Abstract
Active tuberculosis (aTB) remains a major killer from infectious disease, partially due to delayed diagnosis and hence treatment. Classical microbiological methods are slow and lack sensitivity, molecular techniques are costly and often unavailable. Moreover, available immuno-diagnostic tests lack sensitivity and do not differentiate between aTB and latent TB infection (LTBI). Here, we evaluated the performance of the combined measurement of different chemokines/cytokines induced by two different stage-specific mycobacterial antigens, Early-secreted-antigenic target-6 (ESAT-6) and Heparin-binding-haemagglutinin (HBHA), after a short in vitro incubation of either peripheral blood mononuclear cells (PBMC) or whole blood (WB). Blood samples were collected from a training cohort comprising 22 aTB patients, 22 LTBI subjects and 17 non-infected controls. The concentrations of 13 cytokines were measured in the supernatants. Random forest analysis identified the best markers to differentiate M. tuberculosis-infected from non-infected subjects, and the most appropriate markers to differentiate aTB from LTBI. Logistic regression defined predictive abilities of selected combinations of cytokines, first on the training and then on a validation cohort (17 aTB, 27 LTBI, 25 controls). Combining HBHA- and ESAT-6-induced IFN-γ concentrations produced by PBMC was optimal to differentiate infected from non-infected individuals in the training cohort (100% correct classification), but 2/16 (13%) patients with aTB were misclassified in the validation cohort. ESAT-6-induced-IP-10 combined with HBHA-induced-IFN-γ concentrations was selected to differentiate aTB from LTBI, and correctly classified 82%/77% of infected subjects as aTB or LTBI in the training/validation cohorts, respectively. Results obtained on WB also selected ESAT-6- and HBHA-induced IFN-γ concentrations to provided discrimination between infected and non-infected subjects (89%/90% correct classification in the training/validation cohorts). Further identification of aTB patients among infected subjects was best achieved by combining ESAT-6-induced IP-10 with HBHA-induced IL-2 and GM-CSF. Among infected subjects, 90%/93% of the aTB patients were correctly identified in the training/validation cohorts. We therefore propose a two steps strategy performed on 1 mL WB for a rapid identification of patients with aTB. After elimination of most non-infected subjects by combining ESAT-6 and HBHA-induced IFN-γ, the combination of IP-10, IL-2 and GM-CSF released by either ESAT-6 or HBHA correctly identifies most patients with aTB.
Highlights
Tuberculosis (TB) remains a leading cause of death in the world, responsible for high morbidity and mortality worldwide with about 10 million new cases in 2020 and 1.5 million deaths [1]
We previously reported on a combined HBHA- and ESAT-6IGRA performed on either peripheral blood mononuclear cells (PBMC) or whole blood (WB) to diagnose latent TB infection (LTBI) and to partially differentiate LTBI from active TB (aTB) [14–16]
The best discrimination was obtained by HBHA-induced IFN-g, GM-CSF and IL-2, combined with ESAT-6-induced IFN-g and IL-8 (Supplementary Figure 1A). These markers were further evaluated in different combinations by logistic regression analysis for the training cohort. This analysis identified the combination of HBHA- and ESAT-6-induced IFN-g as optimal to differentiate infected from non-infected subjects with 95% correct classification of the individuals (Figure 1A)
Summary
Tuberculosis (TB) remains a leading cause of death in the world, responsible for high morbidity and mortality worldwide with about 10 million new cases in 2020 and 1.5 million deaths [1]. In addition to classical clinical manifestations of aTB, this disease may occur as subclinical TB without suggestive symptoms, or as extrapulmonary TB often pauci-symptomatic in immunocompromised individuals, so that diagnosis strictly based on clinical signs or symptoms is illusive [2]. The classical diagnostic method remains the identification of M. tuberculosis by direct smear microscopy or by culture that are both low in sensitivity and/or slow. People with subclinical TB may likely be missed if TB culture is not performed and this is often the case in asymptomatic individuals. Molecular techniques such as GeneXpert are more sensitive but they are costly and often unavailable in primarycare settings
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