Background/Aims: Crohn’s disease (CD) and intestinal tuberculosis (ITB) remain “difficult-to-differentiate” diseases. We have previously documented peripheral blood frequency of CD4<sup>+</sup>CD25<sup>+</sup>FOXP3<sup>+</sup> T-regulatory cells (Treg) as a biomarker to differentiate CD and ITB. We tried to validate these results in a larger cohort of CD and ITB patients. Methods: Seventy treatment naïve patients of CD (n = 23) and ITB (n = 47) (diagnosed by standard criteria) were recruited prospectively from October 2016 to May 2017. Patients with history of antitubercular therapy in the past were excluded. The frequency of Treg cells in peripheral blood was determined by flow cytometry, and compared between CD and ITB patients. Results: Similar to our previous study, frequency of Treg cells in peripheral blood was significantly increased in ITB as compared to CD patients (40.9 [interquartile range, 33–50] vs. 24.9 [interquartile range, 14.4–29.6], <i>P</i>< 0.001). Further, the receiver operating characteristics curve also showed good diagnostic accuracy with an area under the curve (AUC) of 0.77 (95% confidence interval, 0.65–0.89) and a FOXP3<sup>+</sup> cutoff value of > 31.3% had a sensitivity and specificity of 83% and 82.6% respectively, to differentiate ITB from CD. Even for the indeterminate cases (n = 33), Treg cell frequency had similar diagnostic accuracy with an AUC of 0.85 (95% confidence interval, 0.68–0.95) and a cutoff of 32.37% had sensitivity and specificity of 87% and 95% respectively, to differentiate ITB from CD. Conclusions: The current findings validate that the increased frequency of CD4<sup>+</sup>CD25<sup>+</sup>FOXP3<sup>+</sup> Treg in the peripheral blood can be used as a biomarker with high diagnostic accuracy to differentiate ITB from CD.
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