RESEARCH NOTE. T-cell responses against tuberculin and sensitin in children with tuberculosis and non-tuberculosis mycobacterial lymphadenopathy

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1 Research Notes Teng LJ, Hsueh PR, Tsai JC et al. groesl sequence determination, phylogenetic analysis, and species differentiation for viridans group streptococci. J Clin Microbiol 2002; 40: Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22: Drancourt M, Roux V, Fournier PE, Raoult D. rpob gene sequence-based identification of aerobic gram-positive cocci of the genera Streptococcus, Enterococcus, Gemella, Abiotrophia, and Granulicatella. J Clin Microbiol 2004; 42: Täpp J, Thollesson M, Herrmann B. Phylogenetic relationships and genotyping of the genus streptococcus by sequence determination of the RNase P RNA gene, rnpb. Int J Syst Evol Microbiol 2003; 53: RESEARCH NOTE T-cell responses against tuberculin and sensitin in children with tuberculosis and non-tuberculosis mycobacterial lymphadenopathy K. Magdorf 1, S. D. Schuck 2, S. Leitner 2, U. Wahn 1, S. H. E. Kaufmann 2 and M. Jacobsen 2, 1 Department of Paediatric Pneumology and Immunology, Charité, Universitätsmedizin Berlin and 2 Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany ABSTRACT Multi-colour flow cytometry was applied to determine T-cell-specific interferon-c, interleukin-2 and tumour necrosis factor-a expression in children with tuberculosis and non-tuberculosis mycobacterial lymphadenopathy (-L). In vitro stimulation of peripheral blood mononuclear cells with purified protein derivative from Mycobacterium tuberculosis (tuberculin) and M. avium (sensitin) Corresponding author and reprint requests: M. Jacobsen, Department of Immunology, Max Planck Institute for Infection Biology, Charitéplatz 1, Berlin, Germany jacobsen@bni-hamburg.de Present address: Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany revealed differential recognition of tuberculin and sensitin in both study groups. Ratios of tuberculinspecific and sensitin-specific T-cell proportions in individual patients discriminated between children with tuberculosis or -L. These findings have the potential to improve the differential diagnosis of mycobacterial infections. Keywords Childhood tuberculosis, flow cytometry, intracellular cytokine staining, non-tuberculosis mycobacterial, sensitin, tuberculin Original Submission: 11 January 2008; Revised Submission: 19 March 2008; Accepted: 23 May 2008 Edited by F. Mescart Clin Microbiol Infect 2008; 14: /j x Lymphadenopathy in children can be caused by M. tuberculosis or non-tuberculosis mycobacteria () [1]. Rates of non-tuberculosis mycobacterial lymphadenopathy (-L) in children have increased during recent years in industrialized countries, whereas tuberculosis rates are decreasing [2,3]. Differential diagnosis of tuberculosis and -L has major implications for the treatment procedure but can only be accomplished by bacterial culture lasting at least 3 weeks. Consequently, presumptive diagnosis that relies on clinical findings, patient anamnesis and tuberculin skin test (TST) guides the treatment choice. Different approaches have been evaluated to improve the differential diagnosis of tuberculosis and -L. On the one hand, the TST induration sizes induced by tuberculin have been analysed [4,5]. These studies identified differences in the mean induration size between -infected and M. tuberculosis-infected individuals. However, high variances between individuals and the marked overlap between the study groups rendered induration sizes insufficient as biomarkers, especially in paediatric cases [4]. On the other hand, concomitant TST analyses of tuberculin and sensitin (dual skin testing) have been performed [4,6]. Dual skin testing and calculation of the ratio of responses against each antigen discriminated between and M. tuberculosis infection in adults [6,7], whereas results from studies in children were less convincing [8]. These ambiguous results may be caused by cross-reactivity against the antigens prevalent in both tuberculin and sensitin preparations.

2 1080 Clinical Microbiology and Infection, Volume 14 Number 11, November 2008 Table 1. Clinical characteristics of children with tuberculosis (A I) and children with non-tuberculosis mycobacterial lymphadenopathy (J R) Case Age Gender TST (mm) QuantiFERON Ò Gold T-SPOT Ò Pathogen (from gastric lavage, bronchoscopy, or extirpated lymph nodes) Clinical features A 5 years F 30 Positive Positive M. tuberculosis Primary pulmonary tuberculosis; culture-confirmed B 9 years M 30 Positive Positive M. tuberculosis Primary pulmonary tuberculosis; culture-confirmed C 4 years M 20 Positive Positive M. tuberculosis Primary pulmonary tuberculosis; culture-confirmed D 8 months F 8 Negative Negative M. tuberculosis Primary pulmonary tuberculosis; culture-confirmed E 10 years F 30 Positive Positive M. tuberculosis Primary pulmonary tuberculosis; culture-confirmed F 14 years M 20 Positive Positive M. tuberculosis Primary pulmonary tuberculosis; culture-confirmed G 4 years F 20 Positive Positive M. tuberculosis Primary pulmonary tuberculosis; culture-confirmed H 5 years F 20 Positive Positive M. tuberculosis Multiresistant primary pulmonary tuberculosis; culture-confirmed I 8 years F 20 Positive Positive M. tuberculosis Primary pulmonary tuberculosis; culture-confirmed J 8 years F 20 Negative Negative M. avium Lymphadenopathy; culture-confirmed K 2 years M 15 Negative Negative M. avium Lymphadenopathy; culture-confirmed L 5 years M 10 Negative Negative M. avium Lymphadenopathy; culture-confirmed M 3 years M 10 Negative Negative M. avium Lymphadenopathy; culture-confirmed N 5 years F 6 ND Negative M. avium Lymphadenopathy; culture-confirmed O 6 years M 10 Negative ND M. avium Lymphadenopathy; culture-confirmed P 2 years F 10 Negative Negative M. intracellulare Lymphadenopathy; culture-confirmed Q 2 years F 3 Negative Negative M. avium Lymphadenopathy; culture-confirmed R 3 years F 10 Negative Negative M. intracellulare Lymphadenopathy; culture-confirmed TST, tuberculin skin test; F, female; M, male; ND, not defined. RT23, 2 TU (Statens Serum Institute, Copenhagen, Denmark). Here we used flow cytometry, a highly sensitive and specific method, to determine specific T-cell responses against purified protein derivative from M. tuberculosis (tuberculin) and purified protein derivative from M. avium (sensitin) in children with tuberculosis or -L. Intracellular analyses of cytokines, namely interferon-c (IFN-c), tumour necrosis factor-a (TNF-a), and interleukin-2 (IL-2), were used as readouts after short-term in vitro restimulation. Peripheral blood (3 ml) was obtained from ten children with culture-confirmed tuberculosis and nine children with culture-confirmed -L recruited at the Department of Paediatric Pneumology and Immunology, Charité, in Berlin, Germany. The patients characteristics are summarized in Table 1. This study was approved by the local ethics committee (EA ). Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation, (Biocoll; Invitrogen, Carlsbad, CA, USA) following the manufacturer s instructions. PBMCs ( cells well) were then stimulated with tuberculin, sensitin (each 10 lg ml) (Statens Serum Institute, Copenhagen, Denmark), or Staphylococcus enterotoxin B (1 lg ml) (Sigma-Aldrich, St. Louis, MI, USA), or were unstimulated for 20 h in 200 ll of RPMI (Gibco) containing 5% human serum (Sigma) and 1% L-glutamine (Sigma). During the final 15 h, brefeldin A was added to avoid release of cytokines from the Golgi apparatus. Cells were then fixed and permeabilized using BD Biosciences (New Jersey, USA) cytofix cytoperm, following the manufacturer s instructions, before monoclonal antibody (mab) mixtures were added. These mab mixtures contained anti-ifn-c mab (APC-labelled), anti-il-2 mab (fluorescein isothiocyanate-labelled), and anti-tnf-a mab (Alexa 700-labelled) (all BD Biosciences), combined with anti-cd4 mab (APC-Cy7-labelled), anti-cd3 mab (Pacific Blue-labelled), and anti-cd45ro mab (PE-Cy7- labelled). An LSRII flow cytometer (BD Biosciences) was used for measurement, and FCS express software (De Novo, Los Angeles, CA, USA) was used for data analyses (see Fig. S1 for gating procedures). The Mann Whitney U-test was used to determine significant differences in cytokine-expressing T-cell proportions between study groups, and Student s t-test was applied to compare ratios of stimulation values. These tests were selected on the basis of Kolmogorov Smirnov normality testing with Lilliefors correction. CD4 + T-cells were the predominant cytokineproducing T-cell population after antigen-specific in vitro stimulation (data not shown). The proportions of IFN-c-expressing, IL-2-expressing and TNF-a-expressing T-cells after restimulation with tuberculin in PBMCs from children with tuberculosis were only slightly higher than those in PBMCs from children with -L (Fig. 1a). For sensitin, slightly higher IFN-c-expressing, IL-2- expressing and TNF-a-expressing T-cell propor-

3 Research Notes 1081 (a) IFNγ IL-2 TNFα (b) IFNγ IL-2 TNFα (c) IFNγ IL-2 TNFα (d) IFNγ IL-2 TNFα (e) IFNγ IL-2 Ratios of tuberculin / sensitin specific CD4 + T cells TNFα Fig. 1. Proportions of interferon-c (IFN-c)-expressing, interleukin-2 (IL-2)-expressing and tumour necrosis factor-a (TNFa)- expressing memory T-cells from children with tuberculosis () and non-tuberculosis mycobacterial lymphadenopathy (-L). Multiple scattergraphs indicate IFN-c-expressing (left panel, squares), IL-2-expressing (middle panel, circles), and TNF-a-expressing (right panel, triangle) (separated by dotted lines) T-cell proportions specific for (a) tuberculin and (b) sensitin in peripheral blood mononuclear cells (PBMCs) from children with tuberculosis (dark grey symbols) and those with -L (bright grey symbols). (c, d) Comparisons of individual tuberculin-specific and sensitin-specific T-cell proportions of children with tuberculosis (c) and -L (d) are shown. Symbols from same patients are connected by straight lines. Presentations are as described for (a) and (b). (e) Ratios (tuberculin sensitin) of cytokine-secreting memory T-cell proportions in PBMCs from individual patients are shown. Each symbol represents a ratio from an individual donor. Normality testing was performed using Kolmogorov Smirnov testing with Lilliefors correction. Accordingly, two-sided p values for the Mann Whitney U-test are indicated as p <5) and p <1) in (a d), and Student s t-test values as p <01) in (e). tions were detected in PBMCs of children with -L than in those of children with tuberculosis (p <5; for all cytokines) (Fig. 1b). No differences were detected in the T-cell responses to the superantigen Staphylococcus enterotoxin B between the study groups (data not shown). Owing to the strong variations in cytokine responses of individual donors, the percentages induced by tuberculin and sensitin overlapped markedly between the study groups (Fig. 1a,b). Therefore, comparison of IFN-c-secreting, IL-2- secreting and TNF-a-secreting T-cell proportions

4 1082 Clinical Microbiology and Infection, Volume 14 Number 11, November 2008 specific for tuberculin or sensitin was insufficient to discriminate between children with tuberculosis and those with -L. Next, the CD4 + T-cell responses to tuberculin and sensitin were determined for each individual donor. Significantly higher proportions of tuberculin-specific T-cells were detected in children with tuberculosis (Fig. 1c) and significantly higher proportions of sensitin-specific T-cells in children with -L (Fig. 1d) (p <5; for all cytokines). As the relative proportions of tuberculin-specific and sensitin-specific T-cells correlated strongly in individual patients (p <1 for all three cytokines in both study groups, data not shown), crossreactivity against the antigens is likely, as has been described by others [5]. Consequently, the ratios of cytokine-expressing T-cell proportions induced by tuberculin and sensitin were determined in individual patients from both study groups. The individual cytokine ratios revealed highly significant differences between the study groups for IFN-c (p <01), IL-2 (p <01), and TNF-a (p <01) (Fig. 1e). Notably, ratios of cytokine-expressing T-cell proportions revealed non-overlapping results for all cytokines between children with tuberculosis and those with -L (Fig. 1e). These results were in accordance with studies comparing dual skin testing with tuberculin and sensitin in adults, and demonstrated for the first time that the analysis of T-cell responses against both mycobacterial protein preparations in parallel can contribute to improved differential diagnosis in children [7]. In particular, the high specificity of flow cytometry-based analyses and the enrolment of strictly stratified study groups (Table 1) may account for the contradictory results obtained in previous studies concerning dual skin testing in children [8]. The present study suggests that both sensitin and tuberculin preparations contain exclusive immunogenic proteins. For M. tuberculosis, specific immunodominant proteins have been identified and are already being used in diagnostic tests [9,10]. These IFN-c release assays have shown improved specificity and sensitivity as compared to the TST, but are probably less reliable if used for the diagnosis of immunocompromised patients [11]. Immunity based on serological analyses against components specifically expressed by M. avium has been analysed in patients with tuberculosis as well as in those with -L [12,13], with promising results from a very recent study in adults [13]. Our assay provides the possibility of detecting T-cell immunity specific for M. avium infection. This is a major difference from the IFN-c release assays, which detect M. tuberculosis infection. Therefore, we consider our finding to be an initial step to characterize the influence of co-infection with on tuberculosis and other infectious diseases (e.g. human immunodeficiency virus (HIV)). Especially in regions with high incidences of infections, these co-infections are relevant [14]. Furthermore, in immunocompromised donors (e.g. HIV-infected), false-negative results of the TST and of IFN-c release assays are prevalent, and hence the number of truly M. tuberculosis-infected individuals among these patients is underestimated [11]. Ongoing studies will determine the efficacy of our assay in detecting mycobacterial infections in this group of patients. In conclusion, the combined analyses of cytokine-expressing T-cell proportions specific for tuberculin or sensitin in individual patients rather then the response to one of these antigens discriminated between children with tuberculosis and those with -L. All three cytokines, IFN-c, TNF-a and IL-2, were equally suited for discrimination. We consider our findings an initial step to: (i) identify sensitin-specific components; (ii) analyse the impact of co-infections on other infectious diseases (e.g. in patients with HIV infection); and (iii) develop a fast and reliable method for discrimination between children with tuberculosis and those with -L. ACKNOWLEDGEMENTS We thank M. L. Grossman for carefully reading the manuscript. TRANSPARENCY DECLARATION Part of this work received financial support from the Bill and Melinda Gates Foundation (Grand Challenges 6 to S. H. E. Kaufmann and M. Jacobsen). The authors have no conflicting financial interests. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article:

5 Research Notes 1083 Fig. S1. Gating procedures of flow cytometry analyses to determine tuberculin-specific and sensitin-specific T-cell proportions. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. REFERENCES 1. Marras TK, Daley CL. Epidemiology of human pulmonary infection with nontuberculous mycobacteria. Clin Chest Med 2002; 23: Haverkamp MH, Arend SM, Lindeboom JA, Hartwig NG, van Dissel JT. Nontuberculous mycobacterial infection in children: a 2-year prospective surveillance study in the Netherlands. Clin Infect Dis 2004; 39: Falzon D, Ait-Belghiti F. What is tuberculosis surveillance in the European union telling us? Clin Infect Dis 2007; 44: Haimi-Cohen Y, Zeharia A, Mimouni M, Soukhman M, Amir J. Skin indurations in response to tuberculin testing in patients with nontuberculous mycobacterial lymphadenitis. Clin Infect Dis 2001; 33: von Reyn CF, Horsburgh CR, Olivier KN et al. Skin test reactions to Mycobacterium tuberculosis purified protein derivative and Mycobacterium avium sensitin among health care workers and medical students in the United States. Int J Tuberc Lung Dis 2001; 5: von Reyn CF, Green PA, McCormick D et al. Dual skin testing with Mycobacterium avium sensitin and purified protein derivative: an open study of patients with M. avium complex infection or tuberculosis. Clin Infect Dis 1994; 19: Hersh AL, Tosteson AN, von Reyn CF. Dual skin testing for latent tuberculosis infection: a decision analysis. Am J Prev Med 2003; 24: Larsson LO, Bentzon MW, Lind A et al. Sensitivity to sensitins and tuberculin in swedish children. Part 5: a study of school children in an inland rural area. Tuber Lung Dis 1993; 74: Detjen AK, Keil T, Roll S et al. Interferon-gamma release assays improve the diagnosis of tuberculosis and nontuberculous mycobacterial disease in children in a country with a low incidence of tuberculosis. Clin Infect Dis 2007; 45: Lein AD, von Reyn CF, Ravn P, Horsburgh CR Jr, Alexander LN, Andersen P. Cellular immune responses to esat- 6 discriminate between patients with pulmonary disease due to mycobacterium avium complex and those with pulmonary disease due to Mycobacterium tuberculosis. Clin Diagn Lab Immunol 1999; 6: Hornum M, Mortensen KL, Kamper AL, Andersen AB. Limitations of the QùantiFERON- Gold test in detecting Mycobacterium tuberculosis infection in immunocompromised patients. Eur J Intern Med 2008; 19: Rolinck-Werninghaus C, Magdorf K, Stark K et al. The potential of recombinant antigens esat-6, mpt63 and mig for specific discrimination of Mycobacterium tuberculosis and M. avium infection. Eur J Pediatr 2003; 162: Kitada S, Kobayashi K, Ichiyama S et al. Serodiagnosis of Mycobacterium avium complex pulmonary disease using an enzyme immunoassay kit. Am J Respir Crit Care Med 2007; 177: MacDonell KB, Glassroth J. Mycobacterium avium complex and other nontuberculous mycobacteria in patients with HIV infection. Semin Respir Infect 1989; 4: RESEARCH NOTE Distribution of genes encoding iron uptake systems among enteroaggregative Escherichia coli strains isolated from adults with irritable bowel syndrome B. M. Sobieszczańska Department of Microbiology, University of Medicine, Wrocław, Poland ABSTRACT The distribution of genes encoding different iron acquisition systems in enteroaggregative Escherichia coli (EAEC) from adults with irritable bowel syndrome and from healthy controls was examined using a PCR assay. As many as 95.5% of EAEC carried the chua gene coding for a haem receptor, and the majority of these strains also had yersiniobactin-encoding genes. Apart from yersiniobactin, enterobactin was the siderophore most frequently associated with EAEC among those strains examined. Genes encoding aerobactin and salmochelin siderophores were less frequent in the group of EAEC. Keywords Enteroaggregative Escherichia coli, haem receptor, iron uptake systems, irritable bowel syndrome, yersiniobactin Original Submission: 25 March 2008; Revised Submission: 15 April 2008; Accepted: 28 May 2008 Edited by P. Gerner-Smidt Clin Microbiol Infect 2008; 14: /j x Corresponding author and reprint requests: B. M. Sobieszczańska, Department of Microbiology, University of Medicine, 4 Chałubińskiego Street, Wrocław, Poland mapasobie@provider.pl