Karin Weldingh

Prospective Evaluation of a Whole-Blood Test Using Mycobacterium tuberculosis-Specific Antigens ESAT-6 and CFP-10 for Diagnosis of Active Tuberculosis

ABSTRACT A new immunodiagnostic test based on the Mycobacterium tuberculosis-specific antigens CFP-10/ESAT-6(QFT-RD1) has been launched as an aid in the diagnosis of latent tuberculosis (TB) infection (LTBI). The aim of this study was to evaluate this test for the diagnosis of active TB. Eighty-two patients with suspicion of TB and 39 healthy BCG-vaccinated persons were enrolled. Forty-eight had active TB, 25 did not, and 9 were excluded. Sensitivity and specificity of the test for active TB were evaluated in a prospective blinded manner in patients suspected of TB. The sensitivity of the QFT-RD1 was 85% (40/48; confidence interval [CI], 75 to 96), and it was higher than the sensitivity of microscopy, 42% (20/48; CI, 27 to 56; P = 0.001), and culture, 59% (27/46; CI, 44 to 73; P = 0.009). Of patients with extrapulmonary TB, 92% (12/13) were QFT-RD1 positive, whereas only 31% (4/13) were positive by microscopy and 42% (5/12) by culture (P < 0.05), and 87% (13/15) of those who were negative by both microscopy and culture were QFT-RD1 positive. By combining microscopy and culture with the QFT-RD1 test, sensitivity increased to 96% (CI, 90 to 102). Ten of 25 (40%) non-TB patients were QFT-RD1 positive, resulting in a specificity of 60%. However, 80% (8/10) of these had risk-factors for TB, indicating latent infection in this group. In healthy controls, only 3% (1/39) were QFT-RD1 positive. In conclusion, the QFT-RD1 test is sensitive for diagnosis of TB, especially in patients with negative microscopy and culture. The accuracy of the QFT-RD1 test will vary with the prevalence of LTBI. We suggest that the QFT-RD1 test could be a very useful supplementary tool for the diagnosis of TB.

Healthy Individuals That Control a Latent Infection with Mycobacterium tuberculosis Express High Levels of Th1 Cytokines and the IL-4 Antagonist IL-4δ2

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop disease and identifying what constitutes “protective immunity” is one of the holy grails of M. tuberculosis immunology. It is known that IFN-γ is essential for protection, but it is also apparent that IFN-γ levels alone do not explain the immunity/susceptibility dichotomy. The controversy regarding correlates of immunity persists because identifying infected but healthy individuals (those who are immune) has been problematic. We have therefore used recognition of the M. tuberculosis virulence factor early secretory antigenic target 6 to identify healthy, but infected individuals from tuberculosis (TB)-endemic and nonendemic regions (Ethiopia and Denmark) and have compared signals for cytokines expressed directly ex vivo with the pattern found in TB patients. We find that TB patients are characterized by decreased levels of Th1 cytokines and increased levels of IL-10 compared with the healthy infected and noninfected community controls. Interestingly, the healthy infected subjects exhibited a selective increase of message for the IL-4 antagonist, IL-4δ2, compared with both TB patients or noninfected individuals. These data suggest that long-term control of M. tuberculosis infection is associated not just with elevated Th1 responses but also with inhibition of the Th2 response.

Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection.

Mycobacterium tuberculosis is the infectious agent giving rise to human tuberculosis. The entire genome of M. tuberculosis, comprising approximately 4000 open reading frames, has been sequenced. The huge amount of information released from this project has facilitated proteome analysis of M. tuberculosis. Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was applied to fractions derived from M. tuberculosis culture filtrate, cell wall, and cytosol, resulting in the resolution of 376, 413, and 395 spots, respectively, in silver‐stained gels. By microsequencing and immunodetection, 38 culture filtrate proteins were identified and mapped, of which 12 were identified for the first time. In the same manner, 23 cell wall proteins and 19 cytosol proteins were identified and mapped, with 9 and 10, respectively, being novel proteins. One of the novel proteins was not predicted in the genome project, and for four of the identified proteins alternative start codons were suggested. Fourteen of the culture filtrate proteins were proposed to possess signal sequences. Seven of these proteins were microsequenced and the N‐terminal sequences obtained confirmed the prediction. The data presented here are an important complement to the genetic information, and the established 2‐D PAGE maps (also available at: www.ssi.dk/publichealth/tbimmun) provide a basis for comparative studies of protein expression.

Towards the proteome of Mycobacterium tuberculosis

Human tuberculosis is caused by the intracellular pathogen Mycobacterium tuberculosis. Sequencing of the genome of M. tuberculosis strain H37Rv has predicted 3924 open reading frames, and enabled identification of proteins from this bacterium by peptide mass fingerprinting. Extracellular proteins from the culture medium and proteins in cellular extracts were examined by two‐dimensional gel electrophoresis using immobilized pH gradient technology. By mass spectrometry and immunodetection, 49 culture filtrate proteins and 118 lysate proteins were identified, 83 of which were novel. To date, 288 proteins have been identified in M. tuberculosis proteome studies, and a list is presented which includes all identified proteins (available at http://www.ssi.dk/publichealth/tbimmun). The information obtained from the M. tuberculosis proteome so far is discussed in relation to the information obtained from the complete genome sequence.

