Anne K. Detjen

Mycobacterium tuberculosis-specific CD4+, IFNγ+, and TNFα+ multifunctional memory T cells coexpress GM-CSF

Multifunctional T cells expressing several cytokines in parallel are thought to play a crucial role in protection against different infections. To characterize T cell cytokine patterns associated with disease and protection in Mycobacterium tuberculosis infection we determined the expression of IFNgamma, IL-2, TNFalpha, and GM-CSF in T cell subpopulations from children with tuberculosis (TB) and healthy latently M. tuberculosis-infected children (LTBI) after short-term in vitro restimulation. We identified CD4(+) effector memory T cells (T(EM)) as the major source of all measured cytokines after antigen-specific restimulation. T(EM) from children with TB expressed higher proportions of IFNgamma, TNFalpha, and IL-2 after Mtb restimulation while no differences were detected for GM-CSF between both study groups. GM-CSF secretion strongly depended on antigen-specific stimulation. Analyses of multiple cytokine patterns revealed that the majority of GM-CSF-positive M. tuberculosis-specific memory T cells coexpressed IFNgamma and TNFalpha therefore showing a characteristic feature of multifunctional T cells. We conclude that children with active TB possess higher proportions of IFNgamma-, TNFalpha-, and/or IL-2-positive T(EM) than children with LTBI while GM-CSF coexpression reveals a novel subpopulation within CD4(+) memory T cells not increased in children with active TB.

Short-Term Reproducibility of a Commercial Interferon Gamma Release Assay

ABSTRACT Interferon gamma release assays (IGRAs) have been shown to be sensitive and highly specific for the detection of immune memory against Mycobacterium tuberculosis. Little is known about the reproducibility and within-person variability of these assays. Various aspects of short-term reproducibility of a commercial IGRA, the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, were assessed. The QFT-IT assay was performed twice within 3 days in 27 health care workers in Cape Town, South Africa. Two sets of tests were performed by different operators on day 1, and one set was performed on day 3. Aspects such as interoperator, intraoperator, day-to-day variability, and test-retest variability as well as different the storage methods of plasma were investigated. Seventeen of 27 (63%) of participants had at least one positive QFT-IT text; six had discordant results. The agreement of all aspects studied was high, with kappa values between 0.82 and 1.00 for dichotomous measures, and interclass correlations (ICC) of 0.809 to 0.965 were observed for continuous gamma interferon (IFN-γ) measures. The variability of the magnitude of response was highest comparing measures obtained from individuals on different days (ICC of 0.809). The magnitude of the IFN-γ responses between assays performed for individual participants was variable, with ranges from 0.03 to 11 IU/ml, resulting is discordant results for five participants. The results indicate that the QFT-IT assay is a robust and highly reproducible assay. Considerable intraindividual variability occurs in the magnitude of IFN-γ responses, which may influence the interpretation of serial measures.

Clonal Expansion of CD8+ Effector T Cells in Childhood Tuberculosis

The role of CD8+ T cells in human tuberculosis (TB) remains elusive. We analyzed the T cell repertoire and phenotype in 1) children with active TB (≤4 years), 2) healthy latently Mycobacterium tuberculosis-infected children, and 3) noninfected age-matched (tuberculin skin test-negative) controls. Ex vivo phenotyping of T cell subpopulations by flow cytometry revealed a significant increase in the proportion of CD8+CD45RO−CD62L−CD28−CD27− effector T cells (TEF) in the peripheral blood of children with active TB (22.1 vs 9.5% in latently M. tuberculosis-infected children, vs 8.5% in tuberculin skin test-negative controls). Analyses of TCR variable β-chains revealed markedly skewed repertoires in CD8+ TEF and effector memory T cells. Expansions were restricted to single TCR variable β-chains in individual donors indicating clonal growth. CDR3 spectratyping and DNA sequencing verified clonal expansion as the cause for CD8+ effector T cell enrichment in individual TB patients. The most prominent enrichment of highly similar TEF clones (>70% of CD8+ TEF) was found in two children with active severe TB. Therefore, clonal expansion of CD8+ TEF occurs in childhood TB with potential impact on course and severity of disease.

Proposed management of childhood tuberculosis in low-incidence countries.

The incidence of childhood tuberculosis continues to decline in central Europe, but due to migration from high incidence countries paediatricians will still be confronted with it. The management of childhood tuberculosis in low-incidence, high-income countries differs from most high-incidence countries. The primary measures for preventing the transmission of tuberculosis to children are the detection of adult source cases, detection of latent TB infection (LTBI) in children by history, tuberculin skin testing and, if necessary and recommended, interferon-γ release assays. Children with LTBI should receive preventive therapy. The inclusion of tuberculosis in the differential diagnosis of unclear pulmonary and extrapulmonary disease remains important, and tuberculosis has to be managed according to international standards.

Immunologische Diagnostik der Tuberkulose — Interferon-γ-Tests

Zusammenfassung In Deutschland erkranken derzeit etwa 270 Kinder/Jahr an Tuberkulose (TB). Zwar nimmt die Inzidenz der TB in Deutschland weiter ab, das Krankheitsbild wird aber auch in Zukunft — aufgrund von Migrationsbewegungen — eine wichtige Differenzialdiagnose pulmonaler Infektionen im Kindesalter bleiben. Die Inzidenz von Infektionen mit Umweltmykobakterien dagegen scheint, besonders im Kleinkindalter, zuzunehmen. Das häufigste Krankheitsbild ist die zervikale Lymphadenopathie, meist verursacht durch M.xa0avium. Die Identifizierung des jeweiligen mykobakteriellen Infektionserregers ist wegen der unterschiedlichen therapeutischen Konsequenzen von Bedeutung. Bei klinischem Verdacht auf eine TB-Infektion wird der Tuberkulinhauttest (TST) durchgeführt, dessen Spezifität zur Erkennung einer durch M.xa0tuberculosis verursachten Infektion jedoch unzureichend ist. Aufgrund von Kreuzreaktionen bei Infektionen mit Umweltmykobakterien, aber auch nach BCG-Impfung, kommt es zu falsch-positiven Ergebnissen. Neue immunologische Testverfahren, so genannte Interferon-γ-Tests, zeichnen sich durch ihre Spezifität bei der Unterscheidung der verschiedenen mykobakteriellen Erregergruppen aus. In ihnen wird die immunologische Antwort auf Stimulation mit M.xa0tuberculosis-spezifischen Antigenen, die Ausschüttung von Interferon-γ durch T-Lymphozyten, in vitro gemessen. In Deutschland sind zur Zeit zwei Interferon-γ-Tests erhältlich, der QuantiFERON® Gold In-Tube und der T SPOT™.TB. Diese Tests stellen, insbesondere bei differenzialdiagnostischen Problemfällen, eine sinnvolle Ergänzung zur konventionellen Diagnostik mykobakterieller Infektionen dar.AbstractIn Germany, about 270 children are currently diagnosed with tuberculosis (TB) each year. Although its incidence continues to decline, TB will remain an important differential diagnosis in the presence of pulmonary infections in childhood, partly because of population movements. In contrast, the incidence of infections with non-tuberculous mycobacteria (NTM) appears to be rising, especially among young children. The most common manifestation of NTM infections is cervical lymphadenopathy, in most cases due to Mycobacterium avium. For adequate therapeutic consequences, it is important to differentiate between the different mycobacterial infections. When clinical examination leads to the suspicion of TB infection the first step in diagnosis is the tuberculin skin test (TST). However, due to cross-reactions not only with NTM infections but also with BCG vaccination, the specificity of the tuberculin skin test is not adequate. New immunological tests, known as interferon-γ assays, have demonstrated a high specificity in differentiating between TB- and NTM infections. Those assays measure the interferon-γ secreted by T-lymphocytes as the immunological response to in vitro stimulation with antigens specific for M.xa0tuberculosis. Two interferon-γ assays, QuantiFERON-TB® Gold In-Tube and Txa0SPOT™.TB, are commercially available in Germany. These tests have proved helpful in supplementing conventional diagnostic tests for mycobacterial infections, especially in problematic cases.

The Effect of Deworming on Tests of Tuberculosis Infection in Children With Recent Tuberculosis Exposure: A Randomized Controlled Trial.

Background: Helminth infestations are associated with T-helper cell type 2 (Th2) immune responses, leading to suppression of Th1 responses required to control Mycobacterium tuberculosis infection. We hypothesized that deworming after documented M. tuberculosis exposure might improve Th1 immune responses. Methods: This was a randomized controlled trial comparing the effect of early versus delayed (after 3 months) deworming on tuberculin skin testing (TST) and Quantiferon-Gold-in-tube responses among children from a setting with a known high burden of M. tuberculosis and helminth co-infection in Cape Town, South Africa. Children aged 6 to 15 years with documented M. tuberculosis exposure were enrolled. Ascaris lumbricoides status was measured by Ascaris-specific IgE and stool microscopy. Results: A total of 250 children (mean age, 9.6 years) were enrolled; 11.9% (27/227) were Ascaris stool microscopy positive and 54.2% (135/249) were Ascaris stool and/or IgE positive (Ascaris status). In univariable analysis, deworming at enrollment was not associated with a negative TST at 3 months (odds ratio, 0.61; 95% confidence interval, 0.35–1.07; P = 0.08). In stratified analysis, children with a positive Ascaris status were more likely to be TST negative at 3 months if dewormed early (odds ratio, 0.49; 95% confidence interval, 0.23–1.04; P = 0.06). In multivariable analysis, deworming was not associated with TST status (adjusted odds ratios, 0.62; 95% confidence interval, 0.34–1.10; P = 0.10). There was no association between deworming and Quantiferon-Gold-in-tube status. Conclusions: Deworming in children with recent M. tuberculosis exposure is associated with a trend toward a negative TST result. Timing of deworming might influence interpretation of TST in settings with high burdens of tuberculosis and helminths.

Laryngeal involvement in two severe cases of childhood tuberculosis.

Laryngeal tuberculosis in children is seldom reported in the literature. We present 2 children from Cape Town, South Africa who had disseminated tuberculosis involving the cervical lymph nodes and the larynx. The cases emphasize the pathophysiology, the clinical picture, the bronchoscopic appearance, and the response to therapy in laryngeal tuberculosis.

Klaus Magdorf. 4 July 1942-23 January 2013.

On the 23rd of January 2013, Klaus Magdorf, pediatric pneumologist and infectious diseases specialist, died in Berlin. Until the end of his life, Klaus Magdorf was the ultimate German reference for any clinical problem related to mycobacterial infections in children. Every clinician, who had the honor to know Klaus Magdorf, knew: There is not any better, any more thoughtful advice than his. His career started at the major chest hospital “Heckeshorn” in the 1970s in Berlin, where he continued to work until his retirement. In his early career, tuberculosis incidence in Germany was high (approximately 80 cases/100,000 in 1970). He diagnosed and treated a breathtaking number of TB cases. Thus, few European pediatricians matched his experience in dealing with tuberculosis. With this experience, he advised colleagues not only in Germany, but throughout Europe and beyond. He developed diagnosis and treatment guidelines, and authored numerous articles and textbook chapters on tuberculosis. His research in pharmacokinetics of antituberculosis medicines, dating back to the 1970s, is recognized worldwide. It spurred development of international guidelines on the treatment of TB in children. Other national and international research collaborations focused on TB immunology, diagnostics, and pharmacokinetics. Retirement in 2006 did not slow him down or keep him from engaging in his subject in various national and international research collaborations, focusing more and more on his passion: childhood tuberculosis. Contributing to the recent international recognition of childhood TB as a global health issue is his lifetime achievement. Klaus Magdorf was a pediatrician of the best kind, with ample time and passion for his little patients and their families. His particular foci were, next to tuberculosis, cystic fibrosis and atopic diseases. The more challenging a case, the more determined he became. He never gave up, and always looked beyond the obvious. With deep knowledge of clinical practice and literature, and a detective’s sixth sense, he often found correct diagnosis in cases that seemed impossible to disentangle. When he needed advice, he reached out to colleagues around the world, for whom he in turn was a regular and reliable resource. Many young and experienced colleagues relied on him as a caring and selfless mentor. His mentorship never stopped, both with respect to career development, and when advice on difficult diagnoses and clinical decisions was needed. He is greatly missed by his patients, mentees, and colleagues, not only in Germany, but worldwide. What he leaves with us is the philosophy of his life:

Mendelian susceptibility to mycobacterial disease

ZusammenfassungUnter „Mendelian susceptibility to mycobacterial disease“ (MSDM) versteht man eine genetisch bedingte, erhöhte Anfälligkeit für Infektionen durch opportunistische, niedrig virulente Mykobakterien. Anders als die für das Kleinkindalter typischen „Nontuberculous Mycobacteria“ (NTM)-Infektionen (i. d. R. Lymphadenitiden) sind die mykobakteriellen Infektionen bei Patienten mit „MSMD“ disseminiert oder atypisch lokalisiert, schwer behandelbar und neigen zu Rezidiven. Die erhöhte Suszeptibilität kann u.xa0a. auf verschiedene Defekte zurückgeführt werden, die die IL12-IFNγ-Kaskade beeinträchtigen. Das enge Spektrum mit intrazellulären Erregern ist für die primären Immundefekte in der IL12-IFNγ-Achse pathognomonisch. Im Einzelnen sind Defekte in den beiden Ketten des Interferon-γ-Rezeptors IFNγR1 und IFNγR2, des „Signal transducer and activator of transcription“-1-Moleküls (STAT1), der Interleukin 12p40-Untereinheit (IL-12p40) und der IL-12-Rezeptor-β1-Kette (IL-12Rβ1) beschrieben. Innerhalb der IL12-IFNγ-Achse wurden bisher fünf autosomale Gene identifiziert, deren Mutationen zu MSMD führen können. Innerhalb dieser Gene sind bis jetzt insgesamt 12 verschiedene Defekte durch dominante und rezessive Allele — mit unterschiedlicher Expression und unterschiedlicher funktioneller Aktivität des betroffenen Proteins — beschrieben. Diese Heterogenität bedingt die deutlichen Differenzen in der Ausprägung des Krankheitsbildes und erschwert die Diagnose. Daher ist die molekularbiologische Charakterisierung, obwohl u.xa0U. sehr aufwändig, unerlässlich für die Einschätzung des klinischen Verlaufs und die Einleitung einer optimalen Therapie.Abstract“Mendelian susceptibility to mycobacterial disease” (MSMD) subsumes a clinically elevated predisposition for infection caused by opportunistic, low virulence mycobacteria. In contrast to the typical non-tuberculous mycobacteria (NTM)-infections during childhood (predominantly lymphadenopathy), mycobacterial infections with MSMD are disseminated or atypically located, difficult to treat and recurrent. The increase in susceptibility may be due to mutations in variant genes, which, for example, functionally impair the signaling of the IL-12/IFNγ pathway. The narrow spectrum of intracellular pathogens causing disease in these patients is specific for an immunodeficiency affecting the IL-12/IFNγ pathway. Several mutations have been characterized and functionally identified. Mutations could be detected in genes coding for the interferon-γ receptor ligand-binding chains (IFNγR1and IFNγR2), signal transducer and activator of transcription-1 (STAT-1), interleukin-12 p40 subunit (IL-12 p40), and interleukin-12 receptor β1 chain (IL-12R β1). Up to now, 12 different inheritable disorders in the IL-12/IFNγ pathway have been identified that increase susceptibility to mycobacterial infections. The alleles responsible are either recessive or dominant. They lead to a functional immune defect, which can be either complete or partial. Generally, each mutation functionally affects the immune interaction of the interleukin-12-IFNγ-axis, highlighting the exceptional role of this innate immune response in mycobacterial infection. This diversity of genetic and immunological defects complicates an accurate diagnosis. Nevertheless, an accurate molecular diagnosis is required for optimal treatment and prognosis.