We sought to assess the accuracy and safety of the ESAT6-CFP10 reagent in diagnosing tuberculosis (TB) disease. An open-label, randomized phase 2a trial was conducted in 56 healthy adults and 88 TB patients at one medical centre and one teaching hospital in China. All participants received 0.1, 0.5, 1 or 2 μg ESAT6-CFP10 in their right forearm. Moreover, 56 healthy volunteers and 56 patients were given tuberculin-purified protein derivative (TB-PPD) in their left forearm. The remaining 32 patients were administered placebo. The main outcome measure was induration diameter. An enzyme-linked immunospot (ELISPOT) assay was conducted before the skin test. The ESAT6-CFP10 test caused a higher positivity rate than placebo (81.2% (26/32) vs. 3.1% (1/32); p <0.001). The median maximum induration diameter after ESAT6-CFP10 injection was 17.0 (interquartile range (IQR), 14.0-21.7) mm, similar to that for TB-PPD (17.5 (IQR, 7.0-30.5) mm). The diagnostic accuracy of ESAT6-CFP10 was superior to that of TB-PPD (area under the receiver operating characteristic curve (AUC), 0.870 (95% confidence interval (CI), 0.796-0.944) vs. 0.686 (95% CI, 0.585-0.786); p <0.001). When analysed in all participants, ESAT6-CFP10 had comparable AUC values to the ELISPOT assay (0.849 (95% CI, 0.835-0.952) vs. 0.908 (95% CI, 0.852-0.965)). Local itching (12/144, 8.3%) and pain (26/144, 18.1%) were the main side effects of ESAT6-CFP10. No serious adverse events were reported. The ESAT6-CFP10 skin test appears to be a safe and promising tool; further testing will confirm its efficacy in identifying TB disease.

Tuberculosis (TB) remains one of the most severe infectious diseases. It is still of paramount importance to establish more accurate, rapid, and efficient diagnostic methods. Since infection with Mycobacterium tuberculosis (M. tb) is largely mediated through the respiratory tract, IgA responses against mycobacterial proteins are worthy of investigation for their potential clinical utility. In this study, the IgA response targeting lipoprotein Z (LppZ) was determined by using a homemade ELISA with plasma of TB patients (N = 125), LTBI individuals (N = 92), healthy controls (HCs) (N = 165), as well as TB patients undergoing anti-TB treatment (N = 9). In parallel the antigen-specific IFN-γ release from PBMCs triggered by LppZ and M. tb-specific ESAT-6 or CFP-10 was detected by using an ELISPOT assay. It was found that the LppZ-specific IgA level was dramatically higher in TB patients than in HCs (p < 0.0001). Compared to that before anti-TB treatment, the LppZ-specific IgA level decreased substantially after 2 months of anti-TB treatment (p = 0.0297) and remained at low levels until the end of the treatment. What is more, pulmonary TB patients exhibited significantly higher LppZ-specific IgA-values than extra-pulmonary TB patients (p = 0.0296). Interestingly, the LppZ-specific IgA-values were negatively correlated to the amounts of IFN-γ released in response to LppZ with statistical significance (r = −0.5806, p = 0.0002). LppZ-specific IgA level was also higher in LTBI individuals than in HCs (p < 0.0001). Additionally there were some PPD+ HC individuals with high LppZ-specific IgA levels but the potential of this assay for identifying leaky LTBI in PPD+ HCs needs to be further investigated through follow-up studies. The sensitivity of detecting TB solely with ESAT-6 or CFP-10-specific IFN-γ release was increased by including the LppZ-specific IgA results, respectively, from 86.11 to 100% and 88.89 to 100%; the sensitivity of screening for LTBI was increased from 80.36 to 83.93% and 57.14 to 69.64%, respectively. The higher LppZ-specific IgA responses in TB and LTBI populations than in controls indicated high immunoreactivity to LppZ upon M. tb infection. Although the assay was not efficient enough for independent application in sero-diagnosis, LppZ-specific IgA might become a complementary biomarker for the improvement of TB and LTBI screening.

