Publications by authors named "Robert J Wilkinson"

280 Publications

Radiological and functional evidence of the bronchial spread of tuberculosis: an observational analysis.

Lancet Microbe 2021 Oct;2(10):e518-e526

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, USA.

Background: Direct bronchial spread of tuberculosis was extensively described in pre-antibiotic human pathology literature but this description has been overlooked in the post-antibiotic era, in which most pathology data come from animal models that emphasise the granuloma. Modern techniques, such as [F]2-fluoro-2-deoxy-D-glucose (FDG) PET-CT scans, might provide further insight. Our aim was to understand normal early tuberculosis resolution patterns on pulmonary PET-CT scans in treated patients with tuberculosis who were subsequently cured.

Methods: In this observational analysis, we analysed data from PredictTB, an ongoing, prospective, randomised clinical trial that examined sequential baseline and week 4 FDG-PET-CT scans from participants successfully treated (sputum culture negative 18 months after enrolment) for drug-susceptible pulmonary tuberculosis in South Africa and China. Participants who were aged 18-75 years, GeneXpert MTB/RIF positive for tuberculosis and negative for rifampicin resistance, had not yet started tuberculosis treatment, had not been treated for active tuberculosis within the previous 3 years, and met basic safety laboratory criteria were included and participants with diabetes, HIV infection, or with extrapulmonary tuberculosis including pleural tuberculosis were excluded. Scans were assessed by two readers for the location of tuberculosis lesions (eg, cavities and consolidations), bronchial thickening patterns, and changes from baseline to week 4 of treatment.

Findings: Among the first 124 participants (enrolled from June 22, 2017, to Sept 27, 2018) who were successfully treated, 161 primarily apical cavitary lesions were identified at baseline. Bronchial thickening and inflammation linking non-cavitary consolidative lesions to cavities were observed in 121 (98%) of 124 participants' baseline PET-CT scans. After 4 weeks of treatment, 21 (17%) of 124 participants had new or expanding lesions linked to cavities via bronchial inflammation that were not present at baseline, particularly participants with two or more cavities at baseline and participants from South Africa.

Interpretation: In participants with pulmonary tuberculosis who were subsequently cured, the location of cavitary and non-cavitary lesions at baseline and new lesions at week 4 of treatment suggest a cavitary origin of disease and bronchial spread through the lungs. Bronchial spread from cavities might play a larger role in the spread of pulmonary tuberculosis than has been appreciated. Elucidating cavity lesion dynamics and viability within cavities might better explain treatment outcomes and why some patients are cured and others relapse.

Funding: Bill & Melinda Gates Foundation, European and Developing Countries Clinical Trials Partnership, China Ministry of Science and Technology, National Natural Science Foundation of China, and National Institutes of Health.

Translations: For the Chinese, Afrikaans and Xhosa translations of the abstract see Supplementary Materials section.
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http://dx.doi.org/10.1016/S2666-5247(21)00058-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478663PMC
October 2021

Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria.

Sci Rep 2021 Sep 20;11(1):18661. Epub 2021 Sep 20.

Institute of Infection and Global Health, University of Liverpool, Liverpool, L7 3EA, UK.

Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology-including the tendency to clump, and "differential" culturability-and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA + cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis bacillus Calmette-Guérin (BCG) time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.
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http://dx.doi.org/10.1038/s41598-021-98176-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452731PMC
September 2021

Same-day antiretroviral therapy initiation for people living with HIV who have tuberculosis symptoms: a systematic review.

HIV Med 2021 Sep 15. Epub 2021 Sep 15.

Clinical Research Department, Faculty of Infectious and Tropical Disease, London School of Hygiene and Tropical Medicine, London, UK.

Objectives: Tuberculosis symptoms are very common among people living with HIV (PLHIV) initiating antiretroviral therapy (ART), are not specific for tuberculosis disease and may result in delayed ART start. The risks and benefits of same-day ART initiation in PLHIV with tuberculosis symptoms are unknown.

Methods: We systematically reviewed nine databases on 12 March 2020 to identify studies that investigated same-day ART initiation among PLHIV with tuberculosis symptoms and reported both their approach to TB screening and clinical outcomes. We extracted and summarized data about TB screening, numbers of people starting same-day ART and outcomes.

Results: We included four studies. Two studies deferred ART for everyone with any tuberculosis symptoms (one or more of cough, fever, night sweats or weight loss) and substantial numbers of people had deferred ART start (28% and 39% did not start same-day ART). Two studies permitted some people with tuberculosis symptoms to start same-day ART, and fewer people deferred ART (2% and 16% did not start same-day). Two of the four studies were conducted sequentially; proven viral load suppression at 8 months was 31% when everyone with tuberculosis symptoms had ART deferred, and 44% when the algorithm was changed so that some people with tuberculosis symptoms could start same-day ART.

Conclusions: Although tuberculosis symptoms are very common in people starting ART, there is insufficient evidence about whether presence of tuberculosis symptoms should lead to ART start being deferred or not. Research to inform clear guidelines would help to maximise the benefits of same-day ART.
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http://dx.doi.org/10.1111/hiv.13169DOI Listing
September 2021

Th22 Cells Are a Major Contributor to the Mycobacterial CD4 T Cell Response and Are Depleted During HIV Infection.

J Immunol 2021 09 13;207(5):1239-1249. Epub 2021 Aug 13.

Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa;

HIV-1 infection substantially increases the risk of developing tuberculosis (TB). Mechanisms such as defects in the Th1 response to in HIV-infected persons have been widely reported. However, Th1-independent mechanisms also contribute to protection against TB. To identify a broader spectrum of defects in TB immunity during HIV infection, we examined IL-17A and IL-22 production in response to mycobacterial Ags in peripheral blood of persons with latent TB infection and HIV coinfection. Upon stimulating with mycobacterial Ags, we observed a distinct CD4 Th lineage producing IL-22 in the absence of IL-17A and IFN-γ. Mycobacteria-specific Th22 cells were present at high frequencies in blood and contributed up to 50% to the CD4 T cell response to mycobacteria, comparable in magnitude to the IFN-γ Th1 response (median 0.91% and 0.55%, respectively). Phenotypic characterization of Th22 cells revealed that their memory differentiation was similar to -specific Th1 cells (i.e., predominantly early differentiated CD45ROCD27 phenotype). Moreover, CCR6 and CXCR3 expression profiles of Th22 cells were similar to Th17 cells, whereas their CCR4 and CCR10 expression patterns displayed an intermediate phenotype between Th1 and Th17 cells. Strikingly, mycobacterial IL-22 responses were 3-fold lower in HIV-infected persons compared with uninfected persons, and the magnitude of responses correlated inversely with HIV viral load. These data provide important insights into mycobacteria-specific Th subsets in humans and suggest a potential role for IL-22 in protection against TB during HIV infection. Further studies are needed to fully elucidate the role of IL-22 in protective TB immunity.
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http://dx.doi.org/10.4049/jimmunol.1900984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387408PMC
September 2021

Inflammatory profile of patients with tuberculosis with or without HIV-1 co-infection: a prospective cohort study and immunological network analysis.

