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Diagnosis of Active Tuberculosis in China Using an In-House Gamma Interferon Enzyme-Linked Immunospot Assay
Authors:Xinchun Chen  Qianting Yang  Mingxia Zhang  Michael Graner  Xiuyun Zhu  Nicolas Larmonier  Mingfeng Liao  Weiye Yu  Qunyi Deng  Boping Zhou
Affiliation:Shenzhen Institute of Hepatology, the Affiliated Shenzhen Third Hospital, Guangdong Medical College, Shenzhen 518020, China,1. Shenzhen-Hong Kong Institute of Infectious Disease, Shenzhen Donghu Hospital, Shenzhen 518020, China,2. Department of Neurosurgery, University of Colorado Denver, Aurora, Colorado 80045,3. Department of Pediatrics and Immunobiology, BIO5 Institute and Arizona Cancer Center, University of Arizona, Tucson, Arizona 857214.
Abstract:
Gamma interferon (IFN-γ) release assays have been proven to be useful in the diagnosis of Mycobacterium tuberculosis infection. Nevertheless, their specificity and sensitivity vary among the different populations studied. Here, we evaluate the value of an in-house IFN-γ enzyme-linked immunospot (ELISPOT) assay in the diagnosis of active tuberculosis (TB) in Shenzhen, China, where the prevalence of tuberculosis is severe and Mycobacterium bovis BCG vaccination is mandatory at birth. A total of 305 patients with active tuberculosis, 18 patients with nontuberculosis lung diseases, and 202 healthy controls were recruited in this study. Among them, 156 individuals were simultaneously tested for IFN-γ responses by the commercial QuantiFERON-TB Gold in-tube (QFT-IT) assay. Tuberculin skin tests (TST) were performed with 202 healthy controls. The overall sensitivities of the ELISPOT and QFT-IT assays for active tuberculosis were 83.60% and 80.85%, respectively; the specificities were 76.6% and 73.26%, respectively. The IFN-γ ELISPOT responses, but not those of the TST, were significantly correlated with TB exposure (r = −0.6040, P < 0.0001). The sensitivities of the ELISPOT assay varied for patients with different forms of tuberculosis, with the highest sensitivity for patients with sputum-positive pulmonary tuberculosis (89.89%) and the lowest for those with tuberculous meningitis (62.5%). In conclusion, the IFN-γ ELISPOT assay is a useful adjunct to current tests for diagnosis of active TB in China. The ELISPOT assay is more accurate than TST in identifying TB infections.Tuberculosis (TB) is a leading cause of morbidity and mortality throughout the world, with 95% of cases and 97% of all deaths occurring in high-prevalence countries, such as China, where the prevalence of active TB is as high as 367/100,000 population (10). For the effective and efficient control of TB in these countries, rapid diagnosis and treatment for active-TB patients are the mainstays of the TB control program. However, the current widely used tests, including acid-fast staining of sputum, mycobacterial culture, and antibody test, are not satisfactory for this purpose (4).Recently, commercial immunodiagnostic tests for TB infection have been introduced. These tests are based on the Mycobacterium tuberculosis-specific antigens early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) and include a whole-blood gamma interferon (IFN-γ) enzyme-linked immunosorbent assay (QuantiFERON-TB Gold in-tube [QFT-IT]; Cellestis Ltd, Victoria, Australia) and an enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB; Oxford Immunotec, Oxfordshire, United Kingdom). Both tests have shown promising results in the detection of latent TB infection (LTBI) (1, 11). In addition, some clinical data also suggest the potential to use these IFN-γ assays for the differential diagnoses of active tuberculosis (3, 8, 9). However, the sensitivities and specificities of these assays varied among the different populations studied, due mostly to the different HLA genetic backgrounds, the prevalence of TB infection, and the coverage of Mycobacterium bovis BCG vaccination (11).In contrast to the wide use of IFN-γ assays for the diagnosis of M. tuberculosis infection in Europe and America, the utilization of IFN-γ assays in China is scarce, and no data are available to evaluate the diagnostic value of the QFT-IT assay mainly because of the high cost of these kits. Since China ranks second on the list of 22 countries with the highest tuberculosis burden in the world (11), the aim of this study was to evaluate the usefulness of IFN-γ assays in the diagnosis of active tuberculosis in the Chinese population in mainland China. Thus, we developed and evaluated an in-house IFN-γ ELISPOT assay by using recombinant ESAT-6 protein and peptide pools derived from ESAT-6 and CFP-10 for diagnosis of active TB. We also compared the performance of our ELISPOT assay with that of the commercial QFT-IT assay and analyzed the influence of clinical manifestation on the accuracy of the ELISPOT assay.
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