Clinical Evaluation of a Homemade Enzyme-Linked Immunospot Assay for the Diagnosis of Active Tuberculosis in China

The rapid diagnosis of smear-negative pulmonary tuberculosis (TB) and extrapulmonary TB is a significant problem in clinical practice. We evaluated the usefulness of a homemade enzyme-linked immunospot (ELISPOT) assay for the diagnosis of active TB in China. Seventy-eight healthy volunteers, 60 patients with active TB, and 32 patients with non-TB diseases were evaluated by tuberculin skin test (TST), an ELISPOT assay using a recombinant CFP-10/ESAT-6 fusion protein (rCFP-10/ESAT-6) as a stimulant, and T-SPOT-TB assay. The spot-forming cells (SFC) from 78 healthy subjects containing both PPD-positive and -negative persons was 3.7 ± 6.5. Among 31 diagnosed TB patients, the ELISPOT assay had a sensitivity of 67.7%, compared to a sensitivity of 77.4% for the T-SPOT-TB assay. The ELISPOT assay was more sensitive in smear-positive TB cases (76.9%) than in smear-negative TB cases (61.1%), while T-SPOT-TB had roughly similar sensitivities in smear-positive (76.9%) and smear-negative TB cases (77.8%). The specificity was 90.6% for ELISPOT and 78.1% for T-SPOT-TB among 32 subjects with non-TB diseases. The SFC of TB cases was significantly higher than that of non-TB disease cases, and the SFC of smear-positive TB cases was significantly higher than that of smear-negative TB cases (P < 0.01). We confirmed that the homemade ELISPOT assay appears more specific for the diagnosis of active TB than T-SPOT-TB. ELISPOT assay may be a useful method for the rapid diagnosis of active TB, especially for cases of smear-negative TB.     

Patients with Tuberculosis Disease Have Mycobacterium tuberculosis-Specific CD8 T Cells with a Pro-Apoptotic Phenotype and Impaired Proliferative Capacity,Which Is Not Restored following Treatment

CD8 T cells play a critical role in control of chronic viral infections; however, the role of these cells in containing persistent bacterial infections, such as those caused by Mycobacterium tuberculosis (Mtb), is less clear. We assessed the phenotype and functional capacity of CD8 T cells specific for the immunodominant Mtb antigens CFP-10 and ESAT-6, in patients with pulmonary tuberculosis (TB) disease, before and after treatment, and in healthy persons with latent Mtb infection (LTBI). In patients with TB disease, CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells had an activated, pro-apoptotic phenotype, with lower Bcl-2 and CD127 expression, and higher Ki67, CD57, and CD95 expression, than in LTBI. When CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells were detectable, expression of distinct combinations of these markers was highly sensitive and specific for differentiating TB disease from LTBI. Successful treatment of disease resulted in changes of these markers, but not in restoration of CFP-10/ESAT-6-specific CD8 or CD4 memory T cell proliferative capacity. These data suggest that high mycobacterial load in active TB disease is associated with activated, short-lived CFP-10/ESAT-6-specific CD8 T cells with impaired functional capacity that is not restored following treatment. By contrast, LTBI is associated with preservation of long-lived CFP-10/ESAT-6-specific memory CD8 T cells that maintain high Bcl-2 expression and which may readily proliferate.     

Potential Role of M. tuberculosis Specific IFN-γ and IL-2 ELISPOT Assays in Discriminating Children with Active or Latent Tuberculosis

Background

Although currently available IGRA have been reported to be promising markers for TB infection, they cannot distinguish active tuberculosis (TB) from latent infection (LTBI).

Objective

Children with LTBI, active TB disease or uninfected were prospectively evaluated by an in-house ELISPOT assay in order to investigate possible immunological markers for a differential diagnosis between LTBI and active TB.

Methods

Children at risk for TB infection prospectively enrolled in our infectious disease unit were evaluated by in-house IFN-γ and IL-2 based ELISPOT assays using a panel of Mycobacterium tuberculosis antigens.

