FcγR-mediated SARS-CoV-2 infection of monocytes activates inflammation.

SARS-CoV-2 can cause acute respiratory distress and death in some patients1. Although severe COVID-19 is linked to substantial inflammation, how SARS-CoV-2 triggers inflammation is not clear2. Monocytes and macrophages are sentinel cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D, leading to inflammatory death (pyroptosis) and the release of potent inflammatory mediators3. Here we show that about 6% of blood monocytes of patients with COVID-19 are infected with SARS-CoV-2. Monocyte infection depends on the uptake of antibody-opsonized virus by Fcγ receptors. The plasma of vaccine recipients does not promote antibody-dependent monocyte infection. SARS-CoV-2 begins to replicate in monocytes, but infection is aborted, and infectious virus is not detected in the supernatants of cultures of infected monocytes. Instead, infected cells undergo pyroptosis mediated by activation of NLRP3 and AIM2 inflammasomes, caspase-1 and gasdermin D. Moreover, tissue-resident macrophages, but not infected epithelial and endothelial cells, from lung autopsies from patients with COVID-19 have activated inflammasomes. Taken together, these findings suggest that antibody-mediated SARS-CoV-2 uptake by monocytes and macrophages triggers inflammatory cell death that aborts the production of infectious virus but causes systemic inflammation that contributes to COVID-19 pathogenesis.

The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters.

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release.

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in a minority of patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes and macrophages are sentinel immune cells in the blood and tissue, respectively, that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). We find that about 10% of blood monocytes in COVID-19 patients are infected with SARS-CoV-2. Monocyte infection depends on viral antibody opsonization and uptake of opsonized virus by the Fc receptor CD16. After uptake, SARS-CoV-2 begins to replicate in monocytes, as evidenced by detection of double-stranded RNA and subgenomic RNA and expression of a fluorescent reporter gene. However, infection is aborted, and infectious virus is not detected in infected monocyte supernatants or patient plasma. Instead, infected cells undergo inflammatory cell death (pyroptosis) mediated by activation of the NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD. Moreover, tissue-resident macrophages, but not infected epithelial cells, from COVID-19 lung autopsy specimens showed evidence of inflammasome activation. These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes/macrophages triggers inflammatory cell death that aborts production of infectious virus but causes systemic inflammation that contributes to severe COVID-19 disease pathogenesis.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release.

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in some patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes are sentinel blood cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that ~10% of blood monocytes in COVID-19 patients are dying and infected with SARS-CoV-2. Monocyte infection, which depends on antiviral antibodies, activates NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD cleavage and relocalization. Signs of pyroptosis (IL-1 family cytokines, LDH) in the plasma correlate with development of severe disease. Moreover, expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes triggers inflammation that contributes to severe COVID-19 disease pathogenesis. ONE SENTENCE SUMMARY: Antibody-mediated SARS-CoV-2 infection of monocytes activates inflammation and cytokine release.

Identification of a Distal Locus Enhancer Element That Controls Cell Type-Specific TNF and LTA Gene Expression in Human T Cells.

The human TNF/LT locus genes TNF, LTA, and LTB are expressed in a cell type-specific manner. In this study, we show that a highly conserved NFAT binding site within the distal noncoding element hHS-8 coordinately controls TNF and LTA gene expression in human T cells. Upon activation of primary human CD4+ T cells, hHS-8 and the TNF and LTA promoters display increased H3K27 acetylation and nuclease sensitivity and coordinate induction of TNF, LTA, and hHS-8 enhancer RNA transcription occurs. Functional analyses using CRISPR/dead(d)Cas9 targeting of the hHS-8-NFAT site in the human T cell line CEM demonstrate significant reduction of TNF and LTA mRNA synthesis and of RNA polymerase II recruitment to their promoters. These studies elucidate how a distal element regulates the inducible cell type-specific gene expression program of the human TNF/LT locus and provide an approach for modulation of TNF and LTA transcription in human disease using CRISPR/dCas9.

Cytoplasmic RNA Sensor Pathways and Nitazoxanide Broadly Inhibit Intracellular Mycobacterium tuberculosis Growth.

To establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease.

The FDA-Approved Oral Drug Nitazoxanide Amplifies Host Antiviral Responses and Inhibits Ebola Virus.

Here, we show that the US Food and Drug Administration-approved oral drug nitazoxanide (NTZ) broadly amplifies the host innate immune response to viruses and inhibits Ebola virus (EBOV) replication. We find that NTZ enhances retinoic-acid-inducible protein I (RIG-I)-like-receptor, mitochondrial antiviral signaling protein, interferon regulatory factor 3, and interferon activities and induces transcription of the antiviral phosphatase GADD34. NTZ significantly inhibits EBOV replication in human cells through its effects on RIG-I and protein kinase R (PKR), suggesting that it counteracts EBOV VP35 protein's ability to block RIG-I and PKR sensing of EBOV. NTZ also inhibits a second negative-strand RNA virus, vesicular stomatitis virus (VSV), through RIG-I and GADD34, but not PKR, consistent with VSV's distinct host innate immune evasion mechanisms. Thus, NTZ counteracts varied virus-specific immune evasion strategies by generally enhancing the RNA sensing and interferon axis that is triggered by foreign cytoplasmic RNA exposure, and holds promise as an oral therapy against EBOV.

Treatment of drug-resistant tuberculosis among people living with HIV.

PURPOSE OF REVIEW: This review aims to describe the key principles in treatment of drug-resistant tuberculosis (TB) in people living with HIV, including early access to timely diagnostics, linkage into care, TB treatment strategies including the use of new and repurposed drugs, co-management of HIV disease, and treatment complications and programmatic support to optimize treatment outcomes. These are necessary strategies to decrease the likelihood of poor treatment outcomes including lower treatment completion rates and higher mortality. RECENT FINDINGS: Diagnosis of drug-resistant TB is the gateway into care; yet understanding the utility and the limitations of genotypic methods in this population is necessary. The principles of TB treatment in HIV-infected individuals are similar to those without HIV co-infection, with few exceptions. However, adverse effects with potential significant morbidity may emerge during treatment, and timely antiretroviral therapy is essential to improve mortality in this patient population. Emerging data on the use of new and repurposed drugs and short course multidrug-resistant TB regimens and adherence strategies benefiting this population are reviewed. SUMMARY: The clinical complexity of co-managing drug-resistant TB and HIV, and the higher rate of poor treatment outcomes in this population demand careful clinical management strategies, and multidisciplinary and comprehensive programmatic interventions to optimize treatment success in this vulnerable group.

Initiation, scale-up and outcomes of the Cambodian National MDR-TB programme 2006-2016: hospital and community-based treatment through an NGO-NTP partnership.

INTRODUCTION: Prolonged inpatient multidrug-resistant tuberculosis (MDR-TB) treatment for all patients is not sustainable for high-burden settings, but there is limited information on community-based treatment programme outcomes for MDR-TB. METHODS: The Cambodian Health Committee, a non-governmental organisation (NGO), launched the Cambodian MDR-TB programme in 2006 in cooperation with the National Tuberculosis Program (NTP) including a community-based treatment option as a key programme component. The programme was transferred to NTP oversight in 2011 with NGO clinical management continuing. Patients electing to receive home-based treatment were followed by a dedicated adherence supporter and a multidisciplinary outpatient team of nurses, physicians and community health workers. Patients hospitalised for >1 month of treatment (hospital based) received similar management after discharge. All patients received a standardised second-line MDR-TB regimen and were provided nutritional and adherence support. Outcomes were reviewed for patients completing 24 months of treatment and predictors of treatment success were evaluated using logistic regression. RESULTS: Of 582 patients with MDR-TB who initiated treatment between September 2006 and June 2016, 20% were HIV coinfected, 288 (49%) initiated community-based treatment and 294 (51%) received hospital-based treatment. Of 486 patients with outcomes available, 364 (75%) were cured, 10 (2%) completed, 28 (6%) were lost to follow-up, 3 (0.6%) failed and 77 (16%) died. There was no difference between treatment success in community versus hospital-based groups (adjusted OR (aOR) 1.0, p=0.99). HIV infection, older age and body mass index <16 were strongly associated with decreased treatment success (aOR 0.33, p<0.001; aOR 0.40, p<0.001; aOR 0.40; p<0.001). CONCLUSIONS: Cambodia's NGO-NTP partnership successfully developed and scaled up a model MDR-TB treatment programme. The first large-scale MDR-TB programme in Asia with a significant community-based component, the programme achieved equally high treatment success in patients with community-based compared with hospital-based initiation of MDR treatment.

Diagnostic Potential of Imaging Flow Cytometry.

Imaging flow cytometry (IFC) captures multichannel images of hundreds of thousands of single cells within minutes. IFC is seeing a paradigm shift from low- to high-information-content analysis, driven partly by deep learning algorithms. We predict a wealth of applications with potential translation into clinical practice.

Imaging flow cytometry analysis of intracellular pathogens.

Imaging flow cytometry has been applied to address questions in infection biology, in particular, infections induced by intracellular pathogens. This methodology, which utilizes specialized analytic software makes it possible to analyze hundreds of quantified features for hundreds of thousands of individual cellular or subcellular events in a single experiment. Imaging flow cytometry analysis of host cell-pathogen interaction can thus quantitatively addresses a variety of biological questions related to intracellular infection, including cell counting, internalization score, and subcellular patterns of co-localization. Here, we provide an overview of recent achievements in the use of fluorescently labeled prokaryotic or eukaryotic pathogens in human cellular infections in analysis of host-pathogen interactions. Specifically, we give examples of Imagestream-based analysis of cell lines infected with Toxoplasma gondii or Mycobacterium tuberculosis. Furthermore, we illustrate the capabilities of imaging flow cytometry using a combination of standard IDEAS™ software and the more recently developed Feature Finder algorithm, which is capable of identifying statistically significant differences between researcher-defined image galleries. We argue that the combination of imaging flow cytometry with these software platforms provides a powerful new approach to understanding host control of intracellular pathogens.

A Role for IFITM Proteins in Restriction of Mycobacterium tuberculosis Infection.

The interferon (IFN)-induced transmembrane (IFITM) proteins are critical mediators of the host antiviral response. Here, we expand the role of IFITM proteins to host defense against intracellular bacterial infection by demonstrating that they restrict Mycobacterium tuberculosis (MTb) intracellular growth. Simultaneous knockdown of IFITM1, IFITM2, and IFITM3 by RNAi significantly enhances MTb growth in human monocytic and alveolar/epithelial cells, whereas individual overexpression of each IFITM impairs MTb growth in these cell types. Furthermore, MTb infection, Toll-like receptor 2 and 4 ligands, and several proinflammatory cytokines induce IFITM1-3 gene expression in human myeloid cells. We find that IFITM3 co-localizes with early and, in particular, late MTb phagosomes, and overexpression of IFITM3 enhances endosomal acidification in MTb-infected monocytic cells. These findings provide evidence that the antiviral IFITMs participate in the restriction of mycobacterial growth, and they implicate IFITM-mediated endosomal maturation in its antimycobacterial activity.

Achieving high treatment success for multidrug-resistant TB in Africa: initiation and scale-up of MDR TB care in Ethiopia--an observational cohort study.

BACKGROUND: In Africa, fewer than half of patients receiving therapy for multidrug-resistant TB (MDR TB) are successfully treated, with poor outcomes reported for HIV-coinfected patients. METHODS: A standardised second-line drug (SLD) regimen was used in a non-governmental organisation-Ministry of Health (NGO-MOH) collaborative community and hospital-based programme in Ethiopia that included intensive side effect monitoring, adherence strategies and nutritional supplementation. Clinical outcomes for patients with at least 24 months of follow-up were reviewed and predictors of treatment failure or death were evaluated by Cox proportional hazards models. RESULTS: From February 2009 to December 2014, 1044 patients were initiated on SLD. 612 patients with confirmed or presumed MDR TB had ≥ 24 months of follow-up, 551 (90.0%) were confirmed and 61 (10.0%) were suspected MDR TB cases. 603 (98.5%) had prior TB treatment, 133 (21.7%) were HIV coinfected and median body mass index (BMI) was 16.6. Composite treatment success was 78.6% with 396 (64.7%) cured, 85 (13.9%) who completed treatment, 10 (1.6%) who failed, 85 (13.9%) who died and 36 (5.9%) who were lost to follow-up. HIV coinfection (adjusted HR (AHR): 2.60, p<0.001), BMI (AHR 0.88/kg/m(2), p=0.006) and cor pulmonale (AHR 3.61, p=0.003) and confirmed MDR TB (AHR 0.50, p=0.026) were predictive of treatment failure or death. CONCLUSIONS: We report from Ethiopia the highest MDR TB treatment success outcomes so far achieved in Africa, in a setting with severe resource constraints and patients with advanced disease. Intensive treatment of adverse effects, nutritional supplementation, adherence interventions and NGO-MOH collaboration were key strategies contributing to success. We argue these approaches should be routinely incorporated into programmes.

T cells and adaptive immunity to Mycobacterium tuberculosis in humans.

The adaptive immune response mediated by T cells is critical for control of Mycobacterium tuberculosis (M. tuberculosis) infection in humans. However, the M. tuberculosis antigens and host T-cell responses that are required for an effective adaptive immune response to M. tuberculosis infection are yet to be defined. Here, we review recent findings on CD4(+) and CD8(+) T-cell responses to M. tuberculosis infection and examine the roles of distinct M. tuberculosis-specific T-cell subsets in control of de novo and latent M. tuberculosis infection, and in the evolution of T-cell immunity to M. tuberculosis in response to tuberculosis treatment. In addition, we discuss recent studies that elucidate aspects of M. tuberculosis-specific adaptive immunity during human immunodeficiency virus co-infection and summarize recent findings from vaccine trials that provide insight into effective adaptive immune responses to M. tuberculosis infection.

TB-IRIS, T-cell activation, and remodeling of the T-cell compartment in highly immunosuppressed HIV-infected patients with TB.

OBJECTIVE: To investigate the impact of tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) upon immunological recovery and the T-cell compartment after initiation of TB and antiretroviral therapy (ART). DESIGN AND METHODS: We prospectively evaluated T-cell immunophenotypes by flow cytometry and cytokines by Luminex assays in a subset (n = 154) of highly immunosuppressed HIV-infected patients with TB from the Cambodian Early versus Late Introduction of Antiretrovirals randomized clinical trial. We compared findings from patients who developed TB-IRIS with findings from patients who did not develop TB-IRIS. Data were evaluated with mixed-effect linear regression, Kaplan-Meier estimates, and Wilcoxon rank-sum tests, and q-values were calculated to control for multiple comparisons. RESULTS: Development of TB-IRIS was associated with significantly greater pre-ART frequencies of HLA-DRCD45ROCD4, CCR5CD4, OX40CD4, and Fas effector memory CD8 T cells, and significantly elevated levels of plasma interleukin (IL)-6, IL-1ß, IL-8, and IL-10, and viral load. Post-ART initiation, effector memory CD4 and Fas effector memory CD4 T-cell frequencies significantly expanded, and central memory CD4 T-cell frequencies significantly contracted in patients who experienced TB-IRIS. By week 34 post-TB treatment initiation, effector memory/central memory CD4 T-cell ratios were markedly higher in TB-IRIS versus non-TB-IRIS patients. CONCLUSIONS: A distinct pattern of pre-ART T-cell and cytokine markers appear to poise the immune response of certain patients to develop TB-IRIS. Experience of TB-IRIS is then associated with long-term remodeling of the CD4 T-cell memory compartment towards an effector memory-dominated phenotype. We speculate that these pre and post-ART TB-IRIS-associated immune parameters may contribute to superior immune control of TB/HIV co-infection and better clinical outcome.

