Evaluation of the Performance of an in-House Cyp141–is6110 QPCR System for Tuberculosis Diagnosis

Tuberculosis (TB) is a chronic infectious-contagious disease caused by the Mycobacterium tuberculosis Complex (Mtb). Conventional TB diagnosis is challenging, particularly in extrapulmonary TB cases, due to the paucibacillary nature of samples. In this context, molecular techniques such as Xpert MTB/RIF are sensitive; however, they are standardized for a limited number of biological samples, restricting their use. In-house qPCR stands out as a sensitive, rapid, and more versatile technique for TB detection. Among gene targets, Cytochrome P450 (CyP141) is a complex protein with high sensitivity and specificity, showing strong diagnostic potential. This study aimed to evaluate the performance of a new CyP141 molecular target by in-house qPCR for Mtb detection in patients with and without clinical symptoms of TB. The study was conducted at the Instituto Aggeu Magalhães (Fiocruz Pernambuco) and approved by the Research Ethics Committee (CAAE: 54981421.7.0000.5190 / Opinion n° 5.445.516). Biological samples (sputum, blood, and urine) were collected from 120 participants from SUS hospitals in Pernambuco, divided into TB and non-TB groups. Genomic DNA for CyP141 and IS6110 targets was extracted using a Mini Salting Out protocol with in-house modifications and analyzed by qPCR to assess test performance. Higher sensitivity was observed for the IS6110 target (66.09%), while CyP141 showed sensitivity of 52.14%. Specificity values were similar: 88.46% (CyP141) and 84.31% (IS6110). However, combining both targets in the qPCR system increased sensitivity to 64.96% and specificity to 86.54%, demonstrating good performance, especially in extrapulmonary samples. CyP141 is a promising target for TB diagnosis when combined with IS6110, yielding improved sensitivity and specificity. The combination of both targets shows satisfactory performance, particularly in difficult-to-obtain and paucibacillary samples, and can assist conventional methods, enabling earlier diagnosis and appropriate treatment initiation for TB control in Brazil.