Paradoxical reactions and immune reconstitution inflammatory syndrome in tuberculosis

The coalescence of the HIV-1 and tuberculosis (TB) epidemics in Sub-Saharan Africa has had a significant and negative impact on global health. The availability of effective antimicrobial treatment for both HIV-1 (in the form of highly active antiretroviral therapy (HAART)) and TB (with antimycobacterial agents) has the potential to mitigate the associated morbidity and mortality. However, the use of both HAART and antimycobacterial therapy is associated with the development of inflammatory paradoxical syndromes after commencement of therapy. These include paradoxical reactions (PR) and immune reconstitution inflammatory syndromes (IRIS), conditions that complicate mycobacterial disease in HIV seronegative and seropositive individuals. Here, we discuss case definitions for PR and IRIS, and explore how advances in identifying the risk factors and immunopathogenesis ofthese conditions informs our understanding of their shared underlying pathogenesis. We propose that both PR and IRIS are characterized by the triggering of exaggerated inflammation in a setting of immunocompromise and antigen loading, via the reversal of immunosuppression by HAART and/or antimycobacterials. Further understanding of the molecular basis of this pathogenesis may pave the way for effective immunotherapies for the treatment of PR and IRIS

Determinants of treatment-related paradoxical reactions during anti-tuberculosis therapy: a case control study

BACKGROUND: Inflammatory response following initial improvement with anti-tuberculosis (TB) treatment has been termed a paradoxical reaction (PR). HIV co-infection is a recognised risk, yet little is known about other predictors of PR, although some biochemical markers have appeared predictive. We report our findings in an ethnically diverse population of HIV-infected and uninfected adults. METHODS: Prospective and retrospective clinical and laboratory data were collected on TB patients seen between January 1999-December 2008 at four UK centres selected to represent a wide ethnic and socio-economic mix of TB patients. Data on ethnicity and HIV status were obtained for all individuals. The associations between other potential risk factors and PR were assessed in a nested case-control study. All PR cases were matched two-to-one to controls by calendar time and centre. RESULTS: Of 1817 TB patients, 82 (4.5 %, 95 % CI 3.6-5.5 %) were identified as having a PR event. The frequency of PR was 14.4 % (18/125; 95 % CI 8.2-20.6 %) and 3.8 % (64/1692; 2.9-4.7) for HIV-positive and HIV-negative individuals respectively. There were no differences observed in PR frequency according to ethnicity, although the site was more likely to be pulmonary in those of black and white ethnicity, and lymph node disease in those of Asian ethnicity. In multivariate analysis of the case-control cohort, HIV-positive patients had five times the odds of developing PR (aOR = 5.05; 95 % CI 1.28-19.85, p = 0.028), whilst other immunosuppression e.g. diabetes, significantly reduced the odds of PR (aOR = 0.01; 0.00-0.27, p = 0.002). Patients with positive TB culture had higher odds of developing PR (aOR = 6.87; 1.31-36.04, p = 0.045) compared to those with a negative culture or those in whom no material was sent for culture. Peripheral lymph node disease increased the odds of a PR over 60-fold 4(9.60-431.25, p < 0.001). CONCLUSION: HIV was strongly associated with PR. The increased potential for PR in people with culture positive TB suggests that host mycobacterial burden might be relevant. The increased risk with TB lymphadenitis may in part arise from the visibility of clinical signs at this site. Non-HIV immunosuppression may have a protective effect. This study highlights the difficulties in predicting PR using routinely available demographic details, clinical symptoms or biochemical markers

Variation in C - reactive protein response according to host and mycobacterial characteristics in active tuberculosis

