Tuberculosis (TB) remains a global health priority with an estimated 10 million people worldwide developing TB disease in 2019 (1). Spinal TB is the most common form of osteoarticular TB and involves a chronic inflammatory process that gradually destroys the bony tissue of one or more vertebrae (2). With progression of the disease, those affected may develop serious sequelae such as spinal deformity, spinal instability and neurological deficit (2). These individuals may require costly corrective surgery and are at risk of significant long-term morbidity (2). Conversely, individuals with less advanced disease may be treated with TB medication alone and show largely favorable outcomes (3). Although spinal TB represents only 1-3% of all TB cases (4–6), the absolute number of individuals affected may be considerable in countries with a high burden of TB (7–9), creating a need for vigilance and effective methods of screening and early diagnosis.

Spinal TB is an insidious disease with a variable and non-specific clinical presentation, factors that contribute to a typical diagnostic delay of 4-12 months or more (6–8, 10–12). The most common symptom is chronic back pain, with 83-100% of individuals presenting with this complaint (7, 8, 11–14). However, previous reports suggest that only approximately 23-56% of patients have constitutional symptoms (e.g. fever, weight loss) (7, 8, 11–14) and 7-23% concomitant pulmonary TB (8, 10, 13, 14). With progression of the spinal TB disease, some patients may develop clinical signs such as kyphotic deformity (7, 8, 11–14). However, for the purposes of early diagnosis, there may not always be obvious features to help distinguish spinal TB amidst a high prevalence of mechanical back pain.

Conventional tools to help screen for pathological back pain at primary care level include evaluation of “red flags” and, when available, blood tests for erythrocyte sedimentation rate (ESR) and/or C-reactive protein (CRP) (15, 16). Red flags are patient factors and clinical signs thought to increase the likelihood of serious disease and include items such as unexplained weight loss (17–19). However, the list of potential red flags is lengthy, may vary with different clinical guidelines and is seldom supported by evidence for the diagnostic accuracy of the items (19). For example, there is a paucity of evidence for the effectiveness of red flags in detecting spinal infections (18). Furthermore, this screening approach may have limited sensitivity with a previous study reporting that the absence of red flags did not decrease the probability of a serious spinal pathology diagnosis (20). When considering ESR and CRP, these markers are elevated in most patients with spinal TB but may remain normal in some cases (12). Furthermore, these are non-specific tests of inflammation and patients may present with elevated levels for a variety of other reasons (15, 16).

Given the limitations of existing screening tools, there could be a significant benefit in more accurate point-of-care triage tests to indicate a high probability of spinal TB or diagnostic tools to identify spinal TB, depending on the performance characteristics of such new tools. One approach that may have potential in this regard is to explore a wider variety of diagnostic blood-based biomarkers than ESR and CRP and, in particular, multi-biomarker signatures. This methodology has received considerable attention in the context of pulmonary TB with certain immune biomarkers showing strong diagnostic potential (21–25). For example, in a recent pan-African study, a two-biomarker signature had a sensitivity of 93% and a specificity of 68% for the diagnosis of pulmonary TB regardless of HIV status (26). The ultimate aim of the afore-mentioned research is to develop cost-effective point-of-care triage or diagnostic tests for pulmonary TB in resource-limited settings (22, 27). However, it is possible that a similar approach could be used to assist with other diagnoses such as spinal TB.

Whereas biomarkers for pulmonary TB have been widely reported (21–26), less is known about biomarker profiles in specific forms of extra-pulmonary TB (EPTB), including spinal TB. Furthermore, the few existing studies describing biomarkers in spinal TB did not explore the diagnostic potential of the biomarkers by comparing them between spinal TB and other back pain etiologies (28, 29). The main aim of the current study was to investigate the utility of host biomarkers to distinguish spinal TB from mechanical back pain. A secondary aim was to test the performance of the afore-mentioned two-biomarker signature for pulmonary TB diagnosis (26) when applied to identify spinal TB among the current participants. It was envisaged that the findings of this exploratory study would identify candidate biomarkers for further evaluation as tools for spinal TB screening and diagnosis.

