Reversion and conversion of Mycobacterium tuberculosis IFN-gamma ELISpot results during anti-tuberculous treatment in HIV-infected children.

BACKGROUND: Recent interest has focused on the potential use of serial interferon gamma (IFN-gamma) release assay (IGRA) measurements to assess the response to anti-tuberculous (TB) treatment. The kinetics of IFN-gamma responses to Mycobacterium tuberculosis (MTB) antigens in HIV-infected children during treatment have not however been previously investigated. METHODS: IFN-gamma responses to the MTB antigens, ESAT-6, CFP-10 and PPD were measured by an enzyme-linked immunospot assay (IFN-gamma ELISpot) at presentation and at one, two and six months after starting anti-tuberculous treatment in HIV-infected children with definite or probable TB. Responses at different time points were compared using a Mann-Whitney U test with paired data analysed using the Wilcoxon signed rank test. A Fisher's exact or Chi-squared test was used to compare proportions when test results were analysed as dichotomous outcomes. RESULTS: Of 102 children with suspected TB, 22 (21%) had definite TB and 24 (23%) probable TB. At least one follow up IFN-gamma ELISpot assay result was available for 31 (67%) of the 46 children. In children with definite or probable TB in whom the IFN-gamma ELISpot assay result was positive at presentation, anti-tuberculous treatment was accompanied by a significant decrease in both the magnitude of the IFN-gamma response to individual or combined MTB-specific antigens (ESAT-6 median 110 SFCs/106 PBMC (IQR 65-305) at presentation vs. 15 (10-115) at six months, p = 0.04; CFP-10 177 (48-508) vs. 20 (5-165), p = 0.004, ESAT-6 or CFP-10 median 250 SFCs/106 PBMC (IQR 94-508) vs. 25 (10-165), p = 0.004) and in the proportion of children with a positive IFN-gamma ELISpot assay (Fisher's exact test: ESAT-6 15/0 vs 5/11, p = 0.0002, CFP-10 22/0 vs 8/17, p = 0.0001, ESAT-6 or CFP-10 22/0 vs. 9/17, p= 0.002). However almost half of the children had a positive IFN-gamma ELISpot assay after six months of anti-tuberculous treatment. In addition, there was conversion of the IFN-gamma ELISpot assay result during anti-tuberculous therapy in six of 12 children in whom the initial IFN-gamma ELISpot assay was negative. CONCLUSIONS: In HIV-infected children with definite or probable TB, anti-tuberculosis treatment is accompanied by a reduction in the magnitude of the IFN-gamma ELISpot response to MTB-antigens. However, serial IFN-gamma ELISpot measurements appear to have limited clinical utility in assessing a successful response to anti-tuberculous treatment in HIV infected children.

Childhood tuberculosis is associated with decreased abundance of T cell gene transcripts and impaired T cell function.

The WHO estimates around a million children contract tuberculosis (TB) annually with over 80 000 deaths from dissemination of infection outside of the lungs. The insidious onset and association with skin test anergy suggests failure of the immune system to both recognise and respond to infection. To understand the immune mechanisms, we studied genome-wide whole blood RNA expression in children with TB meningitis (TBM). Findings were validated in a second cohort of children with TBM and pulmonary TB (PTB), and functional T-cell responses studied in a third cohort of children with TBM, other extrapulmonary TB (EPTB) and PTB. The predominant RNA transcriptional response in children with TBM was decreased abundance of multiple genes, with 140/204 (68%) of all differentially regulated genes showing reduced abundance compared to healthy controls. Findings were validated in a second cohort with concordance of the direction of differential expression in both TBM (r2 = 0.78 p = 2x10-16) and PTB patients (r2 = 0.71 p = 2x10-16) when compared to a second group of healthy controls. Although the direction of expression of these significant genes was similar in the PTB patients, the magnitude of differential transcript abundance was less in PTB than in TBM. The majority of genes were involved in activation of leucocytes (p = 2.67E-11) and T-cell receptor signalling (p = 6.56E-07). Less abundant gene expression in immune cells was associated with a functional defect in T-cell proliferation that recovered after full TB treatment (p<0.0003). Multiple genes involved in T-cell activation show decreased abundance in children with acute TB, who also have impaired functional T-cell responses. Our data suggest that childhood TB is associated with an acquired immune defect, potentially resulting in failure to contain the pathogen. Elucidation of the mechanism causing the immune paresis may identify new treatment and prevention strategies.