BACKGROUND: The C - reactive protein (CRP) response is often measured in patients with active tuberculosis (TB) yet little is known about its relationship to clinical features in TB, or whether responses differ between ethnic groups or with different Mycobacterium tuberculosis (M.tb) strain types. We report the relationship between baseline serum CRP prior to treatment and disease characteristics in a metropolitan population with TB resident in a low TB incidence region. METHODS: People treated for TB at four London, UK sites between 2003 and 2014 were assessed and data collected on the following characteristics: baseline CRP level; demographics (ethnicity, gender and age); HIV status; site of TB disease; sputum smear (in pulmonary cases) and culture results. The effect of TB strain-type was also assessed in culture-positive pulmonary cases using VNTR typing data. RESULTS: Three thousands two hundred twenty-two patients were included in the analysis of which 72 % had a baseline CRP at or within 4 weeks prior to starting TB treatment. CRP results were significantly higher in culture positive cases compared to culture negative cases: median 49 mg/L (16-103 mg/L) vs 19 mg/L (IQR 5-72 mg/L), p = <0.001. In those with pulmonary disease, smear positive cases had a higher CRP than smear negative cases: 67 mg/L (31-122 mg/L) vs 24 mg/L (7-72 mg/L), p < 0.001. HIV positive cases had higher baseline CRPs than HIV negative cases: 75 mg/L (26-136 mg/L) vs 37 mg/L (10-88 mg/L), p <0.001. Differing sites of disease were associated with differences in baseline CRP: locations that might be expected to have a high mycobacterial load (e.g. pulmonary disease and disseminated disease) had a significantly higher CRP than those such as skin, lymph node or CNS disease, where the mycobacterial load is typically low in HIV negative subjects. In a multivariable log-scale linear regression model adjusting for host characteristics and M.tb strain type, infection with the East African Indian strain was associated with significantly lower baseline-CRP (fold-change in CRP 0.51 (0.34-0.77), p < 0.01). CONCLUSIONS: Host and mycobacterial factors are strongly associated with baseline CRP response in tuberculosis. This analysis suggests that there are important differences in innate immune response according to ethnicity, Mtb strain type and site of disease. This may reflect differing mycobacterial loads or host immune responses

Validation of Differentially Expressed Immune Biomarkers in Latent and Active Tuberculosis by Real-Time PCR

Tuberculosis (TB) remains a major global threat and diagnosis of active TB ((ATB) both extra-pulmonary (EPTB), pulmonary (PTB)) and latent TB (LTBI) infection remains challenging, particularly in high-burden countries which still rely heavily on conventional methods. Although molecular diagnostic methods are available, e.g., Cepheid GeneXpert, they are not universally available in all high TB burden countries. There is intense focus on immune biomarkers for use in TB diagnosis, which could provide alternative low-cost, rapid diagnostic solutions. In our previous gene expression studies, we identified peripheral blood leukocyte (PBL) mRNA biomarkers in a non-human primate TB aerosol-challenge model. Here, we describe a study to further validate select mRNA biomarkers from this prior study in new cohorts of patients and controls, as a prerequisite for further development. Whole blood mRNA was purified from ATB patients recruited in the UK and India, LTBI and two groups of controls from the UK (i) a low TB incidence region (CNTRLA) and (ii) individuals variably-domiciled in the UK and Asia ((CNTRLB), the latter TB high incidence regions). Seventy-two mRNA biomarker gene targets were analyzed by qPCR using the Roche Lightcycler 480 qPCR platform and data analyzed using GeneSpring™ 14.9 bioinformatics software. Differential expression of fifty-three biomarkers was confirmed between MTB infected, LTBI groups and controls, seventeen of which were significant using analysis of variance (ANOVA): CALCOCO2, CD52, GBP1, GBP2, GBP5, HLA-B, IFIT3, IFITM3, IRF1, LOC400759 (GBP1P1), NCF1C, PF4V1, SAMD9L, S100A11, TAF10, TAPBP, and TRIM25. These were analyzed using receiver operating characteristic (ROC) curve analysis. Single biomarkers and biomarker combinations were further assessed using simple arithmetic algorithms. Minimal combination biomarker panels were delineated for primary diagnosis of ATB (both PTB and EPTB), LTBI and identifying LTBI individuals at high risk of progression which showed good performance characteristics. These were assessed for suitability for progression against the standards for new TB diagnostic tests delineated in the published World Health Organization (WHO) technology product profiles (TPPs)