Lancet Microbe 2021 Aug 18;2(8):e375-e385. Epub 2021 May 18.

Wellcome Centre for Infectious Disease Research in Africa, Institute of Infectious Disease and Molecular Medicine (E Du Bruyn MD, N Rockwood PhD, C Schutz MD, G Meintjes PhD, C Riou PhD, K A Wilkinson PhD, B B Andrade MD, Prof R J Wilkinson FMedSci) and Department of Medicine (E Du Bruyn, C Schutz, G Meintjes, Prof R J Wilkinson), University of Cape Town, Observatory, South Africa; Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil (K F Fukutani PhD, M B Arriaga MSc, J M Cubillos-Angulo MSc, R Tibúrcio MSc, B B Andrade); Multinational Organization Network Sponsoring Translational and Epidemiological Research Initiative, Salvador, Brazil (K F Fukutani, M B Arriaga, J M Cubillos-Angulo, R Tibúrcio, B B Andrade); Curso de Medicina, Faculdade de Tecnologia e Ciências, Salvador, Brazil (K F Fukutani); Department of Infectious Diseases, Imperial College London, London, UK (N Rockwood, Prof R J Wilkinson); Department of Microbiology, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka (N Rockwood); Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA (A Sher PhD); The Francis Crick Institute, London, UK (K A Wilkinson, Prof R J Wilkinson); Universidade Salvador, Laureate Universities, Salvador, Brazil (B B Andrade); Escola Bahiana de Medicina e Saúde Pública, Salvador, Brazil (B B Andrade); Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA (B B Andrade).

Background: HIV-1 mediated dysregulation of the immune response to tuberculosis and its effect on the response to antitubercular therapy (ATT) is incompletely understood. We aimed to analyse the inflammatory profile of patients with tuberculosis with or without HIV-1 co-infection undergoing ATT, with specific focus on the effect of ART and HIV-1 viraemia in those co-infected with HIV-1.

Methods: In this prospective cohort study and immunological network analysis, a panel of 38 inflammatory markers were measured in the plasma of a prospective patient cohort undergoing ATT at Khayelitsha Site B clinic, Cape Town, South Africa. We recruited patients with sputum Xpert MTB/RIF-positive rifampicin-susceptible pulmonary tuberculosis. Patients were excluded from the primary discovery cohort if they were younger than 18 years, unable to commence ATT for any reason, pregnant, had unknown HIV-1 status, were unable to consent to study participation, were unable to provide baseline sputum samples, had more than three doses of ATT, or were being re-treated for tuberculosis within 6 months of their previous ATT regimen. Plasma samples were collected at baseline (1-5 days after commencing ATT), week 8, and week 20 of ATT. We applied network and multivariate analysis to investigate the dynamic inflammatory profile of these patients in relation to ATT and by HIV status. In addition to the discovery cohort, a validation cohort of patients with HIV-1 admitted to hospital with CD4 counts less than 350 cells per μL and a high clinical suspicion of new tuberculosis were recruited.

Findings: Between March 1, 2013, and July 31, 2014, we assessed a cohort of 129 participants (55 [43%] female and 74 [57%] male, median age 35·1 years [IQR 30·1-43·7]) and 76 were co-infected with HIV-1. HIV-1 status markedly influenced the inflammatory profile regardless of ATT duration. HIV-1 viral load emerged as a major factor driving differential inflammatory marker expression and having a strong effect on correlation profiles observed in the HIV-1 co-infected group. Interleukin (IL)-17A emerged as a key correlate of HIV-1-induced inflammation during HIV-tuberculosis co-infection.

Interpretation: Our findings show the effect of HIV-1 co-infection on the complexity of plasma inflammatory profiles in patients with tuberculosis. Through network analysis we identified IL-17A as an important node in HIV-tuberculosis co-infection, thus implicating this cytokine's capacity to correlate with, and regulate, other inflammatory markers. Further mechanistic studies are required to identify specific IL-17A-related inflammatory pathways mediating immunopathology in HIV-tuberculosis co-infection, which could illuminate targets for future host-directed therapies.

Funding: National Institutes of Health, The Wellcome Trust, UK Research and Innovation, Cancer Research UK, European and Developing Countries Clinical Trials Partnership, and South African Medical Research Council.
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http://dx.doi.org/10.1016/s2666-5247(21)00037-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357308PMC
August 2021

Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice.

J Exp Med 2021 Oct 4;218(10). Epub 2021 Aug 4.

Tuberculosis Center, Shanghai Emerging and Re-emerging Infectious Disease Institute, Fudan University, Shanghai, China.

Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. An influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, using complementary genetic models of eosinophil deficiency, we demonstrate that in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice.
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http://dx.doi.org/10.1084/jem.20210469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348215PMC
October 2021

What is the optimum time to start antiretroviral therapy in people with HIV and tuberculosis coinfection? A systematic review and meta-analysis.

J Int AIDS Soc 2021 07;24(7):e25772

Malawi-Liverpool-Wellcome Clinical Research Programme, Blantyre, Malawi.

Background: HIV and tuberculosis are frequently diagnosed concurrently. In March 2021, World Health Organization recommended that antiretroviral therapy (ART) should be started within two weeks of tuberculosis treatment start, at any CD4 count. We assessed whether earlier ART improved outcomes in people with newly diagnosed HIV and tuberculosis.

Methods: We did a systematic review by searching nine databases for trials that compared earlier ART to later ART initiation in people with HIV and tuberculosis. We included studies published from database inception to 12 March 2021. We compared ART within four weeks versus ART more than four weeks after TB treatment, and ART within two weeks versus ART between two and eight weeks, and stratified analysis by CD4 count. The main outcome was death; secondary outcomes included IRIS and AIDS-defining events. We pooled effect estimates using random effects meta-analysis.

