Harriet Mayanja-Kizza

Genome Scan of M. tuberculosis Infection and Disease in Ugandans

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is an enduring public health problem globally, particularly in sub-Saharan Africa. Several studies have suggested a role for host genetic susceptibility in increased risk for TB but results across studies have been equivocal. As part of a household contact study of Mtb infection and disease in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB, by studying three phenotypes. First, we analyzed culture confirmed TB disease compared to latent Mtb infection (LTBI) or lack of Mtb infection. Second, we analyzed resistance to Mtb infection in the face of continuous exposure, defined by a persistently negative tuberculin skin test (PTST-); this outcome was contrasted to LTBI. Third, we analyzed an intermediate phenotype, tumor necrosis factor-alpha (TNFα) expression in response to soluble Mtb ligands enriched with molecules secreted from Mtb (culture filtrate). We conducted a full microsatellite genome scan, using genotypes generated by the Center for Medical Genetics at Marshfield. Multipoint model-free linkage analysis was conducted using an extension of the Haseman-Elston regression model that includes half sibling pairs, and HIV status was included as a covariate in the model. The analysis included 803 individuals from 193 pedigrees, comprising 258 full sibling pairs and 175 half sibling pairs. Suggestive linkage (p<10−3) was observed on chromosomes 2q21-2q24 and 5p13-5q22 for PTST-, and on chromosome 7p22-7p21 for TB; these findings for PTST- are novel and the chromosome 7 region contains the IL6 gene. In addition, we replicated recent linkage findings on chromosome 20q13 for TB (pu200a=u200a0.002). We also observed linkage at the nominal αu200a=u200a0.05 threshold to a number of promising candidate genes, SLC11A1 (PTST- pu200a=u200a0.02), IL-1 complex (TB pu200a=u200a0.01), IL12BR2 (TNFα pu200a=u200a0.006), IL12A (TB pu200a=u200a0.02) and IFNGR2 (TNFα pu200a=u200a0.002). These results confirm not only that genetic factors influence the interaction between humans and Mtb but more importantly that they differ according to the outcome of that interaction: exposure but no infection, infection without progression to disease, or progression of infection to disease. Many of the genetic factors for each of these stages are part of the innate immune system.

Response to M. tuberculosis selected RD1 peptides in Ugandan HIV-infected patients with smear positive pulmonary tuberculosis: a pilot study

BackgroundTuberculosis (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.Methods30 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.ResultsAmong 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.ConclusionIn 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.

Whole blood interferon-gamma responses to mycobacterium tuberculosis antigens in young household contacts of persons with tuberculosis in Uganda.

Background Due to immunologic immaturity, IFN-γ-producing T cell responses may be decreased in young children compared to adults, thus we hypothesized that IFN-γ responses to mycobacterial antigens in household contacts exposed to Mycobacterium tuberculosis (Mtb) would be impaired in young children relative to adults. The objective of this study was to compare whole blood IFN-γ production in response to mycobacterial antigens between children and adults in Uganda. Methodology/Principal Findings We studied household contacts of persons with culture-positive pulmonary tuberculosis (TB) enrolled in a cohort study conducted in Kampala, Uganda. Whole blood IFN-γ production in response to Mtb culture-filtrate antigens was measured by ELISA and compared between infants (<2 years old, nu200a=u200a80), young children (2 <5 years old, nu200a=u200a216), older children (5 <15 years old, nu200a=u200a443) and adults (≥15 years old, nu200a=u200a528). We evaluated the relationship between IFN-γ responses and the tuberculin skin test (TST), and between IFN-γ responses and epidemiologic factors that reflect exposure to Mtb, and the effect of prior BCG vaccination on IFN-γ responses. Young household contacts demonstrated robust IFN-γ responses comparable to those of adults that were associated with TST and known risk factors for infection. There was no effect of prior BCG immunization on the IFN-γ response. Conclusions/Significance Young children in a TB endemic setting can mount robust IFN-γ responses generally comparable to those of adults, and as in adults, these responses correlated with the TST and known epidemiologic risk factors for Mtb infection.