Rapid Diagnosis of Smear-Negative Tuberculosis Using Immunology and Microbiology with Induced Sputum in HIV-Infected and Uninfected Individuals

Rationale and Objectives. Blood-based studies have demonstrated the potential of immunological assays to detect tuberculosis. However lung fluid sampling may prove superior as it enables simultaneous microbiological detection of mycobacteria to be performed. Until now this has only been possible using the expensive and invasive technique of broncho-alveolar lavage. We sought to evaluate an immunoassay using non-invasive induced-sputum to diagnose active tuberculosis. Methods and Results. Prospective cohort study of forty-two spontaneous sputum smear-negative or sputum non-producing adults under investigation for tuberculosis. CD4 lymphocytes specific to purified-protein-derivative of Mycobacterium tuberculosis actively synthesising interferon-gamma were measured by flow cytometry and final diagnosis compared to immunoassay using a cut-off of 0.5%. Sixteen subjects (38%) were HIV-infected (median CD4 count [range] = 332 cells/mu l [103748]). Thirty-eight (90%) were BCG-vaccinated. In 27 subjects diagnosed with active tuberculosis, the median [range] percentage of interferon-gamma synthetic CD4+ lymphocytes was 2.77% [0-23.93%] versus 0% [0-2.10%] in 15 negative for active infection (p<0.0001). Sensitivity and specificity of the immunoassay versus final diagnosis of active tuberculosis were 89% (24 of 27) and 80% (12 of 15) respectively. The 3 positive assays in the latter group occurred in subjects diagnosed with quiescent/latent tuberculosis. Assay performance was unaffected by HIV-status, BCG-vaccination or disease site. Combining this approach with traditional microbiological methods increased the diagnostic yield to 93% (25 of 27) alongside acid-fast bacilli smear and 96% (26 of 27) alongside tuberculosis culture. Conclusions. These data demonstrate for the first time that a rapid immunological assay to diagnose active tuberculosis can be performed successfully in combination with microbiological methods on a single induced-sputum sample

A novel formulation of inhaled sodium cromoglicate (PA101) in idiopathic pulmonary fibrosis and chronic cough: a randomised, double-blind, proof-of-concept, phase 2 trial

