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In this study, we investigated the possible association between TB and Toxoplasma gondii infection. One hundred confirmed TB individuals living in northwest Iran were classified into three subgroups; newly diagnosed patients (NTB), old diagnosed patients (OTB) and multidrug resistance patients (MDR-TB). One hundred healthy subjects in the same age and sex distribution were ethnically matched. Sera samples were screened for anti- Toxoplasma antibodies. Nested-PCR was performed by targeting the B1 and GRA6 genes. The frequency of Toxoplasma infection based on IgG titer was 71.1% in the OTB subgroup and 33% in the control group, indicating significant association between Toxoplasma seropositivity and OTB (P = 0.001). According to phylogenetic network, the type I strain of Toxoplasma was identified in the OTB subgroup (10.1%). We concluded that patients with OTB subgroup are at high risk for acquisition of Toxoplasma infection which could reactivate the latent toxoplasmosis. • Toxoplasma gondii is commonly circulating among tuberculosis patients in northwest Iran. • Chronic tuberculosis patients are at higher risk for acquisition of Toxoplasma infection. • Serological assays should be performed among TB patients, aiming to prevent severe toxoplasmosis. [ABSTRACT FROM AUTHOR]