Interferon- Release Assays for Diagnosis of Tuberculosis Infection in Children

Advances in molecular biology and genomics have led to an alternative to the tuberculin skin test (TST) for the diagnosis of infection with Mycobacterium tuberculosis. Two new in vitro assays have been developed that measure interferon(IFN) released by sensitized T cells after stimulation by M. tuberculosis antigens. Whereas early versions of IFN release assays used purified protein derivative as the antigen, the newer versions use 2 antigens (early secreted antigen target-6 and culture filtrate protein-10) that are more specific for M. tuberculosis. Although several species of nontuberculous mycobacteria (NTM) that rarely cause human disease have early secreted antigen target-6 and culture filtrate protein-10 antigens, these antigens are not shared by CalmetteGuérin bacillus (BCG) strains of Mycobacterium bovis, and common strains of NTM such as Mycobacterium avium complex (Table 1). Two major formats of IFN release assays have been developed. Currently the only one licensed in the United States is QuantiFERON-TB GOLD (QFT; Cellestis Ltd., Carnegie, Australia). The U.S.licensed version uses a 24-well culture plate, whereas a simplified in-tube format is available in countries other than the United States. The patient’s heparinized whole blood is placed in wells, and the stimulating antigens are added. The plasma is then taken off, and to it the appropriate conjugate, then substrate, are added. OD450 of each well is determined, then the results are calculated with the use of a standard analysis software program. A positive (phytohemagglutinin) and negative control is used with each patient sample. The second format that is widely used in Europe is the enzyme-linked immunospot (ELISPOT), commercially available as the T-SPOT.TB test (Oxford Immunotec Ltd., Oxford, United Kingdom). In this test, individual peripheral blood mononuclear cells are isolated and then incubated in the presence of the stimulating antigens. When IFN is produced by individual cells, it binds to specific antibody on the base of the ELISPOT wells, producing spots. These spots are developed and counted, the result being expressed as the number of spot-forming cells, and this is compared with the number of spots in negative and positive controls. The impetus for developing these new tests was the well-known limitations of the TST. The TST requires 2 patient encounters whereas the IFN release assays require only 1. The TST shows cross-reactivity with some strains of NTM and with prior vaccination with BCG, although the degree of cross-reaction is variable and of less significance when the patient has had recent close contact to a case of tuberculosis (TB). The TST is also subject to boosting, or increasing reactivity upon repeat testing in a patient sensitized to 1 or more of the myriad of antigens in purified protein derivative. The IFN release assays are performed in vitro; therefore boosting does not occur. Finally the sensitivity and specificity of the TST are less than desired, and limit its use in certain epidemiologic circumstances. The Centers for Disease Control and Prevention (CDC) in December 2005 published updated guidelines for the use of QFT. The CDC recommends that QFT be used in all circumstances in which the TST is currently used, including contact investigations, evaluation of immigrants and serial testing of health care workers. Although the CDC guidelines acknowledge that there are few data regarding the performance of the QFT in children and adolescents in any clinical or epidemiologic circumstances, the guidelines clearly imply that QFT can be used for children. Subsequent to the issuance of these guidelines, a paper has been published that examines QFT in children. In this study from Australia, 101 children with a high risk of TB infection or disease were studied with a TST and QFT. There was poor correlation of the 2 tests ( statistic 0.3) with 70% of the 37 children with a positive TST having a negative QFT test. There were no instances of a positive QFT assay with a negative TST, and the QFT assay was positive in all 9 children with TB disease. The positive control QFT responses were significantly correlated with age and, in children with TB infection identified by TST, those with a positive QFT were significantly older (12.9 years versus 6.9 years). Disturbingly 17% of the QFT tests yielded inconclusive results due to failure of the negative or positive control assays. More information about use of the QFT assay in children is needed. Several studies have been published that used the ELISPOT technique in children. One study examined a school tuberculosis outbreak in the United Kingdom and showed that, compared with the TST, the ELISPOT assay demonstrated improved sensitivity in children, as reflected by better correlation with the degree of exposure to an active case of tuberculosis. This test also has had a similar performance in human immunodeficiency virus-infected children treated for probable TB disease. A study performed in Turkey on 979 child household contacts of adult patients with sputum-smear positive tuberculosis demonstrated that children From Baylor College of Medicine, Houston, TX Copyright © 2006 by Lippincott Williams & Wilkins ISSN: 0891-3668/06/2510-0941 DOI: 10.1097/01.inf. 0000239685.49999. d3