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

RATIONALEnThe 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.nnnOBJECTIVESnTo determine if M. tuberculosis-specific T cells are generated among healthy children exposed to M. tuberculosis and children with tuberculosis.nnnMETHODSnEnzyme-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).nnnMEASUREMENTS AND MAIN RESULTSnThe 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.nnnCONCLUSIONSnAmong 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.

Contact Investigation for Active Tuberculosis Among Child Contacts in Uganda

BACKGROUNDnTuberculosis is a large source of morbidity and mortality among children. However, limited studies characterize childhood tuberculosis disease, and contact investigation is rarely implemented in high-burden settings. In one of the largest pediatric tuberculosis contact investigation studies in a resource-limited setting, we assessed the yield of contact tracing on childhood tuberculosis and indicators for disease progression in Uganda.nnnMETHODSnChild contacts aged <15 years in Kampala, Uganda, were enrolled from July 2002 to June 2009 and evaluated for tuberculosis disease via clinical, radiographic, and laboratory methods for up to 24 months.nnnRESULTSnSeven hundred sixty-one child contacts were included in the analysis. Prevalence of tuberculosis in our child population was 10%, of which 71% were culture-confirmed positive. There were no cases of disseminated tuberculosis, and 483 of 490 children (99%) started on isoniazid preventative therapy did not develop disease. Multivariable testing suggested risk factors including human immunodeficiency virus (HIV) status (odds ratio [OR], 7.90; P < .001), and baseline positive tuberculin skin test (OR, 2.21; P = .03); BCG vaccination was particularly protective, especially among children aged ≤5 years (OR, 0.23; P < .001). Adult index characteristics such as sex, HIV status, and extent or severity of disease were not associated with childhood disease.nnnCONCLUSIONSnContact tracing for children in high-burden settings is able to identify a large percentage of culture-confirmed positive tuberculosis cases before dissemination of disease, while suggesting factors for disease progression to identify who may benefit from targeted screening.

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 (nu200a=u200a50) 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 (pu200a=u200a0.023). 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.

Inhibition of human immunodeficiency virus-1 (HIV-1) by β-chemokine analogues in mononuclear cells from HIV-1-infected patients with active tuberculosis

Tuberculosis (TB) enhances human immunodeficiency virus‐1 (HIV‐1) activity in patients with dual HIV‐1/TB infection. Therapies that control augmentations of HIV‐1 activity at sites of Mycobacterium tuberculosis (MTB) infection may be useful in inhibition of viral expansion. Regulated upon activation, normal T‐cell expressed and secreted (RANTES) analogues (AOP and NNY) are potent in inhibiting the entry of primary HIV‐1 isolates into host mononuclear cells. These analogues were used to inhibit MTB‐induced HIV‐1 entry in blood monunuclear cells (PBMC) from patients with pulmonary TB, and pleural fluid mononuclear cells (PFMC) from patients with pleural TB. PBMC or PFMC were cultured with and without MTB in presence and absence of RANTES analogues. HIV‐1 strong stop DNA was assessed by real‐time polymerase chain reaction (PCR) as a measure of infection. CCR5 mRNA was assessed by real‐time reverse transcription (RT)‐PCR and by immunostaining and FACS analysis. HIV‐1 infection was induced by MTB in vitro in PBMC from the majority (14 of 20) of HIV‐1/TB subjects, and new infection was inhibited by AOP‐ or NNY‐RANTES. HIV‐1 infection was also inhibited by these reagents in MTB‐induced PFMC from three of three patients with pleural TB. Expression of CCR5 mRNA was significantly induced by MTB in PBMC from patients with pulmonary TB. Further, expression of CCR5 was higher in PFMC compared to PBMC from patients with pleural TB. Also, CCR5 was fourfold higher on CD14+ pleural mononuclear cells than on CD4+ lymphocytes. Blocking new HIV‐1 infection of mononuclear cells may be useful in control of HIV‐1 during dual HIV‐1/TB infection.

Systemic immune activation and microbial translocation in dual HIV/tuberculosis-infected subjects.