Results

Twenty-nine children were classified as uninfected, 21 as LTBI and 25 as active TB cases (including 5 definite and 20 probable cases). Significantly higher IFN-γ ELISPOT responses were observed in infected vs. uninfected children for ESAT-6 (p<0.0001), CFP-10 (p<0.0001), TB 10.3 (p = 0.003), and AlaDH (p = 0.001), while differences were not significant considering Ag85B (p = 0.063), PstS1 (p = 0.512), and HspX (16 kDa) (p = 0.139). IL-2 ELISPOT assay responses were different for ESAT-6 (p<0.0001), CFP-10 (p<0.0001), TB 10.3 (p<0.0001), HspX (16 kDa) (p<0.0001), PstS1 (p<0.0001) and AlaDH (p = 0.001); but not for Ag85B (p = 0.063). Comparing results between children with LTBI and those with TB disease differences were significant for IFN-γ ELISPOT only for AlaDH antigen (p = 0.021) and for IL-2 ELISPOT assay for AlaDH (p<0.0001) and TB 10.3 antigen (p = 0.043). ROC analyses demonstrated sensitivity of 100% and specificity of 81% of AlaDH-IL-2 ELISPOT assay in discriminating between latent and active TB using a cut off of 12.5 SCF per million PBMCs.

Conclusion

Our data suggest that IL-2 based ELISPOT with AlaDH antigen may be of help in discriminating children with active from those with latent TB.     

Phenotypic Profiling of Mycobacterium tuberculosis EspA Point Mutants Reveals that Blockage of ESAT-6 and CFP-10 Secretion In Vitro Does Not Always Correlate with Attenuation of Virulence

The EspA protein of Mycobacterium tuberculosis is essential for the type VII ESX-1 protein secretion apparatus, which delivers the principal virulence factors ESAT-6 and CFP-10. In this study, site-directed mutagenesis of EspA was performed to elucidate its influence on the ESX-1 system. Replacing Trp⁵⁵ (W55) or Gly⁵⁷ (G57) residues in the putative W-X-G motif of EspA with arginines impaired ESAT-6 and CFP-10 secretion in vitro and attenuated M. tuberculosis. Replacing the Phe⁵⁰ (F50) and Lys⁶² (K62) residues, which flank the W-X-G motif, with arginine and alanine, respectively, destabilized EspA, abolished ESAT-6 and CFP-10 secretion in vitro, and attenuated M. tuberculosis. Likewise, replacing the Phe⁵ (F5) and Lys⁴¹ (K41) residues with arginine and alanine, respectively, also destabilized EspA and blocked ESAT-6 and CFP-10 secretion in vitro. However, these two particular mutations did not attenuate M. tuberculosis in cellular models of infection or during acute infection in mice. We have thus identified amino acid residues in EspA that are important for facilitating ESAT-6 and CFP-10 secretion and virulence. However, our data also indicate for the first time that blockage of M. tuberculosis ESAT-6 and CFP-10 secretion in vitro and attenuation are mutually exclusive.     

Transcriptional analysis of ESAT-6 cluster 3 in Mycobacterium smegmatis

Background  

The ESAT-6 (early secreted antigenic target, 6 kDa) family collects small mycobacterial proteins secreted by Mycobacterium tuberculosis, particularly in the early phase of growth. There are 23 ESAT-6 family members in M. tuberculosis H37Rv. In a previous work, we identified the Zur- dependent regulation of five proteins of the ESAT-6/CFP-10 family (esxG, esxH, esxQ, esxR, and esxS). esxG and esxH are part of ESAT-6 cluster 3, whose expression was already known to be induced by iron starvation.     

Combination of Cytokine Responses Indicative of Latent TB and Active TB in Malawian Adults

Background

An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.

Methods

Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.

Results

Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.

Conclusions

CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.     

The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis.     

