens with blood drawn from QuantiFERON-TB Gold In-Tube (QFT-IT) positive healthy household contacts of tuberculosis patients and pulmonary TB patients. Significantly higher level interferon-γ (IFN-γ) was observed, with these novel T cell antigens, in healthy household contacts compared to pulmonary TB subjects (p = 0.0001). In silico analysis also resulted in prediction of 36 promiscuous epitopes from the novel 24 T cell antigens. Population coverage for 4 out of the 36 promiscuous epitopes was >90% [67 VVLLWSPRS (Rv1324), 42 VVGVTTNPS (Rv1448c), 178 MRFLLSAKS (Rv0242c) and 842 IRLMALVEY (Rv3800c)]. Our results shows that these novel antigens and promiscuous epitopes identified from our analysis can further be investigated for their usefulness for subunit vaccine development.

Lack of Mycobacterium tuberculosis–specific interleukin‐17A–producing CD4+ T cells in active disease

Protective immunity to Mycobacterium tuberculosis (Mtb) is commonly ascribed to a Th1 profile; however, the involvement of Th17 cells remains to be clarified. Here, we characterized Mtb-specific CD4(+) T cells in blood and bronchoalveolar lavages (BALs) from untreated subjects with either active tuberculosis disease (TB) or latent Mtb infection (LTBI), considered as prototypic models of uncontrolled or controlled infection, respectively. The production of IL-17A, IFN-γ, TNF-α, and IL-2 by Mtb-specific CD4(+) T cells was assessed both directly ex vivo and following in vitro antigen-specific T-cell expansion. Unlike for extracellular bacteria, Mtb-specific CD4(+) T-cell responses lacked immediate ex vivo IL-17A effector function in both LTBI and TB individuals. Furthermore, Mtb-specific Th17 cells were absent in BALs, while extracellular bacteria-specific Th17 cells were identified in gut biopsies of healthy individuals. Interestingly, only Mtb-specific CD4(+) T cells from 50% of LTBI but not from TB subjects acquired the ability to produce IL-17A following Mtb-specific T-cell expansion. Finally, IL-17A acquisition by Mtb-specific CD4(+) T cells correlated with the coexpression of CXCR3 and CCR6, currently associated to Th1 or Th17 profiles, respectively. Our data demonstrate that Mtb-specific Th17 cells are selectively undetectable in peripheral blood and BALs from TB patients.

Highly Accurate Diagnosis of Pleural Tuberculosis by Immunological Analysis of the Pleural Effusion

Pleural TB is notoriously difficult to diagnose due to its paucibacillary nature yet it is the most common cause of pleural effusions in TB endemic countries such as The Gambia. We identified both cellular and soluble biomarkers in the pleural fluid that allowed highly accurate diagnosis of pleural TB compared to peripheral blood markers. Multi-plex cytokine analysis on unstimulated pleural fluid showed that IP-10 resulted in a positive likelihood ratio (LR) of 9.6 versus 2.8 for IFN-γ; a combination of IP-10, IL-6 and IL-10 resulted in an AUC of 0.96 and positive LR of 10. A striking finding was the significantly higher proportion of PPD-specific IFN-γ+TNF-α+ cell population (PPD-IGTA) in the pleural fluid compared to peripheral blood of TB subjects. Presence of this pleural PPD-IGTA population resulted in 95% correct classification of pleural TB disease with a sensitivity of 95% and specificity of 100%. These data suggest that analysis of the site of infection provides superior diagnostic accuracy compared to peripheral blood for pleural TB, likely due to the sequestration of effector cells at this acute stage of disease.

21 Uric acid and oxidative status in obese subjects

losses of 0.6 to 1.2 g/kg per day have been reported. There is a marked reduction in key micronutrient levels in fevers (vitamin B2), tuberculosis (vitamins A, D, C and E, zinc and iron) and cancer (vitamin C, B2, B6, B12 and D, magnesium and zinc). Almost any nutritional deficiency can further compromise the immune functionality, impacting health outcome, quality of life and recovery from illness. It is therefore, essential to replace the nutritional losses sustained during illness as well as provide additional allowances for convalescence. Increased protein requirements of up to 2 g/day and micronutrient intakes to at least the recommended allowances or higher are indicated. Studies report positive effects of optimal nutritional support with significant increases in body weight, total lean mass and grip strength in TB subjects. Cancer subjects reported improved nutritional status, body weight, un-interrupted course of radiation (in case of subjects undergoing radiotherapy), reduced postoperative complications and improved quality of life as a result of nutrition intervention. Nutritional support should therefore be an integral part of the comprehensive care to optimise treatment for illness like fevers, TB and cancer.