BACKGROUNDnSystemic immune activation is a strong predictor of progression of human immunodeficiency virus type 1 (HIV-1) disease and a prominent feature of infection with Mycobacterium tuberculosis.nnnOBJECTIVEnTo understand the role of systemic immune activation and microbial translocation in HIV/tuberculosis dually infected patients over the full spectrum of HIV-1 immunodeficiency, we studied circulating sCD14 and lipopolysaccharide (LPS) and their relationship to HIV-1 activity.nnnMETHODSnTwo cohorts of HIV/tuberculosis subjects defined by CD4 T-cell count at time of diagnosis of tuberculosis were studied: those with low (<350/μL) and those with high (≥ 350/μL) CD4 T-cell count. Circulating soluble CD14 (sCD14) and LPS were assessed.nnnRESULTSnLevels of sCD14 were higher in HIV/tuberculosis with high (≥ 350/μL) as compared to low CD4 T-cell count (P < .001). Whereas sCD14 levels remained elevated in HIV/tuberculosis subjects with lower CD4 T-cell counts despite treatment of tuberculosis, in HIV/tuberculosis patients with higher CD4 T-cell count (≥ 350/μL), levels declined regardless of whether highly active antiretroviral therapy (HAART) was included with the anti-tuberculosis regimen. Circulating LPS levels in HIV/tuberculosis patients with CD4 T-cell count ≥ 350/μL were unaffected by treatment of tuberculosis with or without HAART.nnnCONCLUSIONnDuring HIV/tuberculosis, systemic immune activation is dissociated from microbial translocation. Changes in circulating sCD14 and LPS are dependent on CD4 T-cell count.

The interaction of monocyte chemoattractant protein-1 and tumour necrosis factor-α in mycobacterium tuberculosis-induced HIV-1 replication at sites of active tuberculosis

Tuberculosis (TB) is a prominent opportunistic infection in HIV‐1‐infected subjects and enhances HIV‐1 replication. TB is associated with excess monocyte chemoattractant protein (MCP)‐1 and tumour necrosis factor (TNF)‐α activity in situ, both of which are implicated in transcriptional activation of HIV‐1. The role of MCP‐1 and TNF‐α in activation of HIV‐1 during TB and by Mycobacterium tuberculosis (MTB) in mononuclear cells from HIV‐1/TB subjects with pleural TB was examined here. Extremely high levels of MCP‐1 (as compared with TNF‐α) protein and mRNA were found in pleural fluid and pleural fluid mononuclear cells. Levels of MCP‐1 mRNA were sustained during in vitro culture of pleural fluid mononuclear cells. Neutralization of MCP‐1 (but not TNF‐α), resulted in inhibition of MTB induced HIV‐1 gag/pol mRNA. Neutralization of both MCP‐1 and TNF‐α, however, abrogated the effect of anti‐MCP‐1 antibody on HIV‐1 mRNA. LMP‐420, a small molecular transcriptional inhibitor of both TNF‐α and MCP‐1 expression, did not reduce MTB‐induced HIV‐1 expression. These data imply that MCP‐1 activity may be critical to activation of HIV‐1 at sites of TB. An interplay of MCP‐1 and TNF‐α is also suggested.

Genetic susceptibility to tuberculosis associated with cathepsin Z haplotype in a Ugandan household contact study

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), causes 9 million new cases worldwide and 2 million deaths annually. Genetic linkage and association analyses have suggested several chromosomal regions and candidate genes involved in TB susceptibility. This study examines the association of TB disease susceptibility with a selection of biologically relevant genes on regions on chromosomes 7 (IL6 and CARD11) and 20 (CTSZ and MC3R) and fine mapping of the chromosome 7p22-p21 region identified through our genome scan. We analyzed 565 individuals from Kampala, Uganda, who were previously included in our genome-wide linkage scan. Association analyses were conducted for 1,417 single-nucleotide polymorphisms (SNP) that passed quality control. None of the candidate gene or fine mapping SNPs was significantly associated with TB susceptibility (p > 0.10). When we restricted the analysis to HIV-negative individuals, 2 SNPs on chromosome 7 were significantly associated with TB susceptibility (p < 0.05). Haplotype analyses identified a significant risk haplotype in cathepsin X (CTSZ; p = 0.0281, odds ratio = 1.5493, 95% confidence interval [1.039, 2.320]).