Results And Discussion: We screened 2468 abstracts, and identified nine trials. Among people with all CD4 counts, there was no difference in mortality by earlier ART (≤4 week) versus later ART (>4 week) (risk difference [RD] 0%, 95% confidence interval [CI] -2% to +1%). Among people with CD4 count ≤50 cells/mm , earlier ART (≤4 weeks) reduced risk of death (RD -6%, -10% to -1%). Among people with all CD4 counts earlier ART (≤4 weeks) increased the risk of IRIS (RD +6%, 95% CI +2% to +10%) and reduced the incidence of AIDS-defining events (RD -2%, 95% CI -4% to 0%). Results were similar when trials were restricted to the four trials which permitted comparison of ART within two weeks to ART between two and eight weeks. Trials were conducted between 2004 and 2014, before recommendations to treat HIV at any CD4 count or to rapidly start ART in people without TB. No trials included children or pregnant women. No trials included integrase inhibitors in ART regimens.

Discussion: Earlier ART did not alter risk of death overall among people living with HIV who had TB disease. For logistical and patient preference reasons, earlier ART initiation for everyone with TB and HIV may be preferred to later ART.
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http://dx.doi.org/10.1002/jia2.25772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294654PMC
July 2021

Study protocol for a phase 2A trial of the safety and tolerability of increased dose rifampicin and adjunctive linezolid, with or without aspirin, for HIV-associated tuberculous meningitis [LASER-TBM].

Wellcome Open Res 2021 1;6:136. Epub 2021 Jun 1.

Department of Neuroradiology, Imaging Department, Royal London Hospital, Barts Health NHS Trust, London, E1 1BB, UK.

Tuberculous meningitis (TBM) is the most lethal form of tuberculosis with a mortality of ~50% in those co-infected with HIV-1. Current antibiotic regimens are based on those known to be effective in pulmonary TB and do not account for the differing ability of the drugs to penetrate the central nervous system (CNS). The host immune response drives pathology in TBM, yet effective host-directed therapies are scarce. There is sufficient data to suggest that higher doses of rifampicin (RIF), additional linezolid (LZD) and adjunctive aspirin (ASA) will be beneficial in TBM yet rigorous investigation of the safety of these interventions in the context of HIV associated TBM is required. We hypothesise that increased dose RIF, LZD and ASA used in combination and in addition to standard of care for the first 56 days of treatment with be safe and tolerated in HIV-1 infected people with TBM. In an open-label randomised parallel study, up to 100 participants will receive either; i) standard of care (n=40, control arm), ii) standard of care plus increased dose RIF (35mg/kg) and LZD (1200mg OD for 28 days, 600mg OD for 28 days) (n=30, experimental arm 1), or iii) as per experimental arm 1 plus additional ASA 1000mg OD (n=30, experimental arm 2). After 56 days participants will continue standard treatment as per national guidelines. The primary endpoint is death and the occurrence of solicited treatment-related adverse events at 56 days. In a planned pharmacokinetic (PK) sub-study we aim to assess PK/pharmacodynamic (PD) of oral vs IV rifampicin, describe LZD and RIF PK and cerebrospinal fluid concentrations, explore PK/PD relationships, and investigate drug-drug interactions between LZD and RIF. Safety and pharmacokinetic data from this study will inform a planned phase III study of intensified therapy in TBM. NCT03927313 (25/04/2019).
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http://dx.doi.org/10.12688/wellcomeopenres.16783.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8283551PMC
June 2021

Development of a fixed module repertoire for the analysis and interpretation of blood transcriptome data.

Nat Commun 2021 07 19;12(1):4385. Epub 2021 Jul 19.

Baylor Institute for Immunology Research, Baylor Research Institute, Dallas, TX, USA.

As the capacity for generating large-scale molecular profiling data continues to grow, the ability to extract meaningful biological knowledge from it remains a limitation. Here, we describe the development of a new fixed repertoire of transcriptional modules, BloodGen3, that is designed to serve as a stable reusable framework for the analysis and interpretation of blood transcriptome data. The construction of this repertoire is based on co-clustering patterns observed across sixteen immunological and physiological states encompassing 985 blood transcriptome profiles. Interpretation is supported by customized resources, including module-level analysis workflows, fingerprint grid plot visualizations, interactive web applications and an extensive annotation framework comprising functional profiling reports and reference transcriptional profiles. Taken together, this well-characterized and well-supported transcriptional module repertoire can be employed for the interpretation and benchmarking of blood transcriptome profiles within and across patient cohorts. Blood transcriptome fingerprints for the 16 reference cohorts can be accessed interactively via:  https://drinchai.shinyapps.io/BloodGen3Module/ .
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http://dx.doi.org/10.1038/s41467-021-24584-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8289976PMC
July 2021

Dysregulation of the Immune Environment in the Airways During HIV Infection.

Front Immunol 2021 30;12:707355. Epub 2021 Jun 30.

Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.

HIV-1 increases susceptibility to pulmonary infection and disease, suggesting pathogenesis in the lung. However, the lung immune environment during HIV infection remains poorly characterized. This study examined T cell activation and the cytokine milieu in paired bronchoalveolar lavage (BAL) and blood from 36 HIV-uninfected and 32 HIV-infected participants. Concentrations of 27 cytokines were measured by Luminex, and T cells were phenotyped by flow cytometry. Blood and BAL had distinct cytokine profiles (p=0.001). In plasma, concentrations of inflammatory cytokines like IFN-γ (p=0.004) and TNF-α (p=0.004) were elevated during HIV infection, as expected. Conversely, BAL cytokine concentrations were similar in HIV-infected and uninfected individuals, despite high BAL viral loads (VL; median 48,000 copies/ml epithelial lining fluid). HIV-infected individuals had greater numbers of T cells in BAL compared to uninfected individuals (p=0.007); and BAL VL positively associated with CD4+ and CD8+ T cell numbers (p=0.006 and p=0.0002, respectively) and CXCL10 concentrations (p=0.02). BAL T cells were highly activated in HIV-infected individuals, with nearly 2-3 fold greater frequencies of CD4+CD38+ (1.8-fold; p=0.007), CD4+CD38+HLA-DR+ (1.9-fold; p=0.0006), CD8+CD38+ (2.8-fold; p=0.0006), CD8+HLA-DR+ (2-fold; p=0.022) and CD8+CD38+HLA-DR+ (3.6-fold; p<0.0001) cells compared to HIV-uninfected individuals. Overall, this study demonstrates a clear disruption of the pulmonary immune environment during HIV infection, with readily detectable virus and activated T lymphocytes, which may be driven to accumulate by local chemokines.
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http://dx.doi.org/10.3389/fimmu.2021.707355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8278481PMC
June 2021

Strategies for the diagnosis and management of meningitis in HIV-infected adults in resource limited settings.

