Production of Proinflammatory Cytokines by CD4+ and CD8+ T Cells in Response to Mycobacterial Antigens among Children and Adults with Tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of pediatric morbidity and mortality. Young children are at high risk of TB following Mtb exposure, and this vulnerability is secondary to insufficient host immunity during early life. Our primary objective was to compare CD4+ and CD8+ T-cell production of proinflammatory cytokines IFN-gamma, IL-2, and TNF-alpha in response to six mycobacterial antigens and superantigen staphylococcal enterotoxin B (SEB) between Ugandan adults with confirmed TB (n = 41) and young Ugandan children with confirmed (n = 12) and unconfirmed TB (n = 41), as well as non-TB lower respiratory tract infection (n = 39). Flow cytometry was utilized to identify and quantify CD4+ and CD8+ T-cell cytokine production in response to each mycobacterial antigen and SEB. We found that the frequency of CD4+ and CD8+ T-cell production of cytokines in response to SEB was reduced in all pediatric cohorts when compared to adults. However, T-cell responses to Mtb-specific antigens ESAT6 and CFP10 were equivalent between children and adults with confirmed TB. In contrast, cytokine production in response to ESAT6 and CFP10 was limited in children with unconfirmed TB and absent in children with non-TB lower respiratory tract infection. Of the five additional mycobacterial antigens tested, PE3 and PPE15 were broadly recognized regardless of TB disease classification and age. Children with confirmed TB exhibited robust proinflammatory CD4+ and CD8+ T-cell responses to Mtb-specific antigens prior to the initiation of TB treatment. Our findings suggest that adaptive proinflammatory immune responses to Mtb, characterized by T-cell production of IFN-gamma, IL-2, and TNF-alpha, are not impaired during early life.

Implementation of point-of-care testing and prevalence of cryptococcal antigenaemia among patients with advanced HIV disease in Mumbai, India.

OBJECTIVES: To describe the implementation of screening for cryptococcal antigenaemia by point-of-care (POC) serum cryptococcal antigen (CrAg) lateral flow assay, measure the prevalence and factors associated with serum cryptococcal antigenaemia in the routine programmatic setting. DESIGN: Cross-sectional study. SETTING: Seventeen publicly funded antiretroviral therapy (ART) centres in Mumbai, India. PARTICIPANTS: Serum CrAg screening was offered to all adolescents (>10 years of age) and adults with advanced HIV disease (AHD) (CD4 <200 cells/mm3 or with WHO clinical stage III/IV) regardless of symptoms of cryptococcal meningitis. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was to describe the implementation of serum CrAg screening and secondary outcome was to measure the prevalence of serum cryptococcal antigenaemia and its risk factors. RESULTS: A total of 2715 patients with AHD were tested for serum CrAg by POC assay. Of these, 25 (0.9%) had a CrAg positive result. Among CrAg-positive patients, only one had symptoms. Serum CrAg positivity was 3.6% (6/169) and 1.6% (6/520) among those presenting with CD4 <100 cells/mm3 in the treatment naïve and treatment experienced group, respectively. On multivariable analysis, CD4 count <100 cells/mm3 (OR: 2.3, 95% CI 1.01 to 5.3; p=0.05) and people living with HIV who were treatment naïve (OR: 2.5, 95% CI 1.04 to 6.0; p=0.04) were significantly associated with a positive serum CrAg result. Lumbar puncture was obtained in 20/25 patients within 4 days (range: 1-4 days) of positive serum CrAg result and one person was confirmed to have meningitis. All serum CrAg-positive patients who had a negative cerebrospinal fluid CrAg were offered pre-emptive therapy. CONCLUSIONS: Implementation of a POC CrAg assay was possible with existing ART centre staff. Initiation of pre-emptive therapy and management of cryptococcal antigenaemia are operationally feasible at ART centres. The Indian National AIDS Control Programme may consider reflexive CrAg screening of all AHD patients with CD4 <100 cells/mm3.

Retaining Patients with Drug-Resistant Tuberculosis on Treatment During the COVID-19 Pandemic - Dharavi, Mumbai, India, 2020-2022.

