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Journal articleLalvani A, Whitworth HS, 2019,
Progress in interferon-gamma release assay development and applications: an unfolding story of translational research
, ANNALS OF TRANSLATIONAL MEDICINE, Vol: 7, ISSN: 2305-5839- Author Web Link
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- Citations: 3
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Journal articleHalliday A, Masonou T, Tolosa-Wright M, et al., 2019,
Immunodiagnosis of active tuberculosis
, Expert Review of Respiratory Medicine, Vol: 13, Pages: 521-532, ISSN: 1747-6348Introduction: There is an unmet clinical need for improved diagnostic tests for active tuberculosis (TB) to provide high sensitivity for all cases, accelerate time to diagnosis and ensure timely and appropriate treatment. Whilst the measurement of M.tb-specific immune responses is widely used for detecting infection in the absence of TB symptoms (i.e. latent TB infection), there is currently no role for immunodiagnostics in active TB disease. This is primarily due to insufficient sensitivity, and an inability to discriminate between active disease and controlled, latent TB infection. Areas covered: In this review, we focus on recent developments in the use of immune-based tests to provide a point of care test for the rule-in or rule-out of active TB. Expert opinion: Recent studies have demonstrated that second-generation IGRAs have the potential to rule-out active TB, particularly in low burden settings. Newer technological platforms, including systems serology and flow cytometry, offer the means to measure specific M.tb specific immune signatures which have been shown to have a high level of accuracy for active TB. However, it is now crucial that new and promising test undergo validation in clinically relevant cohorts which include the full spectrum of TB patients and differential diagnoses.
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Journal articleMasonou T, Hokey DA, Lahey T, et al., 2019,
CD4+ T cell cytokine responses to the DAR-901 booster vaccine in BCG-primed adults: A randomized, placebo-controlled trial
, PLoS ONE, Vol: 14, ISSN: 1932-6203BACKGROUND: DAR-901 is an inactivated whole cell tuberculosis booster vaccine, prepared using a new scalable, broth-grown method from the master cell bank of SRL172, a vaccine previously shown to prevent tuberculosis. This study examined whether DAR-901 (a) induces CD4+ T cell cytokine profiles previously proposed as correlates of protection and (b) has a specific vaccine-induced immunological signature compared to BCG or placebo. METHODS: We analysed CD4+ T cell cytokine immune responses from 10 DAR-901 recipients, 9 BCG recipients and 9 placebo recipients from the Phase I DAR-901 MDES trial. In that study, HIV-negative, IGRA-negative participants with prior BCG immunization were randomized (double-blind) to receive three intradermal injections of DAR-901 or saline placebo or two injections of saline placebo followed by an intradermal injection of BCG. Antigen-specific functional and phenotypic CD4+ T cell responses along with effector phenotype of responder cells were measured by intracellular cytokine staining. RESULTS: DAR-901 recipients exhibited increased DAR-901 antigen-specific polyfunctional or bifunctional T cell responses compared to baseline. Vaccine specific CD4+ IFNγ, IL2, TNFα and any cytokine responses peaked at 7 days post-dose 3. Th1 responses predominated, with most responder cells exhibiting a polyfunctional effector memory phenotype. BCG induced greater CD4+ T cell responses than placebo while the more modest DAR-901 responses did not differ from placebo. Neither DAR-901 nor BCG induced substantial or sustained Th17 /Th22 cytokine responses. CONCLUSION: DAR-901, a TB booster vaccine grown from the master cell bank of SRL 172 which was shown to prevent TB, induced low magnitude polyfunctional effector memory CD4+ T cell responses. DAR-901 responses were lower than those induced by BCG, a vaccine that has been shown ineffective as a booster to prevent tuberculosis disease. These results suggest that induction of higher levels of CD4+ c
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Journal articleHalliday A, Jain P, Hoang L, et al.,
Validation of new technologies for the diagnostic evaluation of active tuberculosis (VANTDET)
, Efficacy and Mechanism Evaluation, ISSN: 2050-4365Background: Tuberculosis (TB) is a devastating disease for which new diagnostic tests are desperately needed. Objective: To validate promising new technologies (namely whole blood transcriptomics, proteomics, flow cytometry and qRT-PCR) and existing signatures for detection of active TB in samples obtained from individuals suspected of active TB. Design: Four sub-studies, each of which used the samples from biobank collected as part of the IDEA study, which was a prospective cohort of patients recruited with suspected TB. Setting: secondary care Participants: Adults (aged ≥ 16 years old) presenting as inpatients or outpatients at 12 NHS hospital trusts in London, Slough, Oxford, Leicester and Birmingham with suspected active TB. Interventions: New tests using either: genome-wide gene expression microarray (transcriptomics); SELDI TOF/ LC-MS (proteomics), flow cytometry, qRT-PCR. Main outcome measures: Area under the curve (AUC), sensitivity and specificity, were calculated to determine diagnostic accuracy. Positive and negative predictive values were calculated in some cases. A decision tree model was developed to calculate the incremental costs and quality-adjusted life-years (QALYs) of changing from current practice to using the novels tests. Results: The project and 4 sub-studies which assessed the previous published signatures measured using each of the new technologies, and a health economic analysis where the best performing tests were evaluated for cost effectiveness. The diagnostic accuracy of the transcriptomic tests ranged from AUC=0.81-0.84 for detecting all TB in our cohort. The performance for detecting culture confirmed TB or pulmonary TB (PTB) was better than for highly probable TB or extrapulmonary TB (EPTB) respectively, but not high enough to be clinically useful. None of the previously described serum proteomic signatures for active TB provided good diagnostic accuracy, not did the candidate rule-out tests. Four of six previously described cell
