Optimizing a Conventional Multiplex PCR for Simultaneous Detection of Granulomatous Skin Infection Agents: Leishmania aethiopica , Mycobacterium leprae , and Mycobacterium tuberculosis

Background Conventional polymerase chain reaction (PCR) assays are well‐established molecular techniques that can be integrated as standard diagnostic tools, especially in referral settings. This study aimed to assess the diagnostic potential of a multiplex PCR (mPCR) assay for the diagnosis of cutaneous leishmaniasis (CL), skin tuberculosis, and leprosy. Method A cross‐sectional study was carried out involving 62 patients in the study group, comprising 45 with CL, 9 with leprosy, 4 with skin tuberculosis, and 4 with coinfections. Additionally, 112 positive control DNA samples were analyzed, including 37 of M. tuberculosis , 46 of M. leprae , and 29 of L. aethiopica . The study assessed sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and detection limits. Results Sensitivity and specificity of the mPCR on positive and negative control samples were 100% (95% CI: 96.8%–100%) and 100% (95% CI: 94.9%–100%), respectively. Its sensitivity and specificity among the study group were 75.8% (95% CI: 63.3%–85.8%) and 100% (95% CI: 94.9%–100.0%), respectively. Conclusions With further validation on more clinical suspects, mPCR has the potential to facilitate diagnosis in settings with coendemic CL, leprosy, and skin tuberculosis.