Mumbai, India's second largest city, has one of the highest prevalences of drug-resistant tuberculosis* (DRTB) in the world. Treatment for DRTB takes longer and is more complicated than treatment for drug-susceptible tuberculosis (TB). Approximately 300 persons receive a new DRTB diagnosis each year in Mumbai's Dharavi slum†; historically, fewer than one half of these patients complete DRTB treatment. As nationwide restrictions to mitigate the COVID-19 pandemic were implemented, a program to facilitate uninterrupted DRTB care for patients receiving treatment was also implemented. A comprehensive tool and risk assessment provided support to DRTB patients and linked those who relocated outside of Dharavi during the pandemic to DRTB care at their destination. During May 2020-September 2022, a total of 973 persons received DRTB treatment in Dharavi, including 255 (26%) who relocated during treatment. Overall, 25 (3%) DRTB patients were lost to follow-up, a rate substantially lower than the rate before the pandemic (18%). Proactive planning and implementation of simple tools retained patients on treatment during periods of travel restrictions and relocations, improving programmatic outcomes. This approach might aid public health programs serving migrant populations or patients receiving treatment for DRTB during public health emergencies.

Building communities of practice through case-based e-learning to prevent and manage TB among people living with HIV-India.

BACKGROUND: Co-management of HIV-TB coinfection remains a challenge globally. Addressing TB among people living with HIV (PLHIV) is a key priority for the Government of India (GoI). In 2016, GoI implemented single-window services to prevent and manage TB in PLHIV. To strengthen HIV-TB service delivery, case-based e-learning was introduced to health care providers at Antiretroviral Therapy centres (ARTc). METHODS: We implemented a hub and spoke model to deliver biweekly, virtual, case-based e-learning at select ARTc (n = 115), from four states of India-Delhi, Uttar Pradesh, Andhra Pradesh and Tamil Nadu. We evaluated feasibility and acceptability of case-based e-learning and its impact on professional satisfaction, self-efficacy, knowledge retention using baseline and completion surveys, session feedback, pre-and post-session assessments. We reviewed routine programmatic data and patient outcomes to assess practices among participating ARTc. RESULTS: Between May 2018 and September 2020, 59 sessions were conducted with mean participation of 55 spokes and 152 participants. For 95% and 88% of sessions ≥ 80% of respondents agreed that topics were clear and relevant to practice, and duration of session was appropriate, respectively. Session participants significantly improved in perceived knowledge, skills and competencies (+ 8.6%; p = 0.025), and technical knowledge (+ 18.3%; p = 0.04) from baseline. Participating ARTc increased TB screening (+ 4.2%, p < 0.0001), TB diagnosis (+ 2.7%, p < 0.0001), ART initiation (+ 4.3%, p < 0.0001) and TB preventive treatment completion (+ 5.2%, p < 0.0001). CONCLUSION: Case-based e-learning is an acceptable and effective modus of capacity building and developing communities of practice to strengthen integrated care. E-learning could address demand for accessible and sustainable continuing professional education to manage complex diseases, and thereby enhance health equity. We recommend expansion of this initiative across the country for management of co-morbidities as well as other communicable and non-communicable diseases to augment the existing capacity building interventions by provide continued learning and routine mentorship through communities of practice.

Utility of the lateral flow urine lipoarabinomannan tuberculosis assay in patients with advanced HIV disease at antiretroviral therapy centres in Mumbai, India.

BACKGROUND: People with Advanced HIV Disease (AHD) are at higher risk of TB coinfection and mortality. However, there are challenges in TB diagnosis with the currently recommended diagnostic tools. WHO recommends lateral flow urine lipoarabinomannan (LF-LAM) assay to assist TB diagnosis among AHD patients. We assessed the utility and acceptability of using urine LF-LAM assay for TB diagnosis among patients at public Antiretroviral Therapy (ART) Centres in Mumbai. METHODS: The cross-sectional study was conducted among adult AHD patients accessing care from 17 ART centres during November,2020-June, 2021. Urine LF-LAM was offered as routine care for eligible patients in combination with standard diagnostic tests. We calculated the proportion of positive LF-LAM results by CD4 categories and TB symptoms and performed multivariable logistic regression to determine the factors associated with LF-LAM positivity. RESULTS: Among 2,390 patients, the majority (74.5%) had CD4 between 101-200 cells/mm3. The mean age was 43.7 years (SD:10.6), 68.6% were male, 8.4% had TB symptoms and 88.0% were on ART. The overall proportion of patients with urine LF-LAM positive results was 6.4%. Among PLHIV with CD4≤100 cells/mm3, the positivity was 43.0% and 7.7% in symptomatic and asymptomatic patients, respectively. Among PLHIV with a CD4>100 cells/mm3, the positivity was 26.7% and 2.7% in symptomatic and asymptomatic patients respectively. Urine LF-LAM positivity was higher among inpatients, ART naïve, patients on treatment for <6 months, symptomatic and in WHO clinical stage III/IV of HIV disease as compared to the reference categories. We detected an additional 131 TB cases with urine LF-LAM in combination with the standard diagnostic tests. CONCLUSION: The study demonstrated the utility of urine LF-LAM for TB diagnosis among AHD patients and the simple, user-friendly test was acceptable as part of routine care. Inclusion of urine LF-LAM test in the current diagnostic algorithm may facilitate early TB diagnosis among AHD patients.

Implementation of the Treat All Policy Among Persons with HIV Infection Enrolled in Care But Not on Antiretroviral Therapy - India, May 2017-June 2018.

Since September 2015, the World Health Organization has recommended antiretroviral therapy (ART) for all persons with human immunodeficiency virus (HIV) infection, regardless of clinical stage or CD4 count (1). This Treat All policy was based on evidence that ART initiation early in HIV infection as opposed to waiting for the CD4 count to decline to certain levels (e.g., <500 cells/mm3, per previous guidelines), was associated with reduced morbidity, mortality, and HIV transmission (2-4). Further, approximately half of persons enrolled in non-ART care that included monitoring for HIV disease progression (i.e., in pre-ART care) were lost to follow-up before becoming ART-eligible (5). India, the country with the third largest number of persons with HIV infection in the world (2.1 million), adopted the Treat All policy on April 28, 2017. This report describes implementation of Treat All during May 2017-June 2018, by India's National AIDS Control Organization (NACO) and partners, by facilitating ART initiation among persons previously in pre-ART care at 46 ART centers supported by the U.S. President's Emergency Plan for AIDS Relief (PEPFAR)* in six districts in the states of Maharashtra and Andhra Pradesh. Partners supported these 46 ART centers in identifying and attempting to contact persons who were enrolled in pre-ART care during January 2014-April 2017, and educating those reached about Treat All. ART center-based records were used to monitor implementation indicators, including ART initiation. A total of 9,898 (39.6%) of 25,007 persons previously enrolled in pre-ART care initiated ART; among these 9,898 persons, 6,315 (63.8%) initiated ART after being reached during May 2017-June 2018, including 1,635 (16.5%) who had been lost to follow-up before ART initiation. NACO scaled up efforts nationwide to build ART centers' capacity to implement Treat All. Active tracking and tracing of persons with HIV infection enrolled in care but not on ART, combined with education about the benefits of early HIV treatment, can facilitate ART initiation.

Comprehensive definition of human immunodominant CD8 antigens in tuberculosis.

Despite widespread use of the Bacillus Calmette-Guerin vaccine, tuberculosis, caused by infection with Mycobacterium tuberculosis, remains a leading cause of morbidity and mortality worldwide. As CD8+ T cells are critical to tuberculosis host defense and a phase 2b vaccine trial of modified vaccinia Ankara expressing Ag85a that failed to demonstrate efficacy, also failed to induce a CD8+ T cell response, an effective tuberculosis vaccine may need to induce CD8+ T cells. However, little is known about CD8, as compared to CD4, antigens in tuberculosis. Herein, we report the results of the first ever HLA allele independent genome-wide CD8 antigen discovery program. Using CD8+ T cells derived from humans with latent tuberculosis infection or tuberculosis and an interferon-γ ELISPOT assay, we screened a synthetic peptide library representing 10% of the Mycobacterium tuberculosis proteome, selected to be enriched for Mycobacterium tuberculosis antigens. We defined a set of immunodominant CD8 antigens including part or all of 74 Mycobacterium tuberculosis proteins, only 16 of which are previously known CD8 antigens. Immunogenicity was associated with the degree of expression of mRNA and protein. Immunodominant antigens were enriched in cell wall proteins with preferential recognition of Esx protein family members, and within proteins comprising the Mycobacterium tuberculosis secretome. A validation study of immunodominant antigens demonstrated that these antigens were strongly recognized in Mycobacterium tuberculosis-infected individuals from a tuberculosis endemic region in Africa. The tuberculosis vaccine field will likely benefit from this greatly increased known repertoire of CD8 immunodominant antigens and definition of properties of Mycobacterium tuberculosis proteins important for CD8 antigenicity.

Adenovirally-Induced Polyfunctional T Cells Do Not Necessarily Recognize the Infected Target: Lessons from a Phase I Trial of the AERAS-402 Vaccine.

The development of a vaccine for Mycobacterium tuberculosis (Mtb) has been impeded by the absence of correlates of protective immunity. One correlate would be the ability of cells induced by vaccination to recognize the Mtb-infected cell. AERAS-402 is a replication-deficient serotype 35 adenovirus containing DNA expressing a fusion protein of Mtb antigens 85A, 85B and TB10.4. We undertook a phase I double-blind, randomized placebo controlled trial of vaccination with AERAS-402 following BCG. Analysis of the vaccine-induced immune response revealed strong antigen-specific polyfunctional CD4+ and CD8+ T cell responses. However, analysis of the vaccine-induced CD8+ T cells revealed that in many instances these cells did not recognize the Mtb-infected cell. Our findings highlight the measurement of vaccine-induced, polyfunctional T cells may not reflect the extent or degree to which these cells are capable of identifying the Mtb-infected cell and correspondingly, the value of detailed experimental medicine studies early in vaccine development.

Mycobacterium tuberculosis specific CD8(+) T cells rapidly decline with antituberculosis treatment.

RATIONALE: Biomarkers associated with response to therapy in tuberculosis could have broad clinical utility. We postulated that the frequency of Mycobacterium tuberculosis (Mtb) specific CD8(+) T cells, by virtue of detecting intracellular infection, could be a surrogate marker of response to therapy and would decrease during effective antituberculosis treatment. OBJECTIVES: We sought to determine the relationship of Mtb specific CD4(+) T cells and CD8(+) T cells with duration of antituberculosis treatment. MATERIALS AND METHODS: We performed a prospective cohort study, enrolling between June 2008 and August 2010, of HIV-uninfected Ugandan adults (n = 50) with acid-fast bacillus smear-positive, culture confirmed pulmonary TB at the onset of antituberculosis treatment and the Mtb specific CD4(+) and CD8(+) T cell responses to ESAT-6 and CFP-10 were measured by IFN-γ ELISPOT at enrollment, week 8 and 24. RESULTS: There was a significant difference in the Mtb specific CD8(+) T response, but not the CD4(+) T cell response, over 24 weeks of antituberculosis treatment (p<0.0001), with an early difference observed at 8 weeks of therapy (p = 0.023). At 24 weeks, the estimated Mtb specific CD8(+) T cell response decreased by 58%. In contrast, there was no significant difference in the Mtb specific CD4(+) T cell during the treatment. The Mtb specific CD4(+) T cell response, but not the CD8(+) response, was negatively impacted by the body mass index. CONCLUSIONS: Our data provide evidence that the Mtb specific CD8(+) T cell response declines with antituberculosis treatment and could be a surrogate marker of response to therapy. Additional research is needed to determine if the Mtb specific CD8(+) T cell response can detect early treatment failure, relapse, or to predict disease progression.

CD8+ T cells provide an immunologic signature of tuberculosis in young children.