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Journal articleWhitworth HS, Badhan A, Boakye AA, et al.,
An observational cohort study to evaluate the clinical utilty of current and second-generation interferon-gamma release-assays in diagnostic evaluation of tuberculosis
, Lancet Infectious Diseases, ISSN: 1473-3099BackgroundThe role of interferon-gamma release assays (IGRAs) in diagnosis of active tuberculosis (TB) is unclear, yet they are commonly used in low-TB-incidence countries. This study sought to resolve this clinical uncertainty by determining the diagnostic accuracy and role of current and second-generation IGRAs in the diagnostic assessment of suspected TB in a low-incidence setting. MethodsThis was a prospective cohort study of 1,060 adults with suspected TB, conducted in routine secondary care in England. Patients were tested for M. tuberculosis (Mtb) infection at baseline using current and second-generation IGRAs, the latter incorporating novel Mtb antigens, and followed up for 6-12m to establish definitive diagnoses. Sensitivity, specificity and positive and negative likelihood ratios (LRs) and predictive values (PVs) of the tests for TB were determined.FindingsTB was diagnosed in 363 (43%) of 845 patients included in analyses. Sensitivity of T-SPOT.TB was 81.4% (95%CI 76.6-85.3%), higher than Quantiferon-Gold In-Tube at 67.3% (95%CI 62.0-72.1%). Second-generation IGRA had higher sensitivity than current tests, at 94.0% (95%CI 90.0–96.4%) for culture-confirmed TB and 89.2% (95%CI 85.2–92.2%) when including highly-probable TB, giving a negative LR for all TB of 0.13 (95%CI 0.10-0.19). Specificity ranged from 86.2% (95%CI 82.3-89.4%) for T-SPOT.TB to 80.0% (95%CI 75.6-83.8%) for second-generation IGRA.InterpretationCurrently-available IGRAs lack sufficient accuracy for diagnostic evaluation of suspected TB. Second-generation tests, however, may have sufficiently high sensitivity, low negative LR and correspondingly high negative PV in low-incidence settings to facilitate prompt rule-out of TB.
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Journal articleEberhardt C, Thillai M, Parker R, et al., 2017,
Proteomic analysis of Kveim reagent identifies targets of cellular immunity in sarcoidosis
, PLOS ONE, Vol: 12, ISSN: 1932-6203Background:Kveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4–6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins.Methods:Kv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay.Results:We observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and contr
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Journal articleSinganayagam A, Manalan K, Connell DW, et al., 2016,
Evaluation of serum inflammatory biomarkers as predictors of treatment outcome in pulmonary tuberculosis
, International Journal of Tuberculosis and Lung Disease, Vol: 20, Pages: 1653-1660, ISSN: 1815-7920OBJECTIVE: To evaluate C-reactive protein (CRP), globulin and white blood cell (WBC) count as predictors of treatment outcome in pulmonary tuberculosis (PTB).METHODS: An observational study of patients with active PTB was conducted at a tertiary centre. All patients had serum CRP, globulin and WBC measured at baseline and at 2 months following commencement of treatment. The outcome of interest was requirement for extension of treatment beyond 6 months.RESULTS: There were 226 patients included in the study. Serum globulin >45 g/l was the only baseline biomarker evaluated that independently predicted requirement for treatment extension (OR 3.42, 95%CI 1.59–7.32, P < 0.001). An elevated globulin level that failed to normalise at 2 months was also associated with increased requirement for treatment extension (63.9% vs. 5.1%, P < 0.001), and had a low negative likelihood ratio (0.07) for exclusion of requirement for treatment extension. On multivariable analysis, an elevated globulin that failed to normalise at 2 months was independently associated with requirement for treatment extension (OR 6.13, 95%CI 2.23–16.80, P < 0.001).CONCLUSIONS: Serum globulin independently predicts requirement for treatment extension in PTB and outperforms CRP and WBC as a predictive biomarker. Normalisation of globulin at 2 months following treatment commencement is associated with low risk of requirement for treatment extension.
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Journal articleRitchie A, Singanayagam A, Manalan K, et al., 2016,
SERUM INFLAMMATORY BIOMARKERS AS PREDICTORS OF TREATMENT OUTCOME IN PULMONARY TUBERCULOSIS
, THORAX, Vol: 71, Pages: A143-A144, ISSN: 0040-6376 -
Journal articleO'Donoghue M, Jarvis H, Drey N, et al., 2016,
THE IMPACT OF TB NICE GUIDANCE ON RESOURCE CAPACITY AND CONTACT SCREENING OUTCOMES: A RETROSPECTIVE, OBSERVATIONAL STUDY WITHIN A CENTRAL LONDON TB CENTRE
, THORAX, Vol: 71, Pages: A142-A142, ISSN: 0040-6376 -
Journal articleMarriott AC, Dennis M, Kane JA, et al., 2016,
Influenza A Virus Challenge Models in Cynomolgus Macaques Using the Authentic Inhaled Aerosol and Intra-Nasal Routes of Infection
, PLOS One, Vol: 11, ISSN: 1932-6203Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections.
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