A systematic review of commercial serological antibody detection tests for the diagnosis of extrapulmonary tuberculosis

Conventional diagnostic tests for tuberculosis have several limitations and are often unhelpful in establishing the diagnosis of extrapulmonary tuberculosis. Although commercial serological antibody based tests are available, their usefulness in the diagnosis of extrapulmonary tuberculosis is unknown. A systematic review was conducted to assess the accuracy of commercial serological antibody detection tests for the diagnosis of extrapulmonary tuberculosis. In a comprehensive search, 21 studies that reported data on sensitivity and specificity for extrapulmonary tuberculosis were identified. These studies evaluated seven different commercial tests, with Anda-TB IgG accounting for 48% of the studies. The results showed that (1) all commercial tests provided highly variable estimates of sensitivity (range 0.00–1.00) and specificity (range 0.59–1.00) for all extrapulmonary sites combined; (2) the Anda-TB IgG kit showed highly variable sensitivity (range 0.26–1.00) and specificity (range 0.59–1.00) for all extrapulmonary sites combined; (3) for all tests combined, sensitivity estimates for both lymph node tuberculosis (range 0.23–1.00) and pleural tuberculosis (range 0.26–0.59) were poor and inconsistent; and (4) there were no data to determine the accuracy of the tests in children or in patients with HIV infection, the two groups for which the test would be most useful. At present, commercial antibody detection tests for extrapulmonary tuberculosis have no role in clinical care or case detection.

Immunogenicity of Novel DosR Regulon-Encoded Candidate Antigens of Mycobacterium tuberculosis in Three High-Burden Populations in Africa

ABSTRACT Increasing knowledge about DosR regulon-encoded proteins has led us to produce novel Mycobacterium tuberculosis antigens for immunogenicity testing in human populations in three countries in Africa to which tuberculosis (TB) is endemic. A total of 131 tuberculin skin test-positive and/or ESAT-6/CFP10-positive, human immunodeficiency virus-negative adult household contacts of active pulmonary TB cases from South Africa (n = 56), The Gambia (n = 26), and Uganda (n = 49) were tested for gamma interferon responses to 7 classical and 51 DosR regulon-encoded M. tuberculosis recombinant protein antigens. ESAT-6/CFP10 fusion protein evoked responses in >75% of study participants in all three countries. Of the DosR regulon-encoded antigens tested, Rv1733c was the most commonly recognized by participants from both South Africa and Uganda and the third most commonly recognized antigen in The Gambia. The four most frequently recognized DosR regulon-encoded antigens in Uganda (Rv1733c, Rv0081, Rv1735c, and Rv1737c) included the three most immunogenic antigens in South Africa. In contrast, Rv3131 induced the highest percentage of responders in Gambian contacts (38%), compared to only 3.4% of Ugandan contacts and no South African contacts. Appreciable percentages of TB contacts with a high likelihood of latent M. tuberculosis infection responded to several novel DosR regulon-encoded M. tuberculosis proteins. In addition to significant similarities in antigen recognition profiles between the three African population groups, there were also disparities, which may stem from genetic differences between both pathogen and host populations. Our findings have implications for the selection of potential TB vaccine candidates and for determining biosignatures of latent M. tuberculosis infection, active TB disease, and protective immunity.

Inhibition of anti-tuberculosis T-lymphocyte function with tumour necrosis factor antagonists.

Reactivation of latent Mycobacterium tuberculosis (Mtb) infection is a major complication of anti-tumour necrosis factor (TNF)-α treatment, but its mechanism is not fully understood. We evaluated the effect of the TNF antagonists infliximab (Ifx), adalimumab (Ada) and etanercept (Eta) on anti-mycobacterial immune responses in two conditions: with ex vivo studies from patients treated with TNF antagonists and with the in vitro addition of TNF antagonists to cells stimulated with mycobacterial antigens. In both cases, we analysed the response of CD4+ T lymphocytes to purified protein derivative (PPD) and to culture filtrate protein (CFP)-10, an antigen restricted to Mtb. The tests performed were lymphoproliferation and immediate production of interferon (IFN)-γ. In the 68 patients with inflammatory diseases (rheumatoid arthritis, spondylarthropathy or Crohns disease), including 31 patients with a previous or latent tuberculosis (TB), 14 weeks of anti-TNF-α treatment had no effect on the proliferation of CD4+ T lymphocytes. In contrast, the number of IFN-γ-releasing CD4+ T lymphocytes decreased for PPD (p < 0.005) and CFP-10 (p < 0.01) in patients with previous TB and for PPD (p < 0.05) in other patients (all vaccinated with Bacille Calmette-Guérin). Treatments with Ifx and with Eta affected IFN-γ release to a similar extent. In vitro addition of TNF antagonists to CD4+ T lymphocytes stimulated with mycobacterial antigens inhibited their proliferation and their expression of membrane-bound TNF (mTNF). These effects occurred late in cultures, suggesting a direct effect of TNF antagonists on activated mTNF+ CD4+ T lymphocytes, and Ifx and Ada were more efficient than Eta. Therefore, TNF antagonists have a dual action on anti-mycobacterial CD4+ T lymphocytes. Administered in vivo, they decrease the frequency of the subpopulation of memory CD4+ T lymphocytes rapidly releasing IFN-γ upon challenge with mycobacterial antigens. Added in vitro, they inhibit the activation of CD4+ T lymphocytes by mycobacterial antigens. Such a dual effect may explain the increased incidence of TB in patients treated with TNF antagonists as well as possible differences between TNF antagonists for the incidence and the clinical presentation of TB reactivation.

Assessing the Serodiagnostic Potential of 35 Mycobacterium tuberculosis Proteins and Identification of Four Novel Serological Antigens

ABSTRACT Improved diagnostic reagents are needed for the detection of Mycobacterium tuberculosis infections, and the development of a serodiagnostic test would complement presently available diagnostic methods. The aim of the present study was to identify novel serological targets for use for the future serodiagnosis of tuberculosis (TB). We cloned and expressed 35 M. tuberculosis proteins as recombinant proteins in Escherichia coli and analyzed their serodiagnostic potentials. By a two-step selection process, four superior seroantigens, TB9.7, TB15.3, TB16.3, and TB51, were identified, none of which has been described before. The four novel antigens were tested with panels of sera from smear-positive and smear-negative TB patients from areas both where TB is endemic and where TB is not endemic, with recognition frequencies ranging from 31 to 93% and with a specificity of at least 97%. The single most potent antigen was TB16.3, which had a sensitivity of 48 to 55% with samples from Danish resident TB patients and a sensitivity of 88 to 98% with samples from African TB patients. Importantly, the TB16.3 and the TB9.7 antigens were recognized by more than 85% of the samples from TB patients coinfected with human immunodeficiency virus, a patient group for which it is in general difficult to detect M. tuberculosis-specific antibodies.

Comparison of in vitro-specific blood tests with tuberculin skin test for diagnosis of latent tuberculosis before anti-TNF therapy

Introduction: Latent tuberculosis infection (LTBI) is detected with the tuberculin skin test (TST) before anti-TNF therapy. We aimed to investigate in vitro blood assays with TB-specific antigens (CFP-10, ESAT-6), in immune-mediated inflammatory diseases (IMID) for LTBI screening. Patients and methods: Sixty-eight IMID patients with (n = 35) or without (n = 33) LTBI according to clinico-radiographic findings or TST results (10 mm cutoff value) underwent cell proliferation assessed by thymidine incorporation and PKH-26 dilution assays, and IFNγ-release enzyme-linked immunosorbent spot (ELISPOT) assays with TB-specific antigens. Results: In vitro blood assays gave higher positive results in patients with LTBI than without (p<0.05), with some variations between tests. Among the 13 patients with LTBI diagnosed independently of TST results, 5 had a negative TST (38.5%) and only 2 a negative blood assays result (15.4%). The 5 LTBI patients with negative TST results all had positive blood assays results. Ten patients without LTBI but with intermediate TST results (6–10 mm) had no different result than patients with TST result ⩽5 mm (p>0.3) and lower results than those with LTBI (p<0.05) on CFP-10+ESAT-6 ELISPOT and CFP-10 proliferation assays. Conclusion: Anti-TB blood assays are beneficial for LTBI diagnosis in IMID. Compared with TST, they show a better sensitivity, as seen by positive results in 5 patients with certain LTBI and negative TST, and better specificity, as seen by negative results in most patients with intermediate TST as the only criteria of LTBI. In the absence of clinico-radiographic findings for LTBI, blood assays could replace TST for antibiotherapy decision before anti-TNF.

Proteomic Profiling of Mycobacterium tuberculosis Identifies Nutrient-starvation-responsive Toxin–antitoxin Systems

In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis of fractionated samples, 1176 proteins were identified from culture filtrates of log phase and nutrient-starved cultures, and the protein levels of 230 proteins were increased in nutrient-starved culture filtrates, whereas those of 208 proteins were decreased. By means of Gene Ontology clustering analysis, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin–antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy. Decreased abundance of proteins involved in amino acid and protein synthesis was apparent, as well as changes in the lipid metabolism. Further analysis of the dataset identified increased abundance of lipoproteins and decreased abundance of ESAT-6 family proteins. Results from the two-dimensional difference gel electrophoresis proteomics demonstrated overall agreement with the LC-MS/MS data and added complementary insights about protein degradation and modification.