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• W1983821346 abstract "Tuberculosis (TB) is a major cause of illness and death worldwide [1]. In 2006, globally nearly 10 million new cases occurred. Particularly, individuals with a weak immune state are at high-risk to attract active TB [2, 3]. Therefore, in 1995, the WHO launched directly observed treatment short course (DOTS) with the aim to achieve improvement of diagnosis and treatment, combined with cost effectiveness in developing countries [4]. Nonetheless, the incidence of TB is still increasing, and multidrug and extensively drug-resistant TB (MDR-TB and XDR-TB) further decrease treatment options [5, 6]. To reach the Millennium Development Goal (MDG), which was defined as ‘stop the increase and start reversing the incidence of TB by 2015’ [7, 8], the WHO introduced a new STOP TB Strategy in 2005 [9]. One of the major problems in the management of TB is the diagnosis and the completion of the long lasting treatment, which affords a high compliance of the patient. So far, TB diagnosis is based on the clinical manifestation of the patient and the microscopic detection of acid fast bacilli and on the detection of growing mycobacteria in culture. There is evidence that fluorescence microscopy is more sensitive and faster than conventional sputum smear microscopy [10], but fluorescence microscopes and expertise in this method is not widely available. However, all conventional methods including PCR are far from ideal, as they are neither specific nor sensitive and they depend on skilled laboratory personal [11]. In addition, culture systems are expensive and the bacterial growth time is around 6 weeks. If diagnosis is rapidly required, e.g. if cerebral TB is suspected, it would be desirable to have minimal turn around times. Furthermore, in areas where TB is endemic, it is desirable to establish reliable diagnostic tests that provide results within a short time on the spot. For decades, the tuberculin skin test (TST) provides a standard method to diagnose TB together with other clinical findings. However, the test cross reacts with antigens, which are common in other mycobacteria than Mycobacterium tuberculosis. Thus, the test is prone to give false positive results following BCG vaccination and environmental exposition to different mycobacterial strains. Furthermore, the sensitivity is low in HIV infected patients, in children with disseminated TB and in malnourished children [12]. In 2001, Lalvani et al. established an enzyme-linked immunospot (ELISpot) that detects INF-γ producing effector T-cells against two Mycobacterium tuberculosis antigens, the 6-kD early secretory antigenic target (ESAT-6) and culture filtrate protein-10 (CFP-10) [13, 14]. Later on, Liebeschuetz et al. [12] showed that ELISpot is more sensitive than the TST to diagnose TB in children living in TB endemic areas. To obtain here a comparatively good sensitivity to diagnose active TB, the TST and the ELISpot should be combined. These revolutionary findings led to a variety of commercial available kits (called interferon-γ release assays; IGRAs) based on ELISpot technique to test the infection with Mycobacterium tuberculosis. IGRAs show an excellent specificity, but until now it is not assessed that immunological tests distinguish active and latent TB [15]. In the present issue of the Journal of Internal Medicine, Nemeth et al. [16] highlight this topic investigating ESAT-6 specific effector T-cells directly by flow cytometry. They examined samples from 25 patients and compared the amount of ESAT-6 specific T-cells isolated from peripheral blood with the number of cytokine secreting T-cells in samples obtained from the infection site. Whilst the ELISpot technique is based on the principle of a sandwich-capture ELISA where secreted INF-γ is detected in the vicinity of individual T-cells, which are not defined in detail [14], flow cytometry allows the measurement of expression of surface markers and secreted cytokines of a single cell at the same time. Nemeth et al. showed that an accumulation of the cells from the infection site compared to the number of antigen specific T-cells isolated from peripheral blood is associated with active TB determined by positive microscopy or culture. In 15 of the examined TB samples, an accumulation of T-cells was found at the infection site, but only in nine of these samples, antigen specific T-cells were discovered in cells isolated from the peripheral blood showing that the examination of T-cells from peripheral blood alone is not sufficient to detect TB cases. As the authors showed by the diversity of samples used during the examination, the measurement of CD4 positive T-cells by flow cytometry seems to be suitable for fluids of each body compartment. Considering that fluids are taken quite often routinely during examination, analysing fluid derived T-cells is a rapid method to detect active TB infection within a short time (as outlined in Fig. 1). This is even more important if life threatening conditions are present. In these cases, this method could help to clarify the situation immediately. For the methodology described in the publication of Nemeth et al., a flow cytometer and experienced personnel performing the test is necessary. An increasing number of flow cytometers is present in TB endemic settings because of the need for quantification of T-cells in HIV positive patients. This could be exploited for TB diagnosis. To measure INF-γ positive T-cells, a three colour staining is sufficient meaning that a comparatively cheap five-channel flow cytometer can be used for the measurement. If further studies emphasize that this method is specific to diagnose active TB it should be considered to become a routinely used diagnostic assay in under resource-constrained conditions. A specific and sensitive diagnosis would be especially valuable to diagnose TB in children, where compliance can be a problem and where extensive TB treatment can have considerable effects on their development (physically and mentally). To diagnose active TB the approach could be as followed: Clinical signs and a positive TST indicate a possibly active TB. To ascertain the diagnosis a sample from the infection site should be taken and analysed by microscopy or culture. Furthermore T-cells isolated from this sample should be investigated by flow cytometry or ELISpot for INF-γ release and further compared to the number of effector T cells isolated from the periphery. If an accumulation at the infection site is observed active TB is very likely and the patient should be treated. In the European Union, the number of TB cases is very low compared to African and Asian countries. In western and central European countries, most of the patients with TB acquired the disease in another country [17]. Considering the ongoing migration worldwide, which increases the possibility to diagnose more TB cases in the future everywhere, a rapid and more sensitive test to diagnose TB is needed. Until now, T-cell diagnosis of fluids, which are gained from the site of infection, is used only sporadically [18-20]. Following stringent evaluation and standardization, it should be possible to establish these tests in a variety of health centres as a routinely used diagnostic tool. This month, Kim et al. published that the ratio of cerebrospinal fluid mononuclear cells and peripheral blood mononuclear cells based on ELISpot data are a hint to distinguish between active and not-active TB [21], which is consistent with the result of the present study presented by Nemeth et al. It is necessary to do further investigations to confirm these results with a higher number of samples and in different settings. Additionally, it would be useful to compare the direct measurement of T-cells by flow cytometry with the measurement of ELISpots in the periphery and at the infection site with respect to feasibility, reproducibility and cost-effectiveness. Because the timeframe of taking the sample and preparing the cells is critical, the transport of blood samples from the physician requiring the analysis to the centre diagnosing the sample is important for both methods. Therefore, it is also necessary to consider that it is important to establish procedures for the right transport and handling of the samples. Altogether, the results published by Nemeth et al. are promising and might become a diagnostic tool to detect active TB patients who need immediate treatment. No conflict of interest was declared." @default.
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• W1983821346 title "Immunological tests to diagnose active tuberculosis" @default.
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