Background Cough can be a debilitating symptom of idiopathic pulmonary fibrosis (IPF) and is difficult to treat. PA101 is a novel formulation of sodium cromoglicate delivered via a high-efficiency eFlow nebuliser that achieves significantly higher drug deposition in the lung compared with the existing formulations. We aimed to test the efficacy and safety of inhaled PA101 in patients with IPF and chronic cough and, to explore the antitussive mechanism of PA101, patients with chronic idiopathic cough (CIC) were also studied. Methods This pilot, proof-of-concept study consisted of a randomised, double-blind, placebo-controlled trial in patients with IPF and chronic cough and a parallel study of similar design in patients with CIC. Participants with IPF and chronic cough recruited from seven centres in the UK and the Netherlands were randomly assigned (1:1, using a computer-generated randomisation schedule) by site staff to receive PA101 (40 mg) or matching placebo three times a day via oral inhalation for 2 weeks, followed by a 2 week washout, and then crossed over to the other arm. Study participants, investigators, study staff, and the sponsor were masked to group assignment until all participants had completed the study. The primary efficacy endpoint was change from baseline in objective daytime cough frequency (from 24 h acoustic recording, Leicester Cough Monitor). The primary efficacy analysis included all participants who received at least one dose of study drug and had at least one post-baseline efficacy measurement. Safety analysis included all those who took at least one dose of study drug. In the second cohort, participants with CIC were randomly assigned in a study across four centres with similar design and endpoints. The study was registered with ClinicalTrials.gov (NCT02412020) and the EU Clinical Trials Register (EudraCT Number 2014-004025-40) and both cohorts are closed to new participants. Findings Between Feb 13, 2015, and Feb 2, 2016, 24 participants with IPF were randomly assigned to treatment groups. 28 participants with CIC were enrolled during the same period and 27 received study treatment. In patients with IPF, PA101 reduced daytime cough frequency by 31·1% at day 14 compared with placebo; daytime cough frequency decreased from a mean 55 (SD 55) coughs per h at baseline to 39 (29) coughs per h at day 14 following treatment with PA101, versus 51 (37) coughs per h at baseline to 52 (40) cough per h following placebo treatment (ratio of least-squares [LS] means 0·67, 95% CI 0·48–0·94, p=0·0241). By contrast, no treatment benefit for PA101 was observed in the CIC cohort; mean reduction of daytime cough frequency at day 14 for PA101 adjusted for placebo was 6·2% (ratio of LS means 1·27, 0·78–2·06, p=0·31). PA101 was well tolerated in both cohorts. The incidence of adverse events was similar between PA101 and placebo treatments, most adverse events were mild in severity, and no severe adverse events or serious adverse events were reported. Interpretation This study suggests that the mechanism of cough in IPF might be disease specific. Inhaled PA101 could be a treatment option for chronic cough in patients with IPF and warrants further investigation

Determinants of recovery from post-COVID-19 dyspnoea: analysis of UK prospective cohorts of hospitalised COVID-19 patients and community-based controls

Background The risk factors for recovery from COVID-19 dyspnoea are poorly understood. We investigated determinants of recovery from dyspnoea in adults with COVID-19 and compared these to determinants of recovery from non-COVID-19 dyspnoea. Methods We used data from two prospective cohort studies: PHOSP-COVID (patients hospitalised between March 2020 and April 2021 with COVID-19) and COVIDENCE UK (community cohort studied over the same time period). PHOSP-COVID data were collected during hospitalisation and at 5-month and 1-year follow-up visits. COVIDENCE UK data were obtained through baseline and monthly online questionnaires. Dyspnoea was measured in both cohorts with the Medical Research Council Dyspnoea Scale. We used multivariable logistic regression to identify determinants associated with a reduction in dyspnoea between 5-month and 1-year follow-up. Findings We included 990 PHOSP-COVID and 3309 COVIDENCE UK participants. We observed higher odds of improvement between 5-month and 1-year follow-up among PHOSP-COVID participants who were younger (odds ratio 1.02 per year, 95% CI 1.01–1.03), male (1.54, 1.16–2.04), neither obese nor severely obese (1.82, 1.06–3.13 and 4.19, 2.14–8.19, respectively), had no pre-existing anxiety or depression (1.56, 1.09–2.22) or cardiovascular disease (1.33, 1.00–1.79), and shorter hospital admission (1.01 per day, 1.00–1.02). Similar associations were found in those recovering from non-COVID-19 dyspnoea, excluding age (and length of hospital admission). Interpretation Factors associated with dyspnoea recovery at 1-year post-discharge among patients hospitalised with COVID-19 were similar to those among community controls without COVID-19. Funding PHOSP-COVID is supported by a grant from the MRC-UK Research and Innovation and the Department of Health and Social Care through the National Institute for Health Research (NIHR) rapid response panel to tackle COVID-19. The views expressed in the publication are those of the author(s) and not necessarily those of the National Health Service (NHS), the NIHR or the Department of Health and Social Care. COVIDENCE UK is supported by the UK Research and Innovation, the National Institute for Health Research, and Barts Charity. The views expressed are those of the authors and not necessarily those of the funders

Large-scale phenotyping of patients with long COVID post-hospitalization reveals mechanistic subtypes of disease

One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain–gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials