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Abstract Background Tuberculosis (TB) is the most frequent co-infection in HIV-infected individuals still presenting diagnostic difficulties particularly in developing countries. Recently an assay based on IFN-gamma response to M. tuberculosis RD1 peptides selected by computational analysis was developed whose presence is detected during active TB disease. Objective of this study was to investigate the response to selected RD1 peptides in HIV-1-infected subjects with or without active TB in a country endemic for TB and to evaluate the change of this response over time. Methods 30 HIV-infected individuals were prospectively enrolled, 20 with active TB and 10 without. Among those with TB, 12 were followed over time. IFN-gamma response to selected RD1 peptides was evaluated by enzyme-linked immunospot (ELISPOT) assay. As control, response to RD1 proteins was included. Results were correlated with immune, microbiological and virological data. Results Among patients with active TB, 2/20 were excluded from the analysis, one due to cell artifacts and the other to unresponsiveness to M. tuberculosis antigens. Among those analyzable, response to selected RD1 peptides evaluated as spot-forming cells was significantly higher in subjects with active TB compared to those without (p = 0.02). Among the 12 TB patients studied over time a significant decrease (p =< 0.007) of IFN-gamma response was found at completion of therapy when all the sputum cultures for M. tuberculosis were negative. A ratio of RD1 peptides ELISPOT counts over CD4+ T-cell counts greater than 0.21 yielded 100% sensitivity and 80% specificity for active TB. Conversely, response to RD1 intact proteins was not statistically different between subjects with or without TB at the time of recruitment; however a ratio of RD1 proteins ELISPOT counts over CD4+ T-cell counts greater than 0.22 yielded 89% sensitivity and 70% specificity for active TB. Conclusion In this pilot study the response to selected RD1 peptides is associated with TB disease in HIV-infected individuals in a high TB endemic country. This response decreases after successful therapy. The potential of the novel approach of relating ELISPOT spot-forming cell number and CD4+ T-cell count may improve the possibility of diagnosing active TB and deserves further evaluation.

Systemic immune activation is critical to the pathogenesis of HIV-1 disease, and is accentuated in HIV/TB co-infected patients. The contribution of immune activation at sites of HIV/TB co-infection to viral activity, CD4 T cell count, and productive HIV-1 infection remain unclear. In this study, we measured markers of immune activation both in pleural fluid and plasma, and in T cells in pleural fluid mononuclear cell (PFMC) and peripheral blood mononuclear cell (PBMC) in HIV/TB co-infected subjects. The relationship between soluble and T cell activation markers with viral load in pleural fluid and blood CD4 T cell count were assessed. The T cell phenotype and activation status of HIV-1 p24 + T cells in PFMC and PBMC from HIV/TB patients were determined. We found that T cell and macrophage-specific and non-specific soluble markers of immune activation, sCD27, sCD163, IL1Ra, and sCD14, were higher in pleural fluid as compared to plasma from HIV/TB co-infected subjects, and higher as compared to pleural fluid from TB mono-infected subjects. Intestinal fatty acid-binding protein, a marker of intestinal tract damage, in plasma from HIV/TB co-infected patients was not different than that in HIV+ subjects. Expression of HLADR and CD38 double positive (HLADR/CD38) on CD4 T cells, and CD69+ on CD8 T cells correlated with pleural fluid viral load, and inversely with blood CD4 T cell count. Higher expression of HLADR/CD38 and CCR5 on CD4 T cells, and HLADR/CD38 and CD69 on CD8 T cells in PFMC were limited to effector memory populations. HIV-1 p24+ CD8 negative (includes CD4 + and double negative T cells) effector memory T cells in PFMC had higher expression of HLADR/CD38, Ki67, and CCR5 compared to HIV-1 p24- CD8 negative PFMC. Cumulatively, these data indicate that sites of HIV/TB co-infection are the source of intense immune activation. [ABSTRACT FROM AUTHOR]

A higher human immunodeficiency virus 1 (HIV-1) viral load at pleural sites infected with Mycobacterium tuberculosis (MTB) than in peripheral blood has been documented. However, the cellular source of productive HIV infection in HIV-1/MTB-coinfected pleural fluid mononuclear cells (PFMCs) remains unclear. In this study, we observed significant quantities of HIV-1 p24 lymphocytes in PFMCs, but not in peripheral blood mononuclear cells (PBMCs). HIV-1 p24 lymphocytes were mostly enriched in DN T cells. Intracellular CD4 expression was detectable in HIV-1 p24 DN T cells. HIV-1 p24 DN T cells showed lower surface expression of human leukocyte antigen (HLA)-ABC and tetherin than did HIV-1 p24 CD4 T cells. Upon in vitro infection of PFMC CD4 T cells from TB mono-infected subjects, Nef- and/or Vpu-deleted HIV mutants showed lower generation of HIV-1 p24 DN T cells than the wild-type virus. These data indicate that productively HIV-1-infected DN T cells, generated through down-modulation of surface CD4, likely by HIV-1 Nef and Vpu, are the predominant source of HIV-1 at pleural sites of HIV/MTB coinfection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB) is the most frequent co-infection in HIV-infected individuals still presenting diagnostic difficulties particularly in developing countries. Recently an assay based on IFN-gamma response to M. tuberculosis RD1 peptides selected by computational analysis was developed whose presence is detected during active TB disease. Objective of this study was to investigate the response to selected RD1 peptides in HIV-1-infected subjects with or without active TB in a country endemic for TB and to evaluate the change of this response over time. Methods: 30 HIV-infected individuals were prospectively enrolled, 20 with active TB and 10 without. Among those with TB, 12 were followed over time. IFN-gamma response to selected RD1 peptides was evaluated by enzyme-linked immunospot (ELISPOT) assay. As control, response to RD1 proteins was included. Results were correlated with immune, microbiological and virological data. Results: Among patients with active TB, 2/20 were excluded from the analysis, one due to cell artifacts and the other to unresponsiveness to M. tuberculosis antigens. Among those analyzable, response to selected RD1 peptides evaluated as spot-forming cells was significantly higher in subjects with active TB compared to those without (p = 0.02). Among the 12 TB patients studied over time a significant decrease (p =< 0.007) of IFN-gamma response was found at completion of therapy when all the sputum cultures for M. tuberculosis were negative. A ratio of RD1 peptides ELISPOT counts over CD4+ T-cell counts greater than 0.21 yielded 100% sensitivity and 80% specificity for active TB. Conversely, response to RD1 intact proteins was not statistically different between subjects with or without TB at the time of recruitment; however a ratio of RD1 proteins ELISPOT counts over CD4+ T-cell counts greater than 0.22 yielded 89% sensitivity and 70% specificity for active TB. Conclusion: In this pilot study the response to selected RD1 peptides is associated with TB disease in HIV-infected individuals in a high TB endemic country. This response decreases after successful therapy. The potential of the novel approach of relating ELISPOT spot-forming cell number and CD4+ T-cell count may improve the possibility of diagnosing active TB and deserves further evaluation. [ABSTRACT FROM AUTHOR]

Background: WHO recommends gametocytocidal, single low-dose primaquine for blocking the transmission of Plasmodium falciparum; however, safety concerns have hampered the implementation of this strategy in sub-Saharan Africa. We aimed to investigate the safety of age-dosed, single low-dose primaquine in children from Uganda and the Democratic Republic of the Congo.Methods: We conducted this randomised, double-blind, placebo-controlled, non-inferiority trial at the Mbale Regional Referral Hospital, Mbale, Uganda, and the Kinshasa Mahidol Oxford Research Unit, Kinshasa, Democratic Republic of the Congo. Children aged between 6 months and 11 years with acute uncomplicated P falciparum infection and haemoglobin concentrations of at least 6 g/dL were enrolled. Patients were excluded if they had a comorbid illness requiring inpatient treatment, were taking haemolysing drugs for glucose-6-phosphate dehydrogenase (G6PD) deficiency, were allergic to the study drugs, or were enrolled in another clinical trial. G6PD status was defined by genotyping for the G6PD c.202T allele, the cause of the G6PD-deficient A- variant. Participants were randomly assigned (1:1) to receive single low-dose primaquine combined with either artemether-lumefantrine or dihydroartemisinin-piperaquine, dosed by bodyweight. Randomisation was stratified by age and G6PD status. The primary endpoint was the development of profound (haemoglobin <4 g/dL) or severe (haemoglobin <5 g/dL) anaemia with severity features, within 21 days of treatment. Analysis was by intention to treat. The sample size assumed an incidence of 1·5% in the placebo group and a 3% non-inferiority margin. The trial is registered at ISRCTN, 11594437, and is closed to new participants.Findings: Participants were recruited at the Mbale Regional Referral Hospital between Dec 18, 2017, and Oct 7, 2019, and at the Kinshasa Mahidol Oxford Research Unit between July 17, 2017, and Oct 5, 2019. 4620 patients were assessed for eligibility. 3483 participants were excluded, most owing to negative rapid diagnostic test or negative malaria slide (n=2982). 1137 children with a median age of 5 years were enrolled and randomly assigned (286 to the artemether-lumefantrine plus single low-dose primaquine group, 286 to the artemether-lumefantrine plus placebo group, 283 to the dihydroartemisinin-piperaquine plus single low-dose primaquine group, and 282 to the dihydroartemisinin-piperaquine plus placebo group). Genotyping of G6PD identified 239 G6PD-c.202T hemizygous males and 45 G6PD-c.202T homozygous females (defining the G6PD-deficient group), 119 heterozygous females, 418 G6PD-c.202C normal males and 299 G6PD-c.202C normal females (defining the non-G6PD-deficient group), and 17 children of unknown status. 67 patients were lost to follow-up and four patients withdrew during the study-these numbers were similar between groups. No participants developed profound anaemia and three developed severe anaemia: from the G6PD-deficient group, none (0%) of 133 patients who received placebo and one (0·66%) of 151 patients who received primaquine (difference -0·66%, 95% CI -1·96 to 0·63; p=0·35); and from the non-G6PD-deficient group, one (0·23%) of 430 patients who received placebo and one (0·25%) of 407 patients who received primaquine (-0·014%, -0·68 to 0·65; p=0·97).Interpretation: Gametocytocidal, age-dosed, single low-dose primaquine was well tolerated in children from Uganda and the Democratic Republic of the Congo who were infected with P falciparum, and the safety profile of this treatment was similar to that of the placebo. These data support the wider implementation of single low-dose primaquine in Africa.Funding: UK Government Department for International Development, UK Medical Research Council, UK National Institute for Health Research, and the Wellcome Trust Joint Global Health Trials Scheme. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB) is the most frequent co-infection in HIV-infected individuals still presenting diagnostic difficulties particularly in developing countries. Recently an assay based on IFN-gamma response to M. tuberculosis RD1 peptides selected by computational analysis was developed whose presence is detected during active TB disease. Objective of this study was to investigate the response to selected RD1 peptides in HIV-1-infected subjects with or without active TB in a country endemic for TB and to evaluate the change of this response over time.Methods: 30 HIV-infected individuals were prospectively enrolled, 20 with active TB and 10 without. Among those with TB, 12 were followed over time. IFN-gamma response to selected RD1 peptides was evaluated by enzyme-linked immunospot (ELISPOT) assay. As control, response to RD1 proteins was included. Results were correlated with immune, microbiological and virological data.Results: Among patients with active TB, 2/20 were excluded from the analysis, one due to cell artifacts and the other to unresponsiveness to M. tuberculosis antigens. Among those analyzable, response to selected RD1 peptides evaluated as spot-forming cells was significantly higher in subjects with active TB compared to those without (p = 0.02). Among the 12 TB patients studied over time a significant decrease (p =< 0.007) of IFN-gamma response was found at completion of therapy when all the sputum cultures for M. tuberculosis were negative. A ratio of RD1 peptides ELISPOT counts over CD4+ T-cell counts greater than 0.21 yielded 100% sensitivity and 80% specificity for active TB. Conversely, response to RD1 intact proteins was not statistically different between subjects with or without TB at the time of recruitment; however a ratio of RD1 proteins ELISPOT counts over CD4+ T-cell counts greater than 0.22 yielded 89% sensitivity and 70% specificity for active TB.Conclusion: In this pilot study the response to selected RD1 peptides is associated with TB disease in HIV-infected individuals in a high TB endemic country. This response decreases after successful therapy. The potential of the novel approach of relating ELISPOT spot-forming cell number and CD4+ T-cell count may improve the possibility of diagnosing active TB and deserves further evaluation. [ABSTRACT FROM AUTHOR]

Background: CD4 T-cells expressing Foxp3 are expanded systemically during active tuberculosis (TB) regardless of HIV-1 co-infection. Foxp3 + CD4 T cells are targets of HIV-1 infection. However, expansion of HIV-1 infected Foxp3 + CD4 T cells at sites of HIV/TB co-infection, and whether they contribute to promotion of HIV-1 viral activity is not known., Methods: Pleural fluid mononuclear cells (PFMC) from HIV/TB co-infected patients with pleural TB were characterized by immune-staining and FACS analysis for surface markers CD4, CD127, CCR5, CXCR4, HLA-DR and intracellular expression of Foxp3, HIVp24, IFN-γ and Bcl-2. Whole PFMC and bead separated CD4 + CD25 + CD127 - T cells were assessed for HIV-1 LTR strong stop (SS) DNA by real-time PCR, which represents viral DNA post cell entry and initiation of reverse transcription., Results: High numbers of HIV-1 p24 positive Foxp3 + and Foxp3 + CD127 - CD4 T cells were identified in PFMC from HIV/TB co-infected subjects. CD4 + Foxp3 + CD127 - T cells displayed high expression of the cellular activation marker, HLA-DR. Further, expression of the HIV-1 co-receptors, CCR5 and CXCR4, were higher on CD4 + Foxp3 + T cells compared to CD4 + Foxp3 - T cells. Purified CD4 + CD25 + CD127 - T cells isolated from PFMC of HIV/TB co-infected patients, were over 90% CD4 + Foxp3 + T cells, and exhibited higher HIV-1 SS DNA as compared to whole PFMC, and as compared to CD4 + CD25 + CD127 - T cells from an HIV-infected subject with pleural mesothelioma. HIV-1 p24 + Foxp3 + CD4 + T cells from HIV/TB patients higher in Bcl-2 expression as compared to both HIV-1 p24 + Foxp3 - CD4 T cells, and Foxp3 + CD4 + T cells without HIV-p24 expression., Conclusion: Foxp3 + CD4 T cells in PFMC from HIV/TB co-infected subjects are predisposed to productive HIV-1 infection and have survival advantage as compared to Foxp3 negative CD4 T cells., Competing Interests: None of the authors have a commercial or other association that might pose a conflict of interest.

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.

At sites of Mycobacterium tuberculosis (MTB) infection, HIV-1 replication is increased during tuberculosis (TB). Here we investigated the role of positive transcription elongation factor (P-TEFb), comprised of CycT1 and CDK9, as the cellular cofactor of HIV-1 Tat protein in transcriptional activation of HIV-1 in mononuclear cells from HIV-1-infected patients with pleural TB. Expression of CycT1 in response to MTB was assessed in mononuclear cells from pleural fluid (PFMC) and blood (PBMC) from HIV/TB patients with pleural TB, and in blood monocytes (MN) from singly infected HIV-1-seropositive subjects. We then examined whether the CDK9 inhibitor, Indirubin 3'-monoxime (IM), was effective in inhibition of MTB-induced HIV-1 mRNA expression. We found higher expression of CycT1 mRNA in PFMCs as compared to PBMCs from HIV/TB-coinfected subjects. MTB induced the expression of CycT1 and HIV-1 gag/pol mRNA in both PFMCs from HIV/TB subjects and MN from HIV-1-infected subjects. CycT1 protein was also induced by MTB stimulation in PFMCs from HIV/TB patients, and both MN and in vitro-derived macrophages. Inhibition of CDK9 by IM in both PFMCs from HIV/TB and MN from HIV-1-infected subjects in response to MTB led to inhibition of HIV-1 mRNA expression. These data imply that IM may be useful as an adjunctive therapy in control of HIV-1 replication in HIV/TB dually infected subjects.

An in vitro mononuclear cell system to model the microenvironment of coinfection with HIV-1 and Mycobacterium tuberculosis (MTB) was developed. This cellular system was used to assess the interaction of MTB-infected monocytes and T cells from dually infected HIV-1/TB patients with pulmonary tuberculosis (TB). Subjects with higher induction of HIV-1 gag/pol mRNA expression after MTB stimulation had increased MTB-specific T cell IFN-gamma and TNF-alpha production. Lack of HIV-1 mRNA induction did not correlate with increased induction of regulatory T cells (T-reg) as measured by MTB-induced Foxp3 mRNA. HIV-1 induction did not significantly correlate with clinical parameters including plasma HIV-1 viral load or CD4(+) T cell count. These data model MTB-induced HIV-1 replication at the microenvironment of MTB reactivation/infection. The data suggest that the magnitude of MTB-specific T cell responses drives local viral pathogenesis regardless of the stage of HIV-1 disease as reflected by plasma viral load or CD4(+) T cell count.

Tuberculosis (TB) is the most common life-threatening infection in human immunodeficiency virus (HIV)-infected persons and frequently occurs before the onset of severe immunodeficiency. Development of TB is associated with increased HIV type 1 (HIV-1) viral load, a fall in CD4 lymphocyte counts, and increased mortality. The aim of this study was to examine how treatment of pulmonary TB affected HIV-1 activity in HIV-1/TB-coinfected subjects with CD4 cell counts of >100 cells/mul. HIV-1/TB-coinfected subjects were recruited in Kampala, Uganda, and were monitored over time. Based upon a significant (0.5 log10 copies/ml) decrease in viral load by the end of treatment, two patient groups could be distinguished. Responders (n = 17) had more rapid resolution of anemia and pulmonary lesions on chest radiography during TB treatment. This group had a significant increase in viral load to levels not different from those at baseline 6 months after completion of TB treatment. HIV-1 viral load in nonresponders (n = 10) with TB treatment increased and at the 6 month follow-up was significantly higher than that at the time of diagnosis of TB. Compared to baseline levels, serum markers of macrophage activation including soluble CD14 decreased significantly by the end of TB treatment in responders but not in nonresponders. These data further define the impact of pulmonary TB on HIV-1 disease. HIV-1 replication during dual HIV-1/TB infection is not amenable to virologic control by treatment of TB alone. Concurrent institution of highly active antiretroviral treatment needs to be evaluated in patients dually infected with pulmonary TB and HIV-1.