结核分枝杆菌ESX-1分泌蛋白EspB结构和功能分析

结核分枝杆菌作为肺结核病的病原菌,在人类中致死率远高于其他病原菌.结核分枝杆菌具有特殊的疏水性细胞壁结构,这种致密的细胞壁结构帮助结核分枝杆菌抵御外界环境压力和来自宿主细胞的毒素.同时,它利用特殊的分泌系统将体内的毒力蛋白输出体外,ESX-1分泌系统就是其中之一.结核分枝杆菌ESX-1系统在结核分枝杆菌进入宿主细胞吞噬小体、逃逸至细胞质以及杀死吞噬细胞这些过程中发挥重要作用.研究表明,在结核分枝杆菌内膜上存在一个由多亚基组成、旨在帮助结核分枝杆菌向外输送分泌蛋白的分泌装置.在这个分泌装置的帮助下,结核分枝杆菌重要的毒力蛋白ESAT-6跨内膜向外分泌,EspB也通过这个内膜上的分泌装置被转运至胞外.EspB存在于静置培养的结核分枝杆菌的胶囊层中,也可在振荡培养的结核分枝杆菌的培养液中被检测.通过X射线晶体衍射分析,我们解析了EspB的晶体结构,相比于其他同源结构,发现了EspB的不同构象,即EspB单体能够自组装成为七聚体的规则结构,联系其与毒力因子ESAT-6具有共分泌的特点,七聚体构象的发现为解释EspB在结核分枝杆菌向外分泌蛋白的过程中发挥的作用提供线索,即EspB具有锚定在结核分枝杆菌胶囊层中,作为运输ESAT-6的孔道而存在的可能.     

Interferon gamma release assay in diagnosis of pediatric tuberculosis: a meta-analysis

Although interferon gamma release assays (IGRAs) have been widely used for the diagnosis of latent and active tuberculosis in adults, a relative lack of validation studies in children has led to caution in their clinical interpretation. This meta-analysis systematically evaluated two IGRAs (ELISA and ELISPOT) and the tuberculin skin test (TST). We searched databases (PubMed, MEDLINE, Ovid) between January 2000 and January 2011 using search terms of latent tuberculosis infection or tuberculosis and interferon gamma release assay, or T-SPOT.TB test, or QuantiFERON-TB Gold, or ESAT-6, or CFP-10, and child, or childhood, or pediatrics. We also collected data by performing a manual search of references from relevant articles and communicating with selected authors. The meta-analysis was conducted with random effects models to account for heterogeneity between selected studies. The sensitivities of all three tests in active tuberculosis were similar. The pooled sensitivity was 70% for ELISA studies, 62% for ELISPOT studies and 71% for TST. Calculated sensitivities for IGRAs and the TST differ in culture-confirmed tuberculosis [ELISA (85%) vs. ELISPOT (76%) vs. TST (85%)] and clinical diagnosed cases [ELISA (64%) vs. ELISPOT (58%) vs. TST (66%)]. The pooled specificity was 100% for ELISA and 90% for ELISPOT, but was much lower for TST [56% in all included studies and 49% in children with bacillus Calmette-Guerin (BCG) vaccination]. The agreement between the TST and IGRAs in non-BCG-vaccinated children is higher than that in BCG-vaccinated children. In the diagnosis of active tuberculosis in children, the TST and IGRAs have similar sensitivity. By contrast, the specificity of IGRAs is far greater than the TST, particularly in children with previous BCG vaccination.     

Evaluation of Interferon-Gamma Release Assays in the Diagnosis of Recent Tuberculosis Infection in Health Care Workers

Background

Health care workers (HCWs) are a group at risk of latent tuberculosis infection (LTBI). The aims of this study were to determine IFN-γ response by QuantiFERON-TB GOLD In Tube (QFN-G-IT) and T-SPOT.TB in HCWs, comparing the results with tuberculin skin test (TST); and to analyze the capacity of IFN-γ tests to detect recent versus remote LTBI with a prolonged stimulation test (PST).