The current study found that patients with spinal TB had experienced symptoms for several months at the time of diagnosis, in keeping with previous reports (7, 8, 12). Furthermore, the study was one of the few (29) to highlight the functional impact of spinal TB with 88% of patients reporting severe disability as a result of the associated back pain. These observations support the need for interventions that facilitate earlier diagnosis of spinal TB, including the potential role of biomarker-based screening.

When investigating the utility of individual biomarkers, Fibrinogen, CRP, IFN-γ, NCAM and Ferritin showed strong potential to distinguish spinal TB from mechanical back pain (AUC ≥ 0.78). Furthermore these biomarkers generally had similar or slightly improved discriminative utility when restricting the analysis to those without HIV infection, those without constitutional symptoms, or those of a comparable age range. Overall, Fibrinogen and CRP were the most promising individual markers, which is notable in that Fibrinogen is one of the major determinants of ESR (15). ESR and CRP are existing screening measures for a potential infective etiology in back pain with levels typically elevated in spinal TB and normal in chronic mechanical back pain (36). ESR was not assessed in the current study and it is unclear whether Fibrinogen has higher discriminative potential than this conventional measure. Nevertheless, the study demonstrated that conventional marker CRP remained one of the most useful individual markers for distinguishing spinal TB from mechanical back pain despite the range of novel biomarkers investigated.

Although Fibrinogen and CRP performed well when differentiating between the current participants, it is likely that these biomarkers would have lower specificity for spinal TB within a primary care context. Furthermore, they may have lower levels earlier in the disease process or remain normal in a proportion of patients with spinal TB (12). Thus, use of a multi-biomarker signature would be expected to improve diagnostic accuracy and help to inform clinical decision-making among a more heterogeneous patient population. The current study identified a five-biomarker signature (CRP, NCAM, Ferritin, CXCL8 and GDF-15) which was able to correctly classify all participants regardless of HIV status. Nevertheless, the ability of this biomarker signature to identify spinal TB requires further validation, including prospective studies in primary care settings, with inclusion of any patient with chronic back pain, regardless of subsequently elucidated etiologies. Future studies should also consider further investigation of biomarkers that were not included in the best-performing biomarker signature, yet appeared in many of the top 20 GDA models. Examples of this include IFN-γ and VEGF-A, which appeared in 17 and 10 of the best 20 models, respectively. PCA explained a relatively low amount of variation in biomarker levels in the current study, suggesting limited potential for linear combinations of biomarkers in this context. Nevertheless, principal component findings supported further investigation of several of the biomarkers identified through other analyses, including Fibrinogen, CRP, VEGF-A and CCL1.

Validation of the current findings in larger studies is particularly important in light of differences in HIV infection, back pain duration and back pain severity between the spinal TB and mechanical back pain groups. Most of the top-performing biomarkers remained significantly associated with spinal TB following adjustment for participant characteristics. However, the sample size and distribution of the data were notable limitations in these analyses. For example, although biomarker associations remained significant in the subgroup without HIV infection, the study was not able to adequately explore the influence of HIV infection on biomarker utility as only two individuals in the mechanical back pain group were HIV infected. Spinal TB is a comparatively uncommon form of TB and it was necessary to conduct the study at a tertiary hospital in order to optimize recruitment. However, limited resources for this exploratory research meant that it was necessary to recruit participants with mechanical back pain from the same setting. As observed in the study, patients with spinal TB may have developed very severe pain by the time of referral to a tertiary hospital and patients with mechanical back pain may have had pain for a long duration before being referred to a tertiary hospital. Overlap in patient characteristics between the different back pain etiologies would be expected to be larger at primary care level, where biomarker-based screening would provide most benefit.