Validating the use of qualitative behavioral assessment as a measure of the welfare of sheep during transport.

We tested the application of qualitative behavioral assessment (QBA) as a welfare assessment tool. Sheep were exposed to road transport treatments, and behavioral expressions were compared between experimental treatments and validated by correlation with physiological measures. We compared journeys differing in ventilation (closed vs. open-sided trailer), flooring (grip vs. nongrip flooring), and driving styles (stop-start vs. continuous driving). Blood samples were collected immediately before loading and after unloading; heart rate and core body temperatures were recorded continuously. Continuous video footage was edited to show individual sheep to observers for QBA using free-choice profiling (observers used their own descriptive terms). There was significant consensus in observers' scores for the sheep in each experiment (p < .001). Observers distinguished between sheep exposed to flooring (p = .014) or driving-style (p = .005) treatments, but not between ventilation treatments. QBA scores were compared (p < .05) with plasma leptin, glucose, and insulin-like growth factor-1 concentrations; white blood cell profiles; red blood cell counts; hematocrit; body temperatures; and heart rate variability. Observer assessments reflected treatment differences, and correlations between behavioral expression and physiological responses were found.

Knowledge about preventing and managing lymphedema: a survey of recently diagnosed and treated breast cancer patients.

Physical therapists, books and oncologist's staff were the most frequently cited sources of lymphedema prevention and management information in a telephone survey of 148 recently treated breast cancer patients. Awareness, current practice and intention to practice 13 recommended prevention behaviors were low, and variations in these indices and in information sources were observed between those reporting any swelling and those who did not. Citing radiation oncologists as an information source reflected significantly higher levels of awareness, practice and intention as compared to not citing them. Also, citing oncologists reflected lower scores on the three indices as compared to not citing them. The results suggest that lymphedema prevention and management information is not getting to breast cancer survivors in a timely fashion and underscore the urgent need to develop and implement appropriate educational strategies. Additionally, research into factors that could motivate survivors to practice the recommended behaviors is warranted.

Breast cancer genomics: normal tissue and cancer markers.

Mammography is a powerful screening tool for early detection of breast cancer, but it has limitations in terms of both specificity and sensitivity. Imaging tools such as MRI that complement mammography are too costly to serve as first-line screens. Recently, progress has been made on blood markers, particularly microRNAs and proteins. There are new methods for protein marker discovery directly in blood, but they are limited in the number of patients that can be examined. An alternative is to discover markers as transcripts in tissues, followed by development of blood protein tests for those that perform best. To identify genes that are overexpressed in malignancy it is paramount to include normal control tissues from healthy individuals. Here we report the identification of potential breast cancer markers, including some that are overexpressed in aggressive disease.

Polymorphic variation in TIRAP is not associated with susceptibility to childhood TB but may determine susceptibility to TBM in some ethnic groups.

Host recognition of mycobacterial surface molecules occurs through toll like receptors (TLR) 2 and 6. The adaptor protein TIRAP mediates down stream signalling of TLR2 and 4, and polymorphisms in the TIRAP gene (TIRAP) have been associated with susceptibility and resistance to tuberculosis (TB) in adults. In order to investigate the role of polymorphic variation in TIRAP in childhood TB in South Africa, which has one of the highest TB incidence rates in the world, we screened the entire open reading frame of TIRAP for sequence variation in two cohorts of childhood TB from different ethnic groups (Xhosa and mixed ancestry). We identified 13 SNPs, including seven previously unreported, in the two cohorts, and found significant differences in frequency of the variants between the two ethnic groups. No differences in frequency between individual SNPs or combinations were found between TB cases and controls in either cohort. However the 558C-->T SNP previously associated with TB meningitis (TBM) in a Vietnamese population was found to be associated with TBM in the mixed ancestry group. Polymorphisms in TIRAP do not appear to be involved in childhood TB susceptibility in South Africa, but may play a role in determining occurrence of TBM.

Comparison of T-SPOT.TB assay and tuberculin skin test for the evaluation of young children at high risk for tuberculosis in a community setting.

OBJECTIVE: We wished to compare the sensitivity of an enzyme-linked immunospot assay (T-SPOT.TB; Oxford Immunotec, Oxford, United Kingdom) and the tuberculin skin test for the detection of tuberculosis infection in very young children being evaluated for active tuberculosis in a rural community setting. METHODS: Children with a history of exposure to tuberculosis and children presenting to a local clinic or hospital with symptoms suggesting tuberculosis were admitted to a dedicated case verification ward. T-SPOT.TB testing was performed, and children were evaluated with a clinical examination, a tuberculin skin test, chest radiographs, and cultures of induced sputum and gastric lavage specimens. The diagnosis was determined by using a clinical algorithm. RESULTS: A total of 243 children (median age: 18 months) were recruited, of whom 214 (88%) had interpretable T-SPOT.TB results. Children > or =12 months of age were more likely than younger children to have positive T-SPOT.TB results, whereas tuberculin skin test results were unaffected by age. The sensitivity of the T-SPOT.TB was no better than that of the tuberculin skin test for culture-confirmed tuberculosis (50% and 80%, respectively) and was poorer for the combined group of culture-confirmed and clinically probable tuberculosis (40% and 52%, respectively). For the 50 children clinically categorized as not having tuberculosis, the specificity of both the T-SPOT.TB and the tuberculin skin test was 84%. CONCLUSIONS: For young children presenting in a community setting after exposure to tuberculosis or with symptoms suggesting tuberculosis, T-SPOT.TB cannot be used to exclude active disease. The sensitivity of this assay may be impaired for very young children.

Detection of tuberculosis in HIV-infected children using an enzyme-linked immunospot assay.

OBJECTIVE: To evaluate an enzyme-linked immunospot assay (ELISPOT) for the diagnosis of tuberculosis (TB) in HIV-infected children with suspected TB and to compare the performance of ELISPOT with the tuberculin skin test (TST). METHODS: Interferon-gamma responses to Mycobacterium tuberculosis-specific antigens were measured by ELISPOT in HIV-infected children with suspected TB. HIV-infected and HIV-uninfected children without TB were taken for comparison. RESULTS: Results were available for 188 children, of whom 139 (74%) were HIV-infected. Of these, 22 were classified as having definite TB: 24 probable TB, 14 possible TB and 128 not having TB. The median (range) age of patients was 20 (10-54.1) months. Median interferon-gamma responses to early-secreted antigenic target-6 and culture filtrate protein-10 were higher in children with definite or probable TB compared with children without TB (P < 0.002). In HIV-infected children with an interpretable ELISPOT result, the ELISPOT was positive in 14/21 (66%) with definite TB. A significantly higher proportion of HIV-infected children with definite or probable TB had a positive ELISPOT compared with a positive TST [25/39 (64%) vs. 10/34 (29%), P = 0.005]. In contrast to TST, results from ELISPOT were not affected by young age or severe immunosuppression. In HIV-infected children without active TB disease, 27% had a positive ELISPOT, suggesting latent TB infection. CONCLUSION: ELISPOT is more sensitive than TST for the detection of active TB in HIV-infected children. However, the sensitivity of current ELISPOT assays is not sufficiently high to be used as a rule out test for TB.

Enhanced anti-mycobacterial immunity in children with erythema nodosum and a positive tuberculin skin test.

Erythema nodosum (EN) may follow a variety of infections, but in regions with a high prevalence of tuberculosis, is frequently associated with a positive tuberculin skin test (TST) and tuberculosis infection. We aimed to investigate the immunological differences between patients with EN as a manifestation of primary tuberculosis, and those with progressive pulmonary tuberculosis (PTB) or asymptomatic infection. We studied the inflammatory response to both mycobacterial and non-mycobacterial antigens in 11 children with EN associated with a positive TST, 22 children with culture-confirmed tuberculosis, and 53 healthy skin test-positive children. In addition, we evaluated functional anti-mycobacterial immunity using an ex vivo assay of mycobacterial growth restriction in five children with EN and 15 with PTB. Patients with EN were distinguished by enhanced mycobacterial growth restriction on the functional assay, which was associated with a markedly increased production of IFNgamma in response to stimulation with purified protein derivative of Mycobacterium tuberculosis. Children presenting with EN and a positive TST show evidence of responses associated with enhanced anti-mycobacterial immunity.