RATIONALE: The immunologic events surrounding primary Mycobacterium tuberculosis infection and development of tuberculosis remain controversial. Young children who develop tuberculosis do so quickly after first exposure, thus permitting study of immune response to primary infection and disease. We hypothesized that M. tuberculosis-specific CD8(+) T cells are generated in response to high bacillary loads occurring during tuberculosis. OBJECTIVES: To determine if M. tuberculosis-specific T cells are generated among healthy children exposed to M. tuberculosis and children with tuberculosis. METHODS: Enzyme-linked immunosorbent spot assays were used to measure IFN-γ production in response to M. tuberculosis-specific proteins ESAT-6/CFP-10 by peripheral blood mononuclear cells and CD8(+) T cells isolated from Ugandan children hospitalized with tuberculosis (n = 96) or healthy tuberculosis contacts (n = 62). MEASUREMENTS AND MAIN RESULTS: The proportion of positive CD8(+) T-cell assays and magnitude of CD8(+) T-cell responses were significantly greater among young (<5 yr) tuberculosis cases compared with young contacts (P = 0.02, Fisher exact test, P = 0.01, Wilcoxon rank-sum, respectively). M. tuberculosis-specific T-cell responses measured in peripheral blood mononuclear cells were equivalent between groups. CONCLUSIONS: Among young children, M. tuberculosis-specific CD8(+) T cells develop in response to high bacillary loads, as occurs during tuberculosis, and are unlikely to be found after M. tuberculosis exposure. T-cell responses measured in peripheral blood mononuclear cells are generated after M. tuberculosis exposure alone, and thus cannot distinguish exposure from disease. In young children, IFN-γ-producing M. tuberculosis-specific CD8(+) T cells provide an immunologic signature of primary M. tuberculosis infection resulting in disease.

New diagnostic methods for tuberculosis.

PURPOSE OF REVIEW: During the last decade, laboratory tests for the detection of Mycobacterium tuberculosis (Mtb) have improved dramatically. Improvements in the ability to detect latent infection with Mtb, disease associated with Mtb, and strains resistant to commonly used antibiotics are reviewed. RECENT FINDINGS: Advances in the detection of Mtb include light-emitting diode fluorescence microscopy, nucleic acid amplification of Mtb and drug-resistant strains, and more rapid liquid culture with adjunct drug susceptibility testing. In the detection of latent tuberculosis infection, interferon [gamma] release assays offer improved accuracy over the tuberculin skin test. SUMMARY: The past 10 years have seen the most rapid growth in new diagnostics for Mtb in over a century. Although these tests offer improvements in the ability to detect Mtb, drug-resistant isolates, and those with latent tuberculosis infection, these improvements are counter-balanced by the need to deploy these tests in areas where Mtb burden is highest.

Interferon-gamma release assays for diagnosing mycobacterium tuberculosis infection in renal dialysis patients.

BACKGROUND AND OBJECTIVES: End-stage renal disease (ESRD) patients are at high risk for tuberculosis (TB). IFN-gamma release assays that assess immune responses to specific TB antigens offer potential advantages over tuberculin skin testing (TST) in screening such patients for Mycobacterium tuberculosis infection. This study sought to determine whether IFN-gamma release assay results are more closely associated with recent TB exposure than TST results. DESIGN, SETTING, PARTICIPANTS, AND MEASURES: Prospective cohort investigation of patients at a hemodialysis center with a smear-positive case of TB. Patients without a history of TB underwent initial and repeat testing with TST, and with the IFN-gamma assays QuantiFERON-TB Gold (QFT-G) and ELISPOT test. Outcome measures included the prevalence of positive test results, identification of factors associated with positive results, and test result discordance. RESULTS: A total of 100 (47% foreign born; median age, 55 yr; age range, 18 to 83 yr) of 124 eligible patients were enrolled. Twenty-six persons had positive TST results, 21 had positive QFT-G results, and 27 had positive ELISPOT results. Patients with TB case contact were likely to have a positive QFT-G result (P = 0.02) and ELISPOT results (P = 0.04), whereas TB case contact was not associated with positive TST results (P = 0.7). Positive TST results were associated with foreign birth (P = 0.04) and having had a TST in the previous year (P = 0.04). CONCLUSIONS: Positive IFN-gamma assay results were more closely associated with recent TB exposure than were positive TST results. QFT-G and ELISPOT might offer a better method for detecting TB infection in ESRD patients.