Previous studies suggest differential cytokine responses in different types of TB (37) and the optimal diagnostic biomarker signatures may likewise vary according to type of TB disease (33). Nevertheless, a test with utility across different types of TB would have practical advantages and it is interesting to compare findings when the inclusion of the same biomarkers allows (33). Two previous studies in pulmonary TB included several of the same biomarkers as in the current study and also used the same analysis platform (22, 27). As with the current study, CRP (22, 27), NCAM (27), IFN-γ (22), Ferritin (27) and Fibrinogen (22) were among the top-performing individual biomarkers in one or both of these previous studies, depending on the other biomarkers included. However, whereas both Fibrinogen and CRP had high diagnostic utility in the current study, Fibrinogen appeared to have lower diagnostic utility in pulmonary TB with one study reporting an AUC of 0.70 for Fibrinogen versus 0.86 for CRP (22). Conversely, it was noted that CXCL10 appeared to have higher diagnostic utility in pulmonary TB than in the current study. CXCL10 has received considerable attention as a diagnostic marker for pulmonary TB with an AUC of 0.78 and 0.83 in the afore-mentioned studies and an overall AUC of 0.93 in a recent meta-analysis (38). However, in the current study, CXCL10 was not significantly different between the spinal TB and mechanical back pain groups and had an AUC of only 0.63. Similarly, CXCL10 levels showed poor utility to discriminate between back pain etiology groupings in the PCA. Variation in biomarker utility between different types of TB may be related to factors such as tissue-specific variation in immunopathogenesis and compartmentalization of immune cells (39).

When considering combinations of biomarkers from previous studies, the existing pulmonary TB biosignature of CRP and CCL1 performed well in the current study, achieving an AUC of 0.95 compared to 0.90 in the original test set of suspected pulmonary TB (26). While this is somewhat encouraging, it was noted that CCL1 did not contribute significantly to the model in the current study and the model AUC was the same as that of CRP alone. In the current study, CCL1 was no longer significantly different between spinal TB and mechanical back pain in subgroups without HIV infection or of similar age range and it is possible that this analyte has somewhat less utility for distinguishing spinal TB than pulmonary TB. While this observation requires investigation in larger studies, it makes the point that testing existing biomarker signatures alongside optimal combinations can provide helpful additional insights (40)

The current study had several limitations including the small sample size and the case-control study design. As previously discussed, the study was also limited by differences in certain participant characteristics, such as HIV infection, and was not able to adequately explore the effect of these characteristics on biomarker utility. Due to the exploratory nature of the study, we chose not to correct for multiple testing. The rationale was that such corrections may risk relevant biomarker associations being missed and not carried forward into future studies. Although many of the current biomarker findings are in keeping with previous literature, there remains a risk of false positive results and this constitutes a further limitation of our study.

Although the findings are preliminary, biomarkers that showed potential, either individually or through inclusion in the biomarker models, could be considered candidates for further investigation in larger studies. Given that back pain originating from spinal TB may not always be accompanied by constitutional symptoms, such future studies could consider recruiting all individuals presenting with more than 6 weeks of back pain in a TB endemic setting. These individuals could then be monitored prospectively to detect those diagnosed with spinal TB and those confirmed with an alternative diagnosis. Such studies should also assess the influence of HIV status, including the severity of HIV/AIDS disease, on the diagnostic accuracy of the biomarkers.

In conclusion, the current case-control study evaluated a novel selection of biomarkers for distinguishing spinal TB from mechanical back pain and identified both individual biomarkers and a five-biomarker signature as candidates in this regard. These preliminary findings require validation in larger studies, including prospective cohort studies to assess the accuracy of candidate biomarkers among patients presenting to primary care health facilities with chronic back pain.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This study involving human participants was reviewed and approved by the Health Research Ethics Committee of Stellenbosch University and by hospital management. The patients provided written informed consent to participate in the study.

TM: Conceptualization, investigation, formal analysis, project administration, visualization, and writing—original draft. JD: Methodology, investigation, and writing—review and editing. GW: Methodology, resources, and writing—review and editing. CB: Investigation and writing—review and editing. JT: Resources, supervision, and writing—review and editing. RL: Supervision and writing—review and editing. NC: Methodology, investigation, resources, and writing—review and editing. All authors contributed to the article and approved the submitted version.

The work was supported by the South African Government through the National Research Foundation (NRF), the South African Research Chairs Initiative (SARChi) in TB Biomarkers (Grant number 86535), the Stellenbosch Spinal Surgery Training Trust, the Stellenbosch University Division of Orthopaedic Surgery Research Fund and the South African Orthopaedic Association (SAOA). However, these parties were not involved in the study design; in the collection, analysis and interpretation of the data; in the writing of the report; or in the decision to submit the article for publication. Furthermore, the views expressed are those of the authors and are not necessarily to be attributed to the funders.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.