Tom G Connell

Performance of a whole blood interferon gamma assay for detecting latent infection with Mycobacterium tuberculosis in children

Background: The diagnosis of latent Mycobacteriumtuberculosis (MTB) infection with a tuberculin skin test (TST) in children is complicated by the potential influence of prior exposure to Bacille Calmette Geurin (BCG) vaccination or environmental mycobacteria. A whole blood assay has recently been developed to quantitatively measure interferon gamma (IFN-γ) production by lymphocytes specific to the MTB antigens ESAT-6 and CFP-10, but its use and assessment in children has been limited. A study was undertaken to compare the performance of the whole blood IFN-γ assay with the TST in diagnosing latent tuberculosis (TB) infection or TB disease in children in routine clinical practice. Methods: One hundred and six children with a high risk of latent TB infection or TB disease were enrolled in the study. High risk was defined as contact with TB disease, clinical suspicion of TB disease, or recent arrival from an area of high TB prevalence. The whole blood IFN-γ assay was undertaken in 101 children. Results: Seventeen (17%) of the 101 assays yielded inconclusive results due to failure of positive or negative control assays. There was poor correlation between the whole blood IFN-γ assay and the TST (kappa statistic 0.3) with 26 (70%) of the 37 children defined as latent TB infection by TST having a negative whole blood IFN-γ assay. There were no instances of a positive whole blood IFN-γ assay with a negative TST. Mitogen (positive) control IFN-γ responses were significantly correlated with age (Spearman’s coefficient = 0.53, p<0.001) and, in children with latent TB infection identified by TST, those with a positive IFN-γ assay were older (median 12.9 v 6.92 years, respectively, p = 0.007). The whole blood IFN-γ assay was positive in all nine children with TB disease. Conclusion: There was poor agreement between the whole blood IFN-γ assay and TST for the diagnosis of latent TB. The whole blood IFN-γ assay may have lower sensitivity than the TST in diagnosing TB infection in children. A significant proportion of whole blood IFN-γ assays fail when used as a screening assay in routine practice.

A three-way comparison of tuberculin skin testing, QuantiFERON-TB gold and T-SPOT.TB in children.

Background There are limited data comparing the performance of the two commercially available interferon gamma (IFN-γ) release assays (IGRAs) for the diagnosis of tuberculosis (TB) in children. We compared QuantiFERON-TB gold In Tube (QFT-IT), T-SPOT.TB and the tuberculin skin test (TST) in children at risk for latent TB infection or TB disease. Methods and Findings The results of both IGRAs were compared with diagnosis assigned by TST-based criteria and assessed in relation to TB contact history. Results from the TST and at least one assay were available for 96 of 100 children. Agreement between QFT-IT and T-SPOT.TB was high (93% agreement, κ = 0.83). QFT-IT and T-SPOT.TB tests were positive in 8 (89%) and 9 (100%) children with suspected active TB disease. There was moderate agreement between TST and either QFT-IT (75%, κ = 0.50) or T-SPOT.TB (75%, κ = 0.51). Among 38 children with TST-defined latent TB infection, QFT-IT gold and T-SPOT.TB assays were positive in 47% and 39% respectively. Three TST-negative children were positive by at least one IGRA. Children with a TB contact were more likely than children without a TB contact to have a positive IGRA (QFT-IT LR 3.9; T-SPOT.TB LR 3.9) and a positive TST (LR 1.4). Multivariate linear regression analysis showed that the magnitude of both TST induration and IGRA IFN-γ responses was significantly influenced by TB contact history, but only the TST was influenced by age. Conclusions Although a high level of agreement between the IGRAs was observed, they are commonly discordant with the TST. The correct interpretation of a negative assay in a child with a positive skin test in clinical practice remains challenging and highlights the need for longitudinal studies to determine the negative predictive value of IGRAs.

Diagnosis of pulmonary tuberculosis in children: new advances.

The global burden of childhood pulmonary TB has been underappreciated, in part due to difficulties in obtaining microbiological confirmation of disease. Most HIV-uninfected children can be diagnosed using a combination of clinical and epidemiological features, tuberculin skin testing and chest radiography, as represented in different scoring systems. However, accurate microbiologic diagnosis has become increasingly important for timely use of effective treatment. Mycobacterial culture confirms the diagnosis of TB and provides drug susceptibility data but is not available in most areas with a high TB prevalence. Moreover, culture has poor sensitivity in children who usually have paucibacillary disease. The HIV epidemic has made definitive diagnosis even more challenging due to nonspecific clinical and radiological signs. In high HIV-prevalence areas, scoring systems have been especially variable, lacking sensitivity and specificity. Newer methods for diagnosis are aimed either at detecting the organism or a specific host immune response. Methods for organism detection have focused on collection of better samples, improved culture techniques, molecular methods or antigen detection. Recent advances include the use of sputum induction for obtaining a more reliable specimen, faster and more sensitive culture methods, and rapid detection of the organism and drug resistance based on nucleic acid amplification. Improved methods for detecting a specific host response have largely focused on the use of IFN-g release assays. Even with newer methods, accurately diagnosing childhood TB may be challenging. Greater efforts to obtain a microbiologic diagnosis should be made in children, even in primary care settings. Further research to develop a more accurate, cost-effective and simple diagnostic test for childhood TB is urgently needed.

The influence of bacille Calmette-Guerin vaccine strain on the immune response against tuberculosis: a randomized trial.

RATIONALE Approximately 100 million doses of bacille Calmette-Guérin (BCG) vaccine are given each year to protect against tuberculosis (TB). More than 20 genetically distinct BCG vaccine strains are in use worldwide. Previous studies suggest that BCG vaccine strain influences the immune response and protection against TB. Current data on which BCG vaccine strain induces the optimal immune response in humans are insufficient. OBJECTIVES To compare the immune response to three different BCG vaccine strains given to infants at birth. METHODS Newborn infants in a tertiary womens hospital were immunized at birth with one of three BCG vaccine strains. A stratified randomization according to the mothers region of birth was used. MEASUREMENTS AND MAIN RESULTS The presence of mycobacterial-specific polyfunctional CD4 T cells measured by flow cytometry 10 weeks after immunization. Of the 209 infants immunized, data from 164 infants were included in the final analysis (BCG-Denmark, n = 54; BCG-Japan, n = 54; BCG-Russia, n = 57). The proportion of polyfunctional CD4 T cells was significantly higher in infants immunized with BCG-Denmark (0.013%) or BCG-Japan (0.016%) than with BCG-Russia (0.007%) (P = 0.018 and P = 0.003, respectively). Infants immunized with BCG-Japan had higher concentrations of secreted Th1 cytokines; infants immunized with BCG-Denmark had higher proportions of CD107-expressing cytotoxic CD4 T cells. CONCLUSIONS There are significant differences in the immune response induced by different BCG vaccine strains in newborn infants. Immunization with BCG-Denmark or BCG-Japan induced higher frequencies of mycobacterial-specific polyfunctional and cytotoxic T cells and higher concentrations of Th1 cytokines. These findings have potentially important implications for global antituberculosis immunization policies and future tuberculosis vaccine trials.

Susceptibility of Mycobacterium bovis BCG vaccine strains to antituberculous antibiotics.

ABSTRACT Mycobacterium bovis BCG is one of the most commonly administered vaccines. Complications, including disseminated BCG disease, are rare but increasingly reported in immunodeficient children. There is growing recognition of the importance of differences between BCG vaccine strains. We determined the susceptibilities of five genetically distinct BCG vaccine strains to 12 antituberculous drugs.

Early Detection of Perinatal Tuberculosis Using a Whole Blood Interferon-γ Release Assay

BACKGROUND The diagnosis of perinatal tuberculosis (TB) is problematic because of its nonspecific presentation, the difficulty of obtaining microbiological confirmation, and the unreliability of the tuberculin skin test. Immunodiagnosis of TB has received new attention with the discovery of Mycobacterium tuberculosis-specific immunodominant antigens (early secreted antigenic target 6 [ESAT-6] and culture filtrate protein 10 [CFP-10]) that are encoded by the RD1 region of the pathogen. A whole blood assay has recently been developed to quantitatively measure interferon- gamma production by lymphocytes specific to these antigens, but its evaluation in the diagnosis of TB in infants and children has been limited to date. METHODS In addition to routine diagnostic evaluation (tuberculin skin tests, culture of early-morning gastric aspirate samples, and chest radiographs), 2 infants with suspected perinatal TB were investigated with a whole blood interferon-gamma release assay. RESULTS The results of the tuberculin skin tests were negative for both patients. The findings of the chest radiographs were abnormal with features suggestive of miliary TB. A whole blood interferon- gamma release assay was performed and yielded positive results within 48 h after admission to the hospital for both patients, prompting early antituberculous treatment. M. tuberculosis was cultured after 6 weeks from gastric aspirate samples collected on admission to the hospital from both infants. At 6 months of age, both infants were thriving and had acheived normal developmental milestones. CONCLUSIONS The advent of interferon- gamma release assays may prove to be useful in the evaluation of infants with suspected perinatal TB.

QuantiFERON-TB Gold: state of the art for the diagnosis of tuberculosis infection?

Tuberculosis (TB) remains a major threat to global health. The recently launched Global Plan to Stop Tuberculosis 2006–2015 highlights the need for accurate, simple and low-cost diagnostic tests for the detection of TB infection. For the first time in decades, new diagnostic tools have emerged that may facilitate this goal. The discovery of Mycobacterium tuberculosis-specific immunodominant antigens has led to the development of interferon γ-release assays that have been shown to have high sensitivity and specificity for TB disease. This review focuses on the QuantiFERON-TB Gold tests and addresses the potential strengths and limitations of the current assays, summarizes the available evidence for their use and identifies areas of future research and development. Although representing an advance in TB diagnostics, with the potential to have a significant impact on global TB control, many issues remain unanswered. The cost of the tests and laboratory requirements may limit their use in developing countries. Most importantly, additional studies are needed in TB-endemic regions, particularly in high-risk persons such as children and individuals who are also co-infected with HIV.

Mycobacteria-Specific Cytokine Responses Detect Tuberculosis Infection and Distinguish Latent from Active Tuberculosis.

RATIONALE Current immunodiagnostic tests for tuberculosis (TB), including the tuberculin skin test and IFN-γ release assay (IGRA), have significant limitations, which include their inability to distinguish between latent TB infection (LTBI) and active TB, a distinction critical for clinical management. OBJECTIVES To identify mycobacteria-specific cytokine biomarkers that characterize TB infection, determine their diagnostic performance characteristics, and establish whether these biomarkers can distinguish between LTBI and active TB. METHODS A total of 149 children investigated for TB infection were recruited; all participants underwent a tuberculin skin test and QuantiFERON-TB Gold assay. In parallel, whole-blood assays using early secretory antigenic target-6, culture filtrate protein-10, and PPD as stimulatory antigens were undertaken, and cytokine responses were determined by xMAP multiplex assays. MEASUREMENTS AND MAIN RESULTS IFN-γ, interferon-inducible protein-10 (IP-10), tumor necrosis factor (TNF)-α, IL-1ra, IL-2, IL-13, and MIP-1β (macrophage inflammatory protein-1β) responses were significantly higher in LTBI and active TB cases than in TB-uninfected individuals, irrespective of the stimulant. Receiver operating characteristic analyses showed that IP-10, TNF-α, and IL-2 responses achieved high sensitivity and specificity for the distinction between TB-uninfected and TB-infected individuals. TNF-α, IL-1ra, and IL-10 responses had the greatest ability to distinguish between LTBI and active TB cases; the combinations of TNF-α/IL-1ra and TNF-α/IL-10 achieved correct classification of 95.5% and 100% of cases, respectively. CONCLUSIONS We identified several mycobacteria-specific cytokine biomarkers with the potential to be exploited for immunodiagnosis. Incorporation of these biomarkers into future immunodiagnostic assays for TB could result in substantial gains in sensitivity and allow the distinction between LTBI and active TB based on a blood test alone.

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.

Advances in the Diagnosis of Pulmonary Tuberculosis in HIV-Infected and HIV-Uninfected Children

The identification of improved diagnostic tests for tuberculosis has been identified as a global research priority. Over the past decade, there has been renewed interest in the development and validation of novel diagnostic tools for pulmonary tuberculosis that are applicable to resource-poor settings. These techniques are aimed primarily at improving detection of the organism or a specific host immune response. Although most studies have focused on determining the accuracy of novel tests in adults, it is likely they will also have the capacity to significantly improve the diagnosis of childhood tuberculosis. Improving the quality of clinical samples obtained from children with suspected tuberculosis remains an important research priority while awaiting validation of novel diagnostic tests. This review will focus on a number of recent developments for the diagnosis of tuberculosis, with a specific emphasis on the application of these new tests to children in settings where tuberculosis is endemic.