Diagnostic performance of real-time PCR for routine diagnosis of leprosy utilizing slit skin smear from clinically diagnosed patients in Ethiopia

Abstract

Background Ethiopia contributes 1.3% (n = 2500) of the estimated global annual incidence of leprosy, a stigmatising neglected tropical disease. Mycobacterium leprae (M. leprae) is non-cultivable on artificial media, challenging diagnosis and research. Diagnostic challenges include low sensitivity of acid-fast bacilli (AFB) microscopy in paucibacillary (PB) cases and variability in immunologic tests. To improve detection, this study evaluated real-time PCR (qPCR) targeting the RLEP region of M. leprae in slit skin smear (SSS) samples. Methods A cross-sectional study conducted from October 2023 to February 2024 examined 124 SSS samples from individuals with clinical leprosy and 12 controls without a history of the disease. Quantitative PCR (qPCR) was performed using SYBR Green chemistry. Statistical analyses are carried out utilising MedCalc to determine sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and 95% confidence intervals. A significance level of p ≤ 0.05 was adopted for all analyses. Results Among 124 patients with clinically diagnosed leprosy, 89 (71.8%) were confirmed positive through histopathology, while 35 (28.2%) were negative. When evaluated against clinical diagnosis, qPCR demonstrated a sensitivity of 91.1% (95% CI: 84.7–95.4%) and a specificity of 100% (95% CI: 73.5–100%). Using histopathology as a reference standard, qPCR showed a sensitivity of 95.5% (95% CI: 88.9–98.8%). A significant correlation was observed between qPCR and histopathological findings (χ2 = 28.052, P < 0.0001). All negative control samples (n = 12) and specimens of other mycobacteria tested negative by qPCR, indicating the assay's high analytical specificity. Conclusion The qPCR exhibits high sensitivity and a strong correlation with histopathology, while agreement with the clinical diagnosis is excellent. Nonetheless, multicenter studies are necessary to further confirm its accuracy and reproducibility in diverse geographic regions before it can be widely adopted in clinical practice.