Expert Opin Pharmacother 2021 Oct 21;22(15):2053-2070. Epub 2021 Jun 21.

Wellcome Centre for Infectious Disease Research in Africa, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Observatory.

Introduction: The incidence of human immunodeficiency virus-1 (HIV-1) associated meningitis has been declining in the post-combination antiretroviral treatment (ART) era, although survival rates remain low for the common causes like tuberculosis and cryptococcal disease. Diagnosis and treatment of meningitis in HIV-1 is complicated by atypical clinical presentations, limited accuracy of diagnostic tests, access to diagnostic tests, and therapeutic agents in low- and middle-income countries (LMIC) and immune reconstitution inflammatory syndrome (IRIS).

Areas Covered: We provide an overview of the common etiologies of meningitis in HIV-1-infected adults, suggest a diagnostic approach based on readily available tests, and review specific chemotherapeutic agents, host-directed therapies, supportive care, timing of ART initiation, and considerations in the management of IRIS with a focus on resource-limited settings. They identify key knowledge gaps and suggest areas for future research.

Expert Opinion: Evidence-based management of HIV-1-associated meningitis is sparse for common etiologies. More readily available and sensitive diagnostic tests as well as standardized investigation strategies are required in LMIC. There is a lack of availability of recommended drugs in areas of high HIV-1 prevalence and a limited pipeline of novel chemotherapeutic agents. Host-directed therapies have been inadequately studied.
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http://dx.doi.org/10.1080/14656566.2021.1940954DOI Listing
October 2021

Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2 specific T cells.

Eur Respir J 2021 Jun 17. Epub 2021 Jun 17.

Wellcome Centre for Infectious Disease Research in Africa and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, South Africa.

Rapid tests to evaluate SARS-CoV-2-specific T cell responses are urgently needed to decipher protective immunity and aid monitoring vaccine-induced immunity. Using a rapid whole blood assay requiring minimal amount of blood, we measured qualitatively and quantitatively SARS-CoV-2-specific CD4T cell responses in 31 healthcare workers, using flow cytometry. 100% of COVID-19 convalescent participants displayed a detectable SARS-CoV-2-specific CD4T cell response. SARS-CoV-2-responding cells were also detected in 40.9% of participants with no COVID-19-associated symptoms or who tested PCR negative. Phenotypic assessment indicated that, in COVID-19 convalescent participants, SARS-CoV-2 CD4 responses displayed an early differentiated memory phenotype with limited capacity to produce IFNɣ. Conversely, in participants with no reported symptoms, SARS-CoV-2 CD4 responses were enriched in late differentiated cells, co-expressing IFNɣ and TNFα and also Granzyme B. This proof-of-concept study presents a scalable alternative to PBMC-based assays to enumerate and phenotype SARS-CoV-2-responding T cells, thus representing a practical tool to monitor adaptive immunity due to natural infection or vaccine trials.
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http://dx.doi.org/10.1183/13993003.00285-2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215505PMC
June 2021

Cytokine release syndrome in a patient with colorectal cancer after vaccination with BNT162b2.

Nat Med 2021 08 26;27(8):1362-1366. Epub 2021 May 26.

Gastrointestinal and Lymphoma Unit, The Royal Marsden NHS Foundation Trust, Sutton, UK.

Patients with cancer are currently prioritized in coronavirus disease 2019 (COVID-19) vaccination programs globally, which includes administration of mRNA vaccines. Cytokine release syndrome (CRS) has not been reported with mRNA vaccines and is an extremely rare immune-related adverse event of immune checkpoint inhibitors. We present a case of CRS that occurred 5 d after vaccination with BTN162b2 (tozinameran)-the Pfizer-BioNTech mRNA COVID-19 vaccine-in a patient with colorectal cancer on long-standing anti-PD-1 monotherapy. The CRS was evidenced by raised inflammatory markers, thrombocytopenia, elevated cytokine levels (IFN-γ/IL-2R/IL-18/IL-16/IL-10) and steroid responsiveness. The close temporal association of vaccination and diagnosis of CRS in this case suggests that CRS was a vaccine-related adverse event; with anti-PD1 blockade as a potential contributor. Overall, further prospective pharmacovigillence data are needed in patients with cancer, but the benefit-risk profile remains strongly in favor of COVID-19 vaccination in this population.
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http://dx.doi.org/10.1038/s41591-021-01387-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363501PMC
August 2021

Aspirin in tuberculous meningitis.

EClinicalMedicine 2021 May 7;35:100871. Epub 2021 May 7.

The Francis Crick Institute, Midland Road, London NW1 1AT, United Kingdom.

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http://dx.doi.org/10.1016/j.eclinm.2021.100871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129888PMC
May 2021

Plasma Pharmacokinetics of High-Dose Oral versus Intravenous Rifampicin in Patients with Tuberculous Meningitis: a Randomized Controlled Trial.

Antimicrob Agents Chemother 2021 07 16;65(8):e0014021. Epub 2021 Jul 16.

Wellcome Centre for Infectious Diseases Research in Africa, Institute for Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.

Higher doses of intravenous rifampicin may improve outcomes in tuberculous meningitis but are impractical in high-burden settings. We hypothesized that plasma rifampicin exposures would be similar between oral dosing of 35 mg/kg of body weight and intravenous dosing of 20 mg/kg, which has been proposed for efficacy trials in tuberculous meningitis. We performed a randomized parallel-group pharmacokinetic study nested within a clinical trial of intensified antimicrobial therapy for tuberculous meningitis. HIV-positive participants with tuberculous meningitis were recruited from South African hospitals and randomized to one of three rifampicin dosing groups: standard (oral 10 mg/kg), high dose (oral 35 mg/kg), and intravenous (20 mg/kg). Intensive pharmacokinetic sampling was done on day 3. Data were described using noncompartmental analysis, and exposures were compared by geometric mean ratios (GMRs). Forty-six participants underwent pharmacokinetic sampling (standard dose,  = 17; high-dose oral, = 15; intravenous,  = 14). The median CD4 count was 130 cells/mm (interquartile range [IQR], 66 to 253 cells/mm). The rifampicin geometric mean area under the concentration-time curve from 0 to 24 h (AUC) values were 42.9 μg · h/ml (95% confidence interval [CI], 24.5 to 75.0 μg · h/ml) for the standard dose, 295.2 μg · h/ml (95% CI, 189.9 to 458.8 μg · h/ml) for the high oral dose, and 206.5 μg · h/ml (95% CI, 154.6 to 275.8 μg · h/ml) for intravenous administration. The rifampicin AUC GMR was 1.44 (90% CI, 0.84 to 2.21) and the maximal concentration of drug in serum () GMR was 0.89 (90% CI, 0.63 to 1.23) for high-dose oral administration with respect to intravenous dosing. The plasma rifampicin AUC was higher after an oral 35-mg/kg dose than with intravenous administration at a 20-mg/kg dose over the first few days of tuberculosis (TB) treatment. The findings support oral rifampicin dosing in future tuberculous meningitis trials.
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http://dx.doi.org/10.1128/AAC.00140-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611291PMC
July 2021

