Kathryn Wood

Enzyme-Linked Immunospot Assay Responses to Early Secretory Antigenic Target 6, Culture Filtrate Protein 10, and Purified Protein Derivative among Children with Tuberculosis: Implications for Diagnosis and Monitoring of Therapy

BACKGROUNDnThe ability to detect tuberculosis-specific lymphocytes by enzyme-linked immunospot (ELISPOT) assay may have important implications for the diagnosis and monitoring of tuberculosis in children, for which routine methods lack sensitivity. We conducted a study to determine the presence and time course of ELISPOT responses in children with tuberculosis.nnnMETHODSnBlood samples were obtained from children with a clinical diagnosis of tuberculosis, and interferon-gamma ELISPOT assays were performed using purified protein derivative (PPD), early secretory antigenic target 6 (ESAT-6), and culture filtrate protein 10 (CFP10) as stimulants. A subset of children were retested after 1, 3, and 6 months of therapy.nnnRESULTSnDetectable responses to ESAT-6 or CFP10 were found in 49 of 70 children with clinical tuberculosis but were more frequently found in those with culture-proven disease (P = .05). The number of subjects with responses to PPD increased after 1 month of therapy (P = .0004) and decreased at 3 and 6 months.nnnCONCLUSIONnTuberculosis-specific ELISPOT testing is a promising tool that should be evaluated as a potential diagnostic test for childhood tuberculosis. We caution against the use of an early decrease in response as a marker of successful antituberculous chemotherapy.

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 ≥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.

HIV-1 Infection Impairs the Bronchoalveolar T-Cell Response to Mycobacteria

RATIONALEnThe risk of developing active tuberculosis in persons with latent Mycobacterium tuberculosis infection is substantially increased shortly after HIV-1 seroconversion. Immune responses in the lung are important to restrict the growth of M. tuberculosis to prevent the development of disease.nnnOBJECTIVESnTo investigate innate and adaptive immune responses to M. tuberculosis in bronchoalveolar lavage from HIV-1-infected persons without active tuberculosis.nnnMETHODSnPeripheral blood was drawn and bronchoalveolar lavage (BAL) performed on healthy, HIV-1-uninfected (n = 21) and HIV-1-infected (n = 15) adults. Growth of M. tuberculosis was assessed in monocytes and alveolar macrophages. Cytokine expression by mycobacteria-specific CD4 and CD8 T cells was measured by intracellular cytokine staining or IFN-gamma ELISpot.nnnMEASUREMENTS AND MAIN RESULTSnMycobacterial growth in monocytes or alveolar macrophages from HIV-1-infected and -uninfected persons did not differ. Total CD4 T-cell frequencies in BAL were lower in HIV-1-infected than in HIV-1-uninfected persons (P < 0.001). Mycobacteria (bacillus Calmette-Guérin)-specific CD4 T-cell responses in BAL were severely impaired: Frequencies of cells expressing IFN-gamma or tumor necrosis factor (TNF)-alpha, as well as polyfunctional cells, expressing IFN-gamma, TNF-alpha, and IL-2 together, were lower in HIV-1-infected persons than in uninfected controls (P < 0.01 for all).nnnCONCLUSIONSnIn addition to a total CD4 T-cell deficit, the function of mycobacteria-specific CD4 T cells is significantly impaired in the lung of HIV-1-infected persons, which may account for the HIV-1-associated elevated risk for developing tuberculosis.

High prevalence of subclinical tuberculosis in HIV-1-infected persons without advanced immunodeficiency: implications for TB screening

Background The prevalence of asymptomatic tuberculosis (TB) in recently diagnosed HIV-1-infected persons attending pre-antiretroviral therapy (ART) clinics is not well described. In addition, it is unclear if the detection of Mycobacterium tuberculosis in these patients clearly represents an early asymptomatic phase leading to progressive disease or transient excretion of bacilli. Objective To describe the prevalence and outcome of subclinical TB disease in HIV-1-infected persons not eligible for ART. Methods The study was conducted in 274 asymptomatic ART-naïve HIV-1-infected persons in Khayelitsha Day Hospital, Cape Town, South Africa. All participants were screened for TB using a symptom screen and spoligotyping was performed to determine genotypes. Results The prevalence of subclinical TB disease was 8.5% (95% CI 5.1% to 13.0%) (n=18; median days to culture positivity 17u2005days), with 22% of patients being smear-positive. Spoligotyping showed a diverse variety of genotypes with all paired isolates being of the same spoligotype, effectively excluding cross-contamination. 56% of patients followed up developed symptoms 3u2005days to 2u2005months later. All were well and still in care 6–12u2005months after TB diagnosis; 60% were started on ART. A positive tuberculin skin test (OR 4.96, p=0.064), low CD4 count (OR 0.996, p=0.06) and number of years since HIV diagnosis (OR 1.006, p=0.056) showed trends towards predicting TB disease. Conclusion This study found a high prevalence but good outcome (retained in care) of subclinical TB disease in HIV-1-infected persons. The results suggest that, in high HIV/TB endemic settings, a positive HIV-1 test should prompt TB screening by sputum culture irrespective of symptoms, particularly in those with a positive tuberculin skin test, longer history of HIV infection and low CD4 count. Operational difficulties in resource-constrained settings with respect to screening with TB culture highlight the need for rapid and affordable point-of-care tests to identify persons with clinical and subclinical TB disease.

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-γ 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-γ 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.

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

BackgroundRecent interest has focused on the potential use of serial interferon gamma (IFN-γ) release assay (IGRA) measurements to assess the response to anti-tuberculous (TB) treatment. The kinetics of IFN-γ responses to Mycobacterium tuberculosis (MTB) antigens in HIV-infected children during treatment have not however been previously investigated.MethodsIFN-γ responses to the MTB antigens, ESAT-6, CFP-10 and PPD were measured by an enzyme-linked immunospot assay (IFN-γ 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 Fishers exact or Chi-squared test was used to compare proportions when test results were analysed as dichotomous outcomes.ResultsOf 102 children with suspected TB, 22 (21%) had definite TB and 24 (23%) probable TB. At least one follow up IFN-γ ELISpot assay result was available for 31 (67%) of the 46 children. In children with definite or probable TB in whom the IFN-γ ELISpot assay result was positive at presentation, anti-tuberculous treatment was accompanied by a significant decrease in both the magnitude of the IFN-γ 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-γ ELISpot assay (Fishers 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-γ ELISpot assay after six months of anti-tuberculous treatment. In addition, there was conversion of the IFN-γ ELISpot assay result during anti-tuberculous therapy in six of 12 children in whom the initial IFN-γ ELISpot assay was negative.ConclusionsIn HIV-infected children with definite or probable TB, anti-tuberculosis treatment is accompanied by a reduction in the magnitude of the IFN-γ ELISpot response to MTB-antigens. However, serial IFN-γ ELISpot measurements appear to have limited clinical utility in assessing a successful response to anti-tuberculous treatment in HIV infected children.

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.

Enhanced diagnosis of HIV-1-associated tuberculosis by relating T-SPOT.TB and CD4 counts.

The sensitivity of the tuberculin skin test is impaired in HIV-1-infected persons. Enzyme-linked immunospot-based detection of immune sensitisation may be less affected. Furthermore, the quantitative response can be related to the CD4 count, potentially improving specificity for active disease. The T-SPOT.TB assay was performed on HIV-1-infected participants, 85 with active tuberculosis (TB) and 81 healthy patients (non-TB). The ratio of the sum of the 6-kDa early secretory antigenic target and culture filtrate protein 10 response to the CD4 count (spot-forming cell (SFC)/CD4) was calculated. Using the manufacturers guidelines, active TB was diagnosed with 76% sensitivity and 53% specificity. Using an SFC/CD4 ratio of 0.12, sensitivity (80%) and specificity (62%) improved. The quantitative T-cell response increased with increasing smear-positivity in the active TB group (p = 0.0008). In the non-TB group, the proportion of persons scored positive by T-SPOT.TB assay was lower in the group with a CD4 count of <200 cells·mm−3 (p = 0.029). The ratio of the summed T-cell response to CD4 count improved the diagnostic accuracy of the T-SPOT.TB assay in HIV-1-infected persons, and a ratio of SFC/CD4 of >0.12 should prompt investigation for active disease. A strong association between the degree of sputum positivity and T-SPOT.TB score was found. The sensitivity of the T-SPOT.TB assay in active disease may be less impaired by advanced immunosuppression.

Risk Factors Associated with Indeterminate Gamma Interferon Responses in the Assessment of Latent Tuberculosis Infection in a High-Incidence Environment

ABSTRACT The performance of gamma interferon (IFN-γ) release assays (IGRA) in the detection of latent tuberculosis (TB) infection is limited by the higher rates of indeterminate results in HIV-infected persons, who bear the brunt of TB disease in some high-burden settings. The objective of the study was to evaluate predictors of indeterminate IGRA results in the overall study population and in HIV-infected persons. The study setting is Khayelitsha, an informal township in the Western Cape of South Africa, with a high burden of TB and HIV infection. A total of 561 asymptomatic persons were recruited from the day hospital and youth centers. A questionnaire was used to collect demographic information, and blood tests, including CD4 counting and a 7-day in-house IGRA, were performed. The overall prevalence of indeterminate IGRA results was 8.6% (48/561), and this was higher in HIV-infected than in HIV-uninfected persons (11.5% [38/330] versus 4.3% [10/231], respectively; P = 0.003). In the overall study population, predictors of indeterminate IGRA results were the presence of HIV infection (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.10 to 5.08) and the presence of a Mycobacterium bovis BCG scar (OR, 2.48; 95% CI, 1.23 to 5.01). Long-term township residents were significantly less likely to have indeterminate results than recent migrants (OR, 0.30; 95% CI, 0.11 to 0.80). Among HIV-infected persons, participants with CD4 counts of >200 cells/mm3 and long-term residents were significantly less likely to have indeterminate IGRA results (OR of 0.21 with a 95% CI of 0.09 to 0.48 and OR of 0.22 with a 95% CI of 0.07 to 0.68, respectively). We evaluated risk factors for indeterminate IGRA results and report a higher rate of indeterminate results among HIV-infected persons, particularly those with lower CD4 counts. Of note, a recent move to the township was associated with a higher risk of indeterminate IGRA results.

Smoking, BCG and Employment and the Risk of Tuberculosis Infection in HIV-Infected Persons in South Africa

Background The increased susceptibility to latent tuberculosis infection (LTBI) of HIV-1-infected persons represents a challenge in TB epidemic control. However few studies have evaluated LTBI predictors in a generalized HIV/TB epidemic setting. Methods The study recruited 335 HIV-infected participants from Khayelitsha, Cape Town between February 2008 and November 2010. Tuberculin skin tests and interferon-gamma release assays were performed on all participants and active TB excluded using a symptom screen, TB microscopy and culture. Results LTBI prevalence was 52.7% and 61.2% (TST and IGRA respectively). Being a recent TB contact (OR 2.07; 95% C.I. 1.15–3.69) was associated with TST positivity. Participants with a CD4>200 had a two-fold higher risk of IGRA positivity compared to those with CD4 counts <200 (OR 2.07; 95% C.I. 0.99–4.34). There was also a 19% increase in IGRA positivity risk for every additional year of schooling and a strong association between years of schooling and employment (pu200a=u200a0.0004). A decreased risk of IGRA positivity was observed in persons with a BCG scar (OR 0.46; 95% C.I. 0.31–0.69) and in smokers (OR 0.47; 95% C.I. 0.23–0.96). Conclusion We report the novel findings of a decreased risk of IGRA positivity in HIV-infected smokers possibly due to decreased interferon production, and in the persons with a BCG scar suggesting a protective role for BCG in this population. We also found an increased risk of TST positivity in employed persons, possibly due to ongoing transmission in public modes of transport.