Immunological aspectsDiagnostic performance of multiplex cytokine and chemokine assay for tuberculosis
Introduction
Despite availability of inexpensive and relatively effective therapy, tuberculosis (TB) continues to be a major global health problem.1, 2 It is estimated that 9.27 million new cases of TB occurred in 2007 and 4.1 million were smear-positive.3 Due to low efficacy of BCG vaccine, control of this disease largely depends on early diagnosis and treatment.
The current TB diagnosis mainly depends on detection of mycobacteria by acid-fast staining and/or fluorescent microscopy, as well as by bacterial culture that can also be used for subsequent drug susceptibility tests.4 Sputum smear microscopy has low sensitivity and Mycobacterium tuberculosis culture takes at least two weeks, normally 4–8 weeks, which often lead to delays in diagnosis and treatment.5 Fast nucleic acid-based assays have been developed, with variable sensitivity for direct identification of M. tuberculosis from clinical samples.6
IFN-γ release assays (IGRAs) have been introduced into clinical practice for the diagnosis of M. tuberculosis infection, and are used together with other tests to assist diagnosis of active TB in many countries.7, 8, 9, 10, 11 The current commercially available IGRA kits, such as T-SPOT.TB and QuantiFERON-TB Gold in-tube, depend on detection of a single cytokine, IFN-γ, which are influenced by many parameters. Due to high rate of latent TB infection (LTBI) in high-burden countries, current IGRAs have relatively low performance in diagnosis of active TB.12 A number of other cytokines produced from specifically stimulated cells were also explored for the diagnosis of TB, such as IP-10, IL-2 and TNF-α, and some were found to be promising biomarkers.13, 14, 15
Microarray analysis of whole blood identified 393 transcripts that can discriminate active TB from latent infection.16 Flow cytometric detection of M. tuberculosis antigen-specific TNF-α,13 and/or IFN-γ/IL-2 responses has been found to be promising in diagnosis of active TB.17 It is reported that proportions of multifunctional CD4+ T cells that express IFN-γ, IL-2 and TNF-α were significantly different in individuals with active TB and latent TB infection, and were associated with responses to anti-TB chemotherapy.18 Therefore, multiple biomarker detection might have the potential to improve the accuracy and sensitivity of TB diagnosis.
Multiplex microbead-based assay allows for detection of many different soluble proteins simultaneously from the same sample.19 To develop new microbead-based multiplex assay for TB diagnosis, we initially examined 16 soluble proteins, including cytokines, chemokines and some other molecules that involve in immune and inflammatory responses to mycobacterial antigen stimulation, and compared their expression levels in patients with active TB and healthy controls. Five cytokines/chemokines including IFN-γ, IP-10, MIG, TNF-α and IL-2 that showed most significant differences between active TB patients and healthy controls were selected for the multiplex assays and evaluated for diagnostic performance in high TB burden settings.
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Study subjects
In this study, 178 patients with active pulmonary TB, 156 healthy control individuals and 35 patients with bacterial pneumonia or lung cancer were recruited. Patients with active pulmonary TB were recruited from the TB Clinical Center of the Institute of Tuberculosis, 309 Hospital, Beijing, China (Table 1). Diagnosis of pulmonary TB was based on positive sputum smears and/or culture results and chest radiography. For patients with negative bacterial culture results, diagnosis was established by
Simultaneous detection of multiple cytokines and chemokines by microbead-based assay
In our preliminary studies, 16 proteins including IL-1α, IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF-α, TGF-β1, IP-10 (CXCL10), MIG (CXCL9), MCP-1 (CCL2), MIP-1β (CCL4), EGF, sCD40L, VEGF-A were measured by using culture supernatant of PBMCs from patients with active pulmonary TB and healthy control individuals after stimulation with ESAT-6 and CFP-10 peptides pool. Five cytokines, IFN-γ, IP-10, MIG, TNF-α and IL-2, that showed most significant differences between the active TB patient and
Discussion
Due to low sensitivity of mycobacterial smear/culture methods for diagnosis of TB, alternative diagnostic methods are explored extensively, especially for smear/culture-negative TB patients.4, 5 IGRA is considered a breakthrough in TB immunodiagnostic and has high specificity and sensitivity for detection of M. tuberculosis infection, however, it was reported that it has relatively low sensitivity in detection of active TB.12, 22 We hypothesize that multiplex biomarker detection might improve
Ethical approval
The study was approved by the Ethics Committee of the Beijing 309 Hospital and informed consent was obtained from all participants.
Funding
The study was supported by grants from National Natural Science Foundation of China (81071318, 81071319 and 81061120518), a grant from Beijing Natural Science Foundation (7092100) and a grant for infectious diseases from Ministry of Health and Ministry of Science and Technology, China to XC (2008ZX10003-012).
Conflict of interest
None.
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- c
Both authors contributed equally to the work.