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Interferon-γ assays for the diagnosis of Tuberculosis infection in children
Dr Nikos Spyridis
Department of Paediatric Infectious Diseases, St George’s hospital, London


The diagnosis of tuberculosis infection in the Paediatric population has always been difficult and challenging for Paediatricians and infectious diseases specialists. Lack of systemic signs and symptoms and the difficulty to culture the pathogen from fluid specimens are some of a big list of difficulties that someone will encounter.

Until a few years ago the main diagnostic tools were limited on a chest radiograph and the tuberculin skin test (TST). TST has well known limitations. It requires two visits, shows cross reactivity with some strains of NTM and with prior vaccination with BCG, although that varies with the degree of suspicion and is less significant when there is recent close contact.

Advances in molecular biology have led to an alternative to TST for the diagnosis of infection with Mycobacterium Tuberculosis (MTB). Two in vitro assays have been developed that measure the release of INF-γ from T cells after stimulation by MTB antigens. The two antigens used are the ESAT-6 and CFP-10 that are more specific for MTB. Although several non tuberculous and low infectivity for humans mycobacterial species have the same antigens, those are not included in the BCG strains of Mycobacterium bovis and Mycobacterium avium. The result of that is the lack of cross reactivity of the test with individuals previously vaccinated or infected by Mycobacterium bovis or avium.

The two INF assays currently used are the Quantiferon- TB Gold and the ELISPOT. The two tests use different techniques.

In Quantiferon (www.cellestis.com fig. 1) whole blood is placed in wells and stimulated by the two antigens mentioned before. Following that the plasma is separated and the appropriate conjugate is added. A standard analysis software program is used to provide with the result.



In the second format (ELISPOT, www.immunotec.com) (fig 2), centrifuged blood separates T cells which are again placed in wells containing the specific antigens. Following the production of INF-γ a monoclonal antibody binds to the stimulated cells and stains them. That will form small dots called “spot forming units” that are counted and compared with a negative and a positive control.



A review by Pai et al (1) enumerated 40 research questions that need answers in order to evaluate the performance of those two tests in the diagnosis of latent TB infection and disease. Most studies have shown that those assays are more specific compared to TST for the diagnosis of LTBI but possibly less sensitive especially in younger children (2). Quantiferon also shows relatively higher rates of indeterminate results because of failure to provide positive and negative controls. There are very few data regarding the performance of the test in HIV subjects although there are some promising results recently. A publication from Dewan et al (3) described some very worrying findings where in a cohort of smear positive adults, only 64% of them had positive Quantiferon tests. Similar results have also been published in another study looking on the INF-γ response to treatment in TB confirmed cases (4). A current study taking place at Great Ormond Street hospital in London is looking at the sensitivity of the test in TB proven disease in the Paediatric population, in an attempt to provide evidence on the ability of the test to detect disease. There are still a few challenging questions to answer like: a) ability of those tests to distinguish disease from infection b) the performance on the test in extrapulmonary TB and c) the time frame between infection and a positive INF-γ result.

In summary, the INF-γ assays are useful diagnostic tools for the detection of TB infection especially when TST is not conclusive. The test also seems to be more sensitive in HIV patients when TB is suspected. There is no study comparing the two available assays although it seems that ELISPOT is more sensitive in the Paediatric population. The effectiveness of the test in diagnosing disease is not established yet and for that it should be used with caution and under specialist advice.

References

1. Pai M, Kalantri S, Dheda K. New tools and emerging technologies for the diagnosis of tuberculosis, part II. Active tuberculosis and drug resistance. Expert. Rev. Mol. Diagn. 2006 May; 6(3): 423-32

2. Lalvani A, Pathan AA, McShane H, Wilkinson RJ, Latif M, Conlon CP, Pasvol G, Hill AV. Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen specific T cells. Am J Respir Crit Care. 2006;6: 423-432

3. Dewan PK, Ginsdalde J, Kawamura LM. Low sensitivity of a whole blood interferon-gamma assay for detection of active tuberculosis. Clin Infect Dis 2007 Jan 1;44 (1): 69-73

4. Pai M, Joshi R, Bandyopadhyay M, Naranq P, Dogra S, Taksandre B, Kalandris S. Infection, 2007 April;35 (2): 98-103

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