cThis trial was conducted to explore the safety of recombinant fusion protein ESAT6-CFP10 as a skin test reagent for the diagnosis of Mycobacterium tuberculosis infection. Twenty-four healthy adult volunteers were recruited and randomized into four groups (groups A to D) to study four increasing doses of ESAT6-CFP10. All subjects in each dose group received an intradermal injection of reagent (0.1 ml) via the Mantoux technique. Then, the vital signs of all subjects were monitored, and skin reactions around injection sites and adverse events were recorded at different detection time points after the skin test. No serious adverse events were observed in this study. A total of 3 subjects had unexpected events. One subject in group A developed subcutaneous hemorrhage 24 h after the skin test, one subject in group B was found with red spots 15 min after the skin test, and another subject in group A showed abnormity during a chest X-ray after the skin test without affecting her health. One of three adverse events (red spots) was probably related to the recombinant ESAT6-CFP10 reagent. A single dose of 1, 5, 10, or 20 g/ml of recombinant ESAT6-CFP10 as a skin test reagent for M. tuberculosis infection diagnosis is well tolerated and safe in China. (This study has been registered at ClinicalTrials.gov under registration no. NCT01999231.)A lthough tuberculosis (TB) has been a curable disease, it still remains a major global problem that seriously threatens human health. According to the WHO Global TB report 2015, there were 9.6 million new cases and nearly 1.5 million TB-related deaths in 2014 (1). Thus, exploring diagnostic techniques with high sensitivity and specificity is crucial for controlling TB development and its drug resistance.TB is caused by infection with Mycobacterium tuberculosis, an intracellular pathogen transmitted by air (2). Several in vitro assay systems based on the detection of immune reactivity against an M. tuberculosis-specific antigen have been established and widely accepted in recent years. Early secreted antigenic target 6-kDa protein (ESAT6) and culture filtrate protein of 10 kDa (CFP10), two major M. tuberculosis-specific antigens, have been reported to play key roles in the virulence of M. tuberculosis and elicit strong T-cell responses (3, 4). Interferon gamma release assays (IGRAs) based on immune recognition against ESAT6 and CFP10 have been used to help diagnose activated and latent M. tuberculosis infection (5). IGRAs have an increased specificity compared to the conventional tuberculin skin test (TST) because the antigens included in IGRAs, namely, ESAT6 and CFP10, have been selected based on their absence in many environmental nontuberculous mycobacteria and the vaccine strain Mycobacterium bovis Bacillus Calmette-Guérin (BCG), which can only be detected in a number of pathogenic mycobacteria species, including M. tuberculosis, and a minority of nontuberculous mycobacteria (Mycobacteria kansasii, Mycobacteria szulgai, Mycobacteria marinum, and Mycobacteria riyadhense), altogether l...

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Mycobacterial lipoproteins are considered to be involved in both virulence and immunoregulatory processes during Mycobacterium tuberculosis (M.tb) infection. In our previous investigations on the immunoreactivity of more than 30 M.tb proteins in active TB patients, we identified mycobacterial lipoprotein Z (LppZ) as one of the most immune dominant antigens. How LppZ triggers immune responses is still unclear. In this study, we analyzed LppZ-mediated innate and adaptive immunity using a murine air pouch model and an M.tb infection model, respectively. We found that LppZ could not only recruit inflammatory cells but also induce the production of proinflammatory cytokines inside the pouches. LppZ could also induce strong Th1 responses following immunization and confer protection against challenge with M.tb virulent strain H37Rv at a similar level to BCG vaccination but with less pathological damage in the lungs. Furthermore, we revealed the presence of LppZ-specific functional CD4+ T cells in the lungs of the challenged mice that were capable of secreting double or triple cytokines, including IFN-γ, IL-2, and TNF-α. Our study thus demonstrates that LppZ is of strong immunogenicity during M.tb infection in both humans and mice and has the ability to trigger effective innate and cellular immunity. Considering the limitations of candidate antigens in the pipeline of TB vaccine development, LppZ-mediated immune protection against M.tb challenge in the mouse model implies its potential application in vaccine development.