Jun Ma,1,* Li Wang,1,* Xubin Zheng,2 Haiyan Cui,1 Wei Sha1– 3 

1Department of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China; 2Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China; 3Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Haiyan Cui; Wei Sha, Department of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China, Tel +861861687019 ; +8613671758200, Email cuihaiyan23@163.com; shfksw@tongji.edu.cn

Purpose: New economic, rapid, and efficient diagnostic methods are desirable for the control of tuberculosis. This study aimed to evaluate the performance of a dual-gene qPCR melting curve assay (DGPMC) in detecting tuberculosis among patients with suspected pulmonary tuberculosis.
Patients and Methods: The DGPMC assay based on rpoB and IS6110 gene sequences has been established for detection of Mycobacterium tuberculosis. A prospective study was conducted among adult patients with suspected pulmonary tuberculosis from June 2021 to September 2023 at Shanghai Pulmonary Hospital, China. All patients received symptom assessment, high-resolution chest CT scan, and bronchoscopy. Bronchoalveolar lavage fluid was collected for mycobacterial culture and acid-fast staining, GeneXpert MTB/RIF, and DGPMC assay. The diagnostic performance of DGPMC assay was evaluated against the composite reference standard.
Results: Overall, 240 patients were included in this trial, including 80 (33.3%) asymptomatic patients. Clinical diagnosis of tuberculosis was confirmed in 191 (79.6%) patients and 49 (20.4%) patients were confirmed without tuberculosis. The overall sensitivity of the DGPMC assay was 55.0% (95% CI: 47.6– 62.1%), and the corresponding specificity was 85.7% (95% CI: 72.1– 93.6%) in the diagnosis of tuberculosis. The sensitivity of DGPMC assay was higher than that of GeneXpert test (55.0% vs 47.1%, P = 0.038). The Youden index and weighted Youden index of the DGPMC assay were 40.7% and 28.4%, respectively. Subgroup analyses demonstrated that the sensitivity was 32.4% (95% CI: 22.3– 44.4%) in the individuals with negative results for both culture and GeneXpert test. The DGPMC assay performed significantly better than the melting curves based on rpoB gene or IS6110 gene alone (P = 0.0000; P = 0.0020).
Conclusion: The DGPMC assay is an alternative tool favorable for the detection of tuberculosis in patients with suspected pulmonary tuberculosis, especially in the patients with low bacterial load.

Keywords: pulmonary tuberculosis, polymerase chain reaction, melting curve, diagnostic performance