Use of interferon gamma release assay to assess latent tuberculosis infection among healthcare workers in Hong Kong

Key Messages 1. Overall baseline interferon gamma release assay positivity was 20.7%. 2. The conversion to interferon gamma release assay positivity at 3 months was 8.85% in the exposed group and 4.54% in the non-exposed group using the conventional cut-off of 0.35 IU/mL. 3. When grey zone results (0.2I-0.7 IU/mL) were included, the proportion of non-specific conversions and reversions could be reduced. 4. Interferon gamma release assay can be an adjunct tool in contact investigation of latent tuberculosis infection in healthcare workers.published_or_final_versio

TUBERCULOSIS IN CHILDREN: NEW FORMS OF DIAGNOSIS

Introdução: A tuberculose ainda é um grave problema de saúde pública em Portugal. Para diminuir o número de casos de tuberculose ativa em populações de baixa e intermédia incidência, é necessário um diagnóstico rápido e um tratamento eficaz. A prova tuberculínica é o método de rastreio recomendado, mas as suas limitações são conhecidas. Em 2001, foi aprovado o primeiro de diversos interferon gamma release assays, considerados úteis no diagnóstico de infeção latente por Mycobacterium tuberculosis, amplamente utilizados na abordagem da tuberculose nos adultos. Objectivos: Sumarizar a informação disponível sobre os interferon gamma release assays, nomeadamente no que diz respeito à técnica; às vantagens no diagnóstico da infeção latente por Mycobacterium tuberculosis; à sensibilidade e especificidade quando aplicados à população pediátrica; à caracterização de fatores interferentes; e ao seu significado na monitorização do tratamento com antituberculosos. Desenvolvimento: Os interferon gamma release assays são testes imunologicamente seletivos, desenvolvidos com base em antigénios do Mycobacterium tuberculosis. Têm sido aplicados na Pediatria, em regiões com diferentes taxas de prevalência de tuberculose, de forma a compará-los com a prova tuberculínica relativamente à sensibilidade e especificidade. Conclusões: A utilização destes testes como forma de rastreio em Pediatria apresenta limitações. São necessários estu- dos a nível nacional que permitam mostrar de que forma a prova tuberculínica e os interferon gamma release assays se devem articular. Atualmente, os interferon gamma release assays ape- nas complementam a prova tuberculínica

The effect of abrupt weaning of suckler calves on the plasma concentrations of cortisol, catecholamines, leukocyte, acute-phase proteins and in vitro interferon-gamma production

End of project reportThe objective of this study was to examine the effect of abrupt weaning (inclusive of social group disruption and maternal separation) on the physiological mediators of stress and measures of immune function. Thirty-eight male and 38 female continental calves were habituated to handling for two weeks prior to bleeding. Calves were blocked on sex, weight and breed of dam and randomly assigned, within block, to either a control (cows remain with calves) or abruptly weaned group (calves removed from cows). Animals were separated into the respective treatment groups at weaning (0 h). Calves were bled at – 168 h, 6 h (males only), 24 h, 48 h and 168 h post weaning. At each sampling time an observer scored the behavioural reaction of calves to sampling. Blood samples were analysed for cortisol, catecholamine concentrations (not sampled at –168 h) and in vitro interferon-gamma production, neutrophil :lymphocyte ratio and acute phase protein concentrations. All continuous data were analysed using a split-plot ANOVA, except that collected at 6 h, which was analysed using a single factor ANOVA model. The effects of weaning, calf sex and time and respective interactions were described. Disruption of the established social groups at 0 h, increased (p<0.001) the plasma cortisol concentration and neutrophil: lymphocyte ratio and reduced the leukocyte concentration (p<0.001) and the in vitro interferon-gamma response to the mitogen concanavalin-A (p<0.001) and keyhole limpet haemocyanin (p<0.001) for weaned and control animals, when compared with –168h. Plasma adrenaline and noradrenaline concentrations were not affected by group disruption. There was no effect of weaning or sex on calf behavioural reaction to handling during blood sampling. Plasma cortisol and adrenaline concentrations were not affected by weaning or sex. Plasma noradrenaline concentration was influenced by weaning x sex (p<0.05) and time x sex (p<0.05). The response increased for male calves with weaning and increased with each sampling time post weaning. For heifers the response was not affected by weaning and plasma concentrations decreased at 168 h post weaning. There was no effect of weaning or sex on leukocyte concentration. The neutrophils : lymphocyte ration increased post weaning (p<0.01) and was affected by sex (p<0.05). Weaning decreased (p<0.05) the in vitro interferon-gamma response to the antigen KLH. There was a time x weaning x sex (p<0.05) interaction for fibrinogen concentration but no effect of treatment on haptoglobin concentration. Abrupt weaning increased plasma cortisol and nor-adrenaline concentrations, which was accompanied by attenuation of in vitro interferon gamma production to novel mitogen and antigen complexes up to 7 days post weaning.European Union Structural Funds (EAGGF

QuantiFERON®-TB gold in-tube performance for diagnosing active tuberculosis in children and adults in a high burden setting.

To determine whether QuantiFERON®-TB Gold In-Tube (QFT) can contribute to the diagnosis of active tuberculosis (TB) in children in a high-burden setting and to assess the performance of QFT and tuberculin skin test (TST) in a prospective cohort of TB suspect children compared to adults with confirmed TB in Tanzania. Sensitivity and specificity of QFT and TST for diagnosing active TB as well as indeterminate QFT rates and IFN-γ levels were assessed in 211 TB suspect children in a Tanzanian district hospital and contrasted in 90 adults with confirmed pulmonary TB. Sensitivity of QFT and TST in children with confirmed TB was 19% (5/27) and 6% (2/31) respectively. In adults sensitivity of QFT and TST was 84% (73/87) and 85% (63/74). The QFT indeterminate rate in children and adults was 27% and 3%. Median levels of IFN-γ were lower in children than adults, particularly children <2 years and HIV infected. An indeterminate result was associated with age <2 years but not malnutrition or HIV status. Overall childhood mortality was 19% and associated with an indeterminate QFT result at baseline. QFT and TST showed poor performance and a surprisingly low sensitivity in children. In contrast the performance in Tanzanian adults was good and comparable to performance in high-income countries. Indeterminate results in children were associated with young age and increased mortality. Neither test can be recommended for diagnosing active TB in children with immature or impaired immunity in a high-burden setting

The role of the A C395 IFNGR1 mutation in determining susceptibility to intracellular infection in Malta

Background: The first human mycobacterial susceptibility gene was identified amongst four children on the island of Malta in 1995. All affected children were homozygous for a nonsense mutation at position 395 of the interferon gamma receptor 1 (IFNGR1) gene, and all but one died of overwhelming mycobacterial infection. The population of Malta has high rates of infection with intracellular pathogens; leishmania, brucellosis and tuberculosis are all endemic, while leprosy, which was previously endemic, has only recently been eradicated. We hypothesised that heterozygous carriers of the IFNGR1 gene mutation, while resistant to infection with poorly pathogenic organisms, may have increased susceptibility to infection with more virulent pathogens. Methodology and Result: Screening patients with a past history of intracellular infection and healthy newborns for the presence of the IFNGR1 A->C395 mutation, using sequence specific primer PCR, did not identify any carriers of the mutation. Conclusion: These results suggest that the IFNGR1 mutation is unlikely to be of public health significance on Malta.peer-reviewe

Natural variation in immune responses to neonatal mycobacterium bovis bacillus calmette-guerin (BCG) vaccination in a cohort of Gambian infants

Background There is a need for new vaccines for tuberculosis (TB) that protect against adult pulmonary disease in regions where BCG is not effective. However, BCG could remain integral to TB control programmes because neonatal BCG protects against disseminated forms of childhood TB and many new vaccines rely on BCG to prime immunity or are recombinant strains of BCG. Interferon-gamma (IFN-) is required for immunity to mycobacteria and used as a marker of immunity when new vaccines are tested. Although BCG is widely given to neonates IFN- responses to BCG in this age group are poorly described. Characterisation of IFN- responses to BCG is required for interpretation of vaccine immunogenicity study data where BCG is part of the vaccination strategy. Methodology/Principal Findings 236 healthy Gambian babies were vaccinated with M. bovis BCG at birth. IFN-, interleukin (IL)-5 and IL-13 responses to purified protein derivative (PPD), killed Mycobacterium tuberculosis (KMTB), M. tuberculosis short term culture filtrate (STCF) and M. bovis BCG antigen 85 complex (Ag85) were measured in a whole blood assay two months after vaccination. Cytokine responses varied up to 10 log-fold within this population. The majority of infants (89-98% depending on the antigen) made IFN- responses and there was significant correlation between IFN- responses to the different mycobacterial antigens (Spearman’s coefficient ranged from 0.340 to 0.675, p=10-6-10-22). IL-13 and IL-5 responses were generally low and there were more non-responders (33-75%) for these cytokines. Nonetheless, significant correlations were observed for IL-13 and IL-5 responses to different mycobacterial antigens Conclusions/Significance Cytokine responses to mycobacterial antigens in BCG-vaccinated infants are heterogeneous and there is significant inter-individual variation. Further studies in large populations of infants are required to identify the factors that determine variation in IFN- responses

IFN-γ and TNF-α synergize to inhibit CTGF expression in human lung endothelial cells.

Connective tissue growth factor (CTGF/CCN2) is an angiogenetic and profibrotic factor, acting downstream of TGF-β, involved in both airway- and vascular remodeling. While the T-helper 1 (Th1) cytokine interferon-gamma (IFN-γ) is well characterized as immune-modulatory and anti-fibrotic cytokine, the role of IFN-γ in lung endothelial cells (LEC) is less defined. Tumour necrosis factor alpha (TNF-α) is another mediator that drives vascular remodeling in inflammation by influencing CTGF expression. In the present study we investigated the influence of IFN-γ and TNF-α on CTGF expression in human LEC (HPMEC-ST1.6R) and the effect of CTGF knock down on human LEC. IFN-γ and TNF-α down-regulated CTGF in human LEC at the promoter-, transcriptional- and translational-level in a dose- and time-dependent manner. The inhibitory effect of IFN-γ on CTGF-expression could be almost completely compensated by the Jak inhibitor AG-490, showing the involvement of the Jak-Stat signaling pathway. Besides the inhibitory effect of IFN-γ and TNF-α alone on CTGF expression and LEC proliferation, these cytokines had an additive inhibitory effect on proliferation as well as on CTGF expression when administered together. To study the functional role of CTGF in LEC, endogenous CTGF expression was down-regulated by a lentiviral system. CTGF silencing in LEC by transduction of CTGF shRNA reduced cell proliferation, but did not influence the anti-proliferative effect of IFN-γ and TNF-α. In conclusion, our data demonstrated that CTGF was negatively regulated by IFN-γ in LEC in a Jak/Stat signaling pathway-dependent manner. In addition, an additive effect of IFN-γ and TNF-α on inhibition of CTGF expression and cell proliferation could be found. The inverse correlation between IFN-γ and CTGF expression in LEC could mean that screwing the Th2 response to a Th1 response with an additional IFN-γ production might be beneficial to avoid airway remodeling in asthma

Tuberculosis diagnostics and biomarkers: needs, challenges, recent advances, and opportunities

Tuberculosis is unique among the major infectious diseases in that it lacks accurate rapid point-of-care diagnostic tests. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely fashion, allowing continued Mycobacterium tuberculosis transmission within communities. Currently recommended gold-standard diagnostic tests for tuberculosis are laboratory based, and multiple investigations may be necessary over a period of weeks or months before a diagnosis is made. Several new diagnostic tests have recently become available for detecting active tuberculosis disease, screening for latent M. tuberculosis infection, and identifying drug-resistant strains of M. tuberculosis. However, progress toward a robust point-of-care test has been limited, and novel biomarker discovery remains challenging. In the absence of effective prevention strategies, high rates of early case detection and subsequent cure are required for global tuberculosis control. Early case detection is dependent on test accuracy, accessibility, cost, and complexity, but also depends on the political will and funder investment to deliver optimal, sustainable care to those worst affected by the tuberculosis and human immunodeficiency virus epidemics. This review highlights unanswered questions, challenges, recent advances, unresolved operational and technical issues, needs, and opportunities related to tuberculosis diagnostics

Interferon-γ acutely augments inhibition of neocortical layer 5 pyramidal neurons

BACKGROUND: Interferon-γ (IFN-γ, a type II IFN) is present in the central nervous system (CNS) under various conditions. Evidence is emerging that, in addition to its immunological role, IFN-γ modulates neuronal morphology, function, and development in several brain regions. Previously, we have shown that raising levels of IFN-β (a type I IFN) lead to increased neuronal excitability of neocortical layer 5 pyramidal neurons. Because of shared non-canonical signaling pathways of both cytokines, we hypothesized a similar neocortical role of acutely applied IFN-γ. METHODS: We used semi-quantitative RT-PCR, immunoblotting, and immunohistochemistry to analyze neuronal expression of IFN-γ receptors and performed whole-cell patch-clamp recordings in layer 5 pyramidal neurons to investigate sub- and suprathreshold excitability, properties of hyperpolarization-activated cyclic nucleotide-gated current (Ih), and inhibitory neurotransmission under the influence of acutely applied IFN-γ. RESULTS: We show that IFN-γ receptors are present in the membrane of rat's neocortical layer 5 pyramidal neurons. As expected from this and the putative overlap in IFN type I and II alternative signaling pathways, IFN-γ diminished Ih, mirroring the effect of type I IFNs, suggesting a likewise activation of protein kinase C (PKC). In contrast, IFN-γ did neither alter subthreshold nor suprathreshold neuronal excitability, pointing to augmented inhibitory transmission by IFN-γ. Indeed, IFN-γ increased electrically evoked inhibitory postsynaptic currents (IPSCs) on neocortical layer 5 pyramidal neurons. Furthermore, amplitudes of spontaneous IPSCs and miniature IPSCs were elevated by IFN-γ, whereas their frequency remained unchanged. CONCLUSIONS: The expression of IFN-γ receptors on layer 5 neocortical pyramidal neurons together with the acute augmentation of inhibition in the neocortex by direct application of IFN-γ highlights an additional interaction between the CNS and immune system. Our results strengthen our understanding of the role of IFN-γ in neocortical neurotransmission and emphasize its impact beyond its immunological properties, particularly in the pathogenesis of neuropsychiatric disorders

Tricks to translating TB transcriptomics.

Transcriptomics and other high-throughput methods are increasingly applied to questions relating to tuberculosis (TB) pathogenesis. Whole blood transcriptomics has repeatedly been applied to define correlates of TB risk and has produced new insight into the late stage of disease pathogenesis. In a novel approach, authors of a recently published study in Science Translational Medicine applied complex data analysis of existing TB transcriptomic datasets, and in vitro models, in an attempt to identify correlates of protection in TB, which are crucially required for the development of novel TB diagnostics and therapeutics to halt this global epidemic. Utilizing latent TB infection (LTBI) as a surrogate of protection, they identified IL-32 as a mediator of interferon gamma (IFNγ)-vitamin D dependent antimicrobial immunity and a marker of LTBI. Here, we provide a review of all TB whole-blood transcriptomic studies to date in the context of identifying correlates of protection, discuss potential pitfalls of combining complex analyses originating from such studies, the importance of detailed metadata to interpret differential patient classification algorithms, the effect of differing circulating cell populations between patient groups on the interpretation of resulting biomarkers and we decipher weighted gene co-expression network analysis (WGCNA), a recently developed systems biology tool which holds promise of identifying novel pathway interactions in disease pathogenesis. In conclusion, we propose the development of an integrated OMICS platform and open access to detailed metadata, in order for the TB research community to leverage the vast array of OMICS data being generated with the aim of unraveling the holy grail of TB research: correlates of protection