Dynamic Variation in the Cellular Origin of HIV Type 1 during Treatment of Tuberculosis in Dually Infected Subjects

HIV-1 replication remains elevated in dually infected HIV-1/TB subjects at completion of antituberculosis therapy. A viral immunocapture assay was used to examine the cellular origin of HIV-1 within plasma from HIV-1/TB subjects at time of diagnosis of pulmonary TB, at end of TB treatment, and 6 months after completion of treatment. Asymptomatic HIV-1-infected subjects without TB (HIV-1/C) served as controls. Both activated immature macrophage (CD36(+)) and CD4 T cell (CD26(+)) compartments contributed to viral load. Changes in the activation status of either cellular compartment paralleled their contribution to viral load. Levels of HIV-1 originating from activated (HLA-DR(+)) cells and from CD36(+) and CD26(+) mononuclear cells resolved to levels observed in HIV-1/C by the end of treatment. HIV-1 isolated by anti-CD3 immunocapture from HIV-1/TB patients remained significantly higher than from HIV-1/C patients at the end of TB treatment and at 12 months follow-up. Therefore, viral production by lymphocytes extends well beyond the completion of TB treatment.

Inhibition of human immunodeficiency virus-1 (HIV-1) by β-chemokine analogues in mononuclear cells from HIV-1-infected patients with active tuberculosis

Tuberculosis (TB) enhances human immunodeficiency virus-1 (HIV-1) activity in patients with dual HIV-1/TB infection. Therapies that control augmentations of HIV-1 activity at sites of Mycobacterium tuberculosis (MTB) infection may be useful in inhibition of viral expansion. Regulated upon activation, normal T-cell expressed and secreted (RANTES) analogues (AOP and NNY) are potent in inhibiting the entry of primary HIV-1 isolates into host mononuclear cells. These analogues were used to inhibit MTB-induced HIV-1 entry in blood monunuclear cells (PBMC) from patients with pulmonary TB, and pleural fluid mononuclear cells (PFMC) from patients with pleural TB. PBMC or PFMC were cultured with and without MTB in presence and absence of RANTES analogues. HIV-1 strong stop DNA was assessed by real-time polymerase chain reaction (PCR) as a measure of infection. CCR5 mRNA was assessed by real-time reverse transcription (RT)-PCR and by immunostaining and FACS analysis. HIV-1 infection was induced by MTB in vitro in PBMC from the majority (14 of 20) of HIV-1/TB subjects, and new infection was inhibited by AOP- or NNY-RANTES. HIV-1 infection was also inhibited by these reagents in MTB-induced PFMC from three of three patients with pleural TB. Expression of CCR5 mRNA was significantly induced by MTB in PBMC from patients with pulmonary TB. Further, expression of CCR5 was higher in PFMC compared to PBMC from patients with pleural TB. Also, CCR5 was fourfold higher on CD14(+) pleural mononuclear cells than on CD4(+) lymphocytes. Blocking new HIV-1 infection of mononuclear cells may be useful in control of HIV-1 during dual HIV-1/TB infection.

Correlates of protective immune response in tuberculous pleuritis

Tuberculous pleuritis (TB) provides a good model to study the correlates of protective immune response at the site of infection. To study the in vivo correlates of immunity, cell subset profile and cytokine assay in plasma (BL) and pleural fluid (PF) of 82 patients were done. Lymphocyte proliferation and cytokine response to mycobacterial antigens were measured in 32 subjects to understand the in vitro correlates. Increase in CD4 + cells and CD4 +/CD8 + ratio with selective concentration of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-12 in PF suggests that the CD4 + population may be of TH1 type. We observed an accelerated lymphoproliferative response to purified protein derivative (PPD) and heat killed Mycobacterium tuberculosis (MTB) in PF cells of both TB and non-TB (NTB) subjects. Interestingly, in in vitro studies, IL-4 levels together with IFN-γ were significantly increased in the supernatants of PF mononuclear cells (PFMC) of TB patients and showed a shift in immune response towards TH0/TH2 type. PPD and MTB antigens induced an enhanced proliferation of PFMC and also increased in vitro IL-4 response together with apoptosis, thus eliciting a dual response.