A distal locus element mediates IFN-γ priming of lipopolysaccharide-stimulated TNF gene expression.

Interferon γ (IFN-γ) priming sensitizes monocytes and macrophages to lipopolysaccharide (LPS) stimulation, resulting in augmented expression of a set of genes including TNF. Here, we demonstrate that IFN-γ priming of LPS-stimulated TNF transcription requires a distal TNF/LT locus element 8 kb upstream of the TNF transcription start site (hHS-8). IFN-γ stimulation leads to increased DNase I accessibility of hHS-8 and its recruitment of interferon regulatory factor 1 (IRF1), and subsequent LPS stimulation enhances H3K27 acetylation and induces enhancer RNA synthesis at hHS-8. Ablation of IRF1 or targeting the hHS-8 IRF1 binding site in vivo with Cas9 linked to the KRAB repressive domain abolishes IFN-γ priming, but does not affect LPS induction of the gene. Thus, IFN-γ poises a distal enhancer in the TNF/LT locus by chromatin remodeling and IRF1 recruitment, which then drives enhanced TNF gene expression in response to a secondary toll-like receptor (TLR) stimulus.

Causes and determinants of mortality in HIV-infected adults with tuberculosis: an analysis from the CAMELIA ANRS 1295-CIPRA KH001 randomized trial.

BACKGROUND: Shortening the interval between antituberculosis treatment onset and initiation of antiretroviral therapy (ART) reduces mortality in severely immunocompromised human immunodeficiency virus (HIV)-infected patients with tuberculosis. A better understanding of causes and determinants of death may lead to new strategies to further enhance survival. METHODS: We assessed mortality rates, causes of death, and factors of mortality in Cambodian HIV-infected adults with CD4 count ≤200 cells/µL and tuberculosis, randomized to initiate ART either 2 weeks (early ART) or 8 weeks (late ART) after tuberculosis treatment onset in the CAMELIA clinical trial. RESULTS: Six hundred sixty-one patients enrolled contributed to 1366.1 person-years of follow-up; 149 (22.5%) died. There were 8.3 deaths per 100 person-years (95% confidence interval [CI], 6.4-10.7) in the early-ART group and 13.8 deaths per 100 person-years (95% CI, 11.2-16.9) in the late-ART group (P = .002). Tuberculosis was the primary cause of death (28%), followed by other HIV-associated conditions (19%). Factors independently associated with mortality in the first 26 weeks were the age, body mass index, hemoglobin, interrupted or ineffective tuberculosis treatment before identification of drug resistance, disseminated tuberculosis, and nontuberculous mycobacterial disease. After 50 weeks in the trial, the most frequent causes of death were non-HIV related or tuberculosis related, including drug toxicity; factors associated with mortality were late ART, loss to follow-up, and absence of cotrimoxazole prophylaxis. CONCLUSIONS: Despite ART introduction, mortality remained high, with tuberculosis as the leading cause of death. Reducing tuberculosis-related mortality remains a challenge in resource-limited settings and requires innovative strategies. Clinical Trials Registration. NCT00226434.

Plasma concentrations, efficacy and safety of efavirenz in HIV-infected adults treated for tuberculosis in Cambodia (ANRS 1295-CIPRA KH001 CAMELIA trial).

OBJECTIVE: To assess efavirenz plasma concentrations and their association with treatment efficacy and tolerance of efavirenz 600 mg daily in HIV-tuberculosis co-infected patients. METHODS: HIV-infected adults with CD4+ T cell count ≤ 200/mm(3) received standard 6-month tuberculosis treatment and antiretroviral therapy including a daily-dose of 600 mg of efavirenz, irrespective of their body weight. Mid-dose blood samples were drawn both on tuberculosis treatment (week +2 and week +6 after antiretroviral therapy initiation, and week 22 of follow-up) and off tuberculosis treatment (week 50 of follow-up). Considered therapeutic range was 1,000 to 4,000 ng/mL. Multivariate analysis was performed to evaluate the association between efavirenz concentration below 1,000 ng/mL and virological failure. Linear regression was used to test the association between efavirenz exposure and CD4+ T cell gain. Severe side effects potentially related to efavirenz were described and their association with efavirenz exposure was tested by multivariate analysis. RESULTS: Efavirenz plasma concentrations were available in 540 patients. Median [interquartile range] efavirenz concentrations were 2,674 ng/mL [1,690-4,533], 2,667 ng/mL [1,753-4,494] and 2,799 ng/mL [1,804-4,744] at week +2, week +6, week 22, respectively, and 2,766 ng/mL [1,941-3,976] at week 50. Efavirenz concentrations were lower at week 50 (off rifampicin) compared to week 22 (on rifampicin) (p<0.001). Late attendance to study visit and low hemoglobinemia were the only factors associated with an increased risk of efavirenz concentration below 1,000 ng/mL. Efavirenz concentration below 1,000 ng/mL was not associated with treatment failure. Efavirenz concentration above 4,000 ng/mL was associated with higher risk of central nervous system side effects (p<0.001) and of hepatotoxicity (p<0.001). CONCLUSION: Body weight and tuberculosis treatment were not associated with low efavirenz concentrations or treatment failure, supporting the 600 mg daily-dose of efavirenz in HIV-tuberculosis co-infected patients. High efavirenz concentrations were related to a higher risk of central nervous system side effects and hepatotoxicity. TRIAL REGISTRATION: ClinicalTrials.gov NCT01300481.

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome after early initiation of antiretroviral therapy in a randomized clinical trial.

OBJECTIVE: To analyze cases of paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) in the CAMbodian Early versus Late Introduction of Antiretrovirals (CAMELIA) randomized trial designed to compare early (2 weeks) versus late (8 weeks) antiretroviral therapy (ART) initiation after tuberculosis treatment onset in Cambodia (NCT00226434). METHODS: ART-naive adults with CD4 cell count of 200 cells/µl or less, newly diagnosed tuberculosis, and at least one follow-up visit after ART initiation were included in this analysis. Each case of suspected TB-IRIS was systematically validated by two physicians not involved in patients' management. Factors associated with occurrence of TB-IRIS were identified using the Cox proportional hazard model. RESULTS: Among 597 patients, 26% experienced TB-IRIS with an incidence rate of 37.9 cases per 100 person-years [95% confidence interval (CI) 32.4-44.4]. Main clinical manifestations included new or worsening lymphadenopathy (77.4%) and fever (68.4%). Chest radiograph revealed new or worsening abnormalities in 53.4%. Symptoms resolved in 95.5% of patients. Six deaths were directly related to TB-IRIS. Initiating ART early increased the risk of TB-IRIS by 2.61 (95% CI 1.84-3.70). Extrapulmonary or disseminated tuberculosis, CD4 cell count of 100 cells/µl or less, and HIV RNA concentration more than 6 log10 copies/ml were also significantly associated with higher risk of TB-IRIS. CONCLUSION: Shortening the delay between tuberculosis treatment onset and ART initiation to 2 weeks was associated with an increased risk of developing TB-IRIS. However, TB-IRIS was generally easily manageable. Given the marked reported survival advantage of early ART initiation after tuberculosis treatment onset, these data indicate that fear of TB-IRIS should not be an impediment to early ART in adults with advanced immunodeficiency in resource-limited, high burden settings.