Tuberculosis (TB) is caused by the Mycobacterium tuberculosis (Mtb) complex. TB is a global health problem, with an estimated 10.6 million new TB cases in 2022 (1). Despite recent molecular developments, diagnosis of TB remains challenging, with one-third of persons with TB globally remaining undiagnosed in 2022 (1). Moreover, about a quarter of the world’s population is estimated to have TB infection (TBI), a persistent immune response to stimulation by Mtb antigens without evidence of TB disease. TBI is essential to the TB elimination strategy (2). However, no specific standard for diagnosing TBI exists. The development of accurate diagnostic tools is necessary and crucial to distinguish between the host immune responses in TBI and TB (3).

TB primarily affects the lungs as pulmonary TB (PTB), and the diagnosis relies on the culture of Mtb, which is considered the gold standard. However, in 2021, only 72.0% of persons diagnosed with TB in the European region received microbiological confirmation, with PTB confirmed in 86.2% of cases (4). Laboratory confirmation of extrapulmonary TB (EPTB) was low even in high-resource settings due to the overlap of TB symptoms with those of other diseases (5, 6), referred to as conditions mimicking TB (CMTB). Further, difficulties in obtaining relevant material and low bacterial load of TB result in suboptimal sensitivity of current diagnostic tools (7). Consequently, new biomarkers to diagnose TB should be less strongly associated with the bacillary load.

The interferon gamma release assays (IGRA) and tuberculin skin test (TST) in Bacillus Calmette–Guérin (BCG) unvaccinated persons can detect immune reactivity toward Mtb with ≥97% specificity (8, 9). However, it is believed that Mtb can be cleared by the innate immune response without a detectable persisting immune response using IGRAs or TSTs and by the acquired immune response with the persisting immune response (10). Currently, there are no tests available to detect this.

Host-based biomarkers have greater potential than pathogen-based biomarkers for diagnosing TB (11). The World Health Organization (WHO) has thus desired non-sputum-based biomarker tests for TB with high specificity (target 98%) and a sensitivity of at least 65% in all groups, including EPTB (12). The host’s response to Mtb is complex and eludes complete characterization (13), but multiple different cytokines, such as interleukin (IL)-4, IL-10, IL-13 (14), interferon (IFN)-γ, IL-12p70 (15), IL-6 (16), IL-8 (17), tumor necrosis factor (TNF)-α (18), IL-1β (19), and IL-2 (20) have been implicated in immune control of Mtb.

However, no single cytokine measurements fulfill the WHO criteria for distinguishing TB from TBI and CMTB (11). Also, no single cytokine biomarker can differentiate between TB and TBI (21). Cytokine levels have successfully been explored to discriminate between PTB and CMTB (22). Thus, better biomarkers distinguishing between the host immune responses in TBI and TB, including EPTB, are needed.

Both innate and adaptive immunity is crucial for Mtb immunity (23, 24). Toll-like receptor (TLR) 2, by forming heterodimers with TLR1 and TLR6, can recognize a range of mycobacterial products (25). TLR4 also recognizes Mtb-derived proteins (mammalian cell entry proteins), lipoproteins, and glycolipids (26). While T-cell immunity toward Mtb may display retarded kinetics (27), T-cell immunity is nevertheless critical for protective immune responses to Mtb (28).

This explorative study aims to determine whether T-cell (CD3- and CD28-stimulated T cells)- and innate immunity (TLR2 and TLR4 induced)-derived cytokines and established biomarkers [hemoglobin, C-reactive protein (CRP), total white blood cell count (WBC), neutrophils, and monocytes] may delineate useful biomarkers to discrimination between 1) TB from CMTB, 2) TB from TBI, and 3) TBI from healthy controls (HC) with a ≥98% specificity as an add-on test to current TB diagnostics.

