TST-Negative Individuals Do Not Lack Anti-Mycobacterial Responsiveness

Tuberculin-skin-test (TST) is widely used for the diagnosis of Tuberculosis. Negative TSTs are occasionally found in individuals vaccinated or infected by Mycobacterium tuberculosis. We show that false negative results are due to a lack of the Tuberculin-specific immune response as replacing tuberculin by unselected M. tuberculosis antigens improved the efficiency of the assay. Tuberculosis (TB) remains a major global public health concern, with one third of the world population infected, 9 million active cases and 2 million deaths every year [1]. Despite major progress in the development of new strategies for treating TB, the disease remains a great challenge for healthcare workers. Most people infected by M. Tuberculosis, the causative agent of TB, remain asymptomatic (latent infection): the bacilli reside within granulomas, the histological hallmark of TB, and only 10% will develop a clinical disease during their life. The tuberculin skin test (TST) is commonly used to help diagnosis of Mycobacterium Tuberculosis infection [2]. Purified protein derivative from M. Tuberculosis (PPD), also called Tuberculin, is injected intradermally, and the induction of a positive skin reaction is generally considered to indicate a latent or active TB, depending on the level of the skin reaction. In addition, positive tests are also found in patients after vaccination with Bacille Calmette-Guerin (BCG), or exposure to environmental mycobacteria. Negative test results may yet occur in a proportion of patients with active TB (10 to 25%) [3, 4]. These negative tests may find a biological explanation, as peripheral blood mononuclear cells (PBMC) from TB patients stimulated with Tuberculin (or PPD), release lower levels of IFNand IL-12 compared to PPD responsive (i.e. TST-positive) healthy subjects [5]. Qualitative TST responses may vary depending on the clinical presentation of TB, especially in children. Finally, it has been established that there is no relationship between tuberculin skin-reactivity, and protection of vaccinated individuals against the development of active Tubercu*Address correspondence to this author at the Department “Molecular Mechanisms of Mycobacterial Infections”, Team “Molecular Physiology of Mycobacterial Granulomas”, IPBS-CNRS UMR5089, 205, route de Narbonne, 31077 Toulouse cedex, France; Tel: (33) 5 61 17 54 63; Fax: (33) 5 61 17 59 94; E-mail: [email protected] These authors contributed equally to the work. losis [6]. Thus, not only the TST does not distinguish between Tuberculosis infection and BCG vaccination, but hence it does not witness a mycobacterial infection (in false negative individuals), and finally does not appear as a correlate of protection either. Recent advances have improved the specificity of the immune identification of latent TB, using a combination of antigens (early secreted antigenic target 6 (ESAT-6), and culture filtrate protein 10 (CFP-10) encoded by the RD1 locus of M. Tuberculosis which is absent from most nonpathogenic mycobacteria, including Bacille-Calmette-Guerin). The sensitivity has also been improved by the ELISPOT (T SPOT-TB assay), an ex vivo assay quantifying the -Interferon produced by T-cells stimulated with ESAT-6 and CFP-10. On another hand, the performance of the recently proposed Quantiferon tests (based on whole blood interferonrelease) has been shown to be negatively affected by patient’s immune deficiency [7]. These serological and cytokine-based assays have yet to demonstrate an enhanced sensitivity and specificity when compared with the TST [8, 9]. In BCGvaccinated individuals, false positive results occur with all these tests, and false negative tests due to anergy or immune deficiency are widely recognised [10-12]. Moreover, cost will be a critical factor in determining the global use of these new assays [13]. Pai and colleagues suggest combining the TST and interferon assays to increase the sensitivity of the PPD-test and the specificity of RD1 antigens. This combination was used to confirm that the BCG vaccination gave significant protection against TB in children [14]. Yet, due to the numerous disadvantages of the TST previously discussed, we wondered whether we could find a better antigen than the Tuberculin to be used to give a more specific delayed-type hyper-sensitivity (DTH) reaction that could work in any individual. In this study, we thus investigated the possibility to replace Tuberculin by a more complex mixture of M. Tuberculosis (strain H37Rv) antigens, obtained by mechanic 54 The Open Pathology Journal, 2008, Volume 2 Godel et al. disruption of the bacilli, which could possibly avoid false negative results. We used an in vitro model of human granulomas to compare the reactivity of human immunity to PPD and M. Tuberculosis antigens. This model reproduces in vitro a delayed-type hypersensitivity (DTH) reaction, as does the TST in vivo. This reaction is produced by the incubation of PBMC from healthy volunteers with sepharose beads coated with mycobacterial antigens, which produces a granuloma-like structure characteristic of the immune response to M. Tuberculosis [15-17]. Human blood samples were collected with informed consent from volunteers who had been BCG-vaccinated in infancy, and tested for their reactivity to PPD. For this purpose, PBMC from 100 volunteers (age 35-58, M/F: 61/39), including 85 PPD-positive individuals (Tubertest ® 5U, 0.1ml) and 15 PPD-negative individuals, were isolated, incubated with beads coated with PPD, or M. Tuberculosis extracts (prepared as described by Puissegur and colleagues [16]), glycin-coated beads serving as negative control for non specific reaction. The ability of the different individuals to develop a granulomatous reaction around the different beads was followed by optical microscopy (data not shown) and scanning electron microscopy (Fig. 1). As expected, granulomas were found for PPD-responders (TST > 5mm), both around beads coated with PPD (Fig. 1A), and beads covered with M. Tuberculosis extracts (Fig. 1B). In the 15 PPD-nonresponders (TST <5mm), no granulomas were ever found after incubation with PPD-coated beads (Fig. 1D), but important granulomatous reaction developed in response to M. Tuberculosis extracts (Fig. 1E). Fig. (1). Scanning electron micrographs of H37Rv surface extract-induced or PPD-induced granulomas. Beads covered with PPD (A,D) or surface extract from H37Rv (B,E) were incubated with PBMC isolated from TST positive (A,B,C) or negatives (D,E,F) volunteers, all BCG vaccinated in infancy. Beads covered with Glycine (C,F) represent the negative control for non specific fixation. After 11 days of culture, granulomas were prepared and observed under a scanning electron microscope. Bar represent 5 m (A,C,D,F) or 50 m (B,E). All the analyses herein presented were performed according to the principles expressed in the Helsinki Declaration, with an informed consent of all