The ability to diagnose TB infection, and distinguish active TB from LTBI by measurement of a limited number of analytes on a small amount of blood in an overnight assay would be a major advance over the currently available TB diagnostic tests. In this study, we have shown for the first time that multiple biomarkers measured in QFT test supernatants have high ability to discriminate between active TB and the absence of active disease. This has significant implications for the diagnostic utility of the QFT test. The top single markers were EGF and sCD40L. Three-marker combinations of EGF with MIP-1β, sCD40L, IL-1α or VEGF showed promising results with the top model comprising EGFNil, EGFAg-Nil and MIP-1βAg-Nil.
The ability of these markers to differentiate between different M. tb
infection states is probably a reflection of successful and unsuccessful immunological responses against the pathogen. The successful control of M. tb
infection by the host immune response is largely dependent on T-cells, macrophages and a balance between pro-inflammatory and regulatory cytokines and chemokines. Pulmonary TB granulomas, including areas of caseous necrosis, are rich in growth factors such as EGF, TGF-α and VEGF and provide good growth environments for mycobacteria, including M. tb
]. In addition to enhancing the growth of mycobacteria within granulomas, Bermudez and co-workers [14
] showed that both M. tb
and M. avium
express receptors for EGF. VEGF, an angiogenesis mediator, has been associated with disease activity in both pleural TB and TB meningitis [16
] and levels decline after successful TB treatment [18
]. Both the unstimulated and TB antigen stimulated levels of these growth factors were higher in TB patients than in LTBI in this study.
MIP-1β and IL-1 are produced by macrophages. MIP-1β is known to modulate macrophage functions, is an important mediator of chronic inflammatory processes [19
], and is a potent macrophage, lymphocyte and specifically activated CD4+ lymphocyte chemo-attractant [20
]. IL-1 favours a TH1 immune response [21
] and has been shown to play an important role in the formation of granulomas [22
] along with TNF-α. Although the Mann-Whitney U test showed no significant differences between the unstimulated levels of MIP-1β, and both the unstimulated and M. tb
antigen stimulated levels of IL-1α in the different TB infection states, multivariate analysis showed that lower levels of both markers were characteristic of active disease.
CD40L, is a costimulatory molecule that is expressed on activated CD4+ T cells and is involved in their activation and development of effector functions [23
]. Mizusawa and co-workers [24
] reported significantly higher plasma levels of sCD40L in patients with cavitary TB lesions, compared to those without such lesions. The median levels of sCD40L were higher in TB patients in the present study. While there was no significant difference in the median unstimulated and the antigen stimulated levels in the non-diseased group, unstimulated levels were higher than the antigen stimulated levels in TB patients. Because patients in this study were not classified according to the extent of disease on X-ray, future studies will have to investigate the effect of disease severity on test performance.
Indeterminate results have been an issue of concern, and are often reported in IGRA studies. They frequently occur in immonocompromised subjects [25
] and have also been observed in children under the age of 5 years [26
]. Previous reports have highlighted the potential roles of IP-10, IL-2 and MCP-2 alongside IFN-γ in diagnosing M. tb
]. These studies revealed that combining IFN-γ and IP-10 measurement in QFT supernatants enhances the sensitivity for diagnosing M. tb
infection and decreases the proportion of indeterminate results [27
]. We also observed very high levels of IP-10 in our 29-plex measurements, and which correlated with IFN-γ levels (r = 0.51, p = 0.009) but like IFN-γ, IP-10, which helps to amplify IFN-γ responses by its effects on macrophages, does not differentiate between active TB and LTBI. IFN-γ or IP-10 detection could be used in a first step of a test based on M. tb
antigen stimulated whole blood culture to diagnose (M. tb
) infection. A second step of the test could be performed if positive IFN-γ/IP-10 results are obtained to measure three-marker combinations of EGF, sCD40L, MIP-1β, VEGF, IL-1α or TGF-α levels by ELISA or multiplex cytokine assays, to differentiate individuals with active TB from LTBI. Future larger studies should evaluate both QFT positive and negative participants to ascertain whether a one-step three-marker test is sufficient to diagnose active TB or whether a two-step strategy consisting of a conventional QFT test followed by a three-marker assay in those with a positive QFT result yields the best results. Either approach may allow diagnosis of TB disease with high accuracy within 24 hours after presentation of a patient at the health care service, with only 3 ml of blood and without the requirement of a second visit by the patient.
The levels of some of the markers investigated in this study (EGF, sCD40L and VEGF) were lower in the TB antigen stimulated than in the unstimulated QFT tubes. The reasons for this difference might relate to the expression kinetics of the different markers after stimulation with the TB antigens. Another explanation could be that markers are consumed due to possible co-expression of soluble or membrane bound receptors after stimulation. The actual mechanism behind this observation is beyond the scope of this small study and may need to be investigated further in future studies. It has been suggested that some heparinized blood collection tubes may contain endotoxin, which may induce cytokine production during subsequent culture. In the present study blood samples were collected in heparinized tubes prior to transfer to the QFT tubes whereas the manufacturers recommend collection directly in the QFT tubes which are endotoxin free. Possible endotoxin contamination, however, would not explain the higher levels of some analytes in unstimulated than in stimulated samples as the blood from each participant would be collected in a single heparinized tube and both samples would be exposed to the same level of contaminants. Furthermore, as the levels in unstimulated samples were generally not very high, it is unlikely that endotoxin could have obscured significant analyte production in antigen stimulated samples. We have previously also observed the same pattern of higher unstimulated than stimulated analyte levels in samples that were collected directly into QFT tubes (unpublished data). Future studies should employ collection directly into QFT tubes.
The main limitation of our study is the relatively small number of study participants and the cross – sectional design. Longitudinal cohort studies will be required with careful clinical characterization of participants into TB infection and disease groups to validate the accuracies and the cut-off values of the markers identified in this study. This will require a prospective study whereby misclassification of active and latent TB by these cytokine combinations is noted. Future studies should also access the utility of the three-marker tests in smear negative TB, extrapulmonary TB, immune compromised subjects (especially HIV infected patients), children and people with other lung infections like acute bacterial pneumonia. Additional biomarkers should also be evaluated as new multiplex assays become available. Additionally, development of suitable point-of-care tests will be needed, using easy-to-use, readily accessible and less costly techniques like ELISA assays (as opposed to Luminex).