Relationship of SARS-CoV-2-specific CD4 response to COVID-19 severity and impact of HIV-1 and tuberculosis coinfection.

J Clin Invest 2021 06;131(12)

Wellcome Centre for Infectious Disease Research in Africa and Institute of Infectious Disease and Molecular Medicine.

T cells are involved in control of coronavirus disease 2019 (COVID-19), but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we used flow cytometry to assess the magnitude, function, and phenotype of SARS coronavirus 2-specific (SARS-CoV-2-specific) CD4+ T cells in 95 hospitalized COVID-19 patients, 38 of them being HIV-1 and/or tuberculosis (TB) coinfected, and 38 non-COVID-19 patients. We showed that SARS-CoV-2-specific CD4+ T cell attributes, rather than magnitude, were associated with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity, and enhanced HLA-DR expression. Moreover, HIV-1 and TB coinfection skewed the SARS-CoV-2 T cell response. HIV-1-mediated CD4+ T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2, and a decrease in the polyfunctional capacity of SARS-CoV-2-specific CD4+ T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mycobacterium tuberculosis-specific CD4+ T cells, with possible implications for TB disease progression. These results corroborate the important role of SARS-CoV-2-specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.
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http://dx.doi.org/10.1172/JCI149125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203446PMC
June 2021

TBDBT: A TB DataBase Template for collection of harmonized TB clinical research data in REDCap, facilitating data standardisation for inter-study comparison and meta-analyses.

PLoS One 2021 26;16(3):e0249165. Epub 2021 Mar 26.

Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.

Clinical tuberculosis research, both within research groups and across research ecosystems, is often undertaken in isolation using bespoke data collection platforms and applying differing data conventions. This failure to harmonise clinical phenotype data or apply standardised data collection and storage standards in turn limits the opportunity to undertake meta-analyses using data generated across multiple research projects for the same research domain. We have developed the Tuberculosis DataBase Template (TBDBT), a template for the well-supported, free and commonly deployed clinical databasing platform, REDCap. This template can be used to set up a new tuberculosis research database with a built-in set of standardised data conventions, to ensure standardised data capture across research projects and programs. A modular design enables researchers to implement only the modules of the database template that are appropriate for their particular study. The template includes core modules for informed consent data, participant demographics, clinical symptoms and presentation, diagnostic imaging and laboratory tests. Optional modules have been designed for visit scheduling and calendar functionality, clinical trial randomisation, study logistics and operations, and pharmacokinetic data. Additional fields can be added as needed. This REDCap template can facilitate collection of high-quality data for tuberculosis research, providing a tool to ensure better data harmonisation, analysis and meta-analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249165PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996972PMC
October 2021

Identification of Reduced Host Transcriptomic Signatures for Tuberculosis Disease and Digital PCR-Based Validation and Quantification.

Front Immunol 2021 2;12:637164. Epub 2021 Mar 2.

Section of Paediatrics, Department of Infectious Disease, Faculty of Medicine, Imperial College London, London, United Kingdom.

Recently, host whole blood gene expression signatures have been identified for diagnosis of tuberculosis (TB). Absolute quantification of the concentrations of signature transcripts in blood have not been reported, but would facilitate diagnostic test development. To identify minimal transcript signatures, we applied a transcript selection procedure to microarray data from African adults comprising 536 patients with TB, other diseases (OD) and latent TB (LTBI), divided into training and test sets. Signatures were further investigated using reverse transcriptase (RT)-digital PCR (dPCR). A four-transcript signature (, and ) measured using RT-dPCR distinguished TB patients from those with OD (area under the curve (AUC) 93.8% (CI 82.2-100%). A three-transcript signature () differentiated TB from LTBI (AUC 97.3%, CI: 93.3-100%), regardless of HIV. These signatures have been validated across platforms and across samples offering strong, quantitative support for their use as diagnostic biomarkers for TB.
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http://dx.doi.org/10.3389/fimmu.2021.637164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7982854PMC
September 2021

Antiretroviral Treatment-Induced Decrease in Immune Activation Contributes to Reduced Susceptibility to Tuberculosis in HIV-1/Mtb Co-infected Persons.

Front Immunol 2021 5;12:645446. Epub 2021 Mar 5.

Tuberculosis Laboratory, The Francis Crick Institute, London, United Kingdom.

Antiretroviral treatment (ART) reduces the risk of developing active tuberculosis (TB) in HIV-1 co-infected persons. In order to understand host immune responses during ART in the context of (Mtb) sensitization, we performed RNAseq analysis of whole blood-derived RNA from individuals with latent TB infection coinfected with HIV-1, during the first 6 months of ART. A significant fall in RNA sequence abundance of the Hallmark IFN-alpha, IFN-gamma, IL-6/JAK/STAT3 signaling, and inflammatory response pathway genes indicated reduced immune activation and inflammation at 6 months of ART compared to day 0. Further exploratory evaluation of 65 soluble analytes in plasma confirmed the significant decrease of inflammatory markers after 6 months of ART. Next, we evaluated 30 soluble analytes in QuantiFERON Gold in-tube (QFT) samples from the Ag stimulated and Nil tubes, during the first 6 months of ART in 30 patients. There was a significant decrease in IL-1alpha and IL-1beta (Ag-Nil) concentrations as well as MCP-1 (Nil), supporting decreased immune activation and inflammation. At the same time, IP-10 (Ag-nil) concentrations significantly increased, together with chemokine receptor-expressing CD4 T cell numbers. Our data indicate that ART-induced decrease in immune activation combined with improved antigen responsiveness may contribute to reduced susceptibility to tuberculosis in HIV-1/Mtb co-infected persons.
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http://dx.doi.org/10.3389/fimmu.2021.645446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973093PMC
September 2021

Evaluation of Host Serum Protein Biomarkers of Tuberculosis in sub-Saharan Africa.

Front Immunol 2021 25;12:639174. Epub 2021 Feb 25.

Department of Infectious Disease, Faculty of Medicine, Imperial College London, London, United Kingdom.

Accurate and affordable point-of-care diagnostics for tuberculosis (TB) are needed. Host serum protein signatures have been derived for use in primary care settings, however validation of these in secondary care settings is lacking. We evaluated serum protein biomarkers discovered in primary care cohorts from Africa reapplied to patients from secondary care. In this nested case-control study, concentrations of 22 proteins were quantified in sera from 292 patients from Malawi and South Africa who presented predominantly to secondary care. Recruitment was based upon intention of local clinicians to test for TB. The case definition for TB was culture positivity for ; and for other diseases (OD) a confirmed alternative diagnosis. Equal numbers of TB and OD patients were selected. Within each group, there were equal numbers with and without HIV and from each site. Patients were split into training and test sets for biosignature discovery. A nine-protein signature to distinguish TB from OD was discovered comprising fibrinogen, alpha-2-macroglobulin, CRP, MMP-9, transthyretin, complement factor H, IFN-gamma, IP-10, and TNF-alpha. This signature had an area under the receiver operating characteristic curve in the training set of 90% (95% CI 86-95%), and, after adjusting the cut-off for increased sensitivity, a sensitivity and specificity in the test set of 92% (95% CI 80-98%) and 71% (95% CI 56-84%), respectively. The best single biomarker was complement factor H [area under the receiver operating characteristic curve 70% (95% CI 64-76%)]. Biosignatures consisting of host serum proteins may function as point-of-care screening tests for TB in African hospitals. Complement factor H is identified as a new biomarker for such signatures.
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http://dx.doi.org/10.3389/fimmu.2021.639174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947659PMC
February 2021

Generation of Liposomes to Study the Effect of Lipids on HIV-1 - and -Infections.

Int J Mol Sci 2021 Feb 16;22(4). Epub 2021 Feb 16.

Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L69 7BE, UK.

Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and () co-infection is an early precipitate to AIDS. We aimed to determine whether strains differentially modulate cellular susceptibility to HIV-1 infection (- and -infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 H37Rv, CDC1551 and lineage 2 HN878, EU127) and non-pathogenic ( BCG and ) strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 - and -infection of TZM-bl cells using single-cycle infectious virus particles. glycolipids did not affect HIV-1 direct infection however, -infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from , H37Rv and EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 -infection. These findings indicate that variant strains of have differential effect on HIV-1 -infection with the potential to influence HIV-1 disease course in co-infected individuals.
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http://dx.doi.org/10.3390/ijms22041945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920488PMC
February 2021

Visualizing the dynamics of tuberculosis pathology using molecular imaging.

J Clin Invest 2021 03;131(5)

Center for Infection and Inflammation Imaging Research.

Nearly 140 years after Robert Koch discovered Mycobacterium tuberculosis, tuberculosis (TB) remains a global threat and a deadly human pathogen. M. tuberculosis is notable for complex host-pathogen interactions that lead to poorly understood disease states ranging from latent infection to active disease. Additionally, multiple pathologies with a distinct local milieu (bacterial burden, antibiotic exposure, and host response) can coexist simultaneously within the same subject and change independently over time. Current tools cannot optimally measure these distinct pathologies or the spatiotemporal changes. Next-generation molecular imaging affords unparalleled opportunities to visualize infection by providing holistic, 3D spatial characterization and noninvasive, temporal monitoring within the same subject. This rapidly evolving technology could powerfully augment TB research by advancing fundamental knowledge and accelerating the development of novel diagnostics, biomarkers, and therapeutics.
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http://dx.doi.org/10.1172/JCI145107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919721PMC
March 2021

T cell-tropic HIV efficiently infects alveolar macrophages through contact with infected CD4+ T cells.

Sci Rep 2021 Feb 16;11(1):3890. Epub 2021 Feb 16.

Ragon Institute of MGH, MIT, and Harvard, Massachusetts General Hospital, Harvard Medical School, Cambridge, MA, USA.

Alveolar macrophages (AMs) are critical for defense against airborne pathogens and AM dysfunction is thought to contribute to the increased burden of pulmonary infections observed in individuals living with HIV-1 (HIV). While HIV nucleic acids have been detected in AMs early in infection, circulating HIV during acute and chronic infection is usually CCR5 T cell-tropic (T-tropic) and enters macrophages inefficiently in vitro. The mechanism by which T-tropic viruses infect AMs remains unknown. We collected AMs by bronchoscopy performed in HIV-infected, antiretroviral therapy (ART)-naive and uninfected subjects. We found that viral constructs made with primary HIV envelope sequences isolated from both AMs and plasma were T-tropic and inefficiently infected macrophages. However, these isolates productively infected macrophages when co-cultured with HIV-infected CD4+ T cells. In addition, we provide evidence that T-tropic HIV is transmitted from infected CD4+ T cells to the AM cytosol. We conclude that AM-derived HIV isolates are T-tropic and can enter macrophages through contact with an infected CD4+ T cell, which results in productive infection of AMs. CD4+ T cell-dependent entry of HIV into AMs helps explain the presence of HIV in AMs despite inefficient cell-free infection, and may contribute to AM dysfunction in people living with HIV.
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http://dx.doi.org/10.1038/s41598-021-82066-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886866PMC
February 2021

Recent Developments in Tuberculous Meningitis Pathogenesis and Diagnostics.

Wellcome Open Res 2019 28;4:164. Epub 2021 Jan 28.

Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam.

The pathogenesis of Tuberculous meningitis (TBM) is poorly understood, but contemporary molecular biology technologies have allowed for recent improvements in our understanding of TBM. For instance, neutrophils appear to play a significant role in the immunopathogenesis of TBM, and either a paucity or an excess of inflammation can be detrimental in TBM. Further, severity of HIV-associated immunosuppression is an important determinant of inflammatory response; patients with the advanced immunosuppression (CD4+ T-cell count of <150 cells/μL) having higher CSF neutrophils, greater CSF cytokine concentrations and higher mortality than those with CD4+ T-cell counts > 150 cells/μL. Host genetics may also influence outcomes with LT4AH genotype predicting inflammatory phenotype, steroid responsiveness and survival in Vietnamese adults with TBM. Whist in Indonesia, CSF tryptophan level was a predictor of survival, suggesting tryptophan metabolism may be important in TBM pathogenesis. These varying responses mean that we must consider whether a "one-size-fits-all" approach to anti-bacillary or immunomodulatory treatment in TBM is truly the best way forward. Of course, to allow for proper treatment, early and rapid diagnosis of TBM must occur. Diagnosis has always been a challenge but the field of TB diagnosis is evolving, with sensitivities of at least 70% now possible in less than two hours with GeneXpert MTB/Rif Ultra. In addition, advanced molecular techniques such as CRISPR-MTB and metagenomic next generation sequencing may hold promise for TBM diagnosis. Host-based biomarkers and signatures are being further evaluated in childhood and adult TBM as adjunctive biomarkers as even with improved molecular assays, cases are still missed. A better grasp of host and pathogen behaviour may lead to improved diagnostics, targeted immunotherapy, and possibly biomarker-based, patient-specific treatment regimens.
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http://dx.doi.org/10.12688/wellcomeopenres.15506.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7739117.3PMC
January 2021

Human whole genome sequencing in South Africa.

Sci Rep 2021 01 12;11(1):606. Epub 2021 Jan 12.

Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, SAMRC Centre for Tuberculosis Research, Stellenbosch University, Cape Town, South Africa.

The advent and evolution of next generation sequencing has considerably impacted genomic research. Until recently, South African researchers were unable to access affordable platforms capable of human whole genome sequencing locally and DNA samples had to be exported. Here we report the whole genome sequences of the first six human DNA samples sequenced and analysed at the South African Medical Research Council's Genomics Centre. We demonstrate that the data obtained is of high quality, with an average sequencing depth of 36.41, and that the output is comparable to data generated internationally on a similar platform. The Genomics Centre creates an environment where African researchers are able to access world class facilities, increasing local capacity to sequence whole genomes as well as store and analyse the data.
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http://dx.doi.org/10.1038/s41598-020-79794-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803990PMC
January 2021

Diagnostic Accuracy of the INSHI Consensus Case Definition for the Diagnosis of Paradoxical Tuberculosis-IRIS.

J Acquir Immune Defic Syndr 2021 04;86(5):587-592

Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Background: The diagnosis of paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) relies on characteristic clinical features synthesized as the International Network for the Study of HIV-associated IRIS (INSHI) case definition. There is no confirmatory laboratory test.

Setting: Site B HIV-TB clinic in Khayelitsha, Cape Town, South Africa.

Methods: Using data of participants with HIV-associated tuberculosis starting antiretroviral treatment from a prospective trial evaluating prednisone for TB-IRIS prevention, we applied latent class analysis to model a gold standard for TB-IRIS. The model-predicted probability of TB-IRIS for each participant was used to assess the performance of the INSHI case definition and compare its diagnostic accuracy with several adapted case definitions.

Results: Data for this analysis were complete for 217 participants; 41% developed TB-IRIS. Our latent class model included the following parameters: respiratory symptoms; night sweats; INSHI major criteria 1, 2, and 4; maximum C-reactive protein >90 mg/L; maximum heart rate >120/min; maximum temperature >37.7°C; and preantiretroviral therapy CD4 count <50 cells/µL. The model estimated a TB-IRIS incidence of 43% and had optimal goodness of fit (χ2 = 337, P = 1.0). The INSHI case definition displayed a sensitivity of 0.77 and a specificity of 0.86. Replacing all the minor INSHI criteria with objectives measures (C-reactive protein elevation, fever, and/or tachycardia) resulted in a definition with better diagnostic accuracy, with a sensitivity of 0.89 and a specificity of 0.88.

Conclusion: The INSHI case definition identifies TB-IRIS with reasonable accuracy. Amending the case definition by replacing INSHI minor criteria with objective variables improved sensitivity without loss of specificity.
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http://dx.doi.org/10.1097/QAI.0000000000002606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938911PMC
April 2021

Isoniazid preventive therapy plus antiretroviral therapy for the prevention of tuberculosis: a systematic review and meta-analysis of individual participant data.

Lancet HIV 2021 01;8(1):e8-e15

Department of Global Health, University of Washington, Seattle, WA, USA; Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, USA.

Background: Isoniazid preventive therapy prevents active tuberculosis in people with HIV, but previous studies have found no evidence of benefit in people with HIV who had a negative tuberculin skin test, and a non-significant effect on mortality. We aimed to estimate the effect of isoniazid preventive therapy given with antiretroviral therapy (ART) for the prevention of tuberculosis and death among people with HIV across population subgroups.

Methods: We searched PubMed, Embase, the Cochrane database, and conference abstracts from database inception to Jan 15, 2019, to identify potentially eligible randomised trials. Eligible studies were trials that enrolled HIV-positive adults (age ≥15 years) taking ART who were randomly assigned to either daily isoniazid preventive therapy plus ART or ART alone and followed up longitudinally for outcomes of incident tuberculosis and mortality. We approached all authors of included trials and requested individual participant data: coprimary outcomes were relative risk of incident tuberculosis and all-cause mortality. We did a single-stage meta-analysis of individual participant data using stratified Cox-proportional hazards models. We did prespecified subgroup analyses by sex, CD4 cell count, and evidence of immune sensitisation to tuberculosis (indicated by tuberculin skin test or interferon-γ release assays [IGRAs]). We also assessed the relative risk of liver injury in an additional prespecified analysis. This study is registered with PROSPERO, CRD42019121400.

Findings: Of 838 records, we included three trials with data for 2611 participants and 8584·8 person-years of follow-up for the outcome of incident tuberculosis, and a subset of 2362 participants with 8631·6 person-years of follow-up for the coprimary outcome of all-cause mortality. Risk for tuberculosis was lower in participants given isoniazid preventive therapy and ART than participants given ART alone (hazard ratio [HR] 0·68, 95% CI 0·49-0·95, p=0·02). Risk of all-cause mortality was lower in participants given isoniazid preventive therapy and ART than participants given ART alone, but this difference was non-significant (HR 0·69, 95% CI 0·43-1·10, p=0·12). Participants with baseline CD4 counts of less than 500 cells per μL had increased risk of tuberculosis, but there was no significant difference in the benefit of isoniazid preventive therapy with ART by sex, baseline CD4 count, or results of tuberculin skin test or IGRAs. 65 (2·5%) of 2611 participants had raised alanine aminotransferase, but data were insufficient to calculate an HR.

Interpretation: Isoniazid preventive therapy with ART prevents tuberculosis across demographic and HIV-specific and tuberculosis-specific subgroups, which supports efforts to further increase use of isoniazid preventive therapy with ART broadly among people living with HIV.

Funding: National Institutes of Health and National Institute of Allergy and Infectious Diseases.
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http://dx.doi.org/10.1016/S2352-3018(20)30299-XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875572PMC
January 2021

Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2 specific T cells.

medRxiv 2020 Nov 3. Epub 2020 Nov 3.

Rapid tests to evaluate SARS-CoV-2-specific T cell responses are urgently needed to decipher protective immunity and aid monitoring vaccine-induced immunity. Using a rapid whole blood assay requiring minimal amount of blood, we measured qualitatively and quantitatively SARS-CoV-2-specific CD4 T cell responses in 31 healthcare workers, using flow cytometry. 100% of COVID-19 convalescent participants displayed a detectable SARS-CoV-2-specific CD4 T cell response. SARS-CoV-2-responding cells were also detected in 40.9% of participants with no COVID-19-associated symptoms or who tested PCR negative. Phenotypic assessment indicated that, in COVID-19 convalescent participants, SARS-CoV-2 CD4 responses displayed an early differentiated memory phenotype with limited capacity to produce IFNγ. Conversely, in participants with no reported symptoms, SARS-CoV-2 CD4 responses were enriched in late differentiated cells, co-expressing IFNγ and TNFα and also Granzyme B. This proof of concept study presents a scalable alternative to PBMC-based assays to enumerate and phenotype SARS-CoV-2-responding T cells, thus representing a practical tool to monitor adaptive immunity in vaccine trials.

Summary: In this proof of concept study, we show that SARS-CoV-2 T cell responses are easily detectable using a rapid whole blood assay requiring minimal blood volume. Such assay could represent a suitable tool to monitor adaptive immunity in vaccine trials.
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http://dx.doi.org/10.1101/2020.10.30.20223099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654914PMC
November 2020

An observational study identifying highly tuberculosis-exposed, HIV-1-positive but persistently TB, tuberculin and IGRA negative persons with M. tuberculosis specific antibodies in Cape Town, South Africa.

EBioMedicine 2020 Nov 7;61:103053. Epub 2020 Oct 7.

DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa. Electronic address:

Background: Mycobacterium tuberculosis (Mtb) infection is inferred from positive results of T-cell immune conversion assays measuring Mtb-specific interferon gamma production or tuberculin skin test (TST) reactivity. Certain exposed individuals do not display T-cell immune conversion in these assays and do not develop TB. Here we report a hitherto unknown form of this phenotype: HIV-1-positive persistently TB, tuberculin and IGRA negative (HITTIN).

Methods: A community-based case-control design was used to systematically screen and identify adults living with HIV (HIV+), aged 35-60 years, who met stringent study criteria, and then longitudinally followed up for repeat IGRA and TST testing. Participants had no history of TB despite living in TB hyper-endemic environments in Cape Town, South Africa with a provincial incidence of 681/100,000. Mtb-specific antibodies were measured using ELISA and Luminex.

Findings: We identified 48/286 (17%) individuals who tested persistently negative for Mtb-specific T-cell immunoreactivity (three negative Quantiferon results and one TST = 0mm) over 206±154 days on average. Of these, 97·2% had documented CD4 counts<200 prior to antiretroviral therapy (ART). They had received ART for 7·0±3·0 years with a latest CD4 count of 505·8±191·4 cells/mm. All HITTIN sent for further antibody testing (n=38) displayed  Mtb-specific antibody titres.

Interpretation: Immune reconstituted HIV+ persons can be persistently non-immunoreactive to TST and interferon-γ T-cell responses to Mtb, yet develop species-specific antibody responses. Exposure is evidenced by Mtb-specific antibody titres. Our identification of HIV+ individuals displaying a persisting lack of response to TST and IGRA T-cell immune conversion paves the way for future studies to investigate this phenotype in the context of HIV-infection that so far have received only scant attention.
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http://dx.doi.org/10.1016/j.ebiom.2020.103053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648124PMC
November 2020

Disease extent and anti-tubercular treatment response correlates with -specific CD4 T-cell phenotype regardless of HIV-1 status.

Clin Transl Immunology 2020 28;9(9):e1176. Epub 2020 Sep 28.

Wellcome Centre for Infectious Disease Research in Africa Institute of Infectious Disease and Molecular Medicine University of Cape Town Observatory South Africa.

Objectives: The development of non-sputum-based assays for tuberculosis (TB) diagnosis and treatment monitoring is a key priority. Recent data indicate that whole blood-based assays to assess the phenotype of (Mtb)-specific CD4 T cells hold promise for this purpose and require further investigation in well-characterised TB cohorts. In this study, we investigated the relationship between the phenotypic signature of Mtb-specific CD4 responses, TB disease extent and treatment response.

Methods: Using flow cytometry, we measured the expression of phenotypic and functional markers (HLA-DR, CD27, CD153, KLRG1, IL-2, MIP-1β, TNF-α and IFN-γ) on Mtb-specific CD4 T-cells in whole blood from 161 participants of varying TB and HIV status. TB disease extent was graded as a continuum using the Xpert value, C-reactive protein, Timika radiographic score and monocyte/lymphocyte ratio.

Results: The phenotypic profile of Mtb-specific CD4 T cells pre-anti-tubercular treatment (ATT) strongly correlated with disease extent, irrespective of HIV status. ATT associated with major changes in the phenotype of Mtb-specific CD4 T cells, with decreased expression of HLA-DR and increased CD27 and CD153 expression. Principal component analysis showed an almost complete separation between latent TB infection (LTBI) and active TB (aTB) pre-ATT groups, whereas the profile of the aTB post-ATT group overlapped with the LTBI group. However, in patients experiencing treatment failure or relapse, no significant changes were observed in Mtb-specific CD4 T-cell phenotype pre- and post-ATT.

Conclusion: Whole blood-based assays of Mtb-specific CD4 T-cell activation and maturation markers can be used as non-sputum-based biomarkers of disease extent and treatment monitoring in TB, regardless of HIV-1 status.
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http://dx.doi.org/10.1002/cti2.1176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520805PMC
September 2020
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