Tuberculosis (TB) is a common and sometimes fatal transmissible disease, especially in developing countries. Approximately thirty percent of the global population is exposed to the acid-fast bacilli causing TB. Of those who are infected with tuberculosis, ~10% percent develop a clinical manifestation of the disease during their lifetime. From a global perspective, approximately twenty percent of TB infected population live in India. The World Health Organization (WHO) has estimated that one million children develop TB annually worldwide which accounts for about 11% of all TB cases . Tuberculous bacilli most commonly infect lungs. Mycobacterium tuberculosis (MTB) may also spread to extrapulmonary sites including the meninges, lymph nodes, genitourinary tract, skeletal system and skin . Tuberculous meningitis (TBM) is the infection of meninges caused by MTB, with a mortality rate of ~30%. Further, those who survive TBM are usually left with severe neurological defects [3–5]. There is an increased risk of TBM in HIV-infected patients as compared to non-HIV infected cases although the clinical manifestations of the disease do not differ between the two groups [6, 7].
Culturing mycobacteria and subsequent microbiological examination is considered a gold standard for the diagnosis of TBM. However, this method is time consuming and insensitive, with a positive outcome achieved only in 25–70% of clinically diagnosed cases . Although PCR assays can be an alternative rapid approach for diagnosis, they cannot differentiate between latent or active forms of the disease. Although nucleic acid amplification test (NAAT) has a high specificity when tested in body fluids, it lacks adequate sensitivity in cases of meningitis and pleuritis .
Amongst the existing molecular markers, Adenosine deaminase isoenzyme-2 (ADA2) has a sensitivity of 100% and a specificity of 86.4% for the detection of TBM in cerebrospinal fluid (CSF) . Adenosine deaminase (ADA) activity in the CSF of TBM patients has been suggested to be useful for early differential diagnosis of TBM . The ADA activity of CSF and plasma have been evaluated as a diagnostic aid in TBM  and ADA activity in CSF was considered to be a simple, useful and rapid diagnostic test for early recognition of TBM in children . However, overexpression of ADA was also often overexpressed in other forms of meningitis including infections with pyogenic bacteria . In the CSF of TBM patients, the presence of 65 kDa heat shock protein antigen might be a marker for early diagnosis of the disease . High levels of CSF lactate and lactate dehydrogenase levels have also been suggested for diagnosing TBM .
Early diagnosis of TBM is considered a key to effective treatment and prognosis. Approximately 90% of the patients are diagnosed in stage II or III . Overall, the diagnosis of TBM still remains a major challenge due to inadequate current diagnostic methods and poor sensitivity and/or specificity of existing markers. Although corticosteroids are used extensively to reduce mortality and neurological disability, it may not be the only solution to reduce the mortality and morbidity . In TBM, a number of pathological changes including meningeal adhesion, infarction, tuberculoma and hydrocephalus may occur leading to neurological sequelae . These sequelae are known to correlate with the stage of meningitis at admission. Patients treated at an early stage have a five times higher chance of recovery than those with advanced disease stages . Therefore, patient’s clinical condition at admission and delay in starting the treatment are important factors for determining their survival . These findings emphasize the need to focus on identifying candidate molecular markers which can be developed as diagnostic tools in the management of TBM.
Mass spectrometry-based quantitative proteomics has emerged as a powerful approach for identifying and studying disease biomarkers and has become one of the essential tools in biomarker discovery [21, 22]. Advances in quantitative mass spectrometry have led to identification and quantitation of biomarkers which serve as indicators of disease progression, prognosis, drug safety and help to elucidate the mechanism of drug treatment . There are various labeling approaches that one can employ to carry out quantitative proteomic measurements. In vitro labeling methods include Isobaric Tags for Relative and Absolute Quantitation (iTRAQ), Isotope-Coded Affinity Tags (ICAT),
O labeling and in vivo methods include Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and
Nlabeling [24, 25]. iTRAQ labeling is an effective method for studying differential protein expression levels in tissue samples. It has been extensively used for biomarker discovery in various disease contexts [26–34].
In this study, we used an iTRAQ-based quantitative proteomic approach to identify differentially expressed proteins from brain tissues of tuberculous meningitis cases as compared to controls. We identified several proteins which are differentially expressed in TBM. These proteins include both novel and previously reported candidate protein markers. We validated some of these candidate biomarkers using immunohistochemical labeling.