We investigated the expression of 10 cytokines, hemoglobin, CRP, WBC, neutrophils, and monocytes after ex vivo stimulation of the innate and adaptive immune systems in patients with TB, TBI, CMTB, and HCs. We tested the ability of these biomarkers to differentiate TB from CMTB and TBI to achieve the minimum diagnostic accuracy specified in the WHO target product profile, which requires a specificity of ≥98%. We found that neutrophil counts combined with six cytokines (LPS-IFN-γ, ZYM-IFN-γ, ZYM-TNF-α, ZYM-IL-1β, LPS-IL-4, and ZYM-IL-6) differentiated TB from CMTB with a sensitivity of 52.2% and a specificity of 100%. The study also found that a test consisting of six cytokines (Null-IFN-γ, Null-IL-8, CD3-IL-6, CD3-IL-8, CD3-IL-13, and ZYM IL-1β) discriminated TB from TBI with a sensitivity of 73.9% and a specificity of 100%. Finally, we found no combined test to discriminate TBI from HC with the desirable specificity.

Of previously established biomarkers for infection and inflammation, we found that CRP, WBC, and neutrophil counts increased in TB and CMTB compared to TBI and HC. Meyer et al. evaluated the diagnostic performance of CRP in diagnosing TB in Uganda, a highly endemic country, in 119 patients referred for TB evaluation (34). Using a 10-mg/L cut-off, they found a sensitivity of 78% and a specificity of 52%. In contrast, our data showed higher levels of CRP in CMTB. These findings depend on the patients included in CMTB. Meyer et al. did not provide specific diagnoses of the persons not diagnosed with TB for further comparison. The levels of hemoglobin and WBC in the TB group and HC were consistent with previous findings (35). We interpret no significant difference between definite and probable/possible TB in any of the 45 biomarkers, and the findings similar specificity, sensitivity, and AUCs of the subgroup analysis of definite TB as a low dependency of bacterial load in cytokine response. Previously, no association has been demonstrated between the bacterial burden in culture-positive PTB and unstimulated Luminex-analyzed plasma levels of IL-1β, IL-4, IL5, IL-6, IL-10, and IL-12 (36). However, increasing plasma levels of unstimulated Bio-Plex-analyzed IFN-γ and TNF-α were observed in culture-positive PTB with increasing microscopy smear-grade positivity (37).

This study is unique in relying on innate and adaptive immune stimulation to delineate cytokine responses in TB, CMTB, TBI and HC, suggesting the benefit of incorporating both immune arms in these distinctions. TB patients were characterized by high levels of innate immune system-stimulated cytokines in TB compared to CMTB, which substantiates the importance of both TLR2 and TLR4 in the immune detection of Mtb (25, 26). In addition, T-cell-derived and spontaneously released cytokines helped differentiate TB from TBI, likely reflecting the greater degree of Mtb immune priming in TB compared to TBI. Cytokine differences in unstimulated tubes were generally minor in the current study, and we found no significant differences between TB and CMTB. This finding indicates that stimulation of cytokines is a valuable tool for differentiating TB from CMTB.

Diagnosis of TB remains challenging despite recent developments in sputum-based diagnostics (38). The elevated levels of IL-12, IL-6, and TNF-α after ZYM stimulation is in line with previously described results (39). Other studies have investigated unstimulated or Mtb-antigen-stimulated cytokines. Ren et al. analyzed 12 pro-inflammatory cytokines in sera of patients with PTB, patients co-infected with TB and chronic pulmonary aspergillosis (CPA), CPA patients with tumors or pneumonia (CPA+), and HC. The study likewise found lower WBC and neutrophil counts in TB patients compared to both CPA+ and co-infected TB and CPA (22). The study examined combinations of IL-1β and IL-8, IL-8 and TNF-α, and IL-1β, IL-8, and TNF-α based on significant p-values to discriminate between TB and CPA+. Interestingly, an AUC of ≥0.974 in all combinations was reported. These findings support our utilization of neutrophil counts combined with IFN-γ, TNF-α, and IL-1β cytokine levels to differentiate TB from CMTB and PTB from EPTB, as well as to a different non-TB disease group.

TruCulture-derived cytokine profiles have been evaluated against QuantiFERON ^® -TB Gold in tube after Mtb stimulation in persons with TB and TBI (40). The study found a significant difference in stimulated IFN-γ response between TBI and TB in the TruCulture assay but not in the QuantiFERON ^® -TB Gold in tube, suggesting that the TruCulture assay may be more sensitive.

In the current setup, the analysis of cytokines is expensive and requires advanced laboratory facilities. Assays have been produced for point-of-care testing (41), and TLR4-stimulated cytokine levels have been stimulated in whole blood for a shorter time (4 h) (42) suggesting that cytokine assays could be produced as a point-of-care test with minimum handling and short turn-around time, which is required to have clinical relevance in a high-TB incidence rate low-resource setting. The combined cytokine test have potential as a non-invasive add-on test to established TB diagnostics to detect TB in patients suspected of TB. It is essential to validate these results in larger patient populations and settings with higher TB incidence rate.

The IFN-γ response specific to Mtb is the basis for TBI diagnostics in IGRAs (43), but IGRAs cannot differentiate between TB, TBI, and CMTB with positive IGRA results. Our findings demonstrate the importance of IFN-y in innate immunity to discriminate between TB and CMTB when stimulated by ZYM and LPS and between TB and TBI. This new combined cytokine test shows the potential in discriminating TBI from TB.

The study strengths include the broad group of persons with CMTB suspected of TB and a broad group of TB patients, which makes the test clinically relevant as a guidance in diagnostics for initiation of antituberculous treatment. Second, including all of probable, possible, and definite TB allows for the combined cytokine tests in persons with paucibacillary TB, as the subgroup analysis of definite TB cases showed similar sensitivity and specificity. For different TB subgroups, the use of standardized adaptive and innate immune stimulation, the latter including the Mtb relevant TLR2 and TLR4 receptors, in conjunction with a standardized cytokine panel, enabled us to delineate adaptive and innate immune sensing mimicking in vivo sensing of Mtb.

The main limitations of this study include the small sample size, broad confidence intervals of sensitivity and specificity, and a lack of validation in settings with high TB incidence rates in immunosuppressed individuals, such as persons living with HIV or inflammatory diseases, and in children. The study sample size and selection of cut-offs after data collection may result in overly optimistic test estimates. Nonetheless, the combinations of IL-1β, IL-8, and TNF-α to discriminate between PTB and CMTB were found in a different setting, using another analysis method, and chosen by p-values (22), in contrast to the recursive feature elimination using a random forest model we used to select cytokines, which support the validation of the findings. Further, different assays have previously shown varying sensitivity and specificity (44), and there is currently no reference standardization of cytokine measurements. We did not measure the WBC in the TruCulture tubes, which could implicate the cytokine production during stimulation with more cytokine production with higher WBC that was present in CMTB.

A limitation of the found index test that discriminates between TB and CMTB is TNF-α inclusion, which may limit its value in patients receiving TNF-α inhibitor therapy. It could be clinically relevant to distinguish between the progression of inflammatory bowel symptoms and TB, and validating these findings should include this patient group.

Cytokines as biomarkers have challenges regarding low trace amounts, short half-lives, complex networks of up- and downregulation in responses to the microcellular milieu and changed levels with repeated thawing (45). We studied ex vivo whole-blood cytokines using the highly sensitive and antigen concentration standardized TruCulture assay, which requires minimal blood volume and technical experience to perform (46). We handled the samples within the same short time frame to reduce bias and thawed the samples no more than three times minimizing the risk of changed cytokine levels. We assessed this in a few samples and used the first analysis of the sample to avoid change in cytokine level.

This explorative study suggests that combinations of six stimulated whole-blood cytokines using TruCulture tubes and established biomarkers can differentiate TB from CMTB, and four stimulated and two unstimulated cytokines can differentiate TB and TBI with a high specificity and acceptable sensitivity. This may serve as a promising non-invasive blood-based add-on test in TB diagnostics in adult patients. The sample size was small, and the findings require broader validation including also immunosuppressed patients and children.

The datasets presented in this article are not readily available because of General Data Protection Regulations; https://www.bbmri-eric.eu/wp-content/uploads/BBMRI-ERIC_FAQs_on_the_GDPR_V2.0-1.pdf. Requests to access the datasets should be directed to anne.ahrens.ostergaard@rsyd.dk.

The studies involving humans were approved by Danish National Committee of Health Research Ethics. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

AA: Writing – original draft, Writing – review & editing. SF: Writing – review & editing. MBa: Writing – review & editing. RL: Writing – review & editing. AK: Writing – review & editing. MBo: Writing – review & editing. IT: Writing – review & editing. TJ: Writing – review & editing. OH: Writing – review & editing. CW: Writing – review & editing. SB: Writing – review & editing. MBl: Writing – review & editing. KA: Writing – original draft, Writing – review & editing. IJ: Writing – original draft, Writing – review & editing.