Tuberculosis immunodiagnosis: delving below the surface.

It is one hundred and thirty years since Franz Ziehl and Friedrich Neelsen developed the rapid stain for acid-fast bacilli; 2 accurate point-of-care diagnosis of active tuberculosis (TB) remains a major unmet clinical need. With the sensitivity of the Ziehl–Neelsen stain in sputum less than 50% and more than 20% of TB cases negative on both acid-fast stain and culture for Mycobacterium tuberculosis, there has long been a yawning gap in the diagnostic toolkit for TB. New rapid molecular methods have recently improved detection of M tuberculosis nucleic acids in sputum providing diagnostic sensitivity that is much higher than sputum-smear microscopy but lower than culture. The longstanding diagnostic gap has stimulated decades of research into immunodiagnosis, mostly serological. Although serological tests for TB are point-of-care, they lack diagnostic accuracy and are devoid of clinical utility. After thorough review of the evidence, and on account of their continued widespread misuse in many high-burden countries, WHO recently tried to bury current commercial serological test kits with a negative endorsement warning against their use. Research into cellular immunodiagnosis has been more fruitful, delivering a tangible advance in clinical practice in the form of interferon-gamma release-assays (IGRA). IGRA detect M tuberculosis infection, providing a new standard-of-care for diagnosis of latent TB infection (LTBI), but they cannot distinguish active TB from LTBI. Hence, their potential role in evaluation of patients with suspected active TB is limited to that of a possible rule-out test of TB, yet currently available IGRA lack sufficient diagnostic sensitivity for this indication, as discussed elsewhere in this issue of Thorax. 7 Are there immune responses that differ sufficiently between active TB and LTBI to enable development of an immunodiagnostic test that is specific for active TB? Quantifying genome-wide host geneexpression yielded a signature of 393 genes whose differential expression between active and latent TB enabled accurate distinction of active TB from most cases of LTBI. Translating such a genomic signature into a clinically useful diagnostic test suitable for use in routine diagnostic laboratories is a priority, but will be a long and arduous process. Could we use existing measures of immune responses more intelligently to tease out responses that predominate in active TB versus LTBI? This requires delving below the ‘surface’ of the humoral immune response to uncover the different components that contribute to the composite read-out that is measured by conventional serological tests. Such tests do not distinguish between antibodies that are currently being produced in vivo in response to an infection and pre-existing antibodies. Serum antibody levels include the cumulative history of antibody responses induced by previous exposure to antigens. However, during an active infection, responding B cells migrate through the circulation as plasmablasts secreting antibodies specific for the pathogen. By separating out peripheral blood white cells from blood (and hence from serum) it is possible to measure newly secreted antibodies produced by circulating activated plasmablasts in vitro, independently of pre-existing antibodies (figure 1), as was first shown for measurement of vaccine-induced responses. This method, known as Antibodies in Lymphocyte Supernatant (ALS), was previously used with BCG vaccine as antigen to detect BCG-specific antibodies in TB patients. It showed promising results as a biomarker for active TB in both adults 10 and children. 12 In this issue of Thorax, Ashenafi et al evaluated this assay in parallel with the tuberculin skin test (TST) and an IGRA (Quantiferon TB-Gold-in-tube) in adult TB patients (with and without HIV to coinfection) and asymptomatic controls with and without LTBI in Ethiopia. By comparing these tests with a definite clinical diagnosis of TB, (which was carefully defined by microbiological, cytological and clinical characteristics), they were able to construct receiver-operating characteristic curves and an optimal cut-off for ALS positivity. Using this, they showed that the ALS assay was sensitive (positive in 91% of all cases of active TB), and could discriminate active TB from LTBI where the test was only positive in 16% of cases; its specificity was 94% in asymptomatic controls without LTBI. As expected, and in contrast with ALS, neither the TST nor the IGRA discriminated active TB from LTBI. Additionally, in a subset of patients, they were able to show that the number of IgG+ plasmablasts in peripheral blood mononuclear cells (PBMCs), as determined by flow cytometry, correlated with the titres of BCG-specific IgG antibodies secreted by the cultured PBMCs in the ALS assay. In the context of the existing literature with ALS in TB, the main advance provided by this study is that the diagnostic sensitivity of this assay is well maintained in HIV coinfection, including advanced immunosuppression. Given the difficulty of reliably diagnosing TB in HIV coinfection, advances in this patient population are urgently needed. Intriguingly, there was a significant inverse correlation between CD3 T cell numbers and BCG-specific IgG titres among TB patients. This paradoxical correlation of increasing diagnostic sensitivity with advancing HIV-induced immunosuppression is analogous to the detection of the M tuberculosis cell wall component lipoarabinomannan (LAM), in urine, where mycobacterial dissemination in immunosuppression results in increased diagnostic sensitivity. An interesting pattern is emerging where certain new diagnostic tests for TB may find clinical application in specific subgroups of patients. By contrast with BCG-specific IgG titres, and as expected and previously documented, M tuberculosis-specific interferon-gamma production measured by Quantiferon TB-Gold-in-tube was significantly reduced in HIV coinfection and decreased with declining CD4 Tcell numbers. So is it time to exhume the cadaver recently buried by WHO report? Much remains to be done before any such disinterment can be considered. The results of the current study are not generalisable. The diagnostic sensitivity reported is for a population of TB patients with sputum-smear negative pulmonary TB, pleural TB and lymph node TB. While including a substantial overall proportion of clinical presentations of TB, the 91% diagnostic sensitivity cannot be extrapolated to other types of extrapulmonary TB, which represent a significant diagnostic challenge, especially in HIV coinfection. Although the specificity of the assay was 94% in asymptomatic controls without Tuberculosis Research Unit, National Heart and Lung Institute, Imperial College London, London, UK; Tuberculosis Service, Imperial College Healthcare NHS Trust, St. Mary’s Hospital, London, UK