The present study conducted a preliminary investigation of the diagnostic value of Rv1733c/Rv1733c SLP, the mycobacterium tuberculosis latency-associated antigen, combined with the EAST-6/CFP-10-Fluorospot for the differential diagnosis of ATB and LTBI.
During LTBI, Mtb is contained within granulomas and is composed mainly of activated macrophages. It recruits inflammatory cells and can be isolated from the infected cells in an organized structure. In this way, it creates an environment associated with containment of Mtb infection, dissemination and multiplication. The immune response inside the granuloma generates a set of particular conditions that include oxygen deprivation, low pH and nutrient starvation, in which Mtb is able to enter a defined non-replicating state [15, 16]. Several in vitro models have shown that Mtb is capable of an extensive repertoire of metabolic realignments that can cause its adaptation to the variety of environmental stresses. However, when the host's immunity or the signals maintaining granuloma structure are reduced, dormant Mtb is activated, which proliferates and disseminates, eventually developing into active tuberculosis [17, 18]. Although bacterial and host factors that induce and maintain latent M. tuberculosis infection are ill defined, recent studies have shown that during the dormancy of Mtb, the expression of dormancy survival regulon (DosR) is upregulated. Through the interaction of dormancy-associated proteins with macrophages, the formatting and maintenance of the granuloma structure is required to allow Mtb to transit into a stage of dormancy . It is worth noting that the immune response to the DosR regulon encoded antigens is associated with the containment during latent phases of M. tuberculosis infection, since the antigens encoded by several DosR regulons can be preferentially recognized by the T cells of LTBI subjects, in order to induce the activation of T cells associated with the control of Mtb infection. This process in turn produces various cytokines, such as IFN-γ, TNF and IL-2, preventing the progression of ATB [20, 21]. Similarly, following injection into the body as a preventive vaccine, the DosR regulon encoded antigens can reduce the Mycobacterium tuberculosis infection in the lung . Therefore, the immune response against these antigens may contribute in controlling latent M. tuberculosis infection and preventing reactivation of tuberculosis [23, 24]. Previous studies have shown that while responsiveness to early secretory antigenic target 6 is an optimal marker of M. tuberculosis infection, a strong response to the dormant antigen is largely restricted to latently infected individuals, offering the possibility of differential immunodiagnosis or therapeutic vaccination against TB .
Leyten et al.  stimulated human PBMCs with 25 types of proteins expressed by DosR regulators. The results indicated that Rv1733c-stimulated LTBI subjects produced higher levels of IFN-γ compared to those of ATB patients. The present study demonstrated that the levels of IFN-γ produced by Rv1733c in the LTBI group were almost equal with those of the ATB group, which was different from Leyten’s results. Furthermore, the results of the present study indicated that the levels of IL-2 produced by T cell stimulation with Rv1733c in the LTBI group were higher than those of the ATB group. In addition, these findings may be attributed to the degree of immune response required for stimulation by Rv1733c being too weak or the small sample size, which resulted in no significant differences between the two groups.
In accordance with the results of Mariateresa Coppola et al. , the present study demonstrated that Rv1733c SLP could be well recognized by T cells of LTBI subjects and produced higher levels of IFN-γ than Rv1733c. Therefore, Rv1733c SLP exhibited stronger immunogenicity and protection ability. Consistent with the findings of Abebech Demissie et al. , the latency-associated antigen (Rv2031c) mainly induced a strong immune response in LTBI individuals, which could be used for differential immune-diagnosis of tuberculosis. In the present study, the frequency of T cells following stimulation with Rv1733c SLP in the ATB and LTBI groups confirmed that Rv1733c SLP induced higher number of T cells to produce higher levels of cytokines in the LTBI group, while the difference in the frequency of single IL-2-secreting T cells was significant between the two groups. Therefore, the latency associated antigen Rv1733c SLP may be possibly used to distinguish between ATB and LTBI.
The results of the ESAT-6/CFP-10-Fluorospot indicated that the frequency and proportion of single IFN-γ-secreting T cells in the ATB group were significantly higher than those in the LTBI group (P < 0.05), whereas the frequency and proportion of single Il-2-secreting T cells in the ATB group were significantly lower than those in the LTBI group (P < 0.05). This is consistent with our previous results . On this basis, the immune response against Rv1733c SLP could improve the diagnosis of latent infection.
Clinical diagnosis is a dynamic process. The pretest probability of the disease can be estimated by clinical analysis based on the comprehensive medical history, physical examination and laboratory results . A certain diagnostic test is performed on the basis of the pretest probability, which may increase or decrease the possibility of initial diagnosis. The disease probability at that time is called the posttest probability. The estimation of the LR  of a diagnostic test can distinguish the posttest probability from the pretest probability, reflecting the extent to which the results of a diagnostic test will increase or decrease the pretest probability of the target disease . The data of the current study indicated that the combination of the ESAT-6/CFP-10-fluorospot with the Rv1733c SLP resulted in an increase in the positive likelihood ratio from 3.64 to 6.3 and in the positive predictive value from 72.22–81.82% when the serial test was used. In contrast to these findings, the parallel test decreased the negative likelihood ratio from 0.17 to 0 and increased the negative predictive value from 88.89–100%. Rv1733c SLP combined with ESAT-6/CFP-10-fluorospot can improve the accuracy of differential diagnosis between ATB and LTBI subjects. Therefore, parallel or sequence tests can be selected to improve sensitivity or specificity, respectively in order to aid the exclusion or diagnosis of ATB.
The advantages of the present study can be highlighted as follows: 1) The representative M. tuberculosis latency associated antigens Rv1733c and Rv1733c SLP were added on the basis of the M. tuberculosis specific antigens ESAT-6 and CFP-10; 2) The IFN-γ/IL-2 Fluorospot method was used to simultaneously detect the secretion of IFN-γ and IL-2 cytokines at the single cell level, saving manpower and blood samples to the greatest extent. The limitations of the present study can be summarized as follows: 1) The small sample size and large confidence intervals, which decrease the accuracy of the results; 2) The case-control study design and the fact that all ATB patients were pathogen-conformed, which may amplify the diagnostic accuracy. The accuracy of the differential diagnosis of ATB and LTBI requires further prospective cohort studies for confirmation.