Methodology/Principal Findings

A total of 147 HCWs were enrolled; 23 of whom were BCG vaccinated. 95 HCWs (64.6%) had a previous positive TST and were not retested; and 52 HCWs had a previous negative TST or were tested for the first time. When we analysed individuals without previous positive TST, the number of positive results for T-SPOT.TB was 12/52 (23.1%); and for QFN-G-IT, 9/52 (17.3%). The global concordance (κ) between T-SPOT.TB and QFN-G-IT with TST was 0.754 and 0.929 respectively. Of individuals with previous positive TST, T-SPOT.TB and QFN-G-IT were negative in 51.6% (49/95) and 62.1% (59/95) respectively, decreasing the concordance to 0.321 and 0.288, respectively. In non-BCG vaccinated HCWs with previous positive TST a positive IFN-γ test was associated with degree of exposure and diameter of TST. PST was performed in 24 HCW with previous positive TST and negative IFN-γ tests. PST was developed in 3 cell cultures stimulated with medium alone, ESAT-6 and CFP-10, respectively. In the third and sixth day of incubation period, part of the supernatants were replaced with complete medium supplemented with (rIL)-2. On day 9, ELISPOT assay was performed. In 14 samples PST was not valid due to not having enough cells. In 8 cases, the response was negative, and in 2 cases positive, suggesting that these patients were infected with Mycobacterium tuberculosis in some point in the past.

Conclusions

Both IFN-γ tests showed a similar number of positive results, and concordance between the tests was excellent. None of the tests was affected by prior BCG vaccination. IFN-γ tests are a useful tool for detecting recent infection in HCW population.     

Combined Antigen-Specific Interferon-γ and Interleukin-2 Release Assay (FluoroSpot) for the Diagnosis of Mycobacterium tuberculosis Infection

Background

To evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis.

Methods

Following ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls.

Results

Correlation of IFN- γ⁺ spot-forming cells in EliSpot-IGRA and FluoroSpot were R² = 0.67 for ESAT-6 and R² = 0.73 for CFP-10. The number of IL-2^- IFN- γ⁺ producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ⁺ secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis.

Conclusions

Combined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice.     

Feasibility and Diagnostic Utility of Antigen-Specific Interferon-γ Responses for Rapid Immunodiagnosis of Tuberculosis Using Induced Sputum

Background

The diagnosis of smear-negative or sputum-scarce tuberculosis (TB) is problematic as culture takes several weeks and representative biological samples are difficult to obtain. RD-1 antigen-specific interferon-γ release assays (IGRAs) are sensitive and specific blood-based tests for the diagnosis of M. tuberculosis infection. The feasibility and diagnostic utility of this rapid immunodiagnostic assay, using cells from induced sputum, is unknown.

Methodology/Principal Findings

Cells isolated from induced sputum were co-cultured with ESAT-6 and CFP-10 antigens using a standardized enzyme-linked immunospot (ELISPOT) assay (T-SPOT®.TB) in 101 consecutively recruited TB suspects or non-TB controls. An optimization phase using 28 samples was followed by a validation phase using samples from 73 participants (20 with definite or probable TB, and 48 with non-TB). Despite optimization of sputum processing 65/73 (89%) of the IGRAs in the validation phase were inconclusive. 44/73 (60%) tests failed due to sputum induction-related factors [sputum induction-related adverse events (n = 5), inadequate sputum volume (n = 8), non-homogenisable sputum (n = 7), and insufficient numbers of cells to perform the assay (n = 24)], whilst 20/73 (27%) tests failed due T-SPOT®.TB assay-related factors [excessive debris precluding reading of spots in the ELISPOT well (n = 6), failure of the positive control (n = 11), or high spot count in the negative control (n = 3)]. Only 8/73 (11%) of the available samples could therefore be correctly categorized (7 definite or probable TB, and 1 non-TB patient). Thus, 13/20 (65%) of the definite or probable TB cases remained undiagnosed.

Conclusions/Significance

Rapid immunodiagnosis of pulmonary TB by antigen-specific IFN-γ ELISPOT responses, using cells from induced sputum, is possible. However, the test, in its current ELISPOT format, is not clinically useful because the majority of the assays are inconclusive.     

Decay Kinetics of an Interferon Gamma Release Assay with Anti-Tuberculosis Therapy in Newly Diagnosed Tuberculosis Cases

Background

Qualitative and quantitative changes in IGRA response offer promise as biomarkers to monitor Tuberculosis (TB) drug therapy, and for the comparison of new interventions. We studied the decay kinetics of TB-specific antigen T-cell responses measured with an in-house ELISPOT assay during the course of therapy.

Methods

Newly diagnosed sputum smear positive TB cases with typical TB chest radiographs were recruited. All patients were given standard anti-TB treatment. Each subject was followed up for 6 months and treatment outcomes were documented. Blood samples were obtained for the ESAT-6 and CFP-10 (EC) ELISPOT at diagnosis, 1-, 2-, 4- and 6-months. Qualitative and quantitative reversion of the ELISPOT results were assessed with McNemar test, conditional logistic regression and mixed-effects hierarchical Poisson models.

Results

A total of 116 cases were recruited and EC ELISPOT was positive for 87% (95 of 109) at recruitment. There was a significant decrease in the proportion of EC ELISPOT positive cases over the treatment period (p<0.001). Most of the reversion occurred between the start and first month of treatment and at completion at 6 months. ESAT-6 had higher median counts compared to CFP-10 at all time points. Counts for each antigen declined significantly with therapy (p<0.001). Reverters had lower median SFUs at the start of treatment compared to non-Reverters for both antigens. Apart from the higher median counts for non-Reverters, no other risk factors for non-reversion were found.

Conclusions

TB treatment induces qualitative and quantitative reversion of a positive in-house IGRA in newly diagnosed cases of active TB disease. As this does not occur reliably in the majority of cured individuals, qualitative and quantitative reversion of an IGRA ELISPOT has limited clinical utility as a surrogate marker of treatment efficacy.     

Evaluation of Rv0220, Rv2958c,Rv2994 and Rv3347c of Mycobacterium tuberculosis for serodiagnosis of tuberculosis

Tuberculosis (TB), the leading cause of death among infectious diseases worldwide, is caused by Mycobacterium tuberculosis (M. tuberculosis). Early accurate diagnosis means earlier prevention, treatment and control of TB. To confirm efficient diagnostic antigens for M. tuberculosis, the serodiagnosis value of four recombinant proteins including Rv0220, Rv2958c, Rv2994 and Rv3347c was evaluated in this study. The specificities and sensitivities of four recombinant proteins were determined based on enzyme‐linked immunosorbent assay (ELISA) by screening sera from smear‐positive pulmonary TB patients (n = 92), uninfected individuals (n = 60) and patients with Mycoplasma pneumoniae (n = 32) that potentially cross‐react with M. tuberculosis. The ELISAs showed that Rv0220, Rv2958c, Rv2994 and Rv3347c exhibited high specificities and sensitivities in detecting immunoglobulin G (IgG) antibody, with 98.3/91.3%, 91.7/85.9%, 93.3/89.1% and 93.3/80.4% respectively. According to the receiver‐operating characteristic (ROC) analysis, the area under the ROC of the target proteins was 0.988, 0.969, 0.929 and 0.945 respectively. Western blot was established to evaluate the immunoreactivities of target proteins to mice and human sera. Results demonstrated that Rv0220, Rv2958c, Rv2994 and Rv3347c could specifically recognize TB‐positive sera and the sera of mice immunized with the corresponding protein. Thus, Rv0220, Rv2958c, Rv2994 and Rv3347c were valuable potential diagnostic antigens for M. tuberculosis.     

Production of TNF-α, IL-12(p40) and IL-17 Can Discriminate between Active TB Disease and Latent Infection in a West African Cohort

Background

Mycobacterium tuberculosis (MTb) infects approximately 2 billion people world-wide resulting in almost 2 million deaths per year. Determining biomarkers that distinguish different stages of tuberculosis (TB) infection and disease will provide tools for more effective diagnosis and ultimately aid in the development of new vaccine candidates. The current diagnostic kits utilising production of IFN-γ in response to TB antigens can detect MTb infection but are unable to distinguish between infection and disease. The aim of this study was to assess if the use of a longer term assay and the analysis of multiple cytokines would enhance diagnosis of active TB in a TB-endemic population.

Methods

We compared production of multiple cytokines (TNF-α, IFN-γ, IL-10, IL-12(p40), IL-13, IL-17 and IL-18) following long-term (7 days) stimulation of whole-blood with TB antigens (ESAT-6/CFP-10 (EC), PPD or TB10.4) from TB cases (n = 36) and their Mycobacterium-infected (TST+; n = 20) or uninfected (TST−; n = 19) household contacts (HHC).

Results and Conclusions

We found that TNF-α production following EC stimulation and TNF-α and IL-12(p40) following TB10.4 stimulation were significantly higher from TB cases compared to TST+ HHC, while production of IFN-γ and IL-13 were significantly higher from TST+ compared to TST- HHC following PPD or EC stimulation. Combined analysis of TNF-α, IL-12(p40) and IL-17 following TB10.4 stimulation resulted in 85% correct classification into TB cases or TST+ HHC. 74% correct classification into TST+ or TST− HHC was achieved with IFN-γ alone following TB10.4 stimulation (69% following EC) and little enhancement was seen with additional cytokines. We also saw a tendency for TB cases infected with M. africanum to have increased TNF-α and IL-10 production compared to those infected with M. tuberculosis. Our results provide further insight into the pathogenesis of tuberculosis and may enhance the specificity of the currently available diagnostic tests, particularly for diagnosis of active TB.     

Analysis of predicted CD8<Superscript>+</Superscript> T cell epitopes from proteins encoded by the specific RD regions of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> for vaccine development and specific diagnosis

A number of regions designated as RD1-RD16 (region of difference) and encompassing 129 open reading frames have been identified between Mycobacterium tuberculosis and Mycobacterium bovis on the one hand and Bacillus Calmette-Guérin on the other. Identification of T cell epitopes from this set of proteins may serve to define candidate antigens with potentials in specific diagnosis and development of new vaccines against TB. All possible nonameric peptide sequences from proteins of these M. tuberculosis specific regions were analyzed in silico for the ability to bind to 33 alleles of class I HLA. These results reveal that of all RD proteins, a significant number of these peptides are predicted to be high-affinity HLA binders (T _1/2 ≥ 100 min), irrespective of the length of the protein, and 67% of the peptides predicted to bind are mono-allelic in their binding. Pathogen peptides that could behave as self- or partially self-peptides in the host were eliminated using a comparative study with the human proteome, thus the number of peptides for analysis was reduced. The predicted epitopes can be tested experimentally for their inclusion in a potential vaccine against tuberculosis and specific diagnosis.     

Monokine induced by interferon gamma and IFN-gamma response to a fusion protein of Mycobacterium tuberculosis ESAT-6 and CFP-10 in Brazilian tuberculosis patients

IFN-gamma responses to Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 have been proposed as specific markers of M. tuberculosis infection. Monokine induced by gamma interferon (MIG/CXCL9) has been shown to be expressed by IFN-gamma stimulated mononuclear cells and to attract activated T-cells through the chemokine receptor CXCR3. Since MIG is induced early in the response to IFN-gamma, measuring MIG may provide an interesting marker to assess downstream IFN-gamma induced responses, in contrast to assays that mainly focus on quantifying production of IFN-gamma per se. We, therefore, investigated MIG and IFN-gamma responses to a fusion protein of ESAT-6 and CFP-10, and compared responses to the conserved mycobacterial antigen 85B (Ag85B) and purified protein derivative (PPD) of M. tuberculosis, in 29 BCG vaccine controls and 24 TB patients. IFN-gamma secreting cells were determined by ELISPOT, and MIG production was measured by ELISA and flow cytometry. Production of MIG in response to ESAT-6/CFP-10, Ag85B and PPD correlated overall with increased numbers of IFN-gamma secreting cells (r=0.55, P<0.0001). A significant increase was noted among patients compared to controls in the secretion of IFN-gamma and MIG following stimulation with ESAT-6/CFP-10 or PPD (P<0.05). Moreover, MIG intracellular expression was higher in TB patients compared to BCG vaccines (P<0.05) in response to ESAT-6/CFP-10 or PPD. We conclude that MIG production correlates significantly with enhanced T-cell IFN-gamma production induced by M. tuberculosis-specific antigens ESAT-6/CFP-10. These results point to MIG as a potential novel biomarker that may be helpful in assessing downstream responses induced by IFN-gamma in TB.     

Interferon gamma response to combinations 38 kDa/CFP-10, 38 kDa/MPT-64, ESAT-6/MPT-64 and ESAT-6/CFP-10, each related to a single recombinant protein of Mycobacterium tuberculosis in individuals from tuberculosis endemic areas

Several antigens of Mycobacterium tuberculosis have been identified and specificity to one or multiple antigens could determine the distinction between protective and pathogenic host reaction. Therefore T cell immune response to combinations 38 kDa/CFP-10, 38 kDa/MPT-64, ESAT-6/MPT-64 and ESAT-6/CFP-10 (each related to a single protein of Mycobacterium tuberculosis) in individuals from tuberculosis endemic areas have been examined. ELISA was used to detect IFN-gamma production in PBMC priming with single proteins and combinations in a panel of 105 individuals: 38 tuberculosis patients (6 untreated and 32 treated) and 67 healthy controls with tuberculin skin test positive or negative (TST). Brazilian TB patients highly recognized ESAT-6 (66%), but combinations improved response in the following order: ESAT-6/MPT-64 (89%) > ESAT-6/CFP-10 (73%) > 38 kDa/CFP-10 (70%), the last combination showing the highest specificity (TST(/) = 42% and TST(-) = 83%). Average IFN-gamma production in TB patients was signifi-cantly higher for 38 kDa/CFP-10 (P = 0.012) and 38 kDa/MPT-64 (P <0.035), when compared to single antigens. None of the combinations was able to discriminate TB patients from TST(+) controls; however, 38 kDa/CFP-10 displayed a borderline significance (P = 0.053). Similar to the ESAT-6/CFP-10 combination, IFN-gamma response to 38 kDa/CFP-10 showed an increased tendency in treated patients, although not signifi-cant (P = 0.16). We demonstrated for the first time that 38 kDa/CFP-10 had prediction sensitivity for TB patients similar to the ESAT-6/CFP-10 combination and also significant response improvement related to the single proteins with more selective reactivity among TST-positive individuals, which could be of potential interest for diagnostic evaluation for tuberculosis infection.     

Selection and Application of ssDNA Aptamers to Detect Active TB from Sputum Samples

Background

Despite the enormous global burden of tuberculosis (TB), conventional approaches to diagnosis continue to rely on tests that have major drawbacks. The improvement of TB diagnostics relies, not only on good biomarkers, but also upon accurate detection methodologies. The 10-kDa culture filtrate protein (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) are potent T-cell antigens that are recognised by over 70% of TB patients. Aptamers, a novel sensitive and specific class of detection molecules, has hitherto, not been raised to these relatively TB-specific antigens.

Methods

DNA aptamers that bind to the CFP-10.ESAT-6 heterodimer were isolated. To assess their affinity and specificity to the heterodimer, aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA). One suitable aptamer was evaluated by ELONA using sputum samples obtained from 20 TB patients and 48 control patients (those with latent TB infection, symptomatic non TB patients, and healthy laboratory volunteers). Culture positivity for Mycobacterium tuberculosis (Mtb) served as the reference standard. Accuracy and cut-points were evaluated using ROC curve analysis.

Results

Twenty-four out of the 66 aptamers that were isolated bound significantly (p<0.05) to the CFP-10.ESAT-6 heterodimer and six were further evaluated. Their dissociation constant (K_D) values were in the nanomolar range. One aptamer, designated CSIR 2.11, was evaluated using sputum samples. CSIR 2.11 had sensitivity and specificity of 100% and 68.75% using Youden’s index and 35% and 95%, respectively, using a rule-in cut-point.

Conclusion

This preliminary proof-of-concept study suggests that a diagnosis of active TB using anti-CFP-10.ESAT-6 aptamers applied to human sputum samples is feasible